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GSBS Dissertations and Theses
Graduate School of Biomedical Sciences
December 1999
Identification of the Human Erythrocyte Glucose
Transporter (GLUT1) ATP Binding Domain: A
Dissertation
Kara B. Levine
University of Massachusetts Medical School
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Levine, KB. Identification of the Human Erythrocyte Glucose Transporter (GLUT1) ATP Binding Domain: A Dissertation. (1999).
University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 247. http://escholarship.umassmed.edu/gsbs_diss/
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IDENTIFICA TION OF THE HUMAN ERYTOCYT GLUCOSE
TRANSPORTER (GLUTl) ATP BINDING DOMAIN
Dissertation Presented
Kara B. Levine
Submitted to the Faculty of the
University of Massachusetts School of Biomedical Sciences, W orces ter
in partial fulfilment of the requirements for the degree of
Doctor of Philosophy
December
1 5
1999
Cellular & Molecular Physiology
Copyright Notice
(Q Kara B. Levine ,
1999
All Rights Reserved
Parts of this dissertation
Levine, K. B., Cloherty, E.
have appeared in:
, Fidyk , N.
Structural and Physiologic
Determinants of
Regulation by Adenosine
37(35): 12221- 12232
Sugar Transport
Biochemistry.
Levine ,
, and Carruthers , A. (1998)
K.
B., Hamil
S.,
Cloherty, E.
Human Erythrocyte
Triphosphate.
, and Carruthers, A. (1999)
Alanine Scanning Mutagenesis of the Human Erythrocyte Glucose
Transport Putative ATP Binding Domain. Submitted.
Dr. Cheryl Scheid, Chair of Committee
Dr. Thomas Honeyman, Member of Committee
Dr. Ann Rittenhouse, Member of Committee
Dr. Kendall Knight, Member of Committee
Dr. Laurie Goodyear, Member of Committee
Dr. Anthony Carruthers, Thesis Advisor
Acknowledgements
First and foremost I would like
part of the learning
of the Carruthers
which mistakes
process. I
would also like to thank the members
often acted as my personal
added bonus of
hands in the lab , I don t think this thesis
I
whom over
sounding board
my extra
would have ever
would also
Kelley DeZutter and Janice Lloyd ,
and tricks needed
Heard
maintaining my sanity. A special
thanks go to Erin Cloherty and Stephanie Hamill
completed without their help.
a
are considered a normal
laboratory. Especially Dr. Karen
the past five years
like to
for teaching
set of
been
acknowledge Julie
me the techniques
to do molecular biology.
I would like to thank
my family for their unending support
throughout this experience we like to
especially like to thank
instiled in
Dr. Anthony
this thesis. I am grateful that he maintains
laboratory environment in
which had the
my mentor
direction and encouragement during
Carruthers for his invaluable
the creation of
to thank
call graduate school.
my parents Nate and Judy
Levine
me an intense desire to succeed. Special thanks
I
would
who
go to
sister Shari , her husband Steve , and
my mece " The
Chloe " who were
always there for advice and to remind me that outside ,
continues to
exist.
a real world
Abstract
The human erythrocyte glucose transport protein (GLUTl)
interacts with ,
and is regulated by,
cytosolic ATP.
This study asks
questions concerning ATP modulation of GLUTI
the following
mediated sugar transport. 1) Which region(s) of GLUTI form the
adenine nucleotide- binding domain? 2)
modulation of sugar transport? 3) Is
sufficient for
sugar transport
What factors
influence A
A TP interaction with GLUTI
regulation?
The first question was addressed through peptide mappmg,
terminal sequencmg, and alanine scannmg mutagenesis
usmg (3 2P) -azidoA TP ,
combination of
strategies to
a photoacti vatable
used a
and photolabeling
examine how glycolytic intermediates, pH ,
affect A
GLUTI
A TP analog. We then
transport measurements
transporter oligomeric structure
of
TP regulation
and
of sugar
transport. Finally, GLUTI was reconstituted into proteoliposomes
determine whether A TP is
function in-vitro.
sufficient for the modulation of GLUTI
Vll
This thesis presents data supporting the hypothesis that residues
332- 335
contribute to the
efficiency of adenine nucleotide
to GLUTl. In addition , we show that AMP ,
acidification , and
the transporter
form antagonize
conversion of
to its dimeric
binding
A
regulation of sugar transport. Finally, we present results that
support the proposal that A
for transport
modulation.
TP interaction with GLUTI is sufficient
Vll
Table of Contents
ACKNOWLEDGMENTS
1V
ABSTRACT
VIll
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
CHAPTER I
INTRODUCTION
Sugar Transport
Regulation
Skeletal Muscle
Cardiac Muscle
vianlNucleated Erythrocytes
ATP Regulation of GLUTl in
Human Erythrocytes
Strategy
CHAPTER II
MATERIALS & METHODS
CHAPTER III
FACTORS INFLUENCING A
MODULATION OF ERYTHROCYTE SUGAR
TRANSPORT
Introduction
Results
Glycolytic Intermediates
Oligomeric Structure
Does ATP Modulate GLUTl
Mediated Sugar Transport Directly?
Discussion
17
CHAPTER IV
PEPTIDE MAPPING THE GLUTI A
BINDING DOMAIN
Introduction
Results
Peptide Mapping
terminal Microsequencing
Discussion
CHAPTER V
ALANINE MUTAGENESIS OF THE GLUTI
CONSENSUS A TP BINDING SEQUENCES
Introduction
Results
Purification of
Human
GLUTl HA His
8 1
Scanning Alanine Mutagenesis of
A TP Consensus Binding
Sequences
glucose Uptake into Cos- 7 cells
AzidoATP Labeling of Wild Type &
Discussion
101
113
SIGNIFICANCE AND FUTURE
DIRECTIONS
120
Mutant hGLUTl
CHAPTER VI
REFERENCES
136
List of Tables
Table
Table 2
Table 3
Effects of glycolytic intermediates on
erythrocyte 30MG uptake
Effect of reductant on A TP-modulation
erythrocyte 30MG uptake
Characteristics of S.
Cerevisiae strain RE700A
transfected with GLUT 1
Table 4
Table 5
GLUT 1 alanine mutants
Antibody binding
With GLUTI
to Cos-
cells transfected
(y-
(y-
List of Figures
Figure
Figure 2
putative topography and
Consensus A TP binding domains.
GLUT
Effects of pH on A TP-modulation of sugar
transport (A) and GLUT I- labeling by
azido-
Figure 3
15
P)A TP (B).
Effects of
pH on the
concentration dependence
of A TP-modulation of sugar transport (A)
and ATP- inhibition of GLUT I- labeling by
azidoP)A TP (B).
Figure 4
Azido- (y_
2P)A TP labeling
5 1
of dimeric and
tetrameric GLUT
Figure 5
A TP stimulates 30MG uptake into
reconstituted , purified GLUT
proteoliposomes.
Figure 6
Peptide mappmg of photolabeled GLUTl.
Figure 7
Peptide mappmg of photolabeled GLUTI.
Figure 8
Summary of peptide mapping analysis of
P)ATP labeled GLUTI (Figures 1 & 2).
Figure 9
GLUTl HA His
expression in Saccharomyces
Cerevisiae.
Figure 10
terminal GLUTI antibody western blot of
RE700A +/- GLUTI HA His
7 1
Figure 11
Purification of GLUT 1 HA His
from Cos-
cells.
Figure 12
9 1
cells
deoxyglucose uptake in Cos-
transfected with GLUT 1 HA His
Figure 13
Photo labeling of parental
GLUT 1 and
transfected wild type and
GLUT 1
in Cos- 7 cells by the sugar export
site ligand (3H) -
Figure 14
mutant human
100
CCB.
Azido( P)ATP labeling of GLUTI HA His
and its corresponding alanine
Figure 15
(wild type and mutant).
106
photolabeling
affinity purified GLUTI HA His and
A TP- inhibition of azidoA TP
of
109
GLUT1 332A
Figure 17
104
Corrected AzidoA TP labeling
GLUTI HA His
Figure 16
mutants.
AzidoA TP labeling of
stripped of
Cos-
cell membranes
peripheral proteins.
112
Xll
Abbreviations
2DOG
Deoxyglucose
30MG
0-methyl-
ATP
glucose
adenosine 5' triphosphate
GLUTI c- terminal
antibody
CCB
cytochalasin- b
Cos- 7
simian kidney cell line
8-an ti body
tetrameric GLUTI exofacial
DTT
di thiothrei tol
Ghosts
sealed erythrocyte
antibody
membranes lacking
cytosol
GLUTI
His
human erythrocyte glucose transport
protein
histidines
hemagglutinin
HA-
anti- HA antibody
GLUTI N- terminal
Ni - NT A
antibody
nickel column media
XIV
PMSF
pheny lmethy lsulfony If! uoride
(serine
protease inhibitor)
PFK
phos phofruc tokinase
RE700A
S. Cerevisiae URA 52 mutant with seven of
its glucose
transporters (HXT 1- 7)
SDS
sodium dodecyl sulfate
Stripped Ghosts
red blood cell ghosts
membrane proteins
stripped of
deleted
peripheral
Chapter
Introduction
selectively permeable barrier ,
all mammalian cells. This barrier
and proteins. The hydrophobic
is
the cell membrane , surrounds
composed primarily of lipids
nature of this
cell membrane allows
non- polar molecules, and only the very smallest polar molecules
pass through by simple
than water such as
diffusion. In
ions, sugars,
metabolites must be transported
specialized integral
contrast, polar molecules larger
ammo acids,
across the
nucleotides
cell membrane by
membrane proteins which function
or carriers. The channels
(selected on the basis of
electrochemical gradient
are water
filled pores that
charge and size)
, and cell
to diffuse
as channels
allow substances
down an
across membranes.
In contrast, carrier proteins transfer bound
membranes by way of conformational changes.
solutes across
Two types of
mediated transport have been described , facilitated and active.
Facilitated transport is characterized by rapid,
bidirectional transport of solutes across the
protein mediated
membrane, down
electrochemical gradient. Active transport couples a metabolic
energy source (i. e. A TP) to
the rapid
protein mediated movement of
solute against a concentration gradient (Carruthers , 1990).
Glucose ,
a hexose monosaccharide , is an essential source
energy m mammalian
and non-mammalian cells. Cellular metabolic
machinery reqUIres D- glucose
1990). A family
of
for the production of A TP (Darnell
facilitative glucose transporters selectively pump
glucose across cell membranes
areas of high concentration
to
with a
net movement of
areas of low concentration (Stein,
1986). Stereoselectivity, saturation kinetics ,
fungal metabolite
sugar from
and
inhibition by the
cytochalasin B (CCB) characterize these facilitative
glucose transporters.
Five mammalian glucose transporter isoforms have been
identified in the facilitative glucose transporter family. These five
isoforms are highly homologous (50- 80%)
yet exhibit distinct
distributions (Gould and Bell , 1990). GLUTl ,
transporter ,
is found
red blood cells ,
(Mueckler et
in virtually
all
tissue
the erythrocyte glucose
mammalian tissues including
brain microvessels , kidney,
colon, and placenta
aI., 1985; Birnbaum et aI. , 1986; Gould and Bell ,
1990).
GLUT2, the liver type transporter ,
intestine, and
is located in liver , kidney, small
-cells (Thorens et aI. , 1988). The presence
pancreatic
of GLUT3 in fibroblasts and blood vessels suggests a ubiquitous
tissue distribution
(Kayano et aI. , 1988). GLUT4, the insulin
sensitive transporter
, is found in skeletal and cardiac muscle as well
as adipocytes (Birnbaum , 1989; Charron
aI. , 1989; James
et aI. ,
1989; Kaestner
fructose transporter , is found
et aI. ,
1989).
GLUT5, a
primarily in the small intestine
lower levels of expression identified in
subcutaneous fat (Kayano et aI. ,
The goal of this
et aI., 1989; Fukumoto et
with
kidney, skeletal muscle, and
1990).
understand all aspects
laboratory is to
of
faciltative glucose transport. To accomplish this, we have chosen to
study GLUT1 ,
transporter.
the
human erythrocyte form of the
Specifically, our interest
GLUTI-mediated sugar transport
of GLUTI make it ideal
is
lies in
facilitative glucose
understanding how
regulated. Certain characteristics
for studying
transport regulation. First
GLUTI comprises 6% of total human red blood cell protein. The ease
with which these erythrocytes can be acquired facilitates the
purification of large quantities
(10-
20 mg/unit
of blood) of
the
transporter for use in
experimental protocols.
cytosolic environment
of red blood
In addition , the
cells can be readily controlled
by reversible hypotonic lysis, thereby permitting analysis of
cytosolic determinants of
Sugar Transport
transport.
Regulation
Certain cell types
, such as skeletal and cardiac
adipocytes, and nucleated
avian
muscle
erythrocytes can metabolize sugar
at rates that exceed transport capacity. Under these conditions
glucose transport becomes the rate- limiting step in sugar utilization
and cells become susceptible .to acute transport regulation (Elbrink
and Bihler ,
1975; Carruthers , 1990).
Compounds that regulate glucose carner function can be divided
into two groups: hormones and metabolic factors. The primary
hormone to which
GLUTs respond is insulin ,
the pancreatic islets in response to
(Vander
1990). Cells
muscle. In contrast ,
exercise ,
which is secreted by
changing blood nutrient levels
that respond to insulin include
adipocytes and
metabolic factors such as anOXIa , ischemia
and metabolic pOIsons , have been shown to affect skeletal
muscle , cardiac muscle, and nucleated aVIan erythrocytes.
In
either
case, exposure to
these substances and conditions stimulates
glucose
uptake (Carruthers, 1990).
Two hypotheses exist which explain
transport acceleration.
following equation:
Transport capacity is
V max = Kcat (E)t
catalytic turnover of an
the
enzyme ,
mechanisms governIng
defined by the
where K cat represents the
and (E)t is equal
concentration. It is apparent from
to
total enzyme
this relationship that an increase
in either transporter catalytic turnover or transporter number
would alter transport capacity (V max)' The recruitment
suggests that transport stimulation is a
redistribution to the
hypothesis
direct result of transporter
' plasma membrane from a
subcellular pool
(increased (E)t ) (Cushman and Wardzala , 1980; Suzuki and Kono,
1980). In
contrast, the activation hypothesis implies' that
acceleration is the
result of an increase
transporters already present in the
transport
in the intrinsic activity of
membrane (increased k cat
(Carruthers, 1990). Individually, carner recruitment or activation
could result in sugar transport stimulation.
However ,
mechanisms could also function coordinately in cells.
these two
Under these
conditions, inactivation and reinternalization would result
transport inhibition
(Carruthers,
in
1990).
The following sections present four model cell types that exhibit
acute regulation of glucose transport
, skeletal and cardiac
muscle
avian erythrocytes, and human red blood cells.
Skeletal Muscle
Skeletal muscle is the primary site of
(Douen et aI. ,
tissue is
1990; Klip and
in-vivo
glucose disposal
Paquet, 1990). Sugar transport
maximally stimulated by insulin ,
as well as
in
this
exercise,
anoxia/hypoxia ,
ischemia , and inhibition of oxidative
phosphorylation ,
all of
which result in an increased demand for A
(Clausen , 1975; Elbrink and Bihler, 1975). Subcellular fractionation
and cytochalasin B binding experiments have shown that
stimulation is characterized by a two- fold
increase in plasma
membrane and t- tubule carrier levels (Hirshman et
Wheeler ,
1988;
aI. , 1991).
Douen et aI. ,
1990; Cartee
et aI. ,
aI., 1988;
1991; Goodyear
Experimentally, striated muscle
has been found to
transporter isoforms: GLUT 1 (Mueckler
1986; Gould and Bell ,
1990) and
et aI., 1985; Birnbaum et aI.,
GLUT4 (Cushman and Wardzala,
1980; Birnbaum, 1989; Charron et aI. ,
GLUT1 ,
contain two
1989; James
et aI., 1989).
detected primarily at the cell surface, is proposed to be
responsible for basal
(James et aI. ,
transport rates
1988). In contrast ,
in unstimulated skeletal muscle
transport stimulation due to anOXIa
and exercise appears to result from GLUT4 translocation to the cell
membrane from
Gao et aI. ,
an intracellular compartment (Douen et aI. ,
1994).
More recent evidence
rilUscle contains a pool of
suggested that skeletal
has
conformationally
masked GLUT4 that
becomes exposed during transport stimulation (Wang et aI. ,
Supplementary experiments have indicated
translocation in
level of
skeletal muscle
that
1959;
is not sufficient to account for the
Morgan et aI. ,
1965; Sternlicht et aI., 1988; Wheeler ,
The implication is that
1961;
to anoxia and
Morgan et aI.
1988; Klip and
transport stimulation in
be due to both an Increase
1996).
GLUT4
transport stimulation observed in response
exercise (Morgan et aI. ,
1990;
in the turnover
Paquet, 1990).
skeletal muscle may
rate of existing
carners
total number
as well as the
- 1988; Klip and
of transporters present (Sternlicht et aI.
Paquet, 1990).
