Download Structure, function and biosynthesis of GLUTI

Structure, Function and
Regulation of Glucose
Transporters
Membrane Group/Hormone Group Joint Colloquium Organized and Edited by G. Holman (School of Biology
and Biochemistry, University of Bath) and Sponsored by Pfizer. Novo Nordisk. SmithKline Beecham and
Zeneca Pharmaceuticals. 66 Ist Meeting held at Bath, 9- I I April 1997.
Structure, function and biosynthesis of GLUT I
M. Mueckler*, R. C. Hresko and M. Sat0
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63 I 10, U.S.A.
Structure and function of GLUT1
[9] using glycosylation-scanning mutagenesis, a
procedure whereby N-linked glycosylation consensus sites are engineered into the predicted
aqueous domains of a membrane protein, and
the ability of a specific site to be glycosylated is
interpreted as reflecting an extracytoplasmic disposition for that site. T h e absence of glycosylation at a specific site is inferred as indicating a
cytoplasmic disposition, although this conclusion
is necessarily more tenuous than the converse
assignment. We used the glycosylated exofacial
linker domain of GLUT4 as the glycosylation
marker. When the glycosylation mutants were
expressed in Xenopus oocytes, the pattern of
glycosylation obtained with the mutants was
exactly as predicted by the topological model for
G L U T l that we proposed on the basis of hydropathic analysis of the deduced amino acid
sequence [S]. These data thus provide strong
experimental support for the original topological
model. Interestingly, however, the results
obtained when the mutants were expressed in
reticulocyte lysate supplemented with canine
pancreatic microsomes differed in a significant
manner from the oocyte results. Although all of
the predicted exoplasmically disposed sites were
fully glycosylated in the reticulocyte system, all of
the predicted cytoplasmically disposed sites
showed an approx. 50% level of glycosylation.
These data suggest that aberrant insertion of a
fraction of the chimaeric protein molecules harbouring the glycosylation marker in cytosolic
domains occurred in the in vitro system, but not
in the in vivo system. Analysis of additional chimaeras demonstrated that the altered topology
T h e G L U T family of 50-60 kDa membrane
glycoproteins consists of five known members,
four of which are involved in the facilitative
transport of glucose across cellular membranes
[l-31. GLUT1, the prototype member of this
family, may be the most extensively studied of all
mammalian membrane transporters. It was one
of the first membrane transporters to be purified
to homogeneity [4] and cloned [5], and its
kinetic properties in the erythrocyte membrane
have been studied for over four decades [6].
Despite this attention, our knowledge of the
structurelfunction properties and biosynthesis of
this molecule, and of membrane transporters in
general, is still rudimentary.
T h e cloning of human G L U T l from HepG2
cells revealed a polypeptide of 492 amino acid
residues with a molecular mass of 54 117 Da [5].
In vitro translation studies indicated that
G L U T l possesses a single N-linked oligosaccharide at
[7]. G L U T l was the first of
what is now a large superfamily [S] of membrane
transport proteins that are predicted to possess
12 a-helical transmembrane segments based on
hydrophobicity analysis of its deduced amino acid
sequence. Until recently, however, this ubiquitous 12-transmembrane-segment
topological
motif was without direct experimental support
and thus remained purely hypothetical.
We tested the topological model for G L U T l
Abbreviations used: ER, endoplasmic reticulum membrane.
*To whom correspondence should be addressed.
95 I
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Biochemical Society Transactions
952
indeed represented aberrant insertion whereby a
local disruption of topology occurred in a fraction
of the molecules containing the glycosylation
marker in cytoplasmic domains [9].
Nothing is known at present about the
tertiary structure of the glucose transporters. We
proposed the simplistic model that five amphipathic helices of G L U T l cluster together in the
membrane to form an aqueous compartment
through which sugars traverse the fatty acyl core
of the lipid bilayer [5,10]. Like most membrane
proteins, the purified red cell transporter has
proved to be recalcitrant to crystallization, and
our knowledge of the structure/function relationships of this molecule is limited to what has been
revealed through mutagenesis studies. Our initial
mutagenesis experiments [ 111 focused on the six
tryptophan residues of GLUT1, because it was
believed that cytochalasin B, an inhibitor of facilitative glucose transport that binds to G L U T l in
the inward-facing conformation, could be
covalently bound to the transporter after photoactivation via one or more tryptophan residues.
Two of the six tryptophan residues of G L U T l
were shown to be critical for full transport function. Substitutions at Trp"' severely reduced the
intrinsic activity of G L U T l , whereas substitutions at Trp3XX
resulted in a more modest reduction in intrinsic transport activity. Glycine or
leucine substitutions at the latter site also caused
a rightward shift in a cytochalasin B transport
inhibition curve, suggesting that Trp"'
is
involved in stabilizing the equilibrium binding of
cytochalasin B to the transporter. It has also been
suggested, on the basis of intrinsic fluorescence
experiments conducted on purified erythrocyte
transporter, that Trp38Xforms part of a dynamic
segment of G L U T l that moves from a position
accessible to the aqueous solvent to a solventinaccessible position after substrate binding [ 121.
