Download Yeast

Yeast as a model to study
eukaryotic transport systems
Hana Sychrová
Department of Membrane Transport
Institute of Physiology, AS CR, v.v.i.
Videnska 1083, 142 20 Prague 4
Tel: (420) 241 062 667
E-mail: [email protected]
Characterization of transport
systems
• Kinetic parameters - KT , Jmax , ( sin/sout ), substrate specificity
• pH optimum, inhibitors, source of energy (active transport)
• Isolation of the gene and sequencing  primary structure and
properties of the protein
• Mutagenesis to identify crucial aa residues, secondary and
tertiary structures
• Protein purification  reconstitution, crystalization
-
existence of similar systems (overlap of substrate specificity)
-
small amount of protein (low expression, short half-life)
-
denaturation of protein during membrane solubilization = loss
of characteristic properties
 expression of the gene in host organisms lacking their own
transporters (yeast Saccharomyces cerevisiae)
Saccharomyces cerevisiae
• eukaryotic cell, 4 - 8 m, gen. time  2 h
• haploid/diploid (2 x 16 chr, sequenced 1996)
 6 000 genes
• simple isolation of mutants, viable with
multiple mutations
• universal transformation systems, easy
disruption and deletion of genes
chromosomal structure
regulationexpression
of cell cycleofand
gene expression
Heterologous
transporters:
biogenesis and degradationcharacterization
of proteins (secretory
of ownpathway)
systems
protein structure/function gene
relationship
deletions  „non-transporting mutants“
interaction between proteins
(two hybrid
system)of transporters
expression
and screening
characterization
cloning of long heterologous DNA fragments
(Yeast A
Chromosome)
rtificial organisms
from closely
related
expression of heterologous proteins
other organisms
Na+/H+-antiporters
All organisms
Na+ out / H+ in
bacteria (EcNhaA crystalized)
yeast (3, PM and organelles)
plants (>20, 2 subfamilies, PM and organelles)
Elimination of surplus cations from cytosolu,
Source of energy – H+ gradient across PM
H+ out / Na+ in
animal cells (NHE1-9, PM and organellles)
Regulation of intracellular pH (elimination of surplus H+ from cytosol),
Source of energy – Na+ gradient across PM
Yeast Na+/H+-antiporters
Nha1p – PM
Kha1p – GA
Nhx1p –endosomes
bacterial
? (Mkh1p)-mitochondria
Vnx1p – vacuole
? - ER
animal plant
Přibylová L. et al. Folia Microbiol. 387, 89 (2006)
Gene NHA1
2985 nt, YLR138w, chr XII, low and constitutive expression
phenotype: nha1 deletion - higher and pH-dependent sensitivity to
Na+, Li+, K+, Rb+
- higher pHin
NHA1 overexpression - higher and pH-dependent tolerance
to Na+, Li+, K+, Rb+
activity: Na+, Li+, K+, Rb+ efflux
localization: plasma membrane
Nha1p-GFP
role:
detoxification (Na+, Li+)
regulation of intracellular pH
K+ homeostasis
cell volume
membrane potential
response to osmotic shock
Nha1 protein
985 aa, 110 kDa, 12 tms
tms – high similarity with Na+/H+ antiporters from other yeast
(Z. rouxii, S. pombe, transpoting only Na+ and Li+)
hydrophilic C-terminus 556 aa (56 %)
1600 mM KCl
extracellular
900 mM NaCl
35 mM LiCl
intracellular
NaCl
KCl
LiCl
C
472 568 680
774
883 915
945
985
Detail analysis of region
915-945 aa
Growth of buB31 se zkrácenými Nha1p na 30 mM LiCl
AY E SETEFERQRRLNALGEMTAPADQDD EEL
Nha1 protein
985 aa, 110 kDa, 12 tms
tms – high similarity with Na+/H+ antiporters from other yeast
(Z. rouxii, S. pombe, transpoting only Na+ and Li
hydrophilic C-terminus 556 aa (56 %)
extracellular
intracellular
●C-terminus is not important
for substrate specificity
●Role of C-terminus in K+ homeostasis,
regulation of intracellular pH,
cell response to osmotic stress,
regulation of membrane potential
Is the ScNha1p substrate
specificity exceptional?
