A Yeast Recombinant Aquaporin Mutant That Is Not Expressed or
Mistargeted in Xenopus Oocyte Can Be Functionally
Analyzed in Reconstituted Proteoliposomes*
Valérie
Lagrée
,
Isabelle
Pellerin
,
Jean-François
Hubert
,
Frédérique
Tacnet§,
Françoise
Le Cahérec
,
Nathalie
Roudier§,
Daniel
Thomas
,
Jean
Gouranton
, and
Stéphane
Deschamps
¶
From the
UPRES-A CNRS, Biologie Cellulaire et
Reproduction, Université de Rennes 1, 35042 Rennes cedex and
§ Département de Biologie Cellulaire et
Moléculaire, Service de Biologie Cellulaire, CEA-Saclay,
91191 Gif sur Yvette cedex, France
 |
ABSTRACT |
We have recently identified AQPcic (for
aquaporin cicadella), an insect
aquaporin found in the digestive tract of homopteran insects and
involved in the elimination of water ingested in excess with the
dietary sap (Le Cahérec, F., Deschamps, S., Delamarche, C.,
Pellerin, I., Bonnec, G., Guillam, M. T., Gouranton, J., Thomas, D., and Hubert, J. F. (1996) Eur. J. Biochem.
241, 707-715). Like many other aquaporins, AQPcic is inhibited by
mercury reagents. In this study, we have demonstrated that residue
Cys82 is essential for mercury inhibition. Another mutant
version of AQPcic (AQP-C134S), expression of which in Xenopus
laevis failed to produce an active molecule, was successfully
expressed in Saccharomyces cerevisiae. Using stopped-flow
analysis of reconstituted proteoliposomes, we demonstrated that the
biological activity and Hg sensitivity of yeast-expressed wild type and
mutant type AQPcic was readily assessed. Therefore, we propose that the
yeast system is a valid alternative to Xenopus oocytes for
studying particular mutants of aquaporin.
 |
INTRODUCTION |
The existence of molecules implicated in water transport across
the cellular membranes has been postulated for many years. The high
permeability of certain cell types (i.e. erythrocytes, epithelial cells of the kidney proximal tubules or collecting duct) are
not readily explained by simple diffusion of water across the lipid
bilayer (1). In 1992, Agre and co-workers identified AQP1 (for
aquaporin 1, initially called
CHIP28), which functions in the regulation of water transport across
the membrane in human erythrocytes (2). In mammals, water channels have
been characterized in cell membranes of a variety of tissues such as
the kidney (AQP1 (Ref. 2), AQP2 (Ref. 3), or AQP3 (Refs. 4 and 5)), the
brain (AQP4; Ref. 6), the salivary glands (AQP5; Ref. 7), and the
testis (AQP7 and AQP8; Refs. 8 and 9) (reviewed in Refs. 10 and 11). In
addition, aquaporins have also been described in plants (12-14) and
Escherichia coli (15).
In a previous study, we characterized the first insect aquaporin, which
we termed AQPcic (for aquaporin
cicadella, initially called P25). AQPcic is present in an
epithelial complex found in the digestive tract (the filter chamber) of
an homopteran sap-sucking insect, Cicadella viridis
(16-19). The filter chamber of this insect is highly specialized in
water transport, permitting excess of ingested water to be rapidly
transferred from the initial midgut to the terminal midgut through a
transepithelial osmotic gradient (20). The cDNA encoding AQPcic was
recently isolated and sequenced (18). Thus far, the functional
characterization of AQPcic has been performed either by injecting
native AQPcic-reconstituted proteoliposomes into Xenopus
oocytes (19) or by microinjection of in vitro transcribed
cRNA into Xenopus oocytes (18). Through this work, we
demonstrated that the Hg sensitivity of AQPcic is lower than that of
AQP1 (18). We postulated that this difference may be due to the
intramembraneous localization of cysteine residue(s) implicated in
mercurial inhibition.
In this study, we identify the mercurial inhibitory site of AQPcic by
utilizing three AQPcic mutants, in which Cys82,
Cys90, and Cys134 are replaced by serine
residues.
