From the Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111
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ABSTRACT |
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The membrane topography of the yeast vacuolar
proton-translocating ATPase a subunit (Vph1p) has been investigated
using cysteine-scanning mutagenesis. A Cys-less form of Vph1p lacking
the seven endogenous cysteines was constructed and shown to have 80%
of wild type activity. Single cysteine residues were introduced at 13 sites within the Cys-less mutant, with 12 mutants showing greater than
70% of wild type activity. To evaluate their disposition with respect
to the membrane, vacuoles were treated in the presence or absence of the impermeant sulfhydryl reagent
4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS) followed
by the membrane permeable sulfhydryl reagent 3-(N-maleimidylpropionyl) biocytin (MPB). Three of the 12 active cysteine mutants were not labeled by MPB. The mutants E3C, D89C, T161C, S266C, N447C, K450C, and S703C were labeled by MPB in an AMS-protectable manner, suggesting a cytoplasmic orientation, whereas
G602C and S840C showed minimal protection by AMS, suggesting a lumenal
orientation. Factor Xa cleavage sites were introduced at His-499,
Leu-560, and Pro-606. Cleavage at 560 was observed in the absence of
detergent, suggesting a cytoplasmic orientation for this site. Based on
these results, we propose a model of the a subunit containing nine
transmembrane segments, with the amino terminus facing the cytoplasm
and the carboxyl terminus facing the lumen.
The vacuolar proton-translocating ATPases (or
V-ATPases)1 are multisubunit
complexes found in a variety of intracellular compartments, such as
clathrin-coated vesicles, endosomes, lysosomes, Golgi-derived vesicles,
chromaffin granules, synaptic vesicles, and the central vacuoles of
yeast, Neurospora, and plants (1-9). Acidification of these
intracellular compartments is in turn essential for a variety of
cellular processes, including receptor-mediated endocytosis, intracellular targeting, protein processing and degradation, and coupled transport. V-ATPases are also present in the plasma membrane of
certain specialized cells, where they function in such processes as
renal acidification (7), bone resorption (10), and pH homeostasis
(11).
The V-ATPases from fungi, plants, and animals are structurally very
similar and are composed of two functional domains (1). The
V1 domain is a peripheral complex of molecular mass of 570 kDa composed of eight different subunits of molecular mass 70-14 kDa
(subunits A-H) that is responsible for ATP hydrolysis. The V0 domain is a 260-kDa integral complex composed of five
subunits of molecular mass 100-17 kDa (subunits a, d, c, c', and c")
that is responsible for proton translocation. This structure is similar to that of the ATP synthases (or F-ATPases) that function in ATP synthesis in mitochondria, chloroplasts and bacteria (12-17), and sequence homology between these classes of ATPase has been demonstrated for both the nucleotide binding subunits (A, B, The 100-kDa subunit of the V-ATPase is an integral membrane protein
possessing an amino-terminal hydrophilic domain and a carboxyl-terminal
hydrophobic domain containing multiple putative membrane spanning
segments (22-24). In yeast, the 100-kDa subunit is encoded by two
genes, VPH1 and STV1 (23, 24). Vph1p is targeted
to the central vacuole, whereas Stv1p is normally targeted to some
other intracellular membrane, possibly Golgi or endosomes (24).
We have previously demonstrated using a combination of site-directed
and random mutagenesis that Vph1p contains several buried charged
residues, mutation of which significantly alters proton transport
activity of the V-ATPase complex (25, 26). These results have led us to
suggest that the 100-kDa subunit is the V-ATPase homolog to the a
subunit of the F-ATPases. Mutational studies have identified several
critical buried charged residues in the last two transmembrane segments
of the F-ATPase a subunit that are important for proton translocation
(27-30), although more recent studies have suggested that some of
these residues can be replaced without complete loss of function
(31).
Although considerable information has been obtained concerning the
membrane topography of the F-ATPase a subunit (32, 33), essentially no
information has yet been reported concerning the folding of the
V-ATPase a subunit. In the present study, we have employed a
combination of cysteine-scanning mutagenesis and chemical modification
together with introduction of factor Xa cleavage sites in putative
loops to investigate the topographical arrangement of the V-ATPase a subunit.
Materials and Strains--
Zymolyase 100T was obtained from
Seikagaku America, Inc. Bafilomycin A1 was a kind gift from Dr.
Karlheinz Altendorf (University of Osnabruck). Factor Xa protease and
protease inhibitors were from Roche Molecular Biochemicals.