Family of Na+/H+ antiporters in yeast
plasma membrane
Antiporter
Number of amino acid residues
N-terminus
tms + loops C-terminus
11
414
43
11
418
377
S. pombe sod2
Z. rouxii ZrSod2-22
total
468
806
S. cerevisiae Nha1
985
12
419
554
C. albicans Cnh1-G23
800
11
419
370
Identity (%) entire sequence / tms + loops / C-terminus
Sc Nha1
Ca Cnh1
Zr Sod2-22
Sc Nha1
Ca Cnh1
Zr Sod2-22
Sp Sod2
100
51.3/ 69.9 / 26.7
57.6 / 76.7 / 34.6
40.8 / 43.0 / 19.6
100
46.1 / 65.5 / 20.0
42.4/ 44.5 / 23.9
100
41.6 / 43.3 / 23.9
Comparison of yeast plasmamembrane Na+/H+-antiporters
Substrate specificity
Expression from the NHA1 promoter in BW31 cells (ena1-4 nha1)
K+
Na+
100
100
Observed tolerance corresponds
80
80
[Na ]in (%)
70
60
to antiporters’ transport activity.
+
+
[K ]in (%)
90
60
40
50
20
40
0
30
0
10
20
Time (min)
C
ScNha1p
30
40
0
10
20
30
40
Time (min)
ZrSod2-22
Spsod2
CaCnh1p
Comparison of yeast plasmamembrane Na+/H+-antiporters
Z. rouxii ZrSod2-22p and S. pombe sod2p do not transport K+ and Rb+.
S. cerevisiae Nha1p and C. albicans Cnh1p have broad substrate
specificity for at least 4 cationts.
 Other yeast species?
 System eliminating surplus intracellular K+
in Z. rouxii and S. pombe ?
 What determines the antiporter’s substrate specificity?
Comparison of yeast plasmamembrane Na+/H+-antiporters
Saccharomyces cerevisiae
Zygosaccharomyces rouxii
Debaryomyces hansenii
Pichia sorbitophila
Candida albicans
Yarrowia lipolytica
Schizosaccharomyces pombe
Yeast with antiporters transporting only Na+, Li+
Yeast with antiporters transporting all alkali metal cations ( Na+, Li+, K+, Rb+)
Identification of genes in silico, cloning, heterologous expression in S.
cerevisiae, localization in PM, characterization of substrate specificity
Comparison of yeast plasmamembrane Na+/H+-antiporters
Saccharomyces cerevisiae
Zygosaccharomyces rouxii
(2 systems)
Debaryomyces hansenii
Pichia sorbitophila
Candida albicans
Yarrowia lipolytica
(2 systems)
Schizosaccharomyces pombe
(2 systems)
Yeast with antiporters transporting only Na+, Li+
Yeast with antiporters transporting all alkali metal cations ( Na+, Li+, K+, Rb+)
All yeast species possess a Na+/H+-antiporter regulating [Kin],
pHin and cell volume
Amino-acid residues determining substrate
specificity of yeast Na+/H+-antiporters
 random mutagenesis (UV, PCR) of Z. rouxii SOD2-22 gene (nontransporting K+ ), expression of mutated versions in S. cerevisiae BW31
(ena1-4∆ nha1∆)
 selection of clones growing in the presence of high [KCl], i.e. expresing
“K+-transporting“ ZrSod2-22p versions
empty vector
ZrSod2-22p(wt)
+
K -transporting ZrSod2-22p (ZrSod-94p)
ScNha1p
-
KCl
NaCl
1000 mM 1400 mM
ZrSOD2-22 with mutations in 5th
tms
Pro145
Ser150
Mutated version
mutation in ZrSod2-22p
(wt  mutant)
CCT
145Pro
 145ThrACT
T5
TTT
232Phe
 232SerTCT
L7-8
ZrSod-512p
CCT
145Pro
 145SerTCT
T5
ZrSod-29p