The degree to which recruitment and activation contribute to
exercise ,
and metabolic- depletion induced transport
anoxia,
stimulation in
skeletal muscle has
Carruthers suggests that this
not
yet been determined.
question might be answered
measunng transport kinetics and carrier
by
capacity in sarcoplasmic
vesicles isolated from basal and stimulated muscle (Carruthers
1990). The structural complexity of skeletal muscle
definitive markers
for subcellular fractions
difficult task that has hindered analysis
and the lack of
make this a technically
(Klip and Paquet , 1990)
;Cardiac Muscle
Ii \ Two major glucose
'are expressed in
transporter isoforms -
cardiac muscle. Levels of
"1990; Kraegen et aI. ,
;h.eart muscle than
in
1993) were found
GLUTI and GLUT4 -
GLUT 1 (Calderhead et aI.
to be 3 to 4 fold greater in
skeletal muscle (Doria et aI. ,
bsence of insulin GLUT4 is
1993). In the
localized to tubulovesicular elements
tadjacent to the sarcolemma and
transverse tubules (Fischer
et
aI.,
1997). Immunohistochemical
techniques have detected GLUTI
cardiomyocyte plasma membranes and intercalated discs (Doria et
aI., 1993). Subcellular fractionation has revealed
quantity of GLUTI is also
(Fischer et aI. ,
to undergo
present in
1995; Fischer
significant
intracellular membranes
et aI. , 1997). Both carners were found
translocation from an intracellular compartment
cell surface in response to
(Kolter et aI. ,
1992; Doria
aI., 1994; Zorzano
et
ischemia , hypoxia or
aI., 1997). This
plasma membrane (James et aI. ,
contractile activity
is contrary to what is proposed
GLUTI is detected only at the
1988). Fischer et al (1997)
identified two intracellular pools of carrier
in
are available for translocation , one of which
GLUTl. The evidence suggests
is insulin insensitive (Fischer et aI. ,
whether transport
1997).
stimulation in heart
cardiomyocytes that
contained significant
that this pool of
surface transporter activation. Wheeler
carriers
It is not yet certain
muscle results
from
transporter recruitment alone , or occurs in combination
transporters in the heart
to the
et aI. , 1993; Sun et aI. , 1994; Wheeler
to occur in skeletal muscle , where
quantities of
that a
with cell
found that glucose
show a 50% increase
in
membrane levels in
response to anOXIa compared to a 150%
(Wheeler, 1988).
In contrast , studies undertaken by Haworth et al
showed that anOXIa
Increases glucose transport
cardiomyocytes 10- 20
in skeletal
increase in glucose uptake
fold (Haworth
in rat
and Berkoff 1986). In
muscle , it appears that translocation
heart, as
may not account for
the degree of transport stimulation seen in response to anoxia
ischemia, and exerCIse. This provides additional evidence that
carrier activation may constitute a necessary
part of the process of
transport stimulation.
vianlNucleated Erythrocytes
In contrast to
human erythrocytes ,
aVIan erythrocytes contain
cytosolic structures , primarily mitochondria and a nucleus
(Harris
and Brown , 1971; Brown , 1975). Sugar transport in avian
erythrocytes is subject to
regulation by cellular metabolic status.
Under basal conditions , protein-mediated net sugar uptake is
detectable. Exposure to anoxia or to ' inhibitors of oxidative
metabolism results in a robust stimulation of net sugar uptake
(Cheung et aI. , 1977; Simons ,
1983). Recent studies have
barely
demonstrated that
avian erythrocyte GLUT1 ,
which is present at the
cell surface at all times (Diamond and Carruthers ,
1993),
functions
as an antiporter under normoxic conditions (Cloherty et aI. ,
This means that sugar
metabolic disruption ,
uptake is coupled to sugar
avian red
uniport (Cloherty et aI. ,
absence of
exit. Following
cell GLUTI can catalyze
sugar
1996) allowing net sugar uptake in the
intracellular sugar. Antiport function
by cellular metabolic status. The mediator
switching mechanism is currently unknown.
proposed that
1996).
appears
unaffected
of this antiport/uniport
Cloherty et al has
regulatory factors exist in the avian erythrocyte
which allosterically inhibit GLUTI uniport. Cloherty further
proposes that AMP dependent protein kinase
is involved in
activation/deactivation pathway (Cloherty et aI. ,
this
1996).
While avian erythrocyte GLUTI-mediated sugar uniport is
controlled more completely
by
cellular metabolic status than is
sugar uniport , the similarity between transport
tissues is
mediated by
and Hinkle , 1985) which
striking. Human erythrocyte
GLUTI (Mueckler et aI. ,
is assembled and
sugar
1985; Wheeler
functions as an allosteric
,'
homotetramer (Hebert
and Carruthers , 1992;
Zottola et aI. ,
1995). In
. freshly isolated erythrocytes, net uptake of 3- 0-methylglucose (a
transported but
nonmetabolized sugar) is slower by as much as 150-
fold than 30MG exchange transport (Cloherty et aI. ,
Following hypotonic cell lysis and cellular resealing
medium, 30MG net uptake
:stimulated by 3 to
(but not exchange
et aI. ,
10- fold (Helgerson
erythrocytes are resealed in
1996).
in A TP- free
transport) is
1989).
A TP-containing saline
When lysed
30MG net
,uptake and exchange are indistinguishable from transport
Jin intact cells (Helgerson et aI., 1989).
observed
This suggests that as with
vian red cell sugar transport, uptake stimulation
in
human
erythrocytes results from accelerated uniport function and that
(cytoplasmic A
TP may regulate cellular glucose antiport/uniport
(Levine et aI. ,
1998).
ATP Regulation of GLUTl in Human Erythrocytes
Human erythrocyte GLUTI is subject
ATP (Carruthers
to
allosteric inhibition by
and Helgerson , 1989; Helgerson
affinity high capacity net sugar
uptake. Maximum rates
et aI., 1989) which
uptake to high
affinity
of exchange transport and net
" j
sugar exit appear unaffected by cellular A TP levels (Helgerson et aI.
-- 1989). The net effect of A
levels (50 to
100
JLM)
TP on 30MG uptake
at subsaturating sugar
is to stimulate transport by
a factor
to the increase in the ratio V ma xi K m(app) while
levels greater than K m(app)
We have demonstrated
that the
a GLUTI conformational
1989).
(saturated transport) is
inhibited.
isolated human erythrocyte
nucleotide binding protein
.glucose transport protein is also
(Carruthers and Helgerson ,
transport
A TP binding to GLUTI promotes
change that
leaves the GLUTI carboxyl-
terminus less accessible to peptide- directed antibodies. This A TP-
dependent conformational change is antagonized by
AMP and ADP
Which appear to function as competitive inhibitors of
A TP binding
to GLUTl. Initial peptide mapping studies identified a range of
,residues from 270- 380 as a likely region where A TP attaches
(Carruthers and Helgerson , 1989). The primary sequence of GLUTI
indicates the presence of three
potential consensus A
TP binding
(Figure 1). Sequence I is a Walker A motif located
etween residues 111- 118 , sequence
hich spans residues
225- 229,
II is a hydrophobic loop motif
and sequence III ,
located at residues
i!!
(Ii
;'-, !"';.
Figure
1.
GLUT1
putative topography and consensus
ATP
binding
domains. The three proposed consensus ATP binding sequences located in
GLUT1 are highlighted in black. Sequence I is a Walker A motif located between
G111 and S117. Sequence II is referred to as a loop motif and stretches from
. K225- K229. Sequence III , a Walker B motif , extends from G332- G343.
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Cytosol
Reg i on
Reg i on
Reg i on
332- 343, is a
Walker B motif which overlaps
region of ATP binding on GLUTI (Walker ,
whether A TP binding
with the proposed
1982 #4492). It is unclear
to GLUTI is sufficient
to mediate sugar
transport regulation in
human red blood cells
molecular species
required.
are
Wheeler has demonstrated that A TP
activity of reconstituted
human GLUTI protein
the
(Wheeler , 1989).
these results. 1)
Required
could be mISSIng or dysfunctional in these
reconstituted vesicles or 2) the
GLUTI used
may exhibit an abnormal A TP response
sugar (Carruthers and Helgerson
however ,
other
does not modulate
number of explanations could account for
cellular components
or whether
in these experiments
or capacity to
1989). It is
transport
more probable
that the problem lies with Wheeler s use of reduced (DTT-
treated) GLUTI which we have shown to exist as a GLUTI dimer
(Hebert and Carruthers , 1992).
Work in our laboratory has shown
ii,
that conversion of
binding and A
tetrameric GLUT 1
TP modulation
These results imply that
A
to its dimeric
form ablates
of transport (Levine et
TP modulation
A
aI., 1998).
of GLUT 1 is dependent
upon transporter quaternary structure. In contrast to
Wheeler
, i
work, our
studies employ the physiologic ,
non-reduced form of
GLUTI ( a GLUTI tetra mer (Hebert and Carruthers ,
responds to A
1992))
TP.
In the present study
we ask: 1) What physiologic factors
influence A TP-modulation of
sugar transport?
2) Is
A TP interaction
with GLUTI sufficient for sugar transport regulation?
the GLUT 1 nucleotide- binding
we use
that
a combination of
3)
Where is
domain? To address these questions,
transport measurements and azidoA
photolabeling strategies to examIne the effects of
glycolytic
intermediates , pH changes , and transporter oligomeric structure
A TP modulation of GLUTl. In addition ,
purified erythrocyte GLUTI are carried
sufficient for modulation of
on
reconstitution assays using
out to determine
if A TP
GLUTI mediated sugar transport.
Finally, peptide mapping, n- terminal sequencing, and alanine
scanning mutagenesis are used to
locate the GLUTI A TP binding
II:
If!
I:i
Our findings
suggest that
A TP regulation
intracellular AMP and acidification
of sugar transport , and that A TP-
interaction with GLUTI is sufficient for transport modulation.
data also support the
GLUTl ,
364
The
hypothesis that tetrameric , not dimeric
is regulated by
ATP. In addition we show that residues
form an integral part of the A
TP binding domain on GLUT
and that epitope tagged human GLUTI expressed
normal with respect to sugar
Finally, alanine substitution
301-
in Cos-
cells is
transport and azidoA TP binding.
at residues 338-
transport, while alanine substitution
340 ablates
sugar
at residues 225- 229 , and 332-
334 increases (32 P)A TP binding.
II!
II'
(y-
, ,
Chapter n
Materials and Methods
Azido
. Materials.
CA. (3H)-
P)ATP was purchased from ICN , Costa Mesa,
Methylglucose was purchased from Dupont NEN,
Wilmington, DE. Rabbit antisera
carboxyl- terminal
(C-
Ab) raised
against a
synthetic
peptide of the rat brain glucose transport protein
(residues 480- 492)
was obtained from East Acres Biologicals
Southbridge MA. Glycoprotein
Detection Kits and
ECL detection
reagents were purchased from Amersham Pharmacia Biotech
Piscataway, NJ. Gradient
Scientific ,
Portsmouth
SDS PAGE gels were purchased from Owl
, NH. Nitrocellulose and Immobilon- P
obtained from Fisher Scientific ,
Medium
Pittsburg,
PA. Dulbecco s Modified
FBS, Penicilin/Streptomycin ,
Were purchased from Gibco BRL
were
and G418 (Geneticin)
Grand Island ,
NY. The
QuickChange Site- Directed Mutagenesis Kit was acquired from
Stratagene , Marco Island , FL. pcDNA3. 1 + vector was supplied
Invitrogen ,
Carlsbad , CA.
Fugene 6 Transfection Reagent ,
ntibody (mouse monoclonal
by
anti-
HA-ab), Endoproteinase Lys- C, and N-
- dodecyl-~- D-maltoside
were obtained from Roche, Indianapolis ,
Nickel- NTA (Ni- NTA) was purchased from Qiagen ,
Valencia ,
IN.
CA.
Centricon concentrators were acquired from Millipore, Bedford, MA.
materials were purchased from Sigma unless stated
otherwise.
Tris medium consisted
Solutions.
of 50
mM Tris- HCI ,
5 mM MgCl
pH 7.4. Sample buffer (2X) contained 0. 125M Tris- HCI pH 6. 8, 4%
SDS , 20%
glycerol, and 50 mM DTT. KCI medium consisted of 150
mM KCI , 10 mM Tris- HCI , 5 mM MgCl , 4 mM EGTA, pH 7.4. Stopper
consisted of ice cold
phloretin
, and 10
Tris HCI ,
1
JlM
KCI medium plus
HgCI
10
JlM
cytochalasin B ,
100
JlM
, pH 7.4. Lysis buffer contained 10
mM EGTA , pH 7. 2.
Phosphate buffered saline (PBS)
:n!
I .u
contained 171 mM NaCI ,
10
mM Na HP0 , 3.4 mM KCI ,
, pH 7. 3. Buffer A included 300 mM NaCI ,
20% Glycerol ,
and 50 mM NaH
A + 0. 1 % N- dodecyl ~-
10
1.84
mM imidazole
. Buffer B was composed of Buffer
maltoside. Buffer C contained 10 mM Tris-
HCI (pH) 7. , 300 mM NaCI ,
25 mM imidazole ,
maltoside.. Elution buffer consisted
of 10
and 0. 1
% N- dodecyl
mM Tris- HCI (pH 7. 5),
.- ---
'.
-,
,,' ..,
500 mM NaCI ,
300
mM imidazole ,
and 0. 1
% N- dodecyl ~-
maltoside. Krebs Ringer Phosphate (KRP) contained 4. 5% NaCl
t-
75% KCI, 7. 85% CaCI
, 19.
1 % MgS0
, and 0. 1 M Na
HP0
7,'r:"
A peptide corresponding
Antisera.
to
GLUT 1
residues
88- 95 was synthesized by the University of Massachusetts Medical
Kchool Peptide Synthesis facility. This peptide was conjugated to
keyhole limpet hemocyanin USIng
a
kit purchased from Pierce.
Rabbit antisera (N- Ab) against this GLUTI peptide were obtained
i';
,d
..f
from Animal Pharm Services
Inc of Healdsburg, CA. Whole IgGs were
"'1','
purified from crude
(Sogin and Hinkle ,
".I.
serum by ammonium sulfate precipitation
:il.
1980).
- 1'
I; .
Red Cells.
Erythrocytes were isolated from whole human blood
by
"i
, I!
repeated wash/centrifugation cycles in
volume of whole blood was mixed with
and centrifuged at 10
000 x g for 5
. Suffy coat were aspirated, and the
ice-cold KCI medium.
One
3 volumes of KCI medium
minutes at 4 o C. Serum and the
wash/centrifugation cycle was
until the supernatant was clear and the buffy coat was no
longer visible. Cells
were resuspended in 20 volumes of KCI
and, if used in subsequent sugar transport assays , were
room temperature
for 30 minutes in order to
medium
incubated at
deplete intracellular
sugar levels.
Erythrocyte
Membrane
40 volumes
lysis medium ,
centrifuged at 22
Washed red cells were lysed in
Ghosts.
incubated on ice for 10 minutes , then
000 x g for 20
was aspirated , and
minutes at 4 o C.
the lysis/centrifugation/aspiration cycle was
repeated. The resulting membranes
KCI medium at 37 o C for
centrifugation at 22
The supernatant
resealed by incubation in
60 minutes and harvested by
000 x g for 20
Glucose Transport
were
Protein.
minutes at 4 o
Glucose transport protein was
purified from human erythrocytes as described previously
and Carruthers
is incorporated
1992). The carrier
co-
purifies with red cell
(Hebert
lipid and
into unsealed proteoliposomes upon removal of
by dialysis
(Carruthers
1986).
Glucose Carrier Labeling
with 8- Azidoly32PjATP.
1989). Briefly, a
as described previously (Carruthers and Helgerson
methanolic solution of
resuspended in 500
(azidoA TP)).
8-azido(y- P)A TP is
IlL Tris medium pH 8.
Purified GLUTI
dried under nitrogen
, 10
5 (90 /lCi
proteoliposomes (500
sedimented by centrifugation at 14 000 x g
Labeling was
/lg
and
final
/lM
protein) are
for 20 minutes and
combined with the methanol- free 8-azido (y_ 32P)A TP solution.
The
:11
:111
suspenSIOn is incubated on ice for 30 minutes by which time A
: 'PI
binding to GLUTI achieves equilbrium. Samples are
placed in a
plastic weigh boat on Ice and irradiated for 90 seconds at 280 nm
a Rayonet
Photochemical
uv
Reactor. Following
irradiation
(photolabeling), GLUTI is washed three times to remove unbound 8azido (y_ 32P)A
TP by centrifugation (14 000 x g for 15 minutes) and
resuspended in 500
IlL Tris medium pH 8. 5.
Photolabeling of Cos-
'recombinant hGLUTI is identical to that described above except for
the following 1) samples
eluted from Ni- NTA were
combined
the methanol- free 8-azido (y_ 32P)A TP at pH 7.4 ,
rradiation was carried out in separate
on Ice. The presence
of
wells of a
and 2)
plastic ELISA
dish
detergent precludes washing the sample to
(y-
remove unbound azidoA TP.
':;
The effects of pH on
azidoA
to GLUTI were examined by varying the pH of the
the range 5. 5
- 8. 5 during
A TP binding to the
medium over
photolabeling. MgCl was present
in all experiments. Photolabeling of
at 5
GLUTI was carried out at
P)A TP that
concentrations of 8-azido
Tris
TP binding
were lower than the Km for
transporter. In this instance , the amount of
bound 8-azido(y- 32P)ATP
is gIven
as:
Bmax .
bound
-(aZldoATPJ
This method makes it possible to detect changes in both the
the Kd
for 8-azido(y-
Glycoprotein
P)ATP binding to GLUTl.
Detection.
carbohydrate was detected
Detection Kit.
B max and
Azido(y- 32P)ATP
using the
photolabeled GLUT 1
Amersham ECL Glycoprotein
Briefly, peptide oligosaccharides are oxidized
mM sodium metaperiodate
then
using 10
labeled using 0. 55 mM biotin
, hydrazide. Subsequent detection
of biotinylated glycoprotein
mploys standard western blotting techniques
using
streptavidin-
peroxidase (1 :5 000) and ECL chemiluminescence.