9-Amino-6-chloro-2-methoxyacridine (ACMA),
3-(N-maleimidylpropionyl) biocytin (MPB), and
4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS) were
purchased from Molecular Probes. The monoclonal antibody 10D7 against
the yeast V-ATPase a subunit (34) was also from Molecular Probes.
NeutrAvidin and immunoblotting reagents were obtained from Pierce.
Escherichia coli and yeast culture media were purchased from
Difco Laboratories. Restriction endonucleases, T4 DNA ligase and other
molecular biology reagents were from Life Technologies, Inc., Promega,
and New England Biolabs. ATP, phenylmethylsulfonyl fluoride, and most
other chemicals were purchased from Sigma.
Yeast strain MM112 (MATa Mutagenesis--
The Cys-less Vph1p was generated by
substitution of all seven endogenous cysteine residues with serine.
Single cysteine mutants were then constructed on the Cys-less Vph1p
background. Mutations were introduced into either the wild type
VPH1 or the Cys-less VPH1 using the Altered Sites
II in vitro mutagenesis system (Promega) following the
manufacturer's protocol. The Cys-less VPH1 and the single
cysteine mutants were then confirmed by DNA sequencing.
The factor Xa cleavage site corresponding to the sequence IEGR was
introduced immediately amino-terminal to His-499 and Pro-606, and a
tandem pair of sites was introduced immediately amino-terminal to
Leu-560. Mutations were introduced into the wild type VPH1 using the Altered Sites II system and confirmed by DNA sequencing.
Transformation and Selection--
Yeast cells (MM112) lacking
functional endogenous Vph1p and Stv1p were transformed using the
lithium acetate method (36) with the wild type VPH1 in
pRS316 as a positive control, pRS316 vector alone as a negative
control, the Cys-less form of VPH1, each of the single
cysteine mutants of VPH1, and the mutants bearing the factor
Xa cleavage sites also in pRS316. The transformants were selected on
uracil minus (Ura Isolation of Vacuolar Membrane Vesicles--
Vacuolar membrane
vesicles were isolated as described previously (25). For the labeling
experiments, vacuolar membranes enriched in V0 domains were
prepared by inclusion of a glucose starvation step after spheroplasting
using the protocol of Kane (38). This allowed immunoprecipitation of
the V0 domain directly using the monoclonal antibody 10D7
(which recognizes the a subunit in the V0 domain but not in
the intact V1V0 complex (34)) and reduced
variability due to dissociation of the V-ATPase complex. Briefly, after
the spheroplasting step, the spheroplasts were incubated in the same
medium but without glucose for 20 min at 30 °C. The subsequent steps
were the same as those employed in the normal vacuolar membrane vesicle
preparation, as described previously (25).
Chemical Labeling and Blocking--
The vacuolar membrane
vesicles were collected by centrifugation at 100,000 × g for 10 min at 4 °C and washed using labeling buffer (10 mM Tris-Mes (pH 7.0), 0.25 mM
MgCl2, and 1.1 M glycerol). Membrane vesicles
were then resuspended in 200 µl of labeling buffer and divided into
two tubes. Where indicated, 0.25% Zwittergent 3-14 was added to
permeabilize the vesicles. AMS (100 µM) was added to one
tube, and both samples were incubated for 5 min at 10 °C. Samples
were then transferred to ice and diluted 5-fold with labeling buffer
followed immediately by addition of 250 µM MPB and
incubation for 15 min at 25 °C. Dilution was employed rather than
washing to stop reaction with AMS to avoid variability due to losses
associated with an additional pelleting step. The labeling reaction was
then stopped by addition of 15 mM 2-mercaptoethanol.
Detergent Solubilization, Immunoprecipitation, and Detection of
MPB Labeling of the a Subunit--
After MPB labeling, vesicles were
pelleted by centrifugation at 100,000 × g for 15 min
and solubilized in ice-cold phosphate-buffered saline containing 1%
C12E9, and the V0 domain was
immunoprecipitated using the mouse monoclonal antibody 10D7 specific
for Vph1p plus protein A-Sepharose. In cases where glucose starvation
was not employed after spheroplasting, immunoprecipitation was carried out using the monoclonal antibody 8B1-F3 (Molecular Probes) specific for the 70-kDa A subunit, which precipitates both V1 and
the intact V-ATPase complex. Samples were then subjected to SDS-PAGE on
10% acrylamide gels and transferred to nitrocellulose membranes for MPB detection as described below.
