From the Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111
Received for publication, September 25, 2002, and in revised form, December 11, 2002
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ABSTRACT |
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The yeast vacuolar ATPase (V-ATPase) contains
three proteolipid subunits: c (Vma3p), c' (Vma11p), and c" (Vma16p).
Each subunit contains a buried glutamate residue that is essential for
function, and these subunits are not able to substitute for each other
in supporting activity. Subunits c and c' each contain four putative transmembrane segments (TM1-4), whereas subunit c" is predicted to
contain five. To determine whether TM1 of subunit c" serves an
essential function, a deletion mutant of Vma16p was constructed lacking
TM1 (Vma16p- The vacuolar H+-ATPases (or
V-ATPases)1 are found in
a variety of intracellular compartments that function in both
endocytic and secretory pathways (1-8). Acidification of these
compartments is essential for many 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, including
osteoclasts (9), renal intercalated cells (10), and neutrophils (11),
where they function in such processes as bone resorption, renal
acidification, and pH homeostasis, respectively.
The V-ATPases from fungi, plants, and animals are structurally very
similar and are composed of two domains (1-8). The V1 domain is a 570-kDa peripheral complex composed of eight different subunits of molecular mass of 70 to 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 to 17 kDa (subunits a, d, c, c', and c") that is responsible for proton translocation. The overall structure of the V-ATPase is therefore similar to that of the F1F0-ATP
synthase (or F-ATPase) that functions in ATP synthesis in mitochondria,
chloroplasts, and bacteria (12-15). Sequence homology between these
classes of ATPase has been identified for both the nucleotide-binding
subunits (16, 17) and the proteolipid subunits (18, 19).
Unlike the F-ATPases, however, which contain a single type of
proteolipid subunit (subunit c), the V-ATPases contain three different
proteolipid subunits (c, c', and c"). All three proteolipid subunits
are highly hydrophobic proteins, and all three are essential for
V-ATPase function (19). In yeast, subunits c, c', and c" are encoded by
the VMA3, VMA11, and VMA16 genes,
respectively. The V-ATPase proteolipid subunits are homologous both to
each other and to the F-ATPase subunit c, from which they appear to have been derived by gene duplication and fusion (18). Thus, the
F-ATPase subunit c is an 8-kDa protein containing two transmembrane segments with an essential aspartate residue present in TM2 (14). Subunits c and c' of the V-ATPase are 16-kDa proteins containing four
putative transmembrane segments with an essential glutamate residue
present in TM4 (18, 19). The N- and C-terminal halves of subunits c and
c' are homologous to each other. Subunit c has been shown recently (20)
to contain the binding site for the specific V-ATPase inhibitor bafilomycin.
By contrast, subunit c" of the V-ATPase is a 21-kDa protein predicted
to have five transmembrane helices (19). Although TM2 to TM5 of subunit
c" are homologous to subunits c and c', TM1 is not similar to anything
in these proteins. Interestingly, the essential glutamate residue of
subunit c" is located in TM3 (19). Previously, we demonstrated that the
C terminus of mouse subunit c is present on the lumenal side of the
membrane, whereas the C terminus of subunit c" is located
on the cytoplasmic side (21). These results suggest that the membrane
organization of subunit c" is different from that of subunit c. In this
study, we address the functional role of the first transmembrane
segment of subunit c", and we report additional information concerning the membrane topology of this protein.
Materials and Strains--
Zymolyase 100T was obtained from
Seikagaku America, Inc. Concanamycin A was purchased from Fluka
Chemical Corp. Protease inhibitors were from Roche Molecular
Biochemicals. The monoclonal antibody 3F10 (directed against the HA
antigen) that is conjugated with horseradish peroxidase was also from
Roche Molecular Biochemicals. The monoclonal antibody 8B1-F3 against
the yeast V-ATPase A subunit (22), the monoclonal antibody 10D7 against
the 100-kDa a subunit (23),
3-(N-maleimidylpropionyl)biocytin (MPB), and
4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS) were from
Molecular Probes. Escherichia coli and yeast culture media
were purchased from Difco. Restriction endonucleases, T4 DNA ligase,
and other molecular biology reagents were from Invitrogen, Promega, and
New England Biolabs. ATP, phenylmethylsulfonyl fluoride, and most other
chemicals were purchased from Sigma. Yeast strains lacking the
proteolipid genes, TNY101 (vma3 Cloning of the VMA3, VMA11, and VMA16
Genes--
VMA3, VMA11, and VMA16
genes were amplified from genomic DNA isolated from yeast strain YPH500
and then cloned into the pRS413 yeast shuttle vector. The sequences of
the cloned genes were confirmed by DNA sequencing using an automated
sequencer from Applied Biosystems. The oligonucleotides primers used
for amplification of these genes are as follows: VMA3 forward,
ggctctagaacttctgcgttattattaataattg, and VMA3 reverse,
gccatcgatgaaatgaggtagtttggatatgaag; VMA11 forward, gatctattgaccaaaacaggtgtggaaac, and VMA11 reverse,
ggaggcctagggttttctttcaagtatacacag; VMA16 forward,
cacatgacgccgatttagaagtttcaatg, and VMA16 reverse, ggtctagatcccaggtctcacggaaatcttatc. An HA tag was introduced at the C
terminus of each protein using a PCR-based recombination method
(21).