If!,
:1'
Proteolysis
of Labeled
Azido(y- 32P)ATP-
Carrier.
and/or
biotin-
labeled GLUTI was sedimented by centrifugation at
14, 000 x g for 20 minutes and resuspended in sample
chymotrypsin ,
endoproteinase Lys-
(protein/enzyme 20: 1
buffer :t
or endoproteinase Glu-
by weight). Proteolysis was allowed to
proceed for 30 minutes at 37 o
and was arrested by addition of
phenylmethylsulfonylfluoride (PMSF ,
0. 2
fragments were separated by gradient
mM). The resulting GLUTI
gel electrophoresis (10- 20%
polyacrylamide SDS gels) and transferred
to
nitrocellulose
membranes for autoradiography and western blotting.
Polyacrylamide
Gel Electrophoresis.
10% acrylamide gels
where separation
of
electrophoresis (10-
Western
Blotting.
as described previously
Proteins were resolved on
(Laemmli
proteolytic fragments was required ,
1970)
gradient gel
20% polyacrylamide gradients) was employed.
Proteolytic fragments containing GLUTI
carboxyl- terminal residues 480- 492
GLUTI N- terminal residues
:!:!
88-
and/or a hemagglutinin
tag (HA) were detected
by western
blot analysis. Peptides separated by SDS PAGE were transferred
electrophoretic ally
to nitrocellulose or immobilon membranes,
which were subsequently
blocked for 1 hour in
PBS containing 15%
Carnation TM nonfat dry milk. Following three five-minute washes in
PBS, membranes were incubated for
(C- Ab and N- Ab)
diluted 1:1000 or (HA-ab) 1:400
2% nonfat dry milk.
antibody, membranes
antibody (protein-
1 hour in pnmary antibody
Following three wash cycles
to remove primary
or goat anti-mouse Horseradish
in
chemiluminescence using
Peroxidase
PBS containing 2% nonfat dry milk.
Detection of antigen/antibody/protein-
was sedimented
PBS containing
were exposed for 45 minutes to secondary
conjugate) diluted 1 :3000
Protein Sequencing.
in
complexes was by
Amersham ECL reagents.
Azido(y- 32P)ATP- Iabeled GLUTI
(100
Jlg)
by centrifugation at 14, 000 x g for 20 minutes then
resuspended in 0. 1 % SDS/Tris- HCI pH 7.4 :!
Lys- C. Digestion was carried
Jlg
endoproteinase
out for 18- 24 hours at 4 o C.
fragments were separated using
GLUTI
mM thioglycolate pre-equilibrated
I.J
gradient gels. Peptides were transferred
membranes and
- Coomassie
electrophoretic
ally to PVDF
following detection by autoradiography
and
blue staining, bands of interest were excised from the
membrane , washed
extensively in
distilled water and subjected to
microsequence analysis by Dr. John Leczyk of the University of
Massachusetts Medical School Protein Chemistry facility.
Net 3- Methylglucose
Uptake. Sugar- depleted cells or ghosts at
ice temperature were exposed
containing (3H)-
to 5 volumes of
0 -methylglucose and variable concentrations
unlabeled 3- 0 -methylglucose.
of 15 seconds to 1 minute ,
of stopper
ice cold KCI medium
Uptake was measured
over
intervals
then 50 volumes (relative to cell volume)
solution were added
to
the cell/ghost
suspension.
, Cells/ghosts were sedimented by centrifugation (14
000 x g for
seconds), washed once in stopper , collected by centrifugation,
, extracted in 500 JlL of 3% perchloric acid. The acid
'" centrifuged , and duplicate samples of the clear
counted. Zero- time uptake
of
and
extract was
supernatant were
points were prepared by addition of
stopper to cells/ghosts prior to addition of medium
containing
sugar and radiolabel. Radioactivity associated with cells/ghosts at
zero., time
was subtracted from the activity
associated with
cells/ghosts following the uptake period. All uptakes were
normalized to
to sugar
equilibrium uptake where cells/ghosts were exposed
medium at 37 o C for 60 minutes prior to addition of
stopper. Uptake assays were performed using solutions and tubes
pre-equilibrated to 4 o
Effects of
Glycolytic
Intermediates
Glycolytic intermediates were introduced into human
Uptake.
erythrocyte ghosts by addition
prior to
37 o
on 3- Methylglucose
to the ice-cold resealing
medium
membrane resealing. The membranes were warmed to
C for 60 min then resealed ghosts were harvested
centrifugation (22
in 50 volumes
000 x g for 20 minutes
by
at 4 O C), and washed once
KCI medium to remove extracellular glycolytic
intermediates. Ghosts were then
uptake measurements
processed for 3- 0-methylglucose
as described
above.
Effects of pH on
A
TP-modulation
3-
of
Membrane ghosts were resealed
uptake.
5 mM MgCl , varying (ATP) and
0. All other
KCI medium containing
and
pH ranging from 5. 5 to
stopper) were also
same pH. Ghosts were then processed for
0-methylglucose uptake
Reconstituted
in
adjusted to a
solutions (uptake media
adjusted to the
O-methylglucose
measurements
as described
above.
sugar transport.
GLUTI-mediated
EggPC (40
mg in hexane) and cholesterol (10 mg) (:: 80:20 molar ratio) were
dissolved in hexane. The organic phase was evaporated
stream of N and remaining trace
vacuo
quantities of
under a
hexane removed
for 3 hr. The resulting lipid film was dissolved in Tris medium
containing 5% (by weight) sodium cholate and mixed with
100
/lg
GLUTI solubilized in 50 mM sodium cholate. This mixture (4 mL)
was dialyzed overnight against 6L detergent- free Tris medium and
the resulting
suspensIOn of
distributed into 0.
was frozen in a
small unilamellar
5 mL fractions
dry ice/isopropyl
temperature then subjected to 2
proteoliposomes
in microcentrifuge
tubes. Each tube
alcohol mixture , thawed at room
additional rounds of
freezelthawing. The resulting large unilammelar proteoliposomes
(mean diameter = 2.
by oil immersion
Jlm
phase
microscoPY) sedimented readily at 14 000 x g and
sugar transport determinations at 24 o C.
was measured into
at 0, 10, 20
100 JlL
= 0.
proteoliposome suspension (2
Jlg
Tris
10
JlM
GLUT!)
CCB (a transport
Equilibrium uptake was measured
JlM).
incubation for 2 hr at 24 o C.
mL ice cold
were used for
30MG uptake (0. 05 mM)
, 30 and 60 sec in Tris medium :t
inhibitor; K(app)
contrast
by
Uptake was arrested by addition
medium containing
10
were sedimented by centrifugation at 14
JlM
of 1
CCB. Proteoliposomes
000 x g for 5
min at 4 o
the supernatant was aspirated and the liposomes were washed agam
m Tris medium containing CCB. Uptake ,
v =
where t is
(30MGJ (cpm
(cpm
time, cpmo, cpm t, and cpm ",
associated with proteoliposomes at
time
v,
was computed
cpm
cpm
correspond to
radioactivity
zero, time t and
equili bri um.
Tissue
Culture.
Modified Eagle
Cos- 7 cells were maintained in Dulbecco
Medium (DMEM) supplemented
with
10% fetal
bovine serum (FBS), 100 units/mL penicilin, and
streptomycin in a 37
Construction
C humidified 5% CO
100 ug/mL
incubator.
Mutant GLUTI- HA His
of Wild Type
Kbp fragment of GLUTI cDNA was previously
A 1.
subc10ned into the
BamHI cloning site of pGEM3Z (pGEM3Z- GT1) (Zottola,
1995). Site
directed mutagenesis via a Stratagene QuickChange Kit was used to
introduce a unique Mfel restriction site at residue 1685
GTI. An HA- His
in
pGEM3Z-
epitope tag was inserted into the coding region of
ill
GLUTI at this location. The presence and orientation of the HA His
tag in the
GLUTI coding region was confirmed by sequencing. The
tagged GLUTI was then
further subc10ned into the
the pcDNA3. 1 + mammalian expression vector.
mutagenesis was undertaken
binding domains found in
of
Scanning alanine
through the three conserved A
GLUTI. Mutants were again
with the Stratagene QuickChange
confirmed by sequencing.
BamHI site
engineered
Kit, and their presence was
Transient
7 cells.
Expression
of Wild- type
Subconfluent Cos- 7
and Mutant
GLUT
in Cos-
cells were transfected with wild
type
and mutant GLUTI HA 6His cDNA using the Fugene 6 transfection
reagent system. Following transfection , cells were maintained in
DMEM for 24 hours, at which time 0. 72 mM G418 (Geneticin) was
added to
the media and the cells were allowed
for an additional 48 hours. For harvesting,
to continue growing
6 culture plates of
(150 mm) were washed twice with PBS + 5 mM EDT
into 20 mL lysis buffer mixed
inhibitor cocktail ,
to Loo & Clark ,
and PMSF.
with
5000 x g for 10 minutes at 4
centrifuged at 100, 000 x
fraction. Membranes were
1 mM eukaryotic cell protease
1993). Briefly, cells
of Wildtype
were
with a Fisher Dyna- Mix , then spun
C. The supernatant was decanted
g for 1 hour
L of Buffer A with 1
resuspended in 300
and Mutant
and
to pellet the crude membrane
mM protease inhibitor cocktail and frozen at -
Purification
A, then scraped
Membranes were prepared according
1993 (Loo et aI. ,
homogenized for five minutes
cells
C until use.
GLUTl. Purification 0
recombinant GLUTI HA His was described in Loo and Clark ,
1995
(Loo and
Clarke, 1995). Briefly, extracts
were solubilized in
solubilization buffer (Buffer A + 1 % N- dodecyl ~g for 15 minutes
spun at 15, 000 x
1.7 mL
maltoside) then
to remove insoluble material.
The supernatant was allowed to react for 1 hour at
C with 2 mls of
4
Ni- NT A column media which was pre-equilibrated with 4
buffer
maltoside). The column was
B (buffer A + 0. 1 % N- dodecyl ~-
washed twice with 8 mL buffer B , and twice with 8 mL buffer
Samples were eluted from the column with 1. 5 mL
and concentrated in centricon- l0
glucose
GLUTI- HA
Uptake
Cos-
were transfected
described previously.
post transfection.
for 2 hours at
Cells with Wild
with wild- type
Mutant
Type
cells in 12 well culture
dishes
and mutant GLUTI- HA His
glucose uptake was measured at 72 hours
Prior to uptake , cells were serum
37 C. Next,
incubated at 37 o C
elution buffer
concentrators.
Subconfluent Cos- 7
6His.
C.
for 15
starved in DME
cells were washed twice with KRP then
minutes in
KRP +/- 10
exposed to 400 j.L (3H)2- deoxyglucose diluted
j.M
CCB. Cells were
1 :40 in KRP (10 j.Ci)
mM unlabeled 2- deoxyglucose. Uptake was measured
containing O.
at various times
, from 5 minutes
washing twice with
to 30 minutes and stopped by
ice cold KRP. Duplicate samples
1 mL
were
counted following extraction with 450 III 0. 1 % Triton- XI00.
zero uptake points
well before
were prepared by adding ice
the addition
of
Time
cold KRP to each
medium containing sugar and radiolabel.
Radioactivity associated with cell
extracts at time zero was
subtracted from the activity associated with cell
the' uptake period. All uptakes were normalized
extracts following
,I
for
protein. Uptake
assays were performed USIng solutions and dishes pre-equilibrated
Cos-
Binding.
well culture dishes as
measured 72
cells were grown and transfected
in 12
previously described. 8-antibody binding was
hours post transfection. a-antisera reacts exclusively
with exofacial
epitopes of
solutions were pre-warmed
at 37
, Ii
I';
il
to 37 o
8-Antibody
Ji, '
C with sheep
tetrameric GLUTl. Unless noted, all
to 37
C. Cells
8-antibody diluted
were incubated for 1 hour
1: 1000 in DMEM +10% fetal
bovine serum. Following two washes in
KRP pH 7.4 ,
cells
were
!'
incubated for 30 minutes with
(125 I)-
protein G diluted 1: 1200 in KRP
(protein G recognizes and interacts with antibodies raised
After two additional washes in KRP ,
following extraction in 500 ilL
of 1
in
sheep).
duplicate samples were counted
% triton in KRP pH 7.4. Protein G
cells in the absence of primary antibody was used
binding to Cos-
to quantitate background radioactivity and was subtracted from
each sample. All measurements were normalized to the
quantity of
protein present in each well.
Cytochalasin
Membranes from Cos- 7
B Binding.
transfected
cells
with GLUTI HA His and mutant 338A were isolated as described
previously. Samples were
resuspended in 700
ilL 6. 25
11M (3H)-
cytochalasin B (CCB), 100 11M phloretin , and 10 11M cytochalasin D
in PBS at pH 7.4
and allowed to equilibrate on ice for 10 minutes.
This suspension was placed
irradiated for 4
in a
plastic weigh boat on ice
minutes at 280 nM in
a Rayonet
Reactor. Following UV irradiation membranes
to remove unbound
(100
CCB and sedimented
by
and
Photochemical
were washed with PBS
centrifugation
000 x g for 15 min). Proteins were resolved on 10%
SDS PAGE
gels and stained
for protein or transferred to nitrocellulose for C-
and HA- Ab western blotting. Gel slices (0. 2 cm) were extracted from
stained gels and incubated
containing 300 ilL 30%
corrected for
C in scintillation fluid
Hz O z before being counted. All samples were
protein concentration.
of
Calculation
overnight at 50
+ and
photolabeling experiments
In some GLUTI
ATP levels.
" pH and (A TP) were varied systematically
at fixed EGTA (4 mM) and MgCl (5 mM) levels. Exogenous
not added , thus
total Ca levels
CaCl was
JI
were limited to Ca-contamination
, I
(approx. 15
tLM;
Mgz. ATP , Mg
(Helgerson et aI., 1989)).
Computation of Mg
ATP , ATP , Mg EGTA , Mgz HEGTA and EGTA levels was
carried out using the WinMaxC program (Bers et aI. ,
equilbrium constants cited by Smith
Smith ,
1974) for H+, Ca
and Martell (Martell and
, Mg + and K+ interaction with EGTA and
ATP adjusted for pH and temperature
dissociation constants (- log)
(4 O
calculated for
are: KEGTACa = 2. 91, KEGTAMg = 2.
are: KEGTACa = 3. 32,
1994) and
KEGTAMg = 3.
C). The apparent
EGT A and A TP at pH 5.
, KATpca = 3. 30, KATPMg
31; at pH 6
, KATpca = 4. 30, KATPMg = 0. 19; at pH 7
1.22; at pH 8
are:
GTACa = 3.
, KEGTAMg = 3. 89, KATpca = 6. 29, KATPMg
are:
GTACa = 4.
, KEGTAMg = 4. 03, KATpca = 8. 27, KATPMg = 2. 39 and at
pH 8. 5 are: K EGTACa = 4. 03,
KEGTAMg = 4. 04,
Our calculations confirm that Mg
pH range 5. 5
KATpca = 9.
, KATPMg = 3. 10.
+ levels decline linearly over the
to 8. 5 (from 2.4 to 0. 5
mM) due to increasing
by EGT A and variable association with A TP. Mgz A TP plus
chelation
HA TP
levels increase from 2. 85 mM at pH 5. 5 to a peak of 3. 5 mM at
0 and thereafter decline to 3. 2
greatest at pH 5.
mM at pH 8. 5. Free ATP levels
are
(1.4 mM), are lowest at pH 7 (0.4 mM) and
(Ji
thereafter increase to 0. 8 mM at pH 8.
Computation
experiments ,
to change
(e. g.
of transport
transport
:HI
and labeling constants.
rates and GLUTI labeling
by A
In some
TP were
shown
in a nonlinear (saturable) manner with increasing (A TP)
Fig. 3). In these instances, best fit curves were generated by
regression using the
software package KaleidaGraph
08d (Synergy Software , Reading, P
relationships:
A) and assuming the following
, ,
Fig 3A pH
k2(ATP)
kl
8, uptake =
(ATP)
dATP
where kl = basal transport in
the absence of A TP, k2 is the increment in
saturating A
TP levels and KdATP
with the transport
transport produced
is the apparent Kd for
k2(ATP)
and K dATPI correspond to those
k3(ATP)
(ATP) K
dATPI
dATP2
(ATP)
parameters for Fig
k3 and K dATPz describe an additional interaction
where kl ,
3A pH 8
with A
k2
but where
TP that
uptake.
Fig 3B pH 5. 5 and 8.
k5(ATP)
, labeling =
k4
iATP
In the absence of unlabeled A TP
produced by saturating A
A TP that
A TP interaction
system.
Fig 3A pH 5. , uptake = kl
reduces net
by
, k5 is
(ATP)
where k4 is labeling
the decrease in
TP levels and KiATP
inhibits labeling half-maximally.
labeling
is the concentration of
Chapter m
Factors Influencing
A
TP Modulation
of Erythrocyte
Sugar Transport
The A TP-sensitivity
of
suggests that regulation of
human erythrocyte sugar transport
glycolysis and sugar transport in
nucleated erythrocytes (Wood and Morgan, 1969;
Whitfield and
Morgan , 1983; Diamond and Carruthers , 1993) and cardiac tissue
(Mansford , 1968; Haworth and Berkoff , 1986)
through a
is coordinated
common regulatory principle. The major site of glycolytic
regulation is phosphofructokinase (PFK) which catalyzes the
committed step in
glycolysis. PFK is subject
to allosteric inhibition
by A TP and by citrate and to stimulation by fructosebisphosphate and by
AMP and ADP (Carpenter and Hand ,
Pyruvate kinase , the enzyme
controlling the outflow from glycolysis
is subject to allosteric inhibition by A
fructose-
1986).