AGT
150Ser
 150ThrACT
T5
ZrSod-211p
ZrSod-94p
 179Thr
T6
 362Ile
T11
GTA
 375IleATA
T11
AGA
519Arg
 519IleATA
C
TTC
618Phe
 618LeuCTC
C
GCA
620Ala
 620ThrACA
C
TGC
 703SerAGC
C
179Ala
ACA
ATG
362Met
375Val
703Cys
ATC
intracellular
C
T5
GCA
extracellular
806
 141SerTCT
ACT
141Thr
ZrSod2-22p
Position
of
mutation
Thr141
S. cerevisiae
BW31 (ena1-4∆ nha1∆)
empty vector
ZrSod2-22p(wt)
ZrSod-211p
ZrSod-512p
ZrSod-29p
ZrSod-94p
ScNha1p
-
NaCl
KCl
1400 mM 1000 mM
Sequence comparison of 5th tms of yeast
plasma membraneNa+/H+ antiporters
Thr141
Z. rouxii
S. cerevisiae
C. albicans
S. pombe
P. sorbithophila
Y. lipolytica 1
Y. lipolytica 2
H. anomala
D. hansenii
C. glabrata
C. tropicalis
134
135
134
135
134
130
134
135
134
135
134
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
I
V
V
L
L
L
M
I
V
I
V
I
M
A
V
V
V
G
S
S
A
S
G
A
A
S
S
S
A
A
A
G
A
S
A
A
A
A
A
C
C
C
C
C
C
C
C
C
C
C
I
I
V
I
V
V
I
V
V
I
V
T
T
T
T
T
T
T
T
T
T
T
A
A
A
S
A
P
A
A
A
A
A
Pro145
T
T
T
T
T
T
T
T
T
T
T
D
D
D
D
D
D
D
D
D
D
D
P
P
P
P
P
P
P
P
P
P
P
V
I
V
V
V
V
V
V
V
I
V
Ser150
L
L
L
L
L
L
L
L
L
L
L
A
A
A
S
A
S
A
A
A
A
A
Q
Q
A
A
S
N
S
A
A
Q
A
S
S
A
L
A
S
A
A
A
S
A
V
V
V
I
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
152
153
152
153
152
148
152
153
134
153
152
Serin or Threonin
Pro145
= transport of K+ and Na+
+ loss of Li+ transport
134
152
148
empty vector
149
5th
144
142
tms
151
135
ZrSod2-22p
138
137
KCl
1400 mM
-
NaCl
1000 mM
LiCl
15 mM
native
P145S
P145T
P145G
P145D
P145K
146
140
147
BW31
150
139
143
136
transformants
K+ efflux
(nmol/mg dry wt/120 min)
empty vector
40±13
ZrSod2-22p
38±12
ZrSod2-22(P145S)p
100±10
ZrSod2-22(P145T)p
78±8
ScNha1p
346±4
Ser150
134
152
148
138
137
149
5th
144
ZrSod2-22p
empty vector
-
KCl
1200 mM
NaCl
1000 mM
LiCl
10 mM
native
S150T
S150V
S150D
S150K
142
tms
151
135
146
140
147
150
139
143
136
Threonin
= transport of K+,Na+ and Li+
Thr141
Serin
= transport of K+, Na+ and Li+
ZrSod2-22p
empty vector
-
KCl
1400 mM
134
152
148
NaCl
1000 mM
LiCl
15 mM
native
ZrSod-94p
T141S
138
137
149
5th
144
142
tms
151
135
146
BW31 transformants
K+ efflux
140
147
150
(nmol/mg dry wt/120 min)
empty vector
40±13
ZrSod2-22p
38±12
ZrSod-94p
214±17
ZrSod2-22(T141S)p
66±4
139
143
136
Serin
-
O
O
C
C
HC
H
H
2 O
134
152
148
138
+
H
N
3
137
149
5th tms
142
151
K+
T141, S150
S141, T150
T141, T150
S141, S150
+/+
+
-
135 O
O
C
146
140
147
C
H
3
HO
C
HC
H
+
150
139
H
N
3
143
136
Threonin
Side chains of Thr141, Pro145 a Ser150 are involved in conformational
changes during binding and/or transport of alkali metal cations via yeast
antiporters of the Nha/Sod type
Expression of mammalian Na+/H+
exchangers (NHE) in yeast
Protein similarity (%)
NHE2
NHE3
NHE1
NHE2
48.4
-
36.3
44.2
All tissues
Epitel – basolateral side
General role, pHin
Kidney, small gut
(apical side)
? Role, absorption?
Kidney, small gut
(apical side)
? Role, absorption?