For MPB detection, the blots were probed with horseradish
peroxidase-conjugated NeutrAvidin and developed using the Supersignal ULTRA chemiluminescent system (Pierce). After MPB detection, blots were
stripped and reprobed with the mouse monoclonal antibody 10D7 against
the 100-kDa subunit to ensure the presence of equal amounts of the a
subunit. For immunoblot analysis, original blots or stripped blots were
probed with 10D7, followed by horseradish peroxidase-conjugated
secondary antibody (Bio-Rad) as described previously (25). Blots were
developed using a chemiluminescent detection method obtained from
Kirkegaard & Perry Laboratories.
Factor Xa Cleavage of Wild Type and Vph1p Mutants Bearing Factor
Xa Cleavage Sites--
Vacuolar membrane vesicles (50 µg protein)
prepared from cells transformed with wild type VPH1 or
VPH1 mutants bearing the factor Xa sites as described above
were pelleted and resuspended in buffer containing 400 mM
potassium iodide, 1.6 mM MgATP, and 20 mM
Tris-Mes (pH 7.0) and incubated on ice for 60 min. The purpose of the
potassium iodide treatment was to remove the V1 domain, thus exposing any factor Xa sites that may be shielded by the presence
of V1. Membrane vesicles were then sedimented at
100,000 × g for 45 min and resuspended in factor Xa
buffer (100 mM NaCl, 1 mM CaCl2, 1 M glycerol, 50 mM Tris-HCl (pH 7.4), 2 mM dithiothreitol). The resuspended vesicles were divided
into three aliquots. One received no factor Xa, whereas two received 2 µg of factor Xa protease. One of the samples receiving the factor Xa
also received 1% C12E9. All samples were
incubated overnight at 4 °C followed by addition of Laemmli sample
buffer and separation of half of each sample on a 9% polyacrylamide
gel. The proteins were then blotted to nitrocellulose and probed with
the monoclonal antibody 10D7 against the 100-kDa subunit followed by
horseradish peroxidase-conjugated secondary antibody (Bio-Rad). We have
observed that the antibody 10D7 recognizes a site in the amino-terminal
half of the protein,2 so that
the size of the fragments generated by factor Xa cleavage that would be
recognized by 10D7 could be predicted. Blots were developed using a
chemiluminescent detection method obtained from Kirkegaard and Perry Laboratories.
Other Procedures--
Protein concentrations were determined by
the method of Lowry et al. (39). ATP-dependent
proton transport was measured in transport buffer (25 mM
MES/Tris, pH 7.2, 5 mM MgCl2) using the fluorescence probe ACMA in the presence or absence of 10 nM
bafilomycin A1 as described previously (40).
SDS-polyacrylamide gel electrophoresis was carried out as described by
Laemmli (41).
Construction of the Cys-less Form of Vph1p--
To probe the
membrane topology of the 100-kDa a subunit of the V-ATPase using a
cysteine modification approach, we first needed to construct a Cys-less
form of Vph1p in which all seven of the endogenous cysteine residues
were replaced. Site-directed mutagenesis of VPH1 was
performed to replace each of the endogenous cysteine residues with
serine. The final construct was confirmed by DNA sequencing.
Transformation of a yeast strain in which both VPH1 and
STV1 are disrupted with the Cys-less form of Vph1p gave rise to a strain showing a wild type growth phenotype. That is, cells were
able to grow at both pH 5.5 and 7.5, even in the presence of elevated
Ca2+. This is in contrast to the deletion strain that
displays a vma Vph1p Mutants Bearing Single, Unique Cysteine Residues--
Unique
cysteine residues were introduced into the Cys-less form of Vph1p at
positions 3, 89, 161, 266, 447, 450, 561, 564, 602, 703, 761, 763, and
840 by site-directed mutagenesis, and the mutations were confirmed by
DNA sequencing. The mutant forms of Vph1p were then expressed in the
deletion strain, and the growth phenotype was analyzed. All mutants
displayed a wild type growth phenotype. Isolation of vacuoles and
measurement of bafilomycin-sensitive ATP-dependent proton
transport (Table I) revealed that all of the single cysteine mutants
except G763C displayed greater than 70% of wild type levels of
activity, with G763C having approximately 50% of wild type activity.