TP and to stimulation
by
bisphosphate (Waygood et aI. , 1976; Rosevear et aI.
1987 ).
Work in this laboratory has shown that A TP is an allosteric
inhibitor of
human erythrocyte GLUTI (Carruthers and Helgerson,
1989; Helgerson et aI. ,
1989). The net
effect of ATP on 30MG uptake
II red blood cell ghosts at subsaturating sugar levels (50- 100
to stimulate transport. In contrast ,
levels (
100
lM) is inhibited
The sugar
transport at saturating sugar
(Helgerson et aI., 1989).
transport experiments that defined this
were measured in red
M) is
A TP effect
blood cell ghosts (human red blood cells
which have been hypotonically lysed) or
inside out red blood cell
vesicles (IOV' s) resealed with and without 4 mM ATP at pH 7.4
(Helgerson et aI. , 1989). Under
these circumstances, normal
parameters such as pH and cellular redox state are artificially
controlled. This is not however the
case under normal physiologic
conditions. For example , sugar transport in cardiac
muscle as well as nucleated
erythrocytes responds to external
stimuli such as muscle contraction ,
oxidative metabolism (Cheung
and skeletal
anoxia , and inhibition of
et aI., 1977; Simons , 1983; Whitfield
and Morgan , 1983; Haworth and Berkoff , 1986; Hansen
Lee et aI., 1995). We were
lacked the types of
et aI., 1995;
concerned that our experimental system
intercellular communication (i.e. local
environmental conditions) that might exist
organs. This led us to ask
modulation of
in intact tissues and
1) What metabolic factors affect A
GLUT1? 2) Does the oligomeric structure of GLUTI
contribute to the A
TP effect?
To answer these questions ,
glycolytic intermediates , oligomeric
structure, and changing pH were tested for their ability to alter
30MG uptake into red blood cell ghosts
addition ,
these
resealed with 4
factors were examined for their capacity
azidoATP photolabeling
of purified
GLUTl. Our findings
that intracellular AMP and acidification antagonize
of sugar transport , and that A
for transport modulation.
that A
mM A TP. In
A
to
affect
suggest
TP regulation
TP interaction with GLUTI is sufficient
Our findings also support the
hypothesis
TP regulates only tetrameric GLUT1.
Results
Glycolytic Intermediates
Glycolysis and GLUTI-mediated transport appear
coordinately in
certain tissues (Cloherty et aI. ,
1996).
to be
regulated
We wanted to
investigate whether GLUTI-mediated sugar transport might be
subject to
regulatory feedback mechanisms similar to those found in
the glycolytic
pathway.
Intracellular (but not extracellular; see (Helgerson et aI. ,
Mg. A TP converts
30MG transport by resealed
erythrocyte ghosts
from a high capacity, low affinity transport system (V max
/lM/min , K m(app)
= 12. 3 :t 0.
system (V mn = 220 :t
net effect of A
1989)
=1069 :t 84
8 mM) to a low capacity, high affinity
19 /lM/min ,
K m(app)
=
386 :t 24
/lM;
n=4). The
TP is to increase 30MG uptake at 0. 1 mM sugar
by 2
to 5 fold (see also (Carruthers and Helgerson, 1989)). We measured
30MG uptake at 0. 1 mM sugar
to determine
how resealing
erythrocyte ghosts with or without glycolytic
basal and A
intermediates
affects
TP-modulated sugar transport.
Table 1 shows
modulation of
that intracellular AMP (2 mM) inhibits
A
TP-
transport. Extracellular AMP has no measurable effect
on basal or A TP-modulated
unexpected becaus
30MG transport.
previous work in
AMP , though unable to mimic
competes with A TP
This result is not
this laboratory has shown that
the action of A
i for access to the
. on transport,
transport system (Carruthers
and Helgerson , 1989). Resealing ghosts with 2
mM ADP stimulates
Table 1:
Effects of
glycolytic
erythrocyte
intermediates
30MG uptake
30MG uptake in JLmol/L
lAddition
cell wa ter/min
0 Mg. A TP
4 mM Mg. A TP
19:t2
49 :t 3
AMP (2 mM)
4:t 1
7:t 1
ADP ( 2 mM)
39 :t 3
no addition
NAD (2 mM)
no
NADH (2 mM)
no
Pyruvate (4 mM)
no
Citrate (2 mM)
no
Lactate (2 mM)
Fructose 1
6 bisphosphate (4 mM)
Fructose 2
6 bisphosphate (4 mM)
no
HP0 (4 mM)
Diphosphoglycerate (2 mM)
no
Phosphocreatine (1 mM)
no
Phosphoenolpyruvate (0. 1 mM)
no
no
Fructose- phosphate (0. 1 mM)
no
no
Glucose- phosphate (0. 1 mM)
Glyceraldehyde- phosphate (0. 1 mM)
Phosphoglycerate (0. 1 mM)
Adenosine (50 JiM)
no
no
no
Erythrocyte ghosts were resealed in KCI medium containing 5 mM MgCI plus
the addition shown and adjusted to pH 7.4.
no
indicates that there is no significant difference between the test
measurement and the measurement observed with no addition. The number of
measurements per condition was 3 or more. Results are shown as mean :t SEM.
basal transport. This result was unexpected because ADP , like AMP
competes' with
A TP for
binding to the
transporter. We hypothesize
that residual adenylate kinase present in red cell
ADP to A TP causing transport
ghosts converts
stimulation (Carru thers
and
Helgerson , 1989). The remaining glycolytic intermediates
and A
analogs (including adenosine) are without effect on basal or A
modulated transport whether applied
extracellular (not
shown) surface
at the cytoplasmic or
of the erythrocyte
The experiments describing effects of glycolytic
basal and A
TP-
membrane.
intermediates on
TP-modulated 30MG uptake were performed at pH 7.4.
Under physiologic conditions in muscle and erythrocytes ,
increased
glycolysis elevates cellular lactate levels resulting in acidosis (Cairns
et aI. ,
1993).
We therefore examined the ability of altered pH
modulate ATP-regulation of
GLUT I-mediated sugar transport. Red
blood cell ghosts were resealed
4 mM Mg. ATP adjusted to pH 5.
in
KCI medium
, 6.
, 7. 0
containing or lacking
or 8. 0.
Both extra-
intracellular media were adjusted to the same pH to avoid pH
and
; :.
changes due to transmembrane proton
demonstrated that
insensitive to
flow. Earlier
studies have
the extracellular sugar uptake site
alterations in external pH (Sen
is remarkably
and Widdas , 1962). KCI
both extra- and intracellular media to
medium was employed in
avoid transmembrane KCI gradients and K+ transport- induced
volume changes.
Figure 2A shows that as pH decreases from 8. 0
modulation of 100 p,M
30MG uptake
to 5. , A
is reduced. Subsequent
experiments (see Fig 3A) show even stronger
, :I
inhibition of A TP-
modulated transport. Basal transport rates are unaffected.
Calculation of free Mg, free
A
TP and Mg. A TP
this inhibition is unrelated to systematic
in the ionization state of A
of A TP-modulation of
levels indicates that
(pH- dependent)
TP (see Materials and Methods). The loss
transport at lower pH could
result from
altered binding kinetics (affinity or capacity of GLUTI for
from the pH dependent
alterations
A
TP) or
binding of a regulatory molecule to the
ATP- GLUTI complex. To examine these possibilities ,
was photolabeled at pH 5.
, 6.
, 7.
, and 8. 5
with
10p,M
purified
GLUTI
azidoATP.
Previous studies from this laboratory have demonstrated that
;i!
Figure
2. Effects of
GLUT1- labeling
pH on A TP-modulation of sugar transport (A) and
by azido-(y_
P)A TP (8).
A Erythrocytes were Iyzed and
resealed in KCI medium containing (8) or lacking (0)
2
mM exogenous ATP.
The pH of the resealing and extracellular media is shown on the abscissa.
Ordinate: initial rates of 100
,uM
30MG uptake (,mol/L cell water/ min). These
results summarize triplicate measurements. Lines drawn through the data points
were computed by the method of least squares. 8 Purified GLUT1 was incubated
in Tris- HCI medium containing
minutes at 4 . C. Each sample was
azido-(y- P)A TP at the pH indicated for 15
10,uM
uv
irradiated for 1 min and 30,ug of labeled
protein resolved on 10% SOS PAGE minigels. Proteins were transferred to
;J
nitrocellulose and labeled bands containing azido(y- P)A TP were identified by
autoradiog raphy
Figure 2
120
100
5 7.
5 8.
GLUTI and azidoA TP associate
and that azidoA
A TP ,
ADP ,
and
reversibly to form a binary complex
TP binding to GLUTI is inhibited
AMP (Carruthers and Helgerson ,
competitively by
1989). Proteins were
resolved on minigels and labeling was quantitated
autoradiography. Figure 2B shows that contrary
ATP incorporation
into
GLUTI is
by
to our expectations
as the pH increases from
reduced
to 8.
To better understand this response to
A TP- dependence of
30MG uptake in resealed
5 and 8. 0. Figure 3A
a saturable
however ,
shows that
100JLM
pH , we measured the
red cell
ghosts at pH
30MG uptake increases in
manner with ATP concentration at pH 8. 0. At pH 5.
transport is maximally stimula ted
inhibited at 2
at 200
JLM
mM A TP. Figure 3B summarizes three
experiments in which azido(y-
out at pH 5. 5 and 8. 0
unlabeled A TP. A
A TP , but
separate
P)ATP labeling of GLUTI was carried
in the presence of increasing concentrations of
TP inhibition of GLUTI labeling by azidoA TP shows
simple competition kinetics at both pH 5. 5 and 8. 0 but
maximum
labeling by
azidoA TP and
(app) for
A TP inhibition
reduced at pH 8. 0. The A TP content
of
of labeling are
erythrocyte ghosts resealed
3. Effects of pHon the concentration- dependence of A TP-
Figure
modulation of sugar transport (A) and ATP- inhibition of GLUT1-
azido-rf
P)A TP (B). A. Erythrocytes
labeling by
were lysed and resealed in KCI medium
containing 5 mM MgCI plus increasing concentrations of ATP at pH 8.
0 (8) or at
pH 5. 5
(0). Ordinate:
min). Abscissa: A
initial rates of 100
tlM
30MG uptake (,mol/L cell water/
TP content of the resealing medium in
tlM.
The results
summarize three separate experiments each made in triplicate. Lines drawn
through the data points were computed by nonlinear regression (see Materials
and Methods). These analyses assume that ATP-modulation of transport at pH 8
is comprised of basal transport
(k1
Michaelis- Menten component where
seen at 0 ATP) plus a simple , ATP- dependent
k2
is the increment in transport produced by
saturating A TP levels and K dATP is the apparent Kd
transport system. The computed constants are:
k1
dATP
= 89
tlM
for A TP interaction with the
= 46 tlM/min
= 30 tlM/min;
ATP. ATP-modulation of transport at pH 5. 5 is similar to that at pH
8 but is characterized by an additional interaction with A
uptake where
k2
k3
TP that reduces net
is the decrement in transport produced by saturating A
TP levels
and K dATP2 is the apparent K for A TP inhibition of transport.
The computed
constants are:
tlM/min; K dATP2
k1
= 199
= 48 tlM/min
tlM
k2
= 259 tlM/min; KdATP1
= 189
tlM
ATP
k3
= 259
ATP. B. ATP- inhibition of GLUT1- photolabeling by
azido-(y- P)ATP at pH 5. 5 (0) and pH 8. 0 (8). Purified GLUT1 was incubated
for 15 minutes at 4 o C in Tris- HCI medium containing 5 mM MgC/
. 15
tlM
azido-
(y_
PJATP at pH 5. 5 or pH 8. 0 in the presence of increasing concentrations of
ATP. Each sample was
uv
irradiated for 1 min and 30 Jig of labeled protein
resolved on 10% SDS PAGE minigels. Peptides were transferred to
nitrocellulose and labeled bands containing (y_
PJA TP
were identified by
autoradiography. Ordinate: relative (y_ PJATP incorporation into GLUT1 as
judged by densitometric analysis of resulting autoradiograms. Abscissa: (A TPJ
present during photolabeling (mM). These data summarize 2 separate
experiments. Lines drawn through the points were computed by nonlinear
regression (see Materials and Methods). This analysis assumes that
k4
is the
extent of labeling in the absence of ATP and that ATP competitively inhibits
labeling
iATP
(k5
is the decrease in labeling produced by saturating A TP levels and
is the concentration of A TP that inhibits labeling half-maximally). The
computed constants are: pH 8.
iATP
= 0. 5:t
iATP
= 1. 3 :t 0. 5 mM ATP.
0. 2
mM ATP; pH 5. 5,
= (1. 1 :t 0. 1) x 10
k4
k4=
(4. 7:t
0. 3)
x 10
k5
k5=
= (1. 0 :t 0. 1) x 10
(4. 1 :t 0. 5) x 10
--
Figure 3
110
C\ 1 00
I.
1000
2000
(ATPJ
5 1 0
41
3 1 0
CL 2 1 0
:6
pH 5.
1 1 0
pH 8.
o 10P
(ATPJ mM
with 4 mM ATP at pH 5. 5
or 8. 0 is not
significantly different (pH 5.
(ATP) = 3. 1 mM; pH 8. , (ATP) = 3. 1 mM; ATP content of ghosts
resealed in the absence of exogenous A TP: pH 5. 5, (A TP) = 0. 07 mM;
pH 8.
, (A
TP) = 0. 069
mM).
Oligomeric Structure
Erythrocyte-resident GLUTI exists as a
dissociates to
form a homodimer upon
extracellular reductant (Hebert
1995). In
homotetramer but
erythrocyte exposure to
and Carruthers , 1992; Zottola et aI.
the present study we ask:
does A
TP modulate both
dimeric
and tetrameric GLUTI-mediated sugar transport? Table 2 shows that
erythrocyte ghost exposure to reductant (2 mM DTT) inhibits
modulation of 30MG uptake
but is without
effect on basal
A
TP-
uptake
measured in the absence of A TP. Reduced capacity to modulate
GLUTI-mediated sugar transport
in reductant- treated erythrocyte
ghosts could result from reduced ATP binding to GLUTI or from
altered interaction of A TP- liganded
GLUTI with regulatory cellular
components. Figure 4 shows that incorporation of 8-azido(y- 32P)A TP
into reduced GLUTI is significantly
impaired.
:.','
E.:7YJ'
Table
Effect
of reductant
of erythrocyte
TP-modulation
30MG uptake
30MG uptake
Condition
Control
2 mM DTT
2 mM A TP
2 mM A TP + 2 mM DTT
pmollL cell water/min
41.2 :t 7.4
57. 3 :t 7.
97. 7:t
11.6
48. 1 :t 3.4
::1
Hypotonically Iyzed erythrocytes were resealed :t 2 mM A TP then incubated :t 2
mM OTT for 30 min at 37 . C. 30MG uptake at 100 JiM 30MG was measured at 4
C. The table summarizes results as mean :t SEM of 3 separate experiments
made in triplicate.
Figure
4. Azido-(y-
P)A TP labeling of
dimeric and tetrameric GLUT1.
Purified GLUT1 was incubated in Tris- HCI medium + 5 mM MgCI :! 4 mM OTT
for 30 minutes at 4 o C. Following labeling with azidoATP
30/lg
samples of
protein were resolved on 10% SOS PAGE minigels. Peptides were transferred to
nitrocellulose and bands containing (y- P)A TP were identified by
autoradiography. The presence (+) or omission (-) of OTT treatment is indicated
above each lane.
, I
#&..
Figure 4
105
82
49
33.
28.
19.
DTT
Does ATP modulate GLUTl mediated sugar transport directly?
Reconstitution of purified erythrocyte
(EggPC and cholesterol in an
lacking 2
GLUTI into liposomes
80:20 molar ratio) containing
mM Mg. A TP provides an in-vitro system that
solely of GLUT1 , A TP , and the lipid
transport into
cytochalasin B (10
consists
bilayer. Inhibition of sugar
GLUTI proteoliposomes
JlM
and
following
treatment with
CCB) insures that we are measunng protein
mediated solute movement across the lipid bilayer as opposed
non-specific leakage. Cytochalasin B (CCB) inhibitable 3\, methylglucose
uptake by reconstituted GLUTI proteoliposomes is
I), robustly stimulated by 2 mM Mg. A
TP (Figure
5).
Discussion
We examined whether A TP regulation
of transport in resealed ghosts
is subject to modulation by glycolytic intermediates
known to
regulate glycolysis. Only AMP antagonizes
30MG uptake. Citrate ,
fructose-
bisphosphate (key regulators of
the action of
A TP on
bisphosphate and fructose-
PFK or pyruvate kinase)
basal or A TP stimulated sugar uptake. Other
are without
intermediates
Figure
proteoliposomes.