The replacement of Cys82 in AQPcic completely abrogates Hg
sensitivity of the protein, suggesting that Cys82 has a
critical role in aquaporin function. We further demonstrate that
certain mutants when expressed in Xenopus oocytes are not correctly processed and therefore propose an alternative system to
analyze such mutants.
 |
EXPERIMENTAL PROCEDURES |
Plasmid Construction and Mutagenesis--
pSP-AQPcic corresponds
to the full-length AQPcic coding sequence subcloned into plasmid
pX
G-ev1 (18). The yeast expression vectors (pYeDP10 and pYeDP60)
were gifts of Dr. Pompon (21, 22). The wild type or mutated forms of
AQPcic were placed under the control of a GAL10-CYC1 promoter and a
phosphoglycerate kinase terminator. The coding region of AQPcic
was amplified by polymerase chain reaction using two primers: Y1,
5'-GGGGAATTCATGGCCGCCGACAAGT-3'; Y2,
5'-CGCAAGCTTGAGCTCGTACACTAGTGTCTGGAGCT-3'.
The polymerase chain reaction primers contain EcoRI and
SacI restriction sites (underlined) used to clone into the
yeast vectors' polylinker. These constructs were called pYeDP10-AQPcic
and pYeDP60-AQPcic.
Mutagenesis on cysteine residues 82, 90, and 134 was performed with the
CLONTECH mutagenesis kit (Promega) using pSP-AQPcic vector as a template. Primer PSE (Table I) contains a mutation that
destroys the ScaI site of pX
G-ev1 (PSE, Table I), and the mutation primers (listed in Table I)
contain the mutated codons. Mutations were confirmed by enzymatic
nucleotide sequencing (U. S. Biochemical Corp.). The coding region
of mutant C134S was then amplified by polymerase chain reaction using
primers Y1 and Y2 and the pSP-AQP-C134S construction as a template and
subcloned in the yeast expression vector pYeDP60 (the construct was
termed pYeDP60-C134S).
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Table I
Oligonucleotides for site-directed mutagenesis of AQPcic
The mismatched bases are underlined, and codons in brackets represent
the mutated amino acid residues. Superscript numbers represent the base
pair number of the AQPcic cDNA coding sequence.
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Water Transport Assays in Xenopus Oocytes--
cRNA injections
into oocytes were performed as described previously (18). Briefly,
oocytes swelling was induced by a 5-fold dilution of extracellular
buffer A (82.5 mM NaCl, 2.5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 2 mM NaHCO3, 10 mM Hepes/NaOH, pH
7.4) and was monitored by videomicroscopy. The osmotic water
permeability coefficient (Pf) was calculated by
Equation 1.
|
(Eq. 1)
|
S is the oocyte surface area (S = 0.045 cm2), V0 the initial volume
(V0 = 9 × 10
4
cm3), V the oocyte volume at a given time
t, Vw the molecular volume of water
(Vw = 18 cm3/mol), and
d(V/V0)/dt the initial rate of oocyte swelling.
Cin is 176 mmol/kg and
Cout 38 mmol/kg. For mercurial inhibition
analysis, oocytes were incubated 15 min in 0.3 mM
HgCl2 prior to osmotic shock. For each experiment,
Xenopus oocyte total membranes were prepared by the method
described in Ref. 24.
Expression of Recombinant Wild Type or Mutated AQPcic in
Saccharomyces cerevisiae--
Studies were performed using the W303.1B
strain of S. cerevisiae (
, leu2, his3, trp1, ura3,
ade2-1, canR, cyr+) as
described previously (25). Two culture conditions were used to
overexpress the recombinant aquaporins. Yeast transformants containing
pYeDP10 vector (called Y10) or pYeDP10-AQPcic (Y10-AQPcic) were grown
at 28 °C for 24 h in a minimal medium (0.7% yeast nitrogen base without amino acids, 0.1% casamino acids, and 2% glucose) to
OD660 nm = 3. To induce heterologous expression of AQPcic, the cells were diluted to 0.1 OD (660 nm) in galactose medium (0.7%
yeast nitrogen base without amino acids, 0.1% casamino acids, and 2%
galactose). Yeast transformants containing, respectively, pYeDP60
vector (Y60), pYeDP60-AQPcic (Y60-AQPcic), or pYeDP60-AQPC134S (Y60-C134S) were grown at 28 °C in a rich medium (1% yeast extract, 1% Bactopeptone, 0.5% glucose) for 36 h. Induction of protein expression was performed by direct addition of galactose in the culture
medium (20 g/liter). The yeast cells were grown at 28 °C with
continuous shaking for 16-20 h.