Western blot analysis of isolated vacuoles using a monoclonal antibody
specific for Vph1p (10D7) showed that all the mutants expressed nearly
wild type levels of Vph1p (data not shown). These results suggest that,
except for G763C, none of the introduced cysteine residues
significantly perturb the structure or function of Vph1p.
Labeling and Protection of Cysteine Mutants of Vph1p--
To
assess the orientation of the cysteine residues introduced into Vph1p
with respect to the membrane, modification by two sulfhydryl reagents
was employed. The first reagent, MPB, is a membrane permeable reagent
that can react with sulfhydryl groups exposed on both sides of the
vacuolar membrane (33, 44). The second reagent, AMS, is a membrane
impermeant reagent that should react only with sulfhydryl groups on
the exposed cytoplasmic surface of the vacuolar membrane (32, 33,
44).
Previous studies indicate that intact vacuoles isolated from yeast are
both uniformly sided with the cytoplasmic side exposed and sealed with
respect to small molecules (45). The specific activity of the V-ATPase
in the vacuolar membrane vesicles employed in the current study (3-4
µmol of ATP/min/mg of protein) is very similar to the values we have
measured in intact vacuoles (46), suggesting that very little of the
V-ATPase is oriented with the cytoplasmic face sequestered inside the
vesicles. Moreover, the vacuolar membrane vesicles used here display a
ratio of proton transport (as measured by ACMA uptake) to
bafilomycin-sensitive ATPase activity that is nearly the same as that
we measure for either intact vacuoles or purified, bovine brain
clathrin-coated vesicles. We have previously shown that coated vesicles
are both tightly sealed and uniformly oriented with the cytoplasmic
surface exposed on the outside (47). These results suggest that the vacuolar membrane vesicles employed here have the same properties.
Labeling of cysteine residues exposed on the cytoplasmic surface of
Vph1p by MPB should be blocked by prior treatment with the impermeant
reagent AMS. By contrast, cysteine residues exposed on the lumenal
surface of the protein should show labeling by MPB that is unchanged by
pretreatment with AMS. This strategy has been successfully employed to
determine the orientation of cysteine residues in several membrane
proteins, including MDR (44) and subunit a of the F-ATPase (32,
33).
Labeling of Vph1p by MPB is measured by detergent solubilization,
immunoprecipitation, SDS-PAGE, and Western blotting using horseradish
peroxidase-conjugated avidin. To avoid possible protection of cysteine
residues by the V1 domain, immunoprecipitation of the
V0 domain was carried out using the monoclonal antibody
10D7 specific for Vph1p. Because 10D7 recognizes the a subunit only in
the free V0 domain (not the intact
V1V0 complex (34)), it is necessary to optimize
the fraction of V0 in the free state. This was accomplished
by including a brief glucose starvation step after spheroplasting of
the yeast, a procedure that Kane (38) has shown causes an increase in
the fraction of free V0 domain in the vacuolar membrane
from approximately 25% to 75%.
Isolated vacuoles were first incubated in the presence or absence of
100 µM AMS for 5 min at 10 °C. These conditions were selected to allow reaction of AMS with cytoplasmically oriented cysteine residues but to minimize its permeation across the membrane and are similar to the conditions previously employed in related studies (32, 33). Vacuoles were then diluted 5-fold and reacted with
250 µM MPB for 15 min at 25 °C. Dilution rather than
washing was employed to stop reaction with AMS to eliminate additional variability in yield associated with pelleting and resuspending the
membranes. The reaction was then stopped by addition of 15 mM 2-mercaptoethanol and the V-ATPase solubilized and
immunoprecipitated as described above.
Preliminary results indicated that four of the introduced cysteine
residues at positions 561, 564, 761, and 763 were not labeled by MPB,
and these mutants were therefore not further pursued. Fig.
1 shows the labeling of the a subunit by
MPB with and without prior treatment with AMS for each of the remaining
nine cysteine mutants, as well as the Cys-less control. Fig.
1A shows the results obtained for the cysteine residues
introduced into the amino and carboxyl-terminal ends of the a subunit,
whereas Fig. 1B shows the results for cysteine residues
introduced into the loop regions. Because labeling of G602C by MPB in
the absence of AMS was reduced approximately 3-fold (by densitometry)
relative to the other loop cysteine residues, a longer exposure was
employed for this mutant so that the intensity of the band in the
absence of AMS would more closely approximate the other cysteine
mutants. This facilitates a more accurate comparison of their relative
labeling in the presence and absence of AMS. Western blotting using the
antibody 10D7 indicated that the reduced labeling of G602C by MPB is
not due to a reduced amount of the a subunit (data not shown).