5. ATP stimulates 30MG uptake into reconstituted , purified GLUT1
GLUT1 (100
Jig)
was reconstituted into eggPC:cholesterol
proteoliposomes and uptake of 50 JiM 30MG was measured at 20 . C in the
presence of 5 mM MgCI :t 2 mM ATP , and in the presence or absence of 20 JiM
cytochalasin B (CCB). Ordinate: 30MG uptake expressed as % of the equilibrium
30MG space of proteoliposomes accessed during 15 sec of uptake; Abscissa:
(ATPJ of medium during reconstitution and uptake determination (0 or 2 mM
ATP). Uptake in the presence of CCB is shown by the solid bars. This figure is
representative of three separate experiments.
Figure 5
OATP
2 mM ATP
were similarly inactive. Adenosine also failed to modulate GLUTI
mediated sugar transport. This suggests
that A
TP is not interacting
with the adenosine receptor , nor is the adenine ring responsible for
the effects of A
TP on GLUT1. ADP ,
however ,
mimics the ability of
A TP to modulate transport. These effects of AMP
transport in erythrocyte ghosts are similar
previously (Carruthers
ADP inhibits A TP
reported
to those
and Helgerson , 1989). External
application of
modulation of sugar efflux from inside out
erythrocyte membranes vesicles and , like AMP ,
action of A
and ADP on sugar
TP which reduces
C- Ab
antagonizes the
binding to purified GLUT 1
(Carruthers and Helgerson , 1989). This suggests that
,adenylate kinase present in
erythrocyte ghosts converts
" This hypothesis is supported by
residual
ADP to A TP.
A TP assays of ADP- Ioaded
ghosts
o.'
, (Carruthers and Helgerson , 1989).
j, regulation of sugar transport
We propose ,
is subject to
therefore , that A TP
inhibition by AMP and
possibly by ADP. The implication is that the glucose
epzyme of glycolysis of the
cell.
transporter -
is characteristically sensitive to the
energy
(y-
All experiments
intermediates on A
performed at fixed
investigating the action of glycolytic
TP modulation
sugar transport
of
were
pH (7.4). Under normal circumstances ,
glycolytic flux gives rise
to elevated
lactic acid levels and
pH (Elliott et aI. , 1992; Cairns et aI. ,
1993; Smith
increased
reduced
et aI. , 1993).
therefore examined the effect of altered pH on basal
and A
regulated sugar transport. To our surprise ,
we observed that while
reduced pH inhibited
sugar transport
A TP-modulation of
azido (y- P)A TP- Iabeling
to increase both
(app) for
of GLUTI was increased. Reduced pH serves
maximum photoincorporation
A TP- inhibition of photolabeling.
transport at pH 8.
increases in a
concentration. However ,
A
Increases with nucleotide
P)A TP and
of
A TP stimulation of
saturable manner with nucleotide
TP stimulation
of
transport at pH 5.
concentrations up to
200
11M
and
thereafter falls with increasing A TP. A TP assays indicate that
intracellular A
TP is not
low pH. Computation of
degraded to a
free Mg
, A
significantly greater extent at
TP and Mg. A
that these observations are not explained
dependent changes in
TP levels
indicates
by systematic, pH-
these species. We conclude
that
intracellular
A TP exerts
antagonistic actions on sugar transport at pH 5.
nature of the inhibitory action is presently
5. The
unknown. Increased
GLUTI labeling by azidoA TP at reduced pH raIses the possibility
that a unIque, transport-antagonistic A
TP binding
acidic pH. Future studies must address this
TP results in
sugar uptake
revealed at
possibility.
Isolated GLUTI is an A TP binding protein.
ghosts with Mg. A
site is
Resealing erythrocyte
stimulation at
subsaturating (30MG) and produces uptake inhibition at saturating
(30MG) (Carruthers
Mg. ATP
and Helgerson , 1989). Addition of 2
to reconstituted GLUTI proteoliposomes results in
stimulation of 30MG uptake
at sub
saturating (50
tLM)
30MG levels.
ATP modulations of purified GLUTI and erythrocyte resident GLUTI
mediated sugar transport
are , therefore , fundamentally
conclude that ATP , GLUT1 ,
similar.
and the lipid bilayer , are sufficient for
A TP modulation of GLUTI function.
This conclusion contrasts with that of
Wheeler who
demonstrated that A TP does not modulate
reconstituted human GLUTI protein
Wheeler
the activity
(Wheeler
of
1989). However
studies employed reduced (DTT- treated) GLUTI which
we
have shown to exist as a GLUTI dimer (Hebert and Carruthers
1992). Our studies employ the
GLUTI (a GLUTI tetramer ,
physiologic , non-reduced form of
see (Hebert and Carruthers , 1992).
therefore asked two questions. 1)
A TP-regulation? 2)
Is reduced
GLUT
susceptible to
Does reduced GLUTI bind A TP? Our studies
shown that reductant
treatment of
erythrocytes converts resident
tetrameric GLUTI to dimeric GLUTI (Zottola et aI. ,
present findings indicate
have
1995) and our
that reductant also ablates A TP-
modulation of 30MG transport. Reductant (DTT) treatment of
purified GLUTI results in the loss of azidoA TP labeling.
results provide a rational
explanation for Wheeler s findings and
suggest strongly that dimeric
to nuc1eotides.
These
GLUTI does
not
present binding sites
They further reinforce the view that red cell-resident
GLUTI exists as the A TP sensitive , GLUTI tetramer (Zottola et aI.
1995 ).
Chapter IV
the GLUTl
Peptide Mapping
ATP
Binding Domain
Isolated human erythrocyte glucose transporter (GLUT1)
functions as an A
1989).
TP binding protein (Carruthers
and Helgerson
Work in this laboratory has shown that the GLUTI carboxyl
terminus becomes less
accessible to peptide- directed
TP exposure.
following A
This A
TP dependent
is antagonized by AMP and ADP that appear
competitive inhibitors of A
out to determine
antibodies
conformational change
to function as
TP binding to GLUTl. In this study we set
the location of the GLUTI nucleotide
binding
domain.
To answer this question we applied peptide mapping
N-terminal sequencing techniques
azidoATP ,
a photoactivatable
suggest that
GLUTI residues
A TP binding
domain.
to
and
GLUTI photolabeled with
P labeled ,
ATP analog. Our findings
301- 364 form an integral part of the
' '
Results
Peptide Mapping
Previous studies from this laboratory have demonstrated that
GLUTI and azidoA TP associate
and that azidoA
reversibly to form a binary complex
TP binding to GLUTI is inhibited
ATP , ADP and AMP (Carruthers and Helgerson
of the azidoA
competitively by
, 1989).
UV
TP/GLUTI complex activates the A TP azido
irradiation
group
sulting in covalent attachment of bound nucleotide to GLUTI. In
the absence of irradiation ,
incorporated into the
no measurable
carrier protein (Carruthers and
1989). Limited proteolysis
photoattachment of azidoA
1989).
Helgerson
studies suggest that the point
TP to GLUTI falls witbin the carboxyl-
terminal half of the protein (residues 270-
Helgerson ,
8-azido(y- P)ATP is
456; see (Carruthers
We have now undertaken
mapping analysis to more precisely define
ttachment of nucleotide to
and
a detailed peptide
the site of covalent
GLUTl.
Peptide mapping studies
of
integral membrane proteins such
as GLUTI are complicated by the typically
hydrophobic nature of
the resulting peptides and by the
presence of N- linked
oligosaccharide. For example , human erythrocyte band 7 proteins
include the
multi-spanning integral membrane glycoproteins
aquaporin (28 kDa) and RhD (Mr = 45
kDa) (Smith and Agre , 1991)
but are resolved as a 32 kDa protein band upon 10% SDS PAGE
(Zottola et aI. ,
1995). Intact
GLUTI (Mr = 55 kDa) is resolved
kDa band (Fig 6A). We therefore decided
markers to
to use
as a 50
GLUTI internal
assist in peptide identification. Peptides containing
linked oligosaccharide
(asn ) were
identified by blot analysis
streptavidin-conjugated HRP) of GLUTI fragments obtained
N(using
from
glucose transporter biotinylated on carbohydrate residues prior to
proteolysis. Pep
tides containing GLUTI residues
88-
and/or 480-
492 (C-residues) were identified by use
antisera (N-
Ab and
C- Ab
peptides corresponding to
95 (N-residues)
of rabbit
respectively) raised against synthetic
these GLUTI domains. Each experiment
(see Figure 6A) thus involves biotinylation
by unsealed GLUTI proteoliposomes ,
photolabeling using azido(y- 32P)A
TP.
of carbohydrate exposed
followed by
The labeled
GLUTI
proteoliposomes
(y_
Figure
6. Peptide mapping of photolabeled
A. Purified GLUT1 (40 j.g
GLUT1.
biotinylated on carbohydrate residues) was photolabeled using
10j.M
azido-
fy_ P)A TP then incubated in sample buffer containing 5 j.g chymotrypsin (30
minutes at 37 . C). Peptides were separated on a 10-
20% SOS PAGE gradient
gel , transferred to nitrocellulose. Azidofy- P)A TP-containing peptides were
detected by autoradiography e p). C- Ab and N- Ab reactive peptides were
detected using immunoblot analysis. Biotinylated carbohydrate (Glyc) was
detected by streptavidin blot analysis. C-
, N-
, 32
p and Glyc analyses were
performed on a sihgle common lane of electrophoretically separated peptides.
Major peptides are assigned a label (a through h) and are indicated by the
leftmost solid bars. The mobility and M of molecular weight standards are
indicated by the solid bars to the right of the figure.
B. EndoLys- C digestion and sequencing of azido-fy-
P)ATP- photolabeled
GLUT1. Labeled , purified GLUT1 (100 j.g) was incubated in 0.
Tris-HCI medium , pH 7.4 containing 5 j.g endoproteinase Lys-
j.g endoproteinase Glu- C (EndoGlu- C;
separated on a 10- 20%
18- 24 hours at 4 O C).
1 % SOS , 50 mM
C (EndoLysC) or 5
Peptides were
SOS PAGE gradient gel , which had been pre-
equilibrated with 1 mM thioglycolate and subsequently transferred to ImmobilonPVOF for staining with Coomassie blue. Bands containing
P)ATP were
, identified
by autoradiography e p). C- Ab and N- Ab reactive peptides were
detected using chemiluminescence immunoblot analysis. Major peptides are
assigned a label (i through 0) and are indicated by the leftmost solid bars. The
mobility and M of molecular weight standards are indicated by the solid bars to
the right of the figure. An intensely labeled 19 kDa band (peptide n) was excised
from the membrane , washed 10 times with distiled water and subjected to
sequence analysis by the UMMC Microsequencing Facility. A single sequence
was obtained as AGVQQPVY A T corresponding to
GLUT1
residues 301- 310.
Figure 6
Ab N- Ab Glyc
33.
28.
19.4
32p
33.
28.
19.4
EndoLys-
EndoGlu-
were subjected
PAGE ,
to limited
transferred to
and/or 32P- Iabelled
proteolysis, peptides are resolved by SDS-
immobilon or nitrocellulose ,
and
biotinylated
peptides are detected by autoradiography.
same membrane filters are
then probed for the presence of
The
peptides
containing N- and C-residues by chemiluminescent immunoblot
analysis.
Figure 6A indicates that not all N- or
contain 32p or
biotinylated carbohydrate. Similarly,
containing peptides include
(V8) digests
labeled GLUTI which were subsequently
One of the
was subjected
not all 32p-
N- or C- Ab-reactive sequence. Figure 6B
shows EndoLys- C and EndoGlu- C
N-residues.
C- Ab-reactive peptides
of 8-azido(y- 32P)ATP
probed for 32p and C- and
32P- Iabeled peptides (figure 6B , peptide n)
to N- terminal
sequence analysis
summarizes additional experiments
intact GLUTI was subjected
to
(see below). Figure 7
in which azidoA TP- Iabeled
digestion by chymotrypsin (Figure
7 A) or by V8 protease (Figure 7B). Major peptides of Figures 6 and 7
have an assigned label and are aligned in Figure 8 according
to size
sequence content
specificity.
(identified by internal markers) and proteolytic
This analysis permits approximate localization of the
(y-
7. Peptide mapping of photolabeled GLUT1. A. Purified
Figure
Jig)
was photolabeled using 10 JiM
azido-(y-
GLUT1 (40
P)A TP then incubated in sample
buffer containing 5 Jig chymotrypsin (30 minutes at 37 . C). Peptides were
separated on a 10- 20% SOS PAGE gradient gel , transferred to nitrocellulose.
P)ATP-containing peptides were detected by autoradiography e
p). N-
reactive peptides were detected using immunoblot analysis. Major peptides are
assigned a label (p through u) and are indicated by the leftmost solid bars. The
mobility and M of molecular weight standards are indicated by the solid bars to
the right of the figure. B. V8- protease digestion of azido-(y- P)A TPphoto/abeled GLUT1. Labeled , purified GLUT1 (100
Jig)
was incubated in 0. 1 %
SOS , 50 mM Tris- HCI medium , pH 7.4 containing 8 or 15
Jig endoproteinase Glu-
C (EndoGluC; , 18- 24 hours at 4 . C). Peptides were separated on a 10PAGE gradient gel and subsequently transferred to Immobilon-
20% SOS
PVOF for staining
with Coomassie blue. Bands containing (y_ P)ATP were identified by
autoradiography e p). C- Ab reactive peptides were detected using
chemiluminescence immunob/ot analysis. Major peptides are assigned a label (v
through bb) and are indicated by the leftmost solid bars. The mobility and M
molecular weight standards are indicated by the solid bars to the right of the
figure.
"' -
.-
Figure 7
p N-
105
f - 33.
- 28.
- 19.
Chymotrypsin
105
-82
33.
-28.
aabb-
va protease (g)
19.
, (y_
Figure
8. Summary of peptide mapping analysis of (y-
P)ATP- labeled
GLUT1 (Figures 6 and 7). The horizontal axis represents GLUT1 residues
492.
1 to
labeled and unlabeled peptides are indicated by filled and open bars
respectively. Data are from Figures 6 and 7 and are cross-referenced by peptide
labels (a - bb). Peptides containing C- Ab reactive sequence overlap with
residues 480 - 492. Peptides containing N-
GLUT1 residues 88 -
GLUT1
Ab reactive sequence overlap with
95. Peptides containing biotinylated
carbohydrate also
contain GLUT1 residue asn . When two peptides are linked by a single vertical
bar , this indicates that localization is ambiguous and the range of possibilities is
shown for the linked peptides. The length of each peptide was computed by
assuming that electrophoretic mobility accurately represents peptide molecular
weight. The shaded vertical box spanning GLUT1 residues 291-
364 represents
that region of GLUT1 that most likely contains covalently attached (y_
The striped bar represents a 19 kDa
(peptide n) of GLUT1 whose N- terminal
PJATP.
PJA TP- Iabeled , endoLysC fragment
residue (A 301 )
was confirmed by
sequencing. Peptides e and y should (but do not) contain (y_
pJA TP and are
shaded differently for comparison. Presumed specificities of proteolysis are:
Endo- LysC , K; V8 protease , 0 , E; chymotrypsin
,F
//,/
;,, / / /
".- , ;,..
Figure 8
45 88 95
291
364
nv /
480 492
/ //1
:J;,,
.-J
':1
YI
45 88 95
291
364
480 492
(y_
site of covalent attachment
364. Recognizing
of
P)A TP to
residues 291 through
that the masses of hydrophobic and glycosylated
proteins obtained by SDS- PAGE analysis
of nucleotide attachment should be
are approximate , this regIOn
viewed with some caution. Two
peptides have properties inconsistent with this conclusion.
Peptide e
(Figure 6A) and peptide y (Figure 7B) cross react with C-
, are
characterized by Mrapp 19. 9
no
and
22.4 kDa respectively
but contain
p. Three observations suggest that these peptides are
detectable
dysfunctional
GLUTI fragments
present in
GLUTI proteoliposomes
pnor to labeling and digestion. 1) These C- Ab-reactive peptides
observed occasionally in
GLUTI preparations
proteolytic enzymes (Hebert and Carruthers ,
Carruthers ,
unpublished
correspond to any
Peptide n
are
that are untreated by
1992); Cloherty and
observations). 2) Peptide
does not
possible V8-specific GLUTI cleavage product. 3)
overlaps with peptides
e and y, contains 32p and
identified definitively by N-terminal sequencing (see
below).
(y-
N- Terminal Microsequencing
To further refine definition of the site
of nucleotide attachment
azido (y- P)A TP labeled GLUTI was subjected to
digestion. Endoproteinase Lys-
Lys-
ester ,
endoproteinase
specifically hydrolyzes amide
and peptide bonds at the carboxylic side of
lysine (Jekel et aI.
1983). Assuming the conditions of
proteolysis reveal each cleavage
site and also favor EndoLys-
produce a 16 kDa
456. Purified ,
GLUTI fragment
azido- A TP labeled
for 18- 24 hours at 4 o
10- 20%
specificity, this
enzyme is expected to
corresponding to
GLUT 1 was exposed to endoLys-
C and the resulting peptides were resolved
the
blots were exposed to
stained with Coomassie blue. Bands
membrane , and
of
film for autoradiography and
interest were excised from the
subjected to N- terminal
microsequence analysis.
Figure 6B indicates that a 19 kDa band (peptide
n) is heavily labeled
P)ATP but lacks recognition sites for both N-
terminal sequence analysis
of
and C- Ab. N-
this peptide yielded 10 pmol of a
single sequence of AGVQQPVY A
301- 310.
on
gradient SDS- PAGE gels. Following transfer to Immobilon-
membranes ,
by
residues 301 -
T corresponding to
GLUTI residues
Discussion
The results of our
291- 364 as
peptide
mappIng studies point to GLUTI residues
the smallest GLUTI cassette
attached azido (y32PJ- A
TP. This
that contains covalently
result is consistent with previous
indirect studies suggesting that GLUTI residues
involved in
nucleotide binding (Carruthers
270- 380
and Helgerson , 1989).