Transformation and Selection--
Yeast cells lacking functional
endogenous Vma3p, Vma11p, or Vma16p were transformed using the lithium
acetate method (24). The transformants were selected on histidine minus
(HIS Isolation of Vacuolar Membrane Vesicles--
Vacuolar membrane
vesicles were isolated using a modification of the protocol described
by Uchida et al. (25). Yeast cells were grown overnight at
30 °C to 1 × 107 cells/ml in 1 liter of selective
medium. Cells were pelleted, washed once with water, and resuspended in
100 ml of 10 mM dithiothreitol and 100 mM
Tris-HCl, pH 9.4. After incubation at 30 °C for 15 min, cells were
pelleted again, washed once with 100 ml of YEPD medium containing 0.7 M sorbitol, 2 mM dithiothreitol, and 100 mM MES-Tris, pH 7.5, resuspended in 100 ml of YEPD medium
containing 0.7 M sorbitol, 2 mM dithiothreitol,
100 mM MES-Tris, pH 7.5, and 2 mg of Zymolase 100T, and
incubated at 30 °C with gentle shaking for 60 min. The resulting
spheroplasts were osmotically lysed, and the vacuoles were isolated by
flotation on two consecutive Ficoll gradients. Protein concentrations
were measured by the BCA protein assay (Pierce).
Analysis of Subunit Expression and V-ATPase
Assembly--
Vacuolar membrane proteins were separated by SDS-PAGE on
8 or 12.5% acrylamide gels. The expression of Vma16p and Vma16p- Immunoprecipitation of the HA-tagged Vma16p from Intact or
Detergent-solubilized Vacuolar Membrane Vesicles--
To determine
whether the C terminus of Vma16p is exposed on the cytoplasmic side of
the membrane, the accessibility of the HA epitope tag introduced at the
C terminus in intact vacuoles was determined. Anti-HA antibody was
added to intact vacuolar membrane vesicle (100 µg) and incubated for
2 h at 4 °C. Vacuolar membranes were washed with the overlay
buffer (20 mM MES-Tris, pH 7.6, 0.25 mM
MgCl2, 1.1 M glycerol) and solubilized with
phosphate-buffered saline containing 1% C12E9, and the
Vma16p::HA was recovered with protein G-Sepharose. As a
control, the anti-HA antibody was added to
C12E9-solubilized vacuolar membranes followed
by precipitation of the Vma16p::HA with protein G-Sepharose.
Samples were separated by SDS-PAGE and transferred to nitrocellulose,
and Vma16p was detected using a peroxidase-conjugated anti-HA antibody
(Roche Molecular Biochemicals) and the Supersignal ULTRA
chemiluminescent system (Pierce).
Chemical Labeling and Blocking of Introduced Cysteine
Residues--
Chemical labeling of introduced cysteine residues by the
membrane-permeant sulfhydryl reagent MPB and blocking by the
membrane-impermeant reagent AMS was performed using a modification of
the protocol described previously (27). Briefly, vacuolar membrane
vesicles were washed using labeling buffer (10 mM
MES-Tris, pH 7.0, 0.25 mM MgCl2, and 1.1 M glycerol) and divided into two tubes. AMS (100 µM) was added to one tube, and both samples were
incubated for 5 min at 15 °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. The labeling reaction was then stopped by addition of 15 mM 2-mercaptoethanol. After MPB labeling, vesicles were
pelleted and solubilized in ice-cold phosphate-buffered saline
containing 1% C12E9. The V0
domain was immunoprecipitated using the mouse monoclonal antibody 10D7
specific for Vph1p plus protein G-Sepharose. Samples were then
subjected to SDS-PAGE on 10% acrylamide gels and transferred to
nitrocellulose membranes. The blots were probed with horseradish
peroxidase-conjugated NeutrAvidin and developed using the Supersignal
ULTRA chemiluminescent system (Pierce).
Other Procedures--
Protein concentrations were determined by
the Lowry method (28). ATPase activity was measured using a coupled
spectrophotometric assay in the presence or absence of 1 µM concanamycin, as described previously (29).
ATP-dependent proton transport was measured in transport
buffer (25 mM MES-Tris, pH 7.2, 5 mM
MgCl2) using the fluorescence probe ACMA
(9-amino-6-chloro-2-methoxyacridine) in the presence or absence of
1 µM concanamycin, as described previously (29). SDS-PAGE
was carried out as described by Laemmli (30).