Purification of AQPcic--
Yeast cells were harvested,
homogenized with glass beads, and shaken manually, and the total
membrane fractions were prepared as described previously (26). AQPcic
was purified using a method adapted from the purification of AQP1 (27,
28). Membrane proteins were solubilized by addition of one volume of
4% N-lauroylsarcosine, 4 mM NaHCO3,
2 mM DTT1 for
2 h at room temperature. The non-solubilized material was pelleted
by centrifugation at 100,000 × g for 45 min at
10 °C. The pellet was resuspended in 0.1 volume of 1.2% OG
(n-octyl
-D-glucopyranoside), 1 mM DTT, 20 mM Tris-HCl, pH 7.4, and AQPcic
solubilization was performed overnight at 4 °C. Preparation was then
centrifuged at 150,000 × g for 45 min at 10 °C, and
the supernatant (containing AQPcic) was recovered and filtered through
a 0.22-µm membrane (Millipore). The material was loaded onto a MonoQ
HR 5/5 anion-exchange column (Amersham Pharmacia Biotech) equilibrated
with chromatography buffer (1.2% OG, 20 mM Tris-HCl, pH
7.4, 1 mM DTT). The column was eluted with a 15-ml gradient
of 0-0.4 M NaCl in the same buffer, and 0.5-ml fractions
were collected. Highly pure AQPcic was eluted at 0.15 M
NaCl.
Proteoliposome Reconstitution and Stopped Flow
Experiments--
To prepare proteoliposomes, lipid solution (23%
cholesterol, 77% phosphatidylcholine/phosphatidic acid (9/1, w/w)) was
dried to a thin film under a stream of nitrogen. The lipid film was dissolved either in 2 ml of 1.2% OG, 50 mM NaCl, 1 mM DTT, 20 mM Tris-HCl, pH 7.4, with or without
aquaporins. The protein/lipid ratio of proteoliposomes was 1/150 (w/w).
The solutions were dialyzed at 4 °C against buffer D (50 mM NaCl, 1 mM DTT, 20 mM Tris-HCl, pH 7.4) and filtered through a 0.2-µm Millex GV membrane
(Nucléopore). AQPcic proteoliposomes or control liposomes were
exposed to an osmotic gradient by rapid mixing with an equal volume of
buffer D containing mannitol, and the Pf were
measured. The concentration of mannitol was sufficient to increase by
2-fold the osmolarity of the mixed solution. Experiments were performed with a stopped-flow spectrophotometer (SFM3, Biologic, Claix, France)
characterized by a dead time of 0.8 ms and a maximal rate of data
acquisition of 10 kHz. The light of a 150-watt mercury-xenon arc lamp
is driven from the monochromator to the observation chamber (8 µl) by
an optical fiber. The increase of the 90° scattered light intensity,
corresponding to water efflux from the liposomes, was followed at
ex = 430 nm. The data obtained from at least 10 determinations were averaged and fitted to single exponential curves
using a software provided by Biologic (Claix, France). The fitting
parameters were used to calculate the initial rate constant
k (s
1) and the Pf
(cm·s
1) was determined according to Equation 2.
|
(Eq. 2)
|
S/V0 (cm
1) is
the ratio of the vesicle surface area to the initial volume,
Vw is the partial molar volume of water (18 cm3/mol),
osm is the osmotic difference between the
initial intra- and extravesicular mannitol concentrations, and
is
the reflexion coefficient of the mannitol (
man = 1; see
Ref. 29).
Electrophoresis and Immunoblotting--
Proteins resolved by
SDS-PAGE (30) were either stained with Coomassie Blue or
electrotransferred onto PVDF membrane (31). Immunodetection was
performed using polyclonal antibodies raised against the native
Cicadella AQPcic protein (17).
 |
RESULTS |
Cysteine 82 Is the Mercurial-sensitive Site of AQPcic--
In
order to identify the mercurial inhibitory site(s) of AQPcic, we
constructed three mutants, which contain substitutions to serine at
positions Cys82 (AQP-C82S), Cys90 (AQP-C90S),
and Cys134 (AQP-C134S) (Fig.