The present study offers significant improvement
analyses because we have employed internal
approximate origin of
assumed
residues), GLUTI residues
linked carbohydrate (asn
480-
markers to indicate the
assigned on
residues 84- 96
(N-
492 (C-residues) and asparagIne-
). Even USIng
these markers , however
GLUTI peptide mapping studies
the frequently anomalous mobility of
is complicated by
GLUTI fragments upon SDS
:PAGE. For example , an azido(yP)- ATP
, fragment with intact carboxylterminus
Jragment. EndoLys-
earlier
proteolytic enzyme
specificity alone. These markers are: GLUTI
interpretation of
over our
proteolytic fragments previously
the basis of peptide mass and
may
labeled endoLysC GLUTI
is resolved as a 23 kDa
specifically hydrolyzes amide ,
ester , and
peptide bonds at the carboxylic side of
in proteolysis (an assumption
lysine. Assuming specificity
supported by sequence analysis
smaller endoLysC GLUTI fragment), this peptide could
cleavage at lysine 300 or
, peptides (21
of a
derive from
lysine 256. The predicted masses of these
kDa and 25. 8
kDa
respectively) differ
significantly
from the observed mass on SDS PAGE.
The smaller azido(yfragment (19. 1 kDa) was
P)- ATP labeled endoLysC GLUTI
subjected to N- terminal
sequence analysis.
I:
This confirmed the origin of the azido(y
P)- ATP
fragment as
labeled GLUTI
ala 01 with hydrolysis occurring at lys 30o
specificity and precise
places the site of
i:
. Assuming
molecular weight determination
, this result
azido(y P)- ATP incorporation within a GLUTI
cassette comprised of residues
weight = 19223 D).
301-
477 (theoretical
molecular
Our peptide mapping studies suggest that
azido(y P)- A TP incorporation
occurs between
and 364. This permits further refinement
GLUTI residues
of the azidoA
TP-
incorporation domain to residues 301 - 364. This GLUTI
contains primary sequence that
shares
291
domain
50% identity with a region of
adenylate kinase previously shown to
form a
hydrophobic strand of
parallel B-
pleated sheet
flanking the adenine-ribose and
triphosphate chain of MgA TP (Fry
et aI. , 1986).
This sequence
adenyl ate kinase
GLUT
GQPTLLL YVDAG
Izl
GRRTLHLIGLAG
33z 343
IS also found in the adenine
nucleotide binding proteins
phosphorylase (Newgard et aI. , 1988),
glycogen
RAD3 (Reynolds et aI. ,
the ATP/ADP translocase (PIano and Winkler ,
1991), the
brown fat uncoupling protein (Casteilla et aI. ,
1989; Cassard
1990), phosphofructokinase (Valdez et aI. ,
subunits of Fj ATPase (Ohta and Kagawa ,
GLUTI domain
to the glucose
while strengthened
of a
of this
remaInS
detailed GLUTI structure. It is
contains the consensus
which is found in a
may be exposed
Walker
number of A TP and GTP
binding proteins (Fry et aI. , 1986). In GLUTl ,
(residues 111- 118)
et aI.
the (X- &
1986). Assignment
, mappIng studies ,
interesting to note that GLUTI also
motif GXXXXGKS/T ,
human
transport nucleotide binding pocket
by our peptide
inferential in the absence
1989) and
1985),
this
sequence
to the exterior of the cell
(Hresko et aI. ,
1994) suggesting that it
intracellular nucleotide binding.
assumes no role in
Chapter
Alanine mutagenesis of the GLUTl consensus
ATP
binding sequences
Peptide mappIng and n- terminal sequencIng studies
laboratory have suggested
that residues 301-
the GLUTI ATP binding domain.
pnmary sequence
that is
This
in
this
364 comprise part of
region of GLUTI contains
50% identical with a region
of
adenylate
kinase previously shown to form a hydrophobic strand of
a parallel
pleated sheet flanking the adenine ribose and triphosphate
of Mg. ATP (Fry et aI. ,
sequences have been
1986).
Two additional consensus ATP binding
identified in GLUTI (Fry et aI. ,
to delineate further the location of the A
we have
the data
also show that
transporter function.
three proposed regions
suggest that residues 332-
the efficiency of adenine nucleotide
1986). In order
TP binding site on GLUTI
mutated all of the residues in these
to alanine. Our findings
chain
binding to
residues 338- 340
335 contribute to
GLUTl. In addition
are necessary
for normal
Results
Purification of
Human GLUTl HA His
GLUTI is a glycosylated membrane protein that contains
linked oligosaccharide
a
single
at asn . The presence of this carbohydrate
makes it difficult to synthesize GLUTI in a bacterial system. We felt
that either a yeast system
fulfill our needs.
or a
transient mammalian system
would
Our data suggest that we were fairly successful in
expressing hGLUTI in a strain of Saccharomyces Cerevisiae
(RE700A) in which seven
(hxt 1- 7) of 20 putative glucose
transporter genes are disrupted
of yeast is unable to
Figure 9B). Following
or
deleted. Unmodified ,
this
strain
survive on standard glucose media (Table
transfection with human GLUT1 ,
clones are able to grow on
source (Figure 9A & 9B).
and
selected
transport glucose as a sole
In addition , separation of
3
carbon
whole cell
extracts for western blotting reveals a 55 Kd protein that reacts
with
GLUTI C- Ab (Figure 10). The data suggest that we have successfully,
heterologously, expressed human GLUTI in yeast.
Unfortunately,
this mutant yeast grows too sl wly and produces insufficient GLUTI
for our purposes.
Table 3: Characteristics of S. Cerevisiae strain RE700A
transfected with
Yeast
Carbon
Source
Wild Type
Glucose
Wild Type
Galactose
Wild Type
Maltose
RE700A
Glucose
RE700A
Galactose
RE700A
Maltose
RE700A + GLUT1
Glucose
RE700A + GLUT1
Galactose
RE700A + GLUT1
Maltose
GLUT1
Glucose
Uptake
Growth
S. Cerevisiae
2 RE700A is aURA 52
mutant with 7 of its glucose transporters (HXT 1deleted or disrupted.
RE700A transfected with HA His epitope tagged human erythrocyte glucose
transporter (GLUT1).
Figure
9. GLUT1 HA His expression in Saccharomyces Cerevisiae. A.
H)0- glucose uptake measured in RE700A +/- tagged GLUT1. Yeast were
grown to an
600
= 0. 6 for uptake. 1- 2 mL of cells were pelleted and washed in
PBS prior to addition of uptake media. All solutions except stop were preheated
to 37 C. Uptake of 100 /lM O- glucose was terminated at 5, 30, and 60 seconds
by washing with ice cold PBS pH 7.4. Following lysis , duplicate samples of
supernatant were counted in a Beckman scintillation counter. B. RE700A +/tagged GLUT1 grown on YP-maltose or minimal media + glucose.
Figure 9
+ GLUT1 HA HIS
- GLUT1 HA HIS
:: 350
-- 250
150
Null
Null
YP- Maltose
Minimal Media + Glc
+/-
Figure 10.
C-terminal GLUT1 antibody western blot of RE700A
GLUT1 HA His Cell extracts were prepared from RE700A +/- tagged GLUT1
grown to an OD 600
of 0. 6.
Prior to extraction , the yeast were washed twice with
ice cold 50mM Tris- HCI pH 7. 5, 10 mM NaN . Cells were combined with 0. 2 mM
glass beads and 100 JlI of protein sample buffer containing 1 mM PMSF. The
mixture was vortexed briefly and 50 JlI of each sample was resolved on 10% SDS
PAGE gels that were transferred to nitrocellulose for western blotting with GLUT1
terminal antibody.
;: .:
Figure
t(5
q, e
(r
105
33.
28.
19.
,-,
We therefore
mammalian transient
decided to develop a
eXpreSSIOn system for GLUTl. Cos-
cells are a simian kidney cell
GLUTl.
line that is capable of correctly processIng
1985; Schurmann et aI. ,
(HA), 6 histidine (His
1997).
We introduced
(Mueckler et aI.,
a hemagglutinin
)' tag to facilitate the purification of the
expressed human GLUTI (GLUTI HA His ). To accomplish this, a
unique Mfel restriction
site
was introduced at residue
Kbp fragment of GLUTI cDNA previously
subc10ned into the
site of pGEM3Z (pGEM3Z- GT1) (Zottola et aI. ,
insertion of an HA His
further subcloned
expression vector.
the His
into the
epitope tag at this
BamHI
1685 in a 1.7
BamHl
1995). Following the
site, the GLUTI was
site of a pcDNA3. 1+ mammalian
The benefits of attaching this tag are twofold;
region binds Nickel- NT A allowing affinity purification
recombinant protein and , 2)
HA can be detected with standard
western blotting techniques , allowing us
parental Cos- 7
of
to differentiate between
GLUTI and tagged human GLUTI (GLUTI HA His
To purify hGLUT1 , Cos- 7 cells were transfected with
pcDNA3. 1 GLUT
1 via the Fugene 6 transfection
Cells were harvested and homogenized
reagent system.
72 hours post transfection
and membranes were
isolated by high-speed
centrifugation in
Beckman TI50. 2
rotor at 100 000 x g (Loo and Clarke , 1994).
the hydrophobic
nature of hGLUT1 ,
detergent ,
n- dodecyl-~
D maltoside ,
purification because ionic
to solubilize the
it is necessary
applied to the Ni- NTA.
membranes before they are
detergents
of the protein to bind to the
A non- ionic
was used to prepare samples for
may interfere with the ability
column (Loo and Clarke ,
solubilized protein was allowed
Due to
1995).
The
to bind to the nickel matrix for one
hour with gentle agitation. The Ni- NTA column was washed with
increasing concentrations
were eluted with a
of imidazole (10- 25
mM),
and samples
solution of 500 mM NaCI and 300 mM imidazole.
Fractions were resolved on 10% SDS- PAGE gels and transferred to
nitrocellulose membranes
for immunoblotting.
Standard western blotting techniques were used
GLUTI present on these membranes. Gels stained
suggest that GLUTI HA His
pure.
Rabbit antisera
corresponding to
to identify the
with Pro- blue
containing eluate is approximately
50%
raised against a synthetic peptide
hGLUTI residues
480- 492
(C- Ab) was used to
locate and identify intact GLUTI in various purification fractions.
Human GLUTI and Cos- 7 GLUTI show significant homology, thus C-
terminal Ab binding
is indicative of
total GLUTI concentration. To
quantitate hGLUT1 , we used anti- HA antisera which binds selectively
to epitope tagged hGLUTI.
Figures llA
& B show both the
resulting
terminal and HA western blots from a standard GLUTI HA His
purification protocol using Cos-
cell membranes. A single
band with an approximate molecular weight of 55
large
KDa is visible
in
the final elution
fraction (eluate (E) on both blots. The presence of
both an intact
terminus and an HA tag suggests that we have
C-
successfully affinity- purified His tagged hGLUTI protein.
Alanine Scanning Mutagenesis of
Fry et al (Fry et aI. ,
potential consensus A
ATP Consensus
1986) showed
TP binding
Binding Sequences
that GLUTI contains
sequences
(Figure 1).
three
Current
GLUTI topology would place consensus site I (Gresidues 111- 117), within transmembrane
II (K- X- V -
domain 3.
amino acids 225- 229) lies within the
loop between domains 6 & 7.
S/T
Finally site III (G-
Consensus site
large cytoplasmic
L (Y ,
I) X-
Figure
11. Purification of GLUT1 HA His from Cos- 7 cells. HA- Ab (A) and G-
Ab (8) immunoblot analysis of samples of GLUT1 HA His transfected cell
membranes obtained during GLUT1 affnity purification on Ni- NT
A. Isolated
Gos-
7 cell membranes were solubilized in n- dodecyl-~- O-maltoside (lane 2) and were
applied to aNi- NT
A column. Following flowthrough of non
bound GLUT1 (lane
3), columns were washed with 300 mM NaGI plus 10 mM imidazole (lane 4), 300
mM NaGI plus 30 mM imidazole (lane 5) and 500 mM NaGI plus 300 mM
imidazole (lane 6). Fractions were collected and proteins were separated on 10%
SOS- PAGE gels and transferred to nitrocellulose for immunoblot analysis. A. The
presence of GLUT1 in each of the samples was detected with G- Ab. Purified
human erythrocyte GLUT1 was placed in the far left lane as a control. GLUT1
runs as a diffuse band with an approximate molecular weight of 55 kOa. Lower
molecular weight bands are proteolytic fragments of GLUT1. B. GLUT1 HA His
was identified with a mouse anti- HA antibody. Fractions correspond to and are
aligned with those in panel A. The mobility of molecular weight standards are
indicated by the solid bars to the left of the panels A and 8.
..-
.. ..::. ..
.::
:=
,-
Figure 11
I- Q) 0
or E
'C
Q)
u. T" ('
'C ..
en
ca
a. C/ .. :s
Ct
9750352922-
143
97503529-
143
1--
residues 332- 343) occupIes both a portion of the cytoplasmic
loop between helices 8 & 9
and transmembrane
mapping studies from this laboratory (Levine et aI. ,
that the point of covalent
Arg 301
-
A TP attachment to GLUT 1
Met 364 . This proposed
sequence III.
Once a
was in place ,
site-
Peptide
domain 9.
region of binding
1998) suggest
is between
overlaps with
procedure for producing and purifying GLUT
directed mutagenesis could be undertaken. To test
the involvement of the three domains in
changed the residues in each of the
GLUTI A TP binding, we
three proposed sites
(Table 4) and determined whether transporter
to alanine
function or
azidoA TP
binding was altered.
D-
glucose Uptake into Cos-
Cells
To assess whether wild type and mutant hGLUTI are functional
we measured 2- deoxy- glucose (2DOG) uptake
into Cos-
cells
transfected with each of our pcDNA3. 1+ GLUTI constructs. 2DOG is
used in this instance because it undergoes phosphorylation and
remams trapped
in
the intracellular space. Cells
expressing
epitope
tagged hGLUTI show a two- fold increase in cytochalasin B (CCB)
Table
Mutant Name
4 -
GLUT1 Alanine Mutants
Original Sequence
111A
111
225A
225
GFSK 114
KSVLK229
332A
332
335A
335
338A
338
341A
341
334
Mutation
111
225
AAAA 114
AAAAA
229
332
AAA 334
335
AAA 337
LIG340
338
AAA
LAG 343
341
AAN
GRR
TLH337
340
inhibitable sugar transport above basal levels (i.e.
measured in untransfected cos- 7
cells ,
transport
or in cells transfected with
pcDNA3. 1 + lacking GLUT1 HA His cDNA) (Figure
12). Transfection
with alanine mutants: lIlA , 225A , 332A , 335A, and
results in a 2- fold stimulation of
hypothesized that this
transport (Figure 12). We
increase was a result
presence at the plasma membrane.
measuring 8-antibody
binding to Cos-
is an approximate
of greater
by
cells +/- hGLUTl. Because
epitope of
measurement of
expression at the membrane. Cos-
GLUT1
We tested this possibility
this antibody recognizes an exofacial
its binding
341A also
tetrameric hGLUT1
transporter
cells transfected with tagged
GLUT 1 exhibit a 2- fold increase in 8-antibody binding above Cos-
cell levels (Table 5). This confirms our original assumption that
transport stimulation is a
direct result of enhanced GLUTI presence
at the plasma membrane.
In contrast, mutant 338A shows only basal transport activity
and is considered
later ,
retains
non- functional.
its ability to bind
This mutant, as we wil discuss
azidoA TP. This
result implies that
Figure
12. 2- deoxyglucose
uptake in Cos- 7 cells transfected with GLUT1
Comparison of 200G (0. 1 mM) uptake at 37 o C by Cos7 cells , Cos-
HA Hisij.
cells transfected with vector (pcONA3. 1) lacking
cells expressing
GLUT1 HA
His
and
GLUT1 HA
GLUT1
His
sequence and by Cos7
alanine mutants. Ordinate:
CCB- inhibitable 200G accumulation (mol per min per Jig total cell protein).
Abscissa: cells in which transport was measured. Results are shown as mean
:tSEM
of triplicate measurements.
Cos-
His6
Cos- 7 pcDNA3.
GLUT1 HA
GLUT1
GLUT1 225A
GLUT1 332A
GLUT1
GLUT1 338A
GLUT1 341A
1 1 0-
21
3 10-
2DODG uptake
4 10-
mol per min per Jlg cell protein
51
Table 5: o- Antibody binding to Cos- 7 Cells transfected with
GLUT1
Antibody Binding
125
cpm
per
Cos- 7
Cells
Cos- 7 Cells + GLUT1
HA His
Cos- 7 Cells + 338A
1 N=
/lg
protein G
SEM
total protein
2658
704
5595
427
2199
experiments carried out in duplicate
alanine substitution
at residues 338-
340 does
not
grossly affect
transporter quaternary structure (i.e. GLUTI is folded correctly
respect to
A TP binding). To test
this hypothesis,
CCB (cytochalasin
B) photoincorporation
into
GLUTI was measured
isolated from unaltered
Cos-
7 cells ,
as well as
with
in membranes
cells that had been
transfected with GLUT1 HA His and 338A. Membranes containing
tagged wild type
GLUTI bound 2- fold more CCB than Cos- 7 cells
alone (Figure 13A). In contrast CCB binding
was equivalent to that found in basal
13B). The absence of
338A membranes
Cos- 7 membranes
CCB binding and the
binding imply that some aspect
in
retention of
of the mutant
(Figure
azidoATP
transporter structure
disrupted.