Transmembrane Helix 1 Is Not Essential for Vma16p
Function--
Fig. 1 shows the sequence
alignment of the three proteolipid subunits of the yeast V-ATPase:
subunit c (Vma3p), subunit c' (Vma11p), and subunit c" (Vma16p). As can
be seen, subunits c and c' both contain four putative transmembrane
helices and share significant sequence homology with each other and
with transmembrane segments 2-5 of subunit c". By contrast, TM1 of
subunit c" is not homologous to any other sequence in the other two
proteolipid subunits. The location of the buried glutamate residue
critical for function of each of the proteolipid subunits is depicted
in Fig. 2a. To address the
functional role of TM1 of subunit c", a deleted form of subunit c" was
constructed (Vma16p-
Previous results (33, 34) have suggested that retention of ~20% of
wild type V-ATPase activity is sufficient to confer on cells a wild
type growth phenotype. It is therefore necessary to measure directly
V-ATPase activity and proton transport in vacuoles isolated from cells
expressing the Vma16p- Accessibility of C Terminus of HA-tagged Vma16p in Intact and
Detergent-solubilized Vacuolar Membrane Vesicles--
Previously, we
demonstrated that the C terminus of the mouse subunit c" appears to be
facing the cytoplasmic side of the membrane in COS-1 cells transfected
with a HA-tagged form of the mouse Vma16p homologue (21). To confirm
this result for the yeast protein, we compared the accessibility of an
HA epitope attached at the C terminus of Vma16p in intact
versus solubilized vacuolar membrane vesicles. One sample of
vacuolar membranes was incubated with an anti-HA antibody followed by
washing, detergent solubilization, and immunoprecipitation of the
complexes containing the bound HA antibody. As a control, vacuolar
membranes were solubilized with detergent first before addition of the
anti-HA antibody and immunoprecipitation. Both samples were then
subjected to SDS-PAGE and Western blotting using the anti-HA antibody.
As shown in Fig. 3, the amount of anti-HA
antibody binding to Vma16p::HA is similar whether the
immunoprecipitating antibody was added before or after detergent
solubilization. The somewhat higher level of antibody binding observed
in the right lane of Fig. 3 may be due to some change in
interaction between the subunits upon detergent solubilization of the
complex or to the loss of some antibody during washing of the membranes
in the case where antibody is added before detergent solubilization.
This result indicates that the antibody-binding site at the C terminus
of Vma16p::HA is exposed in intact vacuolar membrane vesicles
and hence resides on the cytoplasmic side of the membrane.
Labeling of Single Cysteine-containing Mutants of Vma16p by
Membrane-permeant and -impermeant Maleimides--
In order to obtain
additional information about the topology of subunit c", we employed
cysteine mutagenesis and covalent modification by the membrane-permeant
sulfhydryl reagent MPB and the membrane-impermeant sulfhydryl reagent
AMS. This method has been employed previously to study the membrane
folding of subunit a of the F1F0-ATP synthase
of E. coli (35, 36), and we have used this method to study
the topology of subunit a of the yeast V-ATPase (27). In order to apply
this method to subunit c", we first constructed a Cys-less form of
Vma16p by replacing each of the three endogenous cysteine residues
present at positions 67, 105, and 159 with serine (Fig. 1).
Site-directed mutagenesis was performed on the HA-tagged form of Vma16p
described above in order to facilitate detection of the expressed
proteins by Western blot. As can be seen in Fig.
4a, the Cys-less form of the
HA-tagged Vma16p was expressed at normal levels in isolated vacuoles
relative to the wild type HA-tagged Vma16p. Moreover, both Vma1p and
Vph1p were present at normal levels in isolated vacuoles, suggesting
that removal of the endogenous cysteine residues of Vma16p did not
perturb assembly of the V-ATPase. Finally, measurement of
concanamycin-sensitive ATPase activity and ATP-dependent
proton transport indicated that the Cys-less form of Vma1p gave rise to
V-ATPase complexes possessing wild type levels of both ATPase activity
and proton transport (Fig. 4b).
By using this Cys-less, HA-tagged form of Vma16p as the starting point,
seven single-cysteine containing mutants were constructed by
replacement of the endogenous residues at positions Ser-5, Ser-11, Ser-55, Ser-135, Ser-137, Ser-178, and Ser-210 with cysteine by
site-directed mutagenesis. An additional mutant (Q213C) of the untagged
form of Vma16p was also constructed. Because this mutant contained a
cysteine residue at the very C terminus, it was felt that it would be
better to use an untagged form of Vma16p to analyze labeling because of
the possibility of interference with access of the labeling reagents by
the presence of the C-terminal HA tag. These mutant forms of Vma16p
were expressed in the VMA16 deletion strain, and the growth
phenotype was analyzed at pH 7.5 and 5.5. All mutants displayed a wild
type growth phenotype at both pH values (data not shown). Western blot
analysis of vacuoles isolated from the mutant strains (Fig.