1). The mutant or wild type forms of
AQPcic were in vitro transcribed and injected into
Xenopus oocytes, and water permeability coefficients
(Pf) of oocytes were measured after hypoosmotic
shock with or without HgCl2 pretreatment. As shown in Fig.
2A, the Pf
values for wild type AQPcic and mutant types AQP-C82S and AQP-C90S are
similar. In contrast, the replacement of Cys134 by a
serine in AQP-C134S totally inhibited the aquaporin-induced water
permeability (Fig. 2A).

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Fig. 1.
Localization of cysteines residues in AQPcic
and AQP1. Schematic representation of an aquaporin with the 6 putative transmembrane domains. The cysteine residues are indicated and
numbered. for AQPcic and for AQP1. The amino acids sequence of
the B loop of AQPcic is shown and the cysteines 82 and 90 are indicated
in bold type.
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Fig. 2.
Expression of wild type or mutated AQPcic in
Xenopus oocytes. A, The Pf of oocyte
expressing wild type or mutated AQPcic were measured under hypoosmotic
condition with or without a pretreatment in 0.3 mM
HgCl2 for 15 min. The values represent an average of 10-15
measurements (± S.E.). B, Western blot analysis of total
membrane proteins purified from injected oocytes. Total membrane
proteins were prepared from 10-15 oocytes and amount of proteins
equivalent to 1 oocyte was resolved by 12.5% SDS-PAGE. The proteins
were transferred onto PVDF membrane and AQPcic was detected using a
polyclonal antibody raised against the native aquaporin. Lane 1, native
AQPcic; lane 2, water injected; lane 3, AQPcic; lane 4, AQP-C82S; lane
5, AQP-C90S; lane 6, AQP-C134S.
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As described previously, pretreatment of AQPcic-expressing oocyte with
0.3 mM HgCl2 resulted in a total inhibition of
the water permeability (Fig. 2A). Although the C90S
substitution did not modify the HgCl2 sensitivity of
AQPcic, mutation of Cys82 totally abolished the inhibitory
effect of mercurial reagent (Fig. 2A). These results clearly
demonstrate that Cys82 is the Hg-sensitive site in
AQPcic.
To verify the presence and the integrity of the expressed aquaporins,
we performed a Western blot with oocyte membrane proteins, using an
AQPcic antibody (Fig. 2B). The recombinant AQPcic or the two
mutant AQP-C82S and AQP-C90S proteins were expressed in the injected
oocytes (Fig. 2B, lanes 3, 4, and
5, respectively). Although there was a slight difference in
the amount of AQP-C90S and AQP-C82S, the Pf values
measured for these oocytes were similar (Fig. 2A). In
contrast, no protein was present in the membrane (Fig. 2B,
lane 6) or in the cytoplasm (data not shown) prepared from
oocyte injected with C134S cRNA, suggesting a lack (or undetectable) of
protein expression.
In order to understand the lack of AQP-C134S protein expression in
oocytes, we analyzed translation ability of the C134S cRNA in an
in vitro reticulocyte lysate system. Fig.
3 clearly shows that AQP-C134S cRNA was
translatable (lane 3), as well as its wild type counterpart
(lane 1).

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Fig. 3.
In vitro translation of AQPcic
cRNA. The cRNA molecules encoding wild type AQPcic or AQP-C134S
were translated in vitro with the
CLONTECH kit (Promega). Translation products were
resolved on 12.5% SDS-PAGE, transferred onto PVDF membrane and
revealed using AQPcic antibodies (1/1000). Arrow indicates the AQPcic
recombinant protein. Lane 1, AQPcic; lane 2, Water control; lane 3, AQP-C134S.
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These results indicate that a single replacement of Cys134
by a serine residue can alter expression, stability, or targeting of
recombinant aquaporin in Xenopus oocyte.