T h e requirement for conformational flexibility in
helix 10 is also suggested by the presence of
three prolines in this helix, one of which (Pro3")
has been shown by mutagenesis studies to
be required for full transport activity and for
binding of the exofacial ligand 2-N-4-( l-azi2,2,2-trifluoroethyl) benzyl- 1,3-bis- (u-mannos4-yloxy)-2-propylamine (ATP-BMPA) [ 131. Binding of ligands that have a higher affinity for the
inward-facing conformation of G L U T l than for
the outward-facing conformation, such as cytochalasin B, was not affected by substitutions at
We suggested that amide and hydroxy amino
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acid side chains within the putative amphipathic
helices 3, 5, 7, 8 and 11 might provide hydrogenbond donors and acceptors to glucose and thus
form the substrate-binding sites [5]. We tested
five such residues (Asn'"", Gln"", Gln2"", TyrZx2,
TyrZx3)that are conserved in all five G L U T proteins, reasoning that a residue that participates in
the formation of a substrate-binding site must be
conserved in all members of the G L U T family
that transport glucose [14]. Amino acid substitutions at four of the five sites, Asn""' being the
exception, reduced intrinsic trapsport activity
normalized to the quantity of protein expressed
in the oocyte plasma membrane. However, the
effect at Gln'"', which resides within transmembrane helix 5, was particularly striking in that
even the conservative substitution of asparagine
for glutamine reduced intrinsic transport activity
by an order of magnitude. This substitution also
reduced the apparent affinity of the non-transported glucose analogue, ethylidene glucose, for
the transporter by 18-fold. Interestingly, however,
the K, for zero trans influx of 2-deoxyglucose
was minimally changed by this mutation,
whereas the catalytic turnover was decreased by
7.5-fold. These data infer that G1nl6' forms part
of the exofacial glucose-binding site and that the
asparagine substitution alters the specificity of
sugar binding at this site. In addition, the
reduced catalytic turnover suggests that substitutions at this position also affect the rate of a
conformation change of the carrier involved in
net glucose influx. This result is surprising in
that the deletion of only a single methylene
group from a glutamine side chain has such
drastic consequences for transporter function.
These observations constituted the first evidence
that a residue within the N-terminal half of
G L U T l participates in substrate binding. T h e
only other residue identified as participating in
exofacial substrate binding is GlnZx2, which
resides in transmembrane helix 7 [15]. These
data on substrate-binding residues are thus consistent with our original suggestion that amideand hydroxy-containing amino acid residues
within amphipathic helices may comprise the
substrate-binding site(s) [S].
A particularly interesting mutation from a
structural as well as a medical standpoint was
discovered in the GLUT2 gene of a patient with
type-2 diabetes [16]. T h e patient was heterozygous for the Val'97+Ile'y7 mutation, and it is
unclear whether this gene defect contributed to
the development of diabetes in the patient. How-
Structure, Function and Regulation of Glucose Transporters
ever, the mutation completely abolished intrinsic
transport activity of GLUT2 expressed in Xenopus oocytes. T h e same mutation at the equivalent
conserved residue of G L U T l ( ~ a l '-~~' l e ' ~ ' )also
abolished transporter activity in oocytes. What is
so striking about this observation is that such a
subtle structural change, again involving the difference of only a single methylene group at a
single residue, has such a dramatic effect on the
function of the transporter. This observation also
appears somewhat puzzling at first, since Val'"'
lies within transmembrane helix 5 , and it is
unclear what specific role a valine residue could
play in the transport of a hydrophilic molecule
such as glucose, particularly in the hydrophobic
environment of the lipid bilayer where hydrophobic interactions with the valine side chain
cannot contribute to stable inter- or intra-molecular interactions.
Our observations with G1nl6' may provide an
important clue as to why the Va116'+Ile'b' mutation has such a dramatic functional impact despite the caveats mentioned above. Va1l6' lies
approximately one helical turn distant from
Gln'", which we know comprises part of the exofacial substrate-binding site and therefore must
face the hypothetical aqueous channel in the
bilayer formed by the amphipathic transmembrane helices. Thus Val'"' also presumably faces
the aqueous channel and also lies very close to
the substrate-binding site. T h e extra methylene
group at position 165 may be sufficient to impede
hydrogen-bond formation between the glucose
molecule and the amide group of GlnI6'. Preliminary data from our laboratory support this hypothesis in that small amino acid side chains are
better tolerated at position 165 than large side
chains.