4a) revealed wild type levels of the HA-tagged Vma16p
protein for six of the seven tagged mutants (only S210C showed somewhat
lower labeling than wild type). In addition, all of the mutants
(including Q213C) showed normal levels of Vma1p and Vph1p on the
vacuolar membrane. Finally, vacuoles isolated from each of the mutant
strains displayed at least 80% of wild type levels of activity for
both concanamycin-sensitive ATPase activity and
ATP-dependent proton transport (Fig. 4b). These
results indicate that the cysteine substitutions in Vma16p do not
significantly compromise stability, assembly, or activity of the
V-ATPase complex.
We next determined the ability of each of the introduced cysteine
residues to react with the membrane-permeant reagent MPB. Vacuolar
membrane vesicles isolated from each of the mutant strains were reacted
with 250 µM MPB for 15 min at 25 °C followed by
detergent solubilization and immunoprecipitation of the V0
complexes with the monoclonal antibody 10D7 directed against subunit a.
Previous studies have shown that this antibody only recognizes its
epitope on subunit a in the free V0 domain and not in the
intact V1V0 complex (23). The
immunoprecipitated proteins were then separated by SDS-PAGE, and
Western blot analysis was performed using horseradish peroxidase-conjugated NeutrAvidin (Pierce). As shown in Fig.
5a, of the eight single
cysteine-containing mutants of Vma16p, only S5C and S178C showed
significant labeling by MPB, with S5C showing much stronger labeling
than S178C. The remaining cysteine residues appear to be inaccessible
to labeling by MPB, possibly due to shielding of these sites by other
subunits in the V0 domain.
We next tested whether pretreatment of the vacuoles containing the S5C
and S178C mutants with the membrane-impermeant sulfhydryl reagent AMS
was able to prevent labeling of these proteins by MPB. As can be seen
in Fig. 5b, whereas MPB labeling of S5C was effectively
blocked by pretreatment with AMS, labeling of S178C was not. By
contrast, permeabilization of the vacuoles isolated from the S178C
mutant with low concentrations of the detergent Zwittergent 3-14 resulted in effective blocking of MPB labeling by pretreatment with
AMS. Quantitation of the extent of labeling using scanning densitometry
(Alpha Imager 2200) revealed that AMS treatment of S5C reduced labeling
of Vma16p by 80%, whereas AMS treatment of S178C reduced labeling by
only 45%. After detergent permeabilization of the vacuolar membrane,
however, AMS reduced labeling of the S178C mutant by 87%. These
results suggest that the cysteine residue in the S5C mutant has a
cytoplasmic orientation, whereas the cysteine residue in the S178C
mutant is oriented toward the lumenal side of the membrane. The values
obtained for AMS protection of Vma16p mutants are similar to those
observed previously (27) for single cysteine-containing mutants of
Vph1p. Thus, for Vph1p residues interpreted as having a lumenal
orientation (S602C and S840C), AMS pretreatment reduced MPB labeling of
subunit a by 50 and 27%, respectively, similar to the 45% observed
for the S178C mutant of Vma16p. By contrast, for the seven cysteine residues interpreted as having a cytoplasmic orientation in Vph1p, the
protection by AMS of MPB labeling ranged from 72 to 98%, with an
average value of 87(±9)%, similar to the 80% observed for the S5C
mutant of Vma16p.
The proteolipid subunits of the V- and F-ATPases have been shown
by mutagenesis studies to play a critical role in proton translocation
through the integral domains of these complexes (14, 19, 37). NMR
analysis of the single F-ATPase proteolipid subunit (subunit c) reveals
a helical hairpin structure with the essential aspartate residue
present in the middle of the second transmembrane helix (38). X-ray
crystallography (39) and cross-linking studies (40) indicate the
presence of 10 copies of the c subunit in a ring structure, although
this number may be species-dependent (41, 42). The critical
aspartate residue is thought to undergo reversible protonation and
deprotonation during proton translocation through F0 (14),
and the orientation of this residue is believed to change during
rotary catalysis (43).
Unlike the F-ATPases, the V-ATPases require three distinct proteolipid
subunits for function (19). Hydropathy analysis suggests that subunits
c and c' both contain four putative transmembrane helices, whereas
subunit c" contains five (19, 37). Quantitation of the subunit
stoichiometry of the bovine-coated vesicle V-ATPase suggests the
presence of 5-6 copies of subunits c plus c' and a single copy of
subunit c" (44). Immunoprecipitation of epitope-tagged forms of the
yeast proteolipid subunits suggests the presence of single copies of
both subunits c' and c" and multiple copies of subunit c (45).
Combining the results from