As can be seen in Fig. 1, seven of the nine cysteine residues (E3C,
D89C, T161C, S266C, N447C, K450C, and S703C) showed very effective
blocking of MPB labeling by pretreatment with AMS, suggesting that
these residues are exposed on the cytoplasmic surface of the protein.
By contrast, two of the nine cysteine residues (G602C and S840C) showed
very little protection of MPB labeling by AMS, suggesting that these
residues are exposed on the lumenal side of the membrane. These results
were reproducible even when the temperature used in blocking by AMS was
raised to 20 °C.
To test whether G602C and S840C are not protected by AMS due to the
inability of the reagent to cross the membrane, reaction with AMS and
labeling by MPB was repeated for these two mutants and one of the other
cysteine mutants (S703C) in the presence of 0.25% Zwittergent 3-14 in
order to allow access of the AMS to the lumenal side of the membrane.
As can be seen in Fig. 2, the presence of
the detergent allowed AMS to effectively block G602C and S840C from
reaction with MPB, consistent with a lumenal orientation of these
residues.
Analysis of Mutants Bearing Factor Xa Cleavage Sites--
To
further probe the topography of the 100-kDa subunit, factor Xa protease
cleavage sites were introduced into three putative loops at positions
His-499, Leu-560, and Pro-606. Table I shows the effect of introduction
of the four amino acid site (IEGR) preceding His-499 and Pro-606 and
the tandem eight amino acid sequence (IEGRIEGR) preceding Leu-560. As
can be seen, only the single factor Xa site at position 606 significantly reduced proton transport activity (by approximately 55%
relative to wild type), whereas the other two mutants showed near wild
type levels of activity. When tested for cleavage by factor Xa in the
absence and presence of detergent (1% C12E9),
only one of the sites (at position 560) showed significant cleavage. As
can be seen in Fig. 3, treatment of the
mutant bearing the tandem sites at position 560 with factor Xa resulted
in the generation of a 65-kDa fragment in either the absence or
presence of detergent, whereas no such fragment was observed for the
wild type protein. These results suggest that the tandem sites at
position 560 are exposed on the cytoplasmic side of the membrane.
We have used cysteine-scanning mutagenesis together with chemical
modification by membrane permeable and impermeable reagents, as well as
introduction of protease cleavage sites, to analyze the membrane
topography of the 100-kDa a subunit of the yeast V-ATPase. The
cysteine-scanning method depends upon the successful construction of a
functionally active Cys-less form of the protein together with mutants
containing unique cysteine residues that do not significantly perturb
activity. In the case of the a subunit, this required the simultaneous
replacement of seven endogenous cysteine residues. In addition, because
the a subunit is part of a multisubunit complex, the mutations
introduced must not alter the interactions between the a subunit and
the remaining V-ATPase subunits. We observed that both the Cys-less
form of the a subunit and all but one of the single cysteine-containing
mutants give rise to complexes showing near wild type levels of
activity, suggesting that these changes do not significantly alter the
structure or function of the a subunit. In the case of G763C, a loss of
approximately 50% of proton transport activity was observed.
The labeling strategy employed depends upon the membrane permeability
of MPB and the membrane impermeability of AMS. This allows MPB to react
with cysteine residues on both sides of the membrane, whereas AMS
reacts only with cysteine residues exposed on the surface of the
membrane vesicles. In the case of yeast vacuolar membrane vesicles used
in the present study, the exposed surface is the cytoplasmic side of
the membrane. The orientation and sealed state of the vesicles employed
is supported by the similar ratio of proton transport to ATP hydrolysis
that we have observed when compared with other sealed, well oriented
vesicles, including intact yeast vacuoles (46) and purified
clathrin-coated vesicles (47). This strategy has been successfully used
to study the membrane topography of several other integral membrane
proteins, including in particular subunit a of the E. coli
F-ATPase (32, 33). These latter studies have led to the current five
membrane spanning model for the F-ATPase a subunit.
In addition to the cysteine modification approach, we have also
introduced factor Xa cleavage sites into three putative loop regions of
the protein. Although the mutant bearing a factor Xa site at position
606 showed only approximately 50% of wild type levels of activity, the
other two mutants showed nearly normal proton transport, suggesting
minimal perturbation of structure.
The results obtained in the current study have led to the model for the
folding of the V-ATPase a subunit shown in Fig.