Biosynthesis of GLUT I
Perhaps even less is known about the biosynthesis of multispanning membrane proteins
than about their structure. G L U T l was one of
the first multispanning proteins the biosynthesis
of which was studied using synthetic mRNA
transcripts translated in an in vitro translation/
translocation system. A very surprising result was
that G L U T l could insert post-translationally
into microsomes in a process that required
phosphoanhydride bond cleavage, and that
G L U T l contained at least two functional internal signal sequences [ 7,171. This constituted the
first evidence that energy in the form of a
nucleoside triphosphate bond was required for
the insertion of proteins into or across the endoplasmic reticulum membrane (ER). In addition,
these observations contradicted the prevailing
theory that insertion of proteins into or across
the ER was strictly a co-translational process
driven by polypeptide chain elongation. Our data
suggest that large domains of membrane proteins may, at least in some instances, be synthesized before their insertion into the membrane.
However, the detailed mechanism by which
G L U T l or any other polytopic membrane protein inserts into the ER has yet to be elucidated.
Once G L U T l is inserted into the ER, in
most cells it appears to follow a constitutive
route to the plasma membrane, where it contributes to basal glucose uptake. We observed that
G L U T l exhibits a relatively rapid transit time
through the ER in both Xenopus oocytes and
3T3L1 adipocytes (1 h and 5 min respectively
[ 181; transit times through the secretory pathway
are in general much slower in oocytes than in
mammalian cells). GLUT4, on the other hand,
which is approx. 65% identical in sequence with
GLUT1, exhibits much slower ER-to-Golgi
transit times in both cell types (24 h and 20 min
respectively). Because the ER-to-Golgi transit
time usually reflects a rate-limiting step in the
folding of membrane proteins, we investigated
the structural basis for the large difference in
this kinetic parameter between G L U T l and
GLUT4 by analysis of the biosynthesis of chimaeric G L U T l /GLUT4 molecules expressed in
Xenopus oocytes. Unexpectedly, the difference in
transit times was localized to discrete structural
domains in G L U T l and GLUT4. T h e glycosylated exofacial loop and the C-terminal cytoplasmic tail of both G L U T l and GLUT4 largely
determined their transit time behaviour. These
observations suggest that these structural
domains participate in a rate-limiting step in the
folding of the glucose transporter molecules.
Conclusions
Progress is being made towards understanding
the structure of membrane transporters and how
that structure is generated during and after the
membrane insertion process. T h e G L U T l
glucose transporter has served as a useful model
to study these processes. However, major progress in the understanding of G L U T l biosynthesis will ultimately require detailed
structural information at atomic resolution
obtained using high-resolution X-ray crystallography or other spectroscopic methods, a goal
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954
that appears to be technically unfeasible at this
time. In the meantime, however, valuable bits
and pieces of information can be obtained using
the cruder tools of site-directed mutagenesis in
conjunction with kinetic and biochemical analyses.
Work in the author's laboratory is supported in part by
grants from the National Institutes of Health and the
American Diabetes Association.
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Received 7 March 1997
Regulation of GLUT1 in response to cellular stress
S. A. Baldwin*§, L. F. Barrost, M. Griffiths*, J. Ingram*, E. C. Robbins*, A. J. Streets* and J. SaklatvalaS
*Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9]T, U.K., tDepartamento de
Medicina Experimental, Facultad de Medicina, Universidad de Chile, lndependencia 1027, Casilla 70058, Santiago 9,
Chile, and $The Matilda and Terence Kennedy Institute of Rheumatology, I Aspenlea Road, Hammersmith,
London W6 8LH, U.K.
A characteristic feature of the early phase of the
response of mammalian tissues to metabolic
stresses such as hypoxia, ischaemia, osmotic
stress, heat shock, viral infection or exposure to
metabolic poisons is an increase in the rate of
cellular glucose uptake [ 1-31. T h e increase can
be large (up to 12-fold in Clone 9 cells exposed
to azide [4]) and rapid [ t I l 2 <10 min for Clone 9
Abbreviations used: AICAR, 5-arninoimidazole-4carboxamide ribonucleoside; AMPK, AMP-activated
protein kinase; JNK, C-terminal Jun kinase; MAP
kinase, mitogen-activated protein kinase; PI 3-kinase,
phosphatidylinositol 3-kinase; SAPK, stress-activated
protein kinase.
$To whom correspondence should be addressed.
Volume 25
cells exposed to 0.4 M sorbitol (L. F. Barros and
S. A. Baldwin, unpublished work)]. T h i s is an
adaptive response, allowing cells to maintain or
regain their ATP levels when energy demand
increases or oxidative phosphorylation is
impaired, allowing the execution of energyrequiring defence programmes such as the synthesis of molecular chaperones, and thus
promotes cell survival. After the initial phase of
the response (up to 2 h), which is independent of
protein synthesis, a second phase occurs, involving 'Ynthesis
Of a number Of
Proteins,
the heat shock proteins and the
glucose transporter G L U T 1 [4]. Although stress
responses have mainly been studied in cultured
cells, they are of great physiological importance