It is also possible that
membrane is
disrupted. To better understand these results ,
antibody binding
Cos-
cells transfected with mutant
and 2- fold
(Table 5).
8-
studies as previously described were applied to
exhibited the same level
cells ,
GLUTI trafficking to the plasma
338A.
Interestingly, 338A
of antibody binding as untransfected Cos-
less binding than cells containing wild type hGLUTI
Figure
13. Photolabeling of parental GLUT1 and transfected wild type and
mutant human GLUT1 in Cos- 7 cells by the sugar export site ligand (3 HJ-
CCB. Cos- 7 cell membranes were photolabeled in the presence and absence of
1 00
M phloretin. Membranes were washed to remove unincorporated (3 H)- CCB
and membrane proteins were separated by SOS-PAGE. Following Coomassie
staining, each gel lane was sliced into 2 mm sections , dissolved and counted for
associated (3 H)- CCB. Ordinate: phloretin inhibitable CCBincorporation (cpm).
Abscissa: relative mobility (gel slice number). A. CCB incorporation in Cos-
cells , and cells transfected with GLUT1 HA His . B. CCB incorporation in Coscells transfected with GLUT1 HA His and alanine mutant 338A.
100
Figure 13
600
500
.. Cos-
0 WT GLUT1
400
300
200
100
Slice#
2000
o WT GLUT1
. 338A
-g 1500
.. 1000
t. 500
Slice#
101
AzidoATP Labeling of Wild
Mutant hGLUTl
Type
To test whether alanine substitution affects A TP binding
transporter ,
we measured azidoA
TP incorporation
(mutant & wild type) purified from Cos-
cells.
in
GLUTI HA His
Wild type
mutant hGLUTI were affinity- purified from Cos-
to the
and
cells as described
previously. Proteins eluted from the Ni- NTA column were
concentrated using an
hour in a
Amicon centricon- 10 spun at 5000
x g for
Beckman JA- 17 rotor. Samples labeled with azidoA TP were
equilibrated for 30 minutes in 10- 15 IJM azidoATP
irradiated for 90 seconds
at 280
then UV
nM in a Rayonet Photochemical
reactor. Fractions were simultaneously resolved on three 10% SDS-
P AGE gels and transferred
to
nitrocellulose. Two of the
membranes
were used for C- Ab and HA- Ab western blotting. The third blot,
which contained photolabeled hGLUT1 ,
autoradiographic film for 5-
24 hours.
was exposed
Densitometry was used to
quantitate epitope tagged GLUTI and azidoA TP incorporation.
protein concentration for each of the
a Pierce BCA kit ,
applied to all
and a uniform
three gels.
Total
constructs was measured with
quantity of each mutant
protein was
102
Western blots and azidoA TP autoradiographs
for each
alanine
mutant are shown in Figure 14. Lane 3 (4 mM ATP) shows purified
epitope tagged GLUTI (from
presence of 4
mM cold A TP. The absence
suggests that A TP
HA His
lane 4) labeled with azidoA
of radiolabel incorporation
and 8-azidoA TP bind to the same site on GLUTI
. This same effect has been seen previously, using purified
human erythrocyte GLUTI (Carruthers
et aI. ,
TP in the
and Helgerson , 1989; Levine
1998).
AzidoA TP binding in each
incorporation in wild type
(azidoA TP) to
alanine mutant was compared to
GLUTI HA His . The ratio
HA antibody was used as a measure
attachment to tagged
transporter. Mutants lilA ,
341A bound quantities
of
azidoA TP that
found in GLUTI HA His (Figure 15).
of photolabel
of A TP
335A , 338A, and
were equivalent to that
In contrast , mutants
225A and
332A exhibited a 1.
to 2.
incorporation (Figure
15). Three possible explanations for this
increased A
fold Increase in photolabel
TP incorporation are: 1) The small sidechain
reduces steric interactions that normally limit
transporter- binding site.
2) By exchanging
A
TP access
polar
of
alanine
to the
residues with
103
Figure
14. Azido( p)ATP labeling of GLUT1 HA His and its corresponding
alanine mutants. A. Transporter was purified by Ni- NT A affinity chromatography
of membranes isolated from Cos- 7 cells transfected with wild- type or mutant
GLUT1 HA His (see Figure 11). Purified GLUT1 was photolabeled using 10 pM
azido( P)ATP , resolved on three separate 10% SOS PAGE gels (25pg total
protein per lane per gel) and transferred to nitrocellulose for western blotting and
autoradiography. For each group of mutants an internal control of GLUT1 HA His
was also processed. Control protein is visible at the far left of each group of blots.
GLUT1 constructs were identified by C- Ab and HA- Ab immunoblot analysis.
Incorporated affnity label (azidoATP) was detected by autoradiography. Antibody
and ATP binding were quantitated by densitometry using NIH Image. The ratio of
azidoATP labeling to HA- Ab reactivity is shown below the blots. Lane 3 (4 mM
ATP) shows that Ni- NTA purified GLUT1 HA His photolabeling byazidoATP
(lane 4) is inhibited in the presence of 4 mM unlabeled ATP.
,,
..
..
104
Figure 14
GLUT-
111A
4 mM ATP
GLUT1-
225A
332A
GLUT1-
a.
335A
338A
8,
341A
105
Figure
15. Corrected AzidoATP labeling of GLUT1 HA His (wild type and
mutant). AzidoA
TP labeling of tagged GLUT1 (wild type & mutant) is presented
as a ratio of A TP/ HA antibody binding. This distinguishes azidoA TP labeling of
recombinant human GLUT1 HA His from parental GLUT1.
106
Figure 15
111A 225A 332A 335A 338A
341A
107
uncharged alanines ,
A TP binding are
structure of the
sequence
increased. 3)
hydrophobicity and subsequently
Charge alterations loosen the
binding pocket.
GLUTI HA His and 332A were also
analysis of
A TP inhibition
of
subjected to competition
azidoA TP photoincorporation. Affinity-
purified GLUT1 HA His and 332A were photolabeled
with azidoA
in the presence of increasing concentrations of unlabeled A TP.
Membranes were then subjected
nitrocellulose and quantitated
to SDS- PAGE , transferred to
for
azidoA TP incorporation
autoradiography and densitometry. As with
of isolated
purified
(Levine et aI. ,
azidoA
TP photolabeling
human erythrocyte glucose transport
1998), photoincorporation of
by
protein
azidoA TP into affinity
purified GLUT1 HA His and 332A is inhibited half-maximally by 50150 JLM
ATP (Figure 16).
With GLUTI 332A , however,
photoincorporation of azidoA
TP in the
fold greater. This suggests
that increased photoincorporation of
azidoA TP into
absence of A
TP is some 10-
mutant 332A results from increased efficiency of
mutant GLUTI labeling. HA- Ab immunoblot efficiency is unchanged
by azidoA
TP photolabeling of GLUTl.
108
Figure
16. ATP- inhibition of azidoATP photolabeling of affinity purified
GLUT1 HA His6 (0) and GLUT1332 (8).
Purified ,
epitope- taggedGLUT1 was
photolabeled as in Figure 14 in the presence of increasing unlabeled (A TP).
Proteins were resolved by SDS- PAGE and label incorporation and epitopeP-autoradiograms and Ha-
tagged GLUT1 were quantitated by densitometry of
Ab immunoblots. Ordinate: the ratio (32 P) incorporation: HA- Ab reactivity.
Abscissa: (ATP) present during photolabeling in JiM. Curves drawn through the
points were computed by nonlinear regression assuming that inhibition of
photolabeling shows simple saturation kinetics and is characterized by the
. P(ATP)
expression: labelincorporation = N - where N IS label
Kj(app) + (ATP)
incorporation in the absence of unlabeled ATP , P is the amount by which
saturating unlabeled A
TP inhibits label incorporation and Kj(app) is that
concentration of unlabeled ATP which reduces labeling by P/2. The following
constants were obtained: GLUT1 HA His , N= 0. 66 :! 0. 01; P=0.41 :! 0.
54:! 17 JiM
ATP.
ATP; GLUT1 332 3A' N= 11. 2:!
0. 7;
P=11. 3:!
1. 0,
Kj(app) =
, Kj(app) =
127:! 49 JiM
109
Figure 16
000 1500
500
(ATP)
2000
110
Parental Cos- 7
be able to form heterotetramers
GLUTI may
with
GLUTI HA His . This could make it difficult to visualize the effects of
GLUTI mutagenesis on azidoA TP
parental GLUTI and 1
binding. For example , if 3 units of
unit of tagged
alterations in labeling caused
by
GLUT1 form a tetramer
alanine substitution
in the
single
subunit may be partially camouflaged. To examIne this possibility,
Cos- 7 membranes +/- GLUTI HA His
were stripped
membrane proteins with an alkaline wash
azidoA TP.
and labeled with
Samples were resolved on 10% SDS- PAGE
photolabel incorporation was measured.
protein was found
to bind 3- fold
transfected with GLUTI HA His
17). This suggests that
gels and
The same quantity of total
more azidoA TP from cells
than non- transfected cells (Figure
parental Cos- 7
GLUT1 ,
present in
preparations of nickel purified , epitope tagged hGLUT ,
obscure the
peripheral
of
should not
affects of alanine mutagenesis on azidoA TP binding.
III
Figure
17. AzidoATP labeling of Cos- 7 cell membranes stripped of
peripheral proteins. Isolated Cos- 7 cell membranes from control cells or from
cells transfected with GLUT1 HA His were labeled with azidoA TP following
3':=HE"
'''I'"r.",
removal of peripheral proteins by alkaline wash. Samples were resolved on 10%
SDS PAGE gels and transferred to nitrocellulose. Protein contents were
determined using a Pierce SCA assay kit , and 35
g of total protein were loaded
in each lane. Label incorporation was detected by exposure of membranes to
autoradiographic film. AzidoATP binding was quantitated by densitometry using
NIH Image. Membranes from GLUT1 HA His transfected cells contain 3 fold
more azidoA TP label than membranes isolated from nontransfected cells.
::
:',
.;
112
Figure 17
'i
143 Kd
97Kd
50Kd
35Kd
29 Kd
22Kd
,,1
113
Discussion
In this study we ask the
the three proposed
question;
Does alanine substitution at
GLUT 1 consensus A TP binding sequences affect
sugar transport and azidoATP binding? As a
expressed HA His
mutagenesis studies , we have
erythrocyte GLUTI in: CosGLUTI HA His
result of our alanine
cells. Intact Cos-
tagged human
cells expressing
exhibit normal sugar transport under basal
conditions. In addition ,
purification of the protein on a Ni- NT A
column provides a transporter
that is
normal with respect
to A
binding capacity.
We compared 2- deoxyglucose (2DOG) uptake in CosGLUTI HA His
as a measure of
and/or cell surface
epitope tagged GLUT 1
above basal Cos-
function
expression. Transfection with GLUT 1 HA His
(wild type) and 5 of the six mutant
335A , and 341A)
cells +/-
constructs (111A , 225A , 332A
resulted in a two- fold stimulation of
transport
cell levels , and levels observed in cells transfected
with empty pcDNA3. 1 + vector.
This increase
in transport is
probably the result of a 2- fold increase in cell surface
most
transporter
114
expreSSIOn. We tested this hypothesis by
binding to Cos- 7
measurIng 8-antibody
cells +/- GLUTI HA His . We observe that cells
expressmg GLUTI HA His present 2- fold more 8-antibody reactive
cell surface sites than untransfected Cos-
our initial hypothesis
cells. This result
that transport stimulation in
confirms
transfected cells
IS a direct result of greater transporter presence at the
plasma
membrane.
In comparison , cells transfected with
unchanged sugar transport
later ,
retains
rates. This construct
its ability to bind
oligomeric structure
of
mutant 338A display
azidoA
, as we wil discuss
TP. This suggests that the
338A is not affected by alanine substitution
at least with respect to A TP binding. This hypothesis was examined
by measuring (3H)- CCB binding to membranes isolated from Coscells transfected with GLUTI HA His , and 338A. Tagged GLUTI
(wild type) binds 2- fold
more CCB than 338A or Cos- cells. These
results suggest that some structural component
of
mutant 338A is
altered (although ATP is still able to bind). Most probably,
regIOn of
GLUTI that contains
it is
the
the closely related sugar efflux and
CCB binding sites (Sultzman and Carruthers
1999). Alterations in
115
the sugar- binding
site might also explain the inability of 338A to
transport sugar.
Two other possibilities could explain the inability of 338A to
i:- -
transport sugar. 1)
trafficking to the plasma
Transporter
The affinity of 338A for sugar may be altered.
may be disrupted.
2)
8-antibody binding
studies were used
expressed at the plasma
binding in
membrane.
338A transfected
due to the absence of
test whether 338A is
The data show that 8-antibody
cells is the
same as that observed in
alanine substitution at 338- 340
of a
epitope of
tetrameric GLUT1
may disrupt GLUT1 oligomeric
configuration insensitive GLUTI exofacial-
in this protocol may resolve
question.
this
Schurmann et al. observed
that transport was lost in
when conserved residues 333R/334R were converted
(Schurmann et aI. ,
may
binding
338A at the plasma membrane or, because 8-
antibody recognizes an exofacial
structure. The use
to
cells. The lack of 8-antibody
untransfected Cos-
membrane
1997). These
alanine
results contrast with our work that
shows normal transport activity in
difference may be explained
to
GLUT4
GLUTI mutant 332A. This
by their reconstitution of
GLUT4 into
116
vesicles to
measure uptake.
carried out in intact Cos-
cells. Substitution of alanine
results in a loss of charged
responsible for
In their
at this
residues. These amino acids
retaining transporter topography in the
absence ,
exposed to
In companson , our experiments were
333R/334R may
site
might be
membrane.
lose topographical stability when
detergent during reconstitution.
We assumed that the strategy of alanine mutagenesis would
decrease, or ablate , azidoA
TP binding to GLUTI. However this was
not the case; in fact two constructs (225A ,
1.5 to 10-
and 332A) incorporate
fold more azidoATP than GLUTI HA His
mutants. This increase in
does not reflect altered
or the other
labeling was somewhat unexpected
affinity for A
TP but rather ,
and
altered efficiency
of photolabeling.
The overall
and 332A might
increase in
be a
azidoA TP labeling observed in 225A
result of alanine substitution reducing the
steric hindrance that normally limits ATP access to the GLUTI ATP
binding pocket. Alternatively, the
makes consensus sequences II ,
their similarity to the proposed
presence of additional alanines
and
III more hydrophobic. Increasing
consensus
sequences may allow
...
-'
117
more azidoA TP
to bind
transporter charge may
to
result in the loss
interactions (Gallivan and
I/.
the transporter.
Dougherty,
binding. Assuming that increased
a secondary
A
of
alterations in
stabilizing cation-
1999), which could affect
A
TP incorporation does not reflect
action in which mutations
introduced at a
nonspecifically alter ligand binding within
pocket, this
Finally,
distal site
the nucleotide- binding
effect might be consistent with a direct
perturbation of
the nucleotide- binding domain.
,,:1
large range appears to exist in the maXImum amount of
azidoA TP that can be incorporated into mutant 332A. Densitometry
of photolabeled
332A shows a 1.5-
labeling (Figure15), as opposed
inhibition of
to
3 fold increase in azidoA
competition analysis (ATP
azidoA TP incorporation) which suggests
azidoA TP labeling
(Figure 16). This discrepancy
autoradiogram overexposure in
which could alter the
The effects
10- fold higher
may be a result
the photolabeling
experiments
32p detection sensitivity of the
of mutating residues 332- 335 on
of
technique.
GLUT1 A
binding are consistent with N- terminal sequence analysis
of
peptides
obtained upon proteolysis of azido A TP- photolabeled GLUTI. These
118
studies indicate that the
GLUTI A TP binding domain lies between
GLUTI residues 301 and 364 (Levine et aI. ,
1998). Since
GLUTI
sequences 307 to 327 and 338 to 358 are strongly hydrophobic and
thus putative membrane spanning a- helices (Mueckler et aI.,
they are unlikely to be
GLUTI residues
accessible to cytosolic A TP. In
332- 335
fall in the intervening
and the
remainder of
contrast
the Walker B domain
(cytosolic) sequence between these membrane
spanning regions and may be well- positioned
A TP and modulation
1985),
for interaction with
of the sugar translocation pathway.
AzidoA TP and A TP binding
to purified
human erythrocyte
GLUTI are mutually exclusive , suggesting that they bind at
site on the protein (Carruthers
and Helgerson , 1989). To
the same
determine
that this is also the case in epitope tagged GLUT1 , we labeled
purified GLUT1 HA His with azidoATP in the presence
of 4
mM ATP.
The absence of 32 P incorporation confirms the stereospecificity of
azidoA TP binding to tagged GLUTl.
It is probable
oligomerize to form
that parental GLUTI and tagged GLUTI
tetrameric GLUT1. However ,
membranes transfected with GLUTI HA His
Cos-
cell
bind 3- fold more
119
azidoA TP than a comparable amount of
protein from untransfected
membranes. This suggests that the presence of
samples of
parental GLUT1 in
affinity purified epitope tagged GLUTI does
changes in azidoA
mutagenesis.