4. In addition, a hydropathy plot for
Vph1p together with the location of each of the putative membrane
spanning segments is shown in Fig. 5. The
protein is proposed to span the bilayer nine times, with the amino
terminus on the cytoplasmic side of the membrane and the carboxyl
terminus on the lumenal side. All four of the cysteine residues
introduced into the amino-terminal soluble domain showed a labeling
pattern consistent with a cytoplasmic orientation whereas S840C at the
carboxyl terminus showed labeling characteristic of a lumenal
orientation. This suggests that the amino and carboxyl termini are on
opposite sides of the membrane, requiring that the protein span the
bilayer an odd number of times.
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ABSTRACT
INTRODUCTION
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DISCUSSION
REFERENCES
, and
) (18, 19)
and the proteolipid subunits (subunits c, c', and c") (20, 21). No
sequence homology has been identified for any of the remaining subunits.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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vph1::LEU2
stv1::LYS2 his3-(
200 leu2 lys2 ura3-52) and
plasmid MM322 (VPH1 in pRS316) were used to generate and
study VPH1 mutants (24). Yeast cells were grown in yeast
extract-peptone-dextrose or synthetic dropout medium (35).
) plates as described previously (37).
Growth phenotypes of the mutants were assessed on yeast
extract-peptone-dextrose plates buffered with 50 mM
KH2PO4 or 50 mM succinic acid to
either pH 7.5 or pH 5.5. For testing calcium sensitivity, yeast
extract-peptone-dextrose plates buffered with 50 mM MES and
50 mM MOPS, pH 7.5, were supplemented with 50 mM calcium chloride.
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phenotype (i.e. unable to grow
at pH 7.5 or in the presence of elevated Ca2+ (5, 42, 43)).
Isolation of vacuoles from the wild type and Cys-less strains and
measurement of bafilomycin-sensitive ATP-dependent proton
transport revealed that the Cys-less form of Vph1p gave rise to a
V-ATPase complex that possessed approximately 78 ± 16% of wild
type levels of transport activity (Table
I). Thus, the Cys-less Vph1p represents
an appropriate genetic background in which to construct mutants bearing
unique cysteine residues.
Effect of Vph1p mutations on bafilomycin-sensitive,
ATP-dependent proton transport in vacuolar membrane
vesicles
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Fig. 1.
Labeling of a subunit cysteine mutants by MPB
and protection by AMS in the absence of detergent. A,
vacuolar membrane vesicles (35 µg of protein) isolated from the
Cys-less mutant and the single cysteine mutants at the amino and
carboxyl termini (E3C, D89C, T161C, S266C, and S840C) were incubated in
the presence or absence of 100 µM AMS for 5 min at
10 °C, followed by 5-fold dilution and labeling with 250 µM MPB for 15 min at 25 °C. The membranes were then
solubilized with C12E9, and the a subunit
immunoprecipitated using the monoclonal antibody 10D7 and protein
A-Sepharose. Samples were separated by SDS-PAGE, transferred to
nitrocellulose, and probed with horseradish peroxidase-NeutrAvidin.
Blots were developed using the Supersignal Ultra system from Pierce.
B, vacuolar membrane vesicles (45 µg of protein) isolated
from the Cys-less mutant, and each of the a subunit mutants containing
single cysteine residues in the loop regions (N447C, K450C, G602C, and
S703C) were treated as described in A. Because G602C showed
approximately 3-fold lower labeling by MPB (by densitometry) in the
absence of AMS relative to the other loop cysteine residues, a longer
exposure is shown for this mutant such that the intensity of the band
in the absence of AMS is comparable to that observed for the other loop
cysteine mutants. This allows a more direct comparison of the effect of
AMS pretreatment on MPB labeling.
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Fig. 2.
Labeling of G602C, S703C, and S840C mutants
by MPB and protection by AMS in the presence of detergent.
Vacuolar membrane vesicles (45 µg protein) isolated from the G602C,
S703C, and S840C mutants were incubated in the presence or absence of
100 µM AMS followed by dilution and labeling with 250 µM MPB as described in the legend to Fig. 1 except that
0.25% Zwittergent 3-14 (ZW) was added to the samples prior
to addition of AMS where indicated. Solubilization with
C12E9, immunoprecipitation of Vph1p, SDS-PAGE,
and blotting were then performed as described in Fig. 1.
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Fig. 3.