Blood pressure
Sensitivity of yeast cells expressing rNHE
to alkali metal cations
-
strain BW31
(ena1-4 nha1)
200 mM NaCl 800 mM KCl
pNHA1-985GFP
pGRU1
pNHA1NHE1GFP
pPMA1NHE1GFP
pNHA1NHE2GFP
pPMA1NHE2GFP
pNHA1NHE3GFP
-
200 mM NaCl 800 mM KCl
pNHA1-985GFP
pGRU1
pNHA1NHE1GFP
pPMA1NHE1GFP
pNHA1NHE2GFP
pPMA1NHE2GFP
pNHA1NHE3GFP
pPMA1NHE3GFP
pPMA1NHE3GFP
NHE expression is not toxic
NHE2 improves KCl tolerance
NHE3 improves NaCl toleranci
NHE1
NHE2
NHE3
20%
1
sucrose
60%
2 3 4 5 6 7 8 9 10 11 12 13 14
NHE1
NHE2
NHE3
Pma1p
Growth of yeast cells expressing
NHE2p
1
OD 600 nm
YNB
+ 800 mM KCl
0,1
0,01
0,001
0
20
40
60
80
100
time (h)
[pGRU1]
[pNHA1-985]
[pNHA1NHE2]
[pPMA1NHE2]
Mammalian antiporters export alkali metal cations in yeast
cells
Expression of mKir2.1 channel in
S. cerevisiae MAB 2d (ena1-4 nha1 trk1 trk2)
Tok1/Duk1/YcK1
Nsc1
Nha1
?
Na+
K+
?
Pho89
H+
H+
Na+
Pi
Potassium
KCl (mM)
Kir2.1: inward rectified, voltage gated,
K+ specific
Point mutations in Kir2.1 genes → activity
changes → illness
 search for activity modulators
0*
10
30
50
ADP+ Pi
ADP+ Pi
K+
K+
ATP
Na+
5th day
* YNB medium contains approximately 7 mM KCl
Functional expression  restore of growth on low [KCl], higher
sensitivity to high [KCl]
H+
Kha1
Trk1, Trk2
Ena1/Pmr2
100
H+
?
ADP+ Pi
K+
Na+
[pKir2.1]
[pYEx]
7th day
ATP
Nhx1
H+
Pma 1
ATP
K+ vac uole
H+
Na+
300
mKir2.1 expression in MAB2d
growth in liquid culture
pYEx (control; ∆)
pKir2 (♦)
10 mM KCl
100 mM KCl
0,6
0,6
0,5
0,5
0,4
0,3
OD
OD
0,4
0,2
0,3
0,2
0,1
0,1
0
0
2
4
6
time (h)
8
10
12
14
0
0
2
4
6
8
time (h)
Growth rate depends on mKir2.1 channel
activity
Testing activity modulators?
10
12
14
mKir2.1 expression in MAB2d
growth in liquid culture
 96 cultures (150 l), different [KCl], different modulators
mKir2.1 expression in MAB2d
growth in liquid culture
1
2
3
4
5
6
7
8
9
10
11
12
mKir2.1 expression in MAB2d
inhibition effect of Ba2+
BaCl2
16
BW31 expressing mKir2.1 ()
control (pYEx, )
EC50 ~ 1 mM
calculated growth
(arbitrary units)
14
12
10
8
6
MAB 2d expressing mKir2.1 ()
control (pYEx, )
EC50 ~ 5 mM
4
2
0
0
1
10
100
1000
10000
[BaCl2] (µM)
 Useful tool for HTS (high throughput screening) of new
drugs modulating activity of potassium channels
Characterization of intracellular
antiporters
N
En K
+
Mk
a1- a+
N K
H
K K
? +5 AHA H a+ N+
h1
+
+
T+DN +
h
H
+
P
NPh
aT
Vn KN a +
K o
+ x
+1
H
x1 +a+ P
K + k
+
K
1
i
1
A A
h
?a N
D T
P HP P
S
+
1
ma
C
+
1 P
1
Trk
1-2
Kha1p – Golgi apparatus, growth at high pHout
detoxification of Hygromycin B
i
Nhx1p - endosomes, regulation of cytosolic pH, protein trafficking
detoxification of HygromycinB
sequestration of Na+ and K+ into vacuoles
Strains:
LMB01 ena1-4∆ nha1∆ kha1∆
AB11c ena1-4∆ nha1∆ nhx1∆
Phenotype:
no growth at pHout> 7,0 HygS
↑sensitivity to NaCl, KCl HygS
 expression of plant orthologous genes
Expression of A. thaliana and O. sativa
antiporters in S. cerevisiae
OsNhx1p
Expression
Strain AB11c ena1-4∆ nha1∆ nhx1∆
NaCl
HygB
OsNhx1-GFPp
+
+++
AtChx17p
Expression
Strain LMB01 ena1-4∆ nha1∆ kha1∆
pH 4,5
pH 7,0
GFP-AtChx17p
ScKha1p
AtChx17p
 strains with kha1 and nhx1 deletions are suitable for characterization
of heterologously expressed transporters from organelles of higher
eukaryots