TP binding that are a direct
not obscure
result of alanine
'--
120
Chapter
Significance
& Future Directions
This thesis examInes several hypotheses. First , we tested
capacity of
various physiologic factors
TP modulation
to influence A
of GLUTI mediated sugar transport. Specifically, we looked
effects of glycolytic intermediates,
oligomeric structure. Next we asked ,
is
the
ATP interaction with GLUTI
nucleotide binding domain
erythrocyte glucose transporter.
at
intracellular pH , and transporter
sufficient for sugar transport modulation? Third , we
location of the
the
investigated the
on the human
Finally, having narrowed
the
possible location of the ATP binding site on GLUT1 ,
we analyzed
whether alanine mutagenesis altered sugar transport
and A
binding.
We have concluded that only AMP ,
ATP
regulation of
GLUTl.
and possibly
ADP ,
inhibit
Previous work in this laboratory has
shown that AMP , and ADP , though unable to mimic the effects of
A TP compete with A TP
for access to the transport system
(Carruthers and Helgerson , 1989). This suggests that the adenosine
;;..
121
ri;
binding, while phosphate chain
moiety is responsible for
dictates the ability of a compound to
length
modulate GLUT 1 activity. The
r. -
F;t::
present study shows that
modulation of
adenosine alone is insufficient for
human GLUTI function.
An increase
in
glycolysis can lead to an
elevation in
lactate levels and subsequent acidosis (Cairns et aI. ,
encouraged us to examine
how alterations
in
cellular
1993). This
intracellular pH affect
A TP regulation of GLUTI. From these studies, we observed that as
pH decreases from 8.
A TP modulation of GLUT1 is inhibited.
0 to
We propose that this loss
of A
TP regulation results from altered
binding kinetics (affinity or capacity
pH dependent
of
GLUTI for A TP) or from the
binding of a regulatory molecule to the A TP- GLUTI
complex.
The data indicate that pH affects
A TP-
Surprisingly, we found that low pH increases
the
(app) for
A TP- inhibition
et al. (Merino et aI. ,
(32P)A
of photolabeling is
1997) has shown
structure of the nucleotide- binding
Specifically, low pH was
GLUTI binding kinetics.
TP binding
while
unchanged. Merino
that pH also modulates the
domain on
A TPase.
found to increase access to the A
TP binding
122
site. A similar mechanism could explain the increase
binding that we have
observed in
in azidoA
GLUTI as pH is lowered from 8.
to 5.
To better understand how low pH inhibits A TP regulation of
GLUT1 ,
we examined the ATP dependence of 30MG uptake
cell ghosts at pH 5. 5 and 8. 0. Uptake was found to increase
saturable manner at pH 8. 0.
into red
in a
In contrast, 30MG uptake at pH 5. 5
was
maximally stimulated at 200 JlM A TP and inhibited at 2 mM A TP.
Degradation of A
TP at low pH was ruled out as a cause of this
unexpected result.
We have concluded that A TP acts in, an inhibitory fashion
sugar transport at low pH.
incorporation at pH 5. 5,
on
In view of the increase seen In photolabel
transport
inhibition was unexpected. This
result alluded to the possibility that low pH unmasks
a unIque
transport-antagonistic ATP binding site on GLUTl. The exposure of
such a site would account
the inhibition
of
for both the increase in A
TP binding and
transport seen under acidic conditions.
Transport
inhibition could also be the result of GLUTI interacting with a
dependent inhibitory protein.
123
We have shown that GLUT1 reconstituted into
proteoliposomes containing 2
stimulation at sub
that GLUT1 ,
exhibits transport
saturating 30MG. We have therefore
the lipid bilayer
required for A
mM A TP
, and ATP are the
concluded
only components
TP modulation of GLUTI function. This same system
could be used to rule out or confirm the presence of a
regulatory protein.
If transport inhibition is seen in
pH inducible
this system at
pH 5. 5, we can say with some certainty that a regulatory protein
not the agent
arresting transport at low pH.
Wheeler (1989) was unable to recreate
the A
TP effect in a
reconstituted system. His ' experimental design used the reduced
dimeric form of
physiologic or
Carruthers
the transporter.
In contrast , our
tetrameric form of the transporter (Hebert and
1992).
This laboratory has observed that exposure to
reductant (dithiothreitol- DTT) inhibits
uptake.
studies employ the
A
TP modulation
of 30MG
Specifically, conversion of tetrameric GLUTI to its
form results in loss
between the two
regulated by A
of azidoA
TP labeling. This explains
data sets. We conclude
TP.
that
the
dimeric
disparity
tetrameric GLUTI is
,t'
f,
124
Although GLUT1 functions
as an A
TP binding protein ,
location of its nucleotide binding domain is
experiments have suggested
;Jmj
.n
that
to GLUTI falls within the range
the
currently unknown. Past
covalent incorporation
of azidoA TP
of C- terminal residues (270- 456)
(Carruthers and Helgerson , 1989). The
peptide mapping studies
carried out in this thesis identify residues 291- 364
as a likely
component of the
GLUTI azidoA TP-attachment site. The strong
hydrophobicity of
GLUTI presents
molecular weight of
this uncertainty,
carbohydrate ,
a problem for estimating the
GLUTI fragments
by gel
mobility. To balance
we used internal markers (C- terminus ,
and azidoA
N- terminus
TP), and proteolytic enzyme specificity to
identify peptide fragments more definitively. More recent work in
our laboratory has shown that A TP increases GLUTI resistance to
proteolytic digestion (Cloherty and Carruthers,
observations). This may offer an explanation
unpublished
for the
encountered obtaining complete proteolytic digests
difficulty
of azidoA
labeled GLUTl.
Digestion of
purified erythrocyte GLUTI with
endoproteinase
125
Lys-
peptide fragment whose first ten
produces a
identified by N'-terminal sequencIng as GLUT1
This sequence and
the peptide mapping data
amInO acids were
residues 301- 310.
allow us to further
refine the proposed azidoA TP-attachment site on GLUTI to
cassette that contains residues 301- 364.
a
Importantly, this range of
residues overlaps with a proposed Walker B motif in
GLUT1.
We have shown that A TP competitively inhibits azidoA TP binding
to purified
GLUT1 (Carruthers and Helgerson ,
that azidoA
TP and A TP bind to the same site on the transporter. The
1989). This
proposed pocket structure of a nucleotide- binding
implies
domain (Fry et
aI., 1986) presents certain problems when using 8-azidoA TP for
peptide mappIng. Specifically the azido moiety that
of adenine may crosslink
site.
For this
reason
8-azidoA TP
8
to a peptide adjacent to the actual binding
GLUTI peptide mapping experiments
2-azidoA TP should also be performed in
GLUTI segments
sits on carbon
contributing to the
2-azidoA TP crosslink
of 2-azido
labeled GLUTI might
pattern of
labeled peptides.
using
an effort to identify
other
nucleotide binding pocket. If
to adjacent peptides , digestion
produce a significantly different
;::!.
126
To further
delineate the location of the A
constructed alanine
mutants for each of
consensus sequences on GLUT 1
TP binding site, we
the three
proposed
and measured azidoA TP
labeling of
and sugar transport by, each mutant GLUTl. This required
development of a
the
GLUTI expression system. Unfortunately, the
yeast strain we created (RE700A + GLUTI HA His
) grew too slowly
and produced insufficient GLUTI for our purposes.
clones are being screened to
and human GLUT
select one with a high
Currently, more
rate of growth
expression.
This problem led us to develop
expressIOn system for
a
transient mammalian
human GLUTI usmg Cos-
cells.
Purification
of HA His tagged GLUTI on a nickel column produced large
quantities of a protein which run as a
diffuse band with
approximate molecular weight of 55 kDa.
GLUTI C-
, and HA-
, identified this as
erythrocyte glucose transporter.
found to be normal
Western blotting with
with respect
transporter was normal with
epitope tagged human
In addition , in-vivo transporter was
to sugar transport , and
respect to azidoA
the isolated
TP binding.
B:.
127
Transfection of Cos- 7
and five of the
cells with epitope tagged wild type GLUT1
SIX alanine mutants, resulted in a 2- fold increase in
DOG uptake above basal Cos- 7 cell levels.
8-
Antibody binding studies
have shown that cells transfected with GLUT1 HA His present a 2- fold
excess of
GLUTI at the plasma membrane relative to untransfected
cells. This suggests
that the
transport stimulation we observed
cells expressing epitope tagged GLUT1 ,
transporter expression at the
cannot rule out the
stimulate transport,
Cos-
in Cos-
is the result of increased
cell membrane.
As a cautionary note , one
possibility that these newly expressed proteins
in part , by having a higher rate of catalytic turnover
than parental GLUT1. However ,
quantitatively the change
increased
transport parallels
in cell surface binding of a
antiserum (8- Ab) directed
against exofacial epitopes of
GLUT I-mediated 2DODG transport
1986). The measurements
in
GLUTl. Km(app)
for
human red cells is 15 mM (Stein
reported in
the present study were made at 0.
mM 2DOG. Transport is, therefore, given by
uptake
polyclonal
max f2DODG)
m(app)
::-\%';
128
where V max
cellular 2DOG transport capacity) = kcat (GLUTILell surface
(the
where kcat is the intrinsic catalytic turnover of GLUTI. Since bo th
transport and (GLUTl)ceii surface are
doubled by Cos-
GLUT1 HA His this suggests that the k / Km(app)
HA His
cell transfection with
ratio for expressed
GLUTI
cells is not significantly different from that of Cos- 7 cell
in Cos-
endogenous GLUT1.
338A was the only mutant whose catalytic activity may have
been affected by alanine substitution. 30MG uptake by this
construct was not significantly different from uptake in
untransfected Cos-
cells. This was suprising because these
residues
(338- 340) are located in a proposed transmembrane domain of
GLUTI and are ,
like alanine
, uncharged. The fact that this
retains its ability to bind azidoA TP suggests
structure is unchanged with
In contrast , phloretin
55 kDa protein
mutant
that its oligomeric
respect to the nucleotide binding site.
inhibitable photoincorporation of CCB into a
in membranes expressing mutant 338A is
not
significantly different from that observed with membranes isolated
from non- transfected cells.
a portion of mutant
The absence
338A structure
of
CCB binding implies that
is disrupted.
Because this
129
mutant retains azidoA TP binding,
the disrupted region in
the
CCB labeling data suggest that
question encompasses the proposed GLUT
CCB binding and sugar efflux sites , which are closely related
(Sultzman and Carruthers
1999).
The lack of 2- deoxyglucose
uptake by the 338 mutant could
result from disabled sugar transport, a result
loss of
consistent with the
CCB binding. Alternatively, the transporter may not be
expressed correctly at the plasma membrane.
GLUT 1 that is
structurally intact , could
studies because total solubilized
to the
Cos-
Incorrectly trafficked
still bind
azidoA TP in
these
cell membranes are applied
nickel column for epitope tagged GLUT 1 purification.
To test this possibilty, cell surface 8:-antibody binding was
measured in Cos-
cells transfected with mutant 338A. The results
show that mutant 338A expressing
antibody as Cos-
cells bind the
cells alone , but 2-
expressing wild type
same amount of
fold fewer IgGs than cells
epitope tagged GLUTl. The lack of o-an ti body
binding may be due to the absence of 338A at the
plasma
membrane or alternatively, alanine substitution at 338- 340 may
convert tetrameric GLUT1 to
dimeric GLUT1.
In
this instance 8-
130
antibody, which recognizes
an exofacial epitope of
GLUT1, would be unable to recogmze the
protein. If this is the case
the affinity of mutant 338A for 2DOG or its ability
2DOG may be altered. Immunohistochemistry
question.
of the following: 1) dysfunctional
GLUTI trafficking to the plasma
structure in a small region of
membrane , 2) abnormal oligomeric
the transporter, and/or 3) an altered
structure.
Originally; we hypothesized
amIno acid to
alanine would knock out ,
were found to bind 1.5 to 2.
essential
that converSIOn of an
or reduce , azidoA
to the transporter. Contrary to this expectation ,
His
this
configuration
conclude that the lack of sugar transport in mutant
338A is a result of one or more
quaternary
to translocate
using a
insensitive exofacial GLUTI antibody may resolve
We therefore
tetrameric
fold more azidoA
TP binding
225A and
TP than
332A
GLUTI HA
or the other alanine mutants. It is possible that the charge
alterations in these mutants loosen the structure
resulting in increased access of the N
of the transporter
group of azidoA
TP to the
A
binding pocket. Another explanation is based on the small side
chain found on alanine. In both 225A and 332A ,
alanine replaces
~~~~
131
polar amino acids
that contain large side chains. Removal of
barriers could reduce steric hindrances controlling access
nucleotide binding pocket. Another possibility is related
This stretch
hydrophobic nature of Sequence III.
GLUTI was found
to
contain 50%
is highly hydrophobic
to the
to the
amino acids in
homology to a Walker B box which
(Fry et aI. ,
alanines for polar residues in
of
these
1986). Substitution of
GLUTI (i.e. 332- 334
the hydrophobic nature of this regIOn.
uncharged
GRR), Increases
By making GLUTI sequence
III more like a true Walker B motif, we may have enhanced azidoA TP
binding to the transporter. In the absence of a change in the k
A TP inhibition
332A ,
of
azidoA TP labeling in GLUT
1 HA His
for
and mutant
it is doubtful that the increased label incorporation that we
have observed in
mutants 225A ,
transporter affinity for
and 332A is due
ATP. The reactive N
to
altered
of azidoATP may simply
be in closer proximity to (i. e. have increased
reacting
probability of
with) side chains in the pocket.
Another obstacle that concerned
us was the
potential for
parental GLUTI oligomerization with tagged GLUTI (both
and wild type). GLUT1 HA His
containing Cos-
mutant
cell membranes
132
bound 3- fold
more azidoA TP than membranes from cells
tagged GLUT1. We propose that the presence
of
lacking
parental GLUTI in
these preparations should not obscure the affects
of alanine
mutagenesis.
We were particularly interested to observe that azidoA TP
incorporation into
increases in a
purified human erythrocyte GLUTI at low pH
manner similar to that found with epitope
GLUTI mutant 332A purified from Cos- cells. Ki(app)
inhibition of
azidoA TP GLUTI HA His
indicating that transporter affinity for A
mutation of
ATP- inhibition
ATP-
labeling is not reduced
TP is not
these residues. AzidoA TP labeling of
increased 5 to 10- fold by lowering
for
tagged
pH from 8. 5
increased by
red
to 5.
cell GLUTI is
5 yet Ki(app)
for
of labeling is unchanged (Levine et aI., 1998).
Histidine is the best candidate
as pH is lowered from 8. 5
amino acid
to 5. 5.
for side-chain protonation
GLUT1 contains
5
histidine
residues of which four (H 160, 239, 337 and 484) are located within
cytoplasmic domains. One of these ,
GLUT 1 Walker B
H 337 , is found in the
putative
domain although direct involvement in pH-
dependent modulation of azidoA
TP binding efficiency
wil
require
133
confirmation by substitution mutagenesis. Why would His
protonation mImIC (alanineh
332 GRR?
substitution for
One clue may
tg'
be obtained from the
hydrophobic
GLUT1 sequence preceding the
putative Walker B mqtif. This sequence contains a phenylalanine
that could pair with Arg333
or Arg334 by cation-n
(Gallivan and Dougherty,
1999). These interactions, which attain
approaching - 4 kcal/mol , can
structure (Gallivan and
Dougherty,
interactions
significantly stabilize
protein
1999). If the neighboring His
::iJ.
':(.\:1
iL'
'J. .
were protonated at reduced pH ,
interactions by charge
by mutagenesis
it might disrupt Phe- Arg
repulsion of Arg
or protonation of His
structure permitting
cation-n
. Removal of Arg 333
or Arg 334
? could relax local GLUTI
uv
closer interaction of the
activated azidoA
nitrene with GLUTI side chains or backbone. This structural
relaxation could increase
the efficiency
of label
without changing the affinity of the transporter
While speculative ,
photoincorporation
for
nucleotide.
this hypothesis warrants experimental
scrutiny.
Fluorescence resonance energy transfer (FRET) is another
technique that can be used to further
GLUT 1 nucleotide binding domain.
delineate the location of the
We have collaborated with a
134
visiting orgamc synthetic chemist (Dr. Boris Yagen ,
University, Jerusalem) to synthesize a fluorescent A
Hebrew
TP derivative
anthraniloyl- A TP (AN- A TP). AN- A TP has an absorbance maximum at
343 nm and
AN-
an emISSIOn maXImum at approximately 450 nm.
ATP binding to 2
with k
tLM
GLUTI is half
constants of 0.
and koff
3 per
tLM
maximal at 7
tLM
AN- ATP
per min and 2. 1
per min
respectively (Cloherty, Yagen and Carruthers, unpublished). AN- ATP
also mimics the
abilty of
transport in red
cells.
ATP to modulate GLUT I-mediated
Using the intrinsic tryptophan fluorescence of GLUT1 (excitation
at 298 nm ,
emission
max 332 nm) to excite AN- A TP bound
GLUT1 , we have observed fluorescence resonance energy transfer
(FRET) between GLUT1 tryptophan residues and bound
AN- A TP.
AN- A TP fluorescence requires GLUTI tryptophan fluorescence
because in
the absence of this energy
extremely low fluorescence.
location of the
source , AN- A
Using this
TP shows
technique, we can map the
GLUTI nucleotide- binding domain as
tryptophan residues
within the
it relates to
transporter. By utilizing
column purification of epitope tagged human
Ni- NT
GLUT 1 , we can
A
135
synthesize and isolate GLUT1 mutants in which tryptophan
1l-
converted to glycine or leucine , thereby identifying tryptophan
residues in close proximity to
i ,
has been
bound AN- A TP
;;?
136
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