Cleavage of a Vph1p mutant bearing tandem
factor Xa sites at position 560 by factor Xa. Vacuolar membrane
vesicles (50 µg of protein) isolated from wild type (WT)
cells or cells bearing a mutant form of Vph1p containing tandem factor
Xa sites at position 560 (2fXa-560) were treated with
potassium iodide and MgATP to remove the V1 domain and then
incubated in the absence or presence of C12E9
(1.0%) and factor Xa (2 µg of protein) as described under
"Experimental Procedures." The samples were then separated by
SDS-PAGE, transferred to nitrocellulose, and blotted with the
monoclonal antibody 10D7 specific for Vph1p as described. The fragment
observed for the Vph1p mutant bearing the tandem factor Xa sites at
position 560 has a molecular mass of approximately 65 kDa,
corresponding to the predicted molecular mass of the amino-terminal
fragment generated by factor Xa cleavage at position 560.
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Fig. 4.
Model of the transmembrane topology of the
V-ATPase a subunit. Cysteine residues introduced into Vph1p at
positions Glu-3, Asp-89, Thr-161, Ser-266, Asn-447, Lys-450, and
Ser-703 displayed a cytoplasmic labeling pattern and are shown in
shaded circles, whereas cysteines introduced at Gly-602 and
Ser-840 show labeling characteristic of a lumenal orientation and are
shown in shaded squares. Cysteines that are not labeled by
MPB at positions 561, 564, 761, and 763 are shown with solid
dots. The location of the factor Xa cleavage site (Leu-560) that
is cleaved by factor Xa in intact vacuolar membranes vesicles
(i.e. is cytoplasmically oriented) is shown with an
arrow, whereas the two cleavage sites that are not cleaved
by factor Xa in the absence or presence of detergent (His-499 and
Pro-606) are shown by ×. The position of mutations previously shown to
affect activity of the V-ATPase (25, 26) are shown in open
circles, whereas the position of mutations causing defects in
assembly of the V-ATPase complex are shown in open squares.
We have also constructed several mutations at Arg-735, including R735K,
R735L, and R735A, because an arginine near the carboxyl terminus of the
F-ATPase a subunit appears to play a role in proton transport (29, 31).
All of the Arg-735 mutants show a defect in either stability of the a
subunit or assembly of the V-ATPase complex (data not shown), so that
we are not able to assess the role of this residue in proton transport
by the V-ATPase. The site of trypsin cleavage of the bovine a subunit
from the cytoplasmic side of the membrane (48) is also shown by an
arrow. The borders of the putative transmembrane segments
are indicated by the residue numbers.
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Fig. 5.
Hydropathy plot of Vph1p and location of
putative transmembrane segments. The amino acid sequence of Vph1p
(24) was analyzed using the method of Kyte and Doolittle (49), and the
resultant hydropathy plot shown. The location of the nine putative
transmembrane segments are also shown and labeled
I--IX. There are four consensus
N-linked glycosylation sites (Asn-X-Ser/Thr) in
Vph1p at Asn-113, Asn-280, Asn-324, and Asn-374. Because all four sites
are located in the amino-terminal soluble domain, which our labeling
data clearly shows is cytoplasmic, it is unlikely that any of these
four sites are glycosylated in vivo. In fact, it has not
clearly been demonstrated that Vph1p is glycosylated in yeast. If Vph1p
is glycosylated, it is possible that carbohydrate is attached at
O-linked sites, for which there is not a clear consensus
sequence.
In our original folding model for the a subunit (25), we had postulated that the amino-terminal soluble domain was exposed on the lumenal side of the membrane based upon the increased labeling observed for the bovine a subunit using membrane impermeant reagents when the membrane was disrupted with detergents (47). This result may be explained by a change in conformation of the protein in the presence of detergent such that previously shielded sites on the cytoplasmic side of the membrane become exposed. In addition, because the a subunit is not synthesized with an amino-terminal leader sequence, a cytoplasmic orientation for the amino terminus is more consistent with what is known concerning the requirements for translocation of hydrophilic protein segments across the endoplasmic reticulum membrane during biosynthesis.
A lumenal orientation of the carboxyl terminus is also at odds with our previously proposed model, in which we suggested a cytoplasmic orientation for the carboxyl terminus (26). This was based on the identification by random mutagenesis of a cluster of five mutations between residues Leu-800 and Gly-814 that disrupted attachment of the V1 and V0 domains, suggesting that this region may be important in assembly of the V-ATPase complex (26). It is nevertheless possible that disruption of assembly in these mutants results from conformational changes in the structure of the 100-kDa subunit or the V0 domain that prevent attachment of V1 to V0. Interestingly, our current model places nearly all of the residues that have been observed to disrupt assembly, including Asp-425, Lys-538, and Arg-735, as well as the five mutations between residues 800 and 814 on the lumenal side of the membrane. It is possible that these residues may form a lumenal domain that senses the intravesicular pH and conveys this information through conformational changes in the 100-kDa subunit to the cytoplasmic domain of the complex.
With respect to the loop regions, four of the introduced cysteine residues (at positions 561, 564, 761, and 763) are not labeled by MPB, suggesting that they are shielded from reaction by interaction with other V0 subunits or other regions of the 100-kDa subunit itself. It is also possible that one or more of these cysteine residues may be shielded from reaction with MPB by interaction with the lipid bilayer. Of the remaining introduced cysteine residues, three appear to have a cytoplasmic orientation (N447C, N450C, and S703C) whereas G602C appears to be lumenal. The cytoplasmic orientation of residues 447 and 450 requires that the hydrophobic region between residues 405 and 443 span the bilayer twice rather than once (as originally postulated (25)), placing Asp-425 on the lumenal side of the membrane. A lumenal orientation for Gly-602 and a cytoplasmic orientation for Ser-703 is consistent with a single membrane span between these sites. That Ser-703 is cytoplasmic is also consistent with the observation that the bovine V-ATPase a subunit can be cleaved by low concentrations of trypsin at a site immediately upstream of this residue in intact coated vesicles (48). Thus, the large soluble loop between residues 653 and 727 is likely to be oriented toward the cytoplasm.
Of the three factor Xa sites introduced into the 100-kDa subunit, protease cleavage was observed only for the tandem sites introduced at position 560. The fact that this cleavage happened in intact vacuolar membrane vesicles as well as in the presence of detergent indicates that this site is exposed on the cytoplasmic side of the membrane. For the sites introduced at positions 499 and 606, no cleavage by factor Xa was observed in either the presence or absence of detergent, again suggesting that these sites may be shielded through interaction with other regions of the protein. Nevertheless, the observation that all three of these mutants show significant proton transport activity suggests that all three sites are located in loop regions between transmembrane segments in the a subunit. This conclusion comes from results obtained with lac permease, where it has been observed that factor Xa sites introduced into loops between transmembrane segments generally do not affect activity, whereas sites introduced within transmembrane segments invariably lead to loss of both correct folding and transport activity.3
It is important to emphasize that the model shown in Fig. 4 is only a
working model for the topography of the V-ATPase a subunit based upon
the first available data presented here concerning the disposition of
this polypeptide with respect to the membrane. The labeling and
proteolysis approaches taken in the current study, like all other
methods of analyzing the topography of membrane proteins, are subject
to several possible sources of error. For instance, it is possible that
local differences in the environment of individual cysteine residues,
in addition to their disposition with respect to the membrane, may
influence their relative reactivity toward the two sulfhydryl reagents
employed. We have shown that permeabilization of the membranes with
detergent allows the lumenally oriented cysteine residues to be
effectively blocked by AMS. Nevertheless, it will be important that the
proposed model be further tested, both by introduction of additional
labeling and cleavage sites and by employing other approaches to
determine topography, such as epitope tagging and preparation of
site-specific antibodies that recognize the native protein. The current
study, however, represents a first step in determining the folding
pattern of this important V-ATPase subunit.
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ACKNOWLEDGEMENTS |
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We thank Drs. Ronald Kaback and Jianhua Wu for valuable discussions, Drs. Julie Long and Steve Vik for sharing their labeling protocol, and Dr. Yin Chen for his assistance in preparing the figures.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM34478. Fluorescence facilities were provided by National Institutes of Health Grant DK 34928.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: Dept. of Cellular and
Molecular Physiology, Tufts University School of Medicine, 136 Harrison
Ave., Boston, MA 02111. Tel.: 617-636-6939; Fax: 617-636-0445.
2 X.-H. Leng, T. Nishi, and M. Forgac, unpublished observations.
3 R. Kaback, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: V-ATPase, vacuolar proton-translocating ATPase; ACMA, 9-amino-6-chloro-2-methoxyacridine; MPB, 3-(N-maleimidylpropionyl) biocytin; AMS, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 2-(N-morpholino)propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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