AQP-C134S Mutant Can Be Overexpressed in Yeast--
Yeast cells
were transformed with constructs derived from pYeDP10 vector (yeast Y10
and Y10-AQPcic) or constructs derived from pYeDP60 vector (yeast Y60,
Y60-AQPcic, and Y60-C134S). Heterologous protein expression was induced
by transferring the cells in a galactose culture medium for 16-20 h at
28 °C. In minimal medium conditions, yeast concentrations reach
values of 2-3.107 cells/ml, whereas values of
9-10.107 cells/ml are obtained in rich medium culture. To
verify expression of wild type or mutated aquaporins, total membrane
proteins were prepared. Western blot analyses revealed a single 25-kDa
polypeptide in Y10-AQPcic, Y60-AQPcic, and Y60-C134S membrane fractions
(Fig. 4, lanes 3,
4, and 6, respectively), the electrophoretic
mobility of which is similar to that of native AQPcic protein (Fig. 4, lane 1). No immunoreactive band was observed in total
membranes prepared from control yeast (Fig. 4, lanes 2 and
5). Interestingly, the mutation of Cys134
modified neither the expression nor the stability of the protein when
it was expressed in yeast cells.

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Fig. 4.
Expression of wild type or C134S AQPcic in
S. cerevisiae. Yeast cells were transformed with the
recombinant expression vectors (yeast Y10-AQPcic and Y60-AQPcic) and
Y60-C134S as described in "Experimental procedures". Control yeast
cells were transformed with the non recombinant expression vector
(yeast Y10 and Y60). Proteins from total membrane extracts were
prepared and resolved on 12.5% SDS-PAGE. The gel was transferred on
PVDF membrane and probed with antibodies anti-AQPcic. Lane 1, 2.2 µg
of native AQPcic; Lane 2, 20 µg of control-yeast (Y10) membrane
proteins; lane 3, 20 µg of Y10-AQPcic membrane proteins; lane 4, 15 µg of Y60-AQPcic membrane proteins; lane 5, 15 µg of control yeast
Y60 and lane 6, 15 µg of Y60-C134S membrane proteins.
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AQPcic and AQP-C134S Can Be Easily Purified--
Treatment of the
Y60-AQPcic (Fig. 5, lane 3) or
Y60-C134S membranes with N-lauroylsarcosine solubilized most
of the membrane proteins except the aquaporin, which remained in the
insoluble fraction (Fig. 5, lanes 4 and 6).
AQPcic or AQP-C134S were extracted from the insoluble fraction by
addition of 1.2% OG (Fig. 5, lanes 5 and 7).
Such procedure allowed us to purify significant amounts of 90% pure
AQPcic. AQPcic and AQP-C134S were further purified by anion exchange
chromatography (Fig. 5, lanes 9 and 10).

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Fig. 5.
Purification of recombinant wild type or
mutated AQPcic. Total membrane extracts from control yeast (TMY),
AQPcic expressing yeast (TMYcic) or AQPcic-C134S yeast (TMYC134S) were
prepared and solubilized with 2% (w/v) N-lauroylsarcosine.
Insolubilized material was recovered, resuspended in 1.2% OG
(n-octyl- -D glucopyranoside). The solubilized proteins were
recovered as described in "Experimental procedures". Proteins from
various stages were resolved on SDS-PAGE and analyzed by Coomassie blue
staining. Lane 1, Molecular mass markers; lane 2, native AQPcic; lane
3, TMYcic proteins; lane 4, TMYcic proteins insolubilized with
N-lauroylsarcosine; lane 5, N-lauroylsarcosine
insolubilized TMYcic proteins solubilized with OG; lane 6, TMYC134S
proteins insolubilized with N-lauroylsarcosine; lane 7, N-lauroylsarcosine insolubilized TMYC134S proteins
solubilized with OG; lane 8, N-lauroylsarcosine
insolubilized TMY proteins solubilized with OG; lane 9 FPLC purified
AQPcic protein; lane 10, purified AQP-C134S protein.
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AQP-C134S Mutant Is Functional--
Purified AQPcic or AQP-C134S
were reconstituted into proteoliposomes, the radii of which were
measured by electron microscopy after negative staining (the values are
respectively 216 ± 46 nm and 199 ± 71 nm, n = 40). The osmotic water permeability coefficient (Pf) was determined by rapidly increasing the
extravesicular osmolarity in a stopped-flow spectrophotometer.
Experiments were performed on proteoliposomes reconstituted with
recombinant proteins prepared from both Y10-AQPcic and Y60-AQPcic
transformed cells. As shown on Fig. 6, no
significant permeability difference was observed between the two AQPcic
proteoliposomes (Pf = 10.36 ± 0.7 10
3 cm/s and 10.65 ± 0.9 10
3 cm/s,
respectively at 20 °C; Fig. 6A (mean of three independent experiments)). The Pf of the AQPcic reconstituted
proteoliposomes was significantly increased when compared with the
permeability of control liposomes (Pf = 3.2 ± 0.3 10
3 cm/s at 20 °C (n = 3), Fig.
6A). A similar increase of Pf was
observed with the protein AQP-C134S reconstituted into proteoliposomes (Pf = 9.1 ± 0.3 10
3 cm/s at
20 °C (n = 3), Fig. 6A).

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Fig. 6.
Water permeability of control, AQPcic, or
AQP-C134S proteoliposomes and mercurial sensitivity. Control
liposomes, AQPcic proteoliposomes or AQP-C134S proteoliposomes were
abruptly exposed to a two fold increase of osmolarity in a stopped-flow
apparatus and the resulting time course increase of the light
scattering intensity (indicating decreased volume) was monitored. Each
curve is an average of ~10 measurements. The signal was fitted to an
exponential function to calculate Pf. A. Stopped-flow analysis of control liposomes, AQPcic reconstituted
proteoliposomes (with AQPcic prepared from Y10-AQPcic or Y60-AQPcic
cells) or AQP-C134S reconstituted proteoliposomes (0.001 mg/ml of
protein/ml). B. Mercurial sensitivity of recombinant wild
type or mutated AQPcic. Stopped-flow experiments were performed on
liposomes or proteoliposomes after treatment in 1 mM
HgCl2 for 15 min.
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To investigate the effects of mercurial reagents on AQPcic
permeability, some experiments were performed in the presence of HgCl2. Addition of 1 mM HgCl2 for
15 min dramatically reduced the Pf of AQPcic- or
AQP-C134S proteoliposomes to values of 3.36 ± 0.1 10
3 cm/s and 3.27 ± 0.15 10
3 cm/s,
respectively (Fig. 6B), similar to control liposomes.
However, the addition of HgCl2 had no effect on control
liposomes water permeability (Fig. 6B).
Determination of Pf for the control liposomes or
AQPcic proteoliposomes were then performed at varying temperatures (data not shown). Measurements of Arrhenius activation energies (Ea) indicated an Ea of 12.98 kcal/mol for liposomes and 4.49 kcal/mol for AQPcic-proteoliposomes.
The low Ea value calculated for
AQPcic-proteoliposomes clearly indicates that the incorporated proteins
facilitate the water transport. These results provide additional
evidence that the recombinant AQPcic is responsible of the water
permeability in reconstituted proteoliposomes.
 |
DISCUSSION |
In this study, we have identified the cysteine residue involved in
the mercurial sensitivity of AQPcic. We then showed that a single point
mutation of aquaporin can abolish its expression in Xenopus
oocyte. Furthermore, we have successfully expressed and purified
recombinant aquaporin in yeast cells.
With the exception of a few water channels (e.g. AQP4, Ref.
5), most aquaporins are inhibited by mercurial agents that bind the SH
group of cysteine amino acids. Some of the cysteines involved in the Hg
sensitivity were identified by site-directed mutagenesis
(Cys189 on E loop for AQP1 (Ref. 24), Cys181
and Cys182 in the E loop for AQP2 and AQP5 (Refs. 32 and 7,
respectively), or Cys118 in
-TIP (Ref. 33)). Our
previous results have shown that the sensitivity of AQPcic to
HgCl2 was lower compared with that of AQP1 and that the
reversibility of AQPcic Hg inhibition by
-mercaptoethanol was
partial (18). The absence of cysteine residue in the E loop area of
AQPcic might explain the differential sensitivity of the two proteins.
Among cysteine residues of AQPcic, Cys82, Cys90
localized close to the NPA box in the B loop, and Cys134 in
the C loop appeared most likely as potential Hg-binding sites. Other
cysteine residues are localized in/or close to the transmembrane domains and thus are unlikely to be bound by mercury (see Fig. 1). Our
data demonstrate that a single mutation of Cys82 in serine
abolishes the HgCl2 inhibition of AQPcic, identifying cysteine 82 as the Hg-binding site. Agre and collaborators (34) have
constructed a double mutant of AQP1 (A73C/C189S), in which intracellular Ala73 from the B loop and extracellular
Cys189 from the E loop were replaced, respectively, by a
cysteine and a serine residue (Fig. 1). When expressed in
Xenopus oocytes, the Hg sensitivity of this double mutant
(A73C/C189S) was two-thirds that of wild type AQP1 (34). According to
the hourglass model (34), the residue Cys82 of AQPcic and
its equivalent Ala73 in AQP1 are localized deep within the
pore. The intramembraneous position of these amino acids might explain
the reduced accessibility of the Hg-binding site and, thus, the lower
sensitivity of AQPcic or AQP1 mutant to a mercurial reagent. In
contrast, Shi and Verkman (35) have mutated the Gly72 of
AQP4 in a cysteine residue (in AQP4, Gly72 is the
equivalent of Ala73 in AQP1 and Cys82 in
AQPcic). The AQP4-G72C mutant presents a significantly greater sensitivity to HgCl2 than AQPcic or AQP1 mutant. Similarly,
replacement of Ala210 by cysteine in AQP4 (the equivalent
of Cys189 in AQP1) did not confer mercury sensitivity (5).
These results support the hypothesis that the structures of the channel
apertures in AQPcic are closer to AQP1 than to AQP4 ones.
Our data showed that the C134S injected oocytes had a
Pf value resembling to the water-injected oocytes.
However, the absence of expression of the mutated protein and/or its
instability in Xenopus oocytes might explain the lack of
aquaporin function. To analyze the function of the AQP-C134S mutant, we
have overexpressed wild type or mutated AQPcic in the yeast S. cerevisiae. The presence of a functional AQP-C134S in the yeast
membranes as well as the wild type AQPcic attests that a single
replacement of Cys134 in serine changes neither the
expression of the protein nor its stability in yeast. It thus appears
that this substitution does not provoke the same changes in the
expressing machinery of the two cells, which suggests that oocyte and
yeast cells behave differently when overexpressing a foreign membrane
protein. Xenopus oocytes have been largely used as an
experimental system for expression and functional analysis of
heterologous proteins (see, e.g., review in Refs. 36-38),
but the intracellular transport or plasma membrane targeting remains
largely unexplained in these cells. In order to study aquaporin
function, localization and quantification of the protein of interest
are crucial steps. Unexpressed or misrouted aquaporin mutants in
Xenopus oocyte system have been described previously for
some AQP1 or AQP2 mutants. The only partial integration of several
(potentially functional) AQP2 mutants in oocyte plasma membrane (39,
40), the mistargeting of some AQP1 mutants (34), or the low expression
of AQP1-C189W (24) are responsible of the reduced Pf
values of injected oocytes. Consequently, a functional analysis of such
kind of aquaporin mutants in Xenopus oocytes is definitely
not conceivable.
In contrast to the limited knowledge on membrane proteins targeting in
higher eukaryote cells, the yeast intracellular transport of secretory
or membrane proteins is extensively studied (see, e.g.,
Refs. 41-43). Different observations suggest that, in S. cerevisiae, only a subset of preproteins that are translocated across the endoplasmic reticulum membrane require the function of a
signal recognition particle (SRP) (44). For instance, yeast cells are
viable in the absence of SRP and SRP receptor and may "adapt" over
time and thereby gain the ability to translocate many proteins (45),
indicating that SRP-mediated targeting is not the only route through
which proteins enter secretory pathway. This "adaptation" of yeast
cells possibly explain the easy production of high level of recombinant
membrane aquaporins in S. cerevisiae.
In this paper, we propose a rapid and efficient way to successfully
perform functional studies on wild type and/or mutant aquaporins by
overcoming problems encountered by mistargeted aquaporins in
Xenopus oocytes.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Alain Viel, Pierre Falson, and
Pierre Ripoche for helpful discussion. We are very grateful to Annie
Cavalier for the immunocytochemistry experiments. We thank Louis
Communier for photography.
 |
FOOTNOTES |
*
This work was supported by the Langlois Foundation (Rennes,
France).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. Fax:
33-2-99-28-14-77; E-mail: stephane.deschamps{at}univ-rennes1.fr.
1
The abbreviations used are: DTT, dithiothreitol;
OG, n-octyl
-D-glucopyranoside; PAGE,
polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride;
SRP, signal recognition particle.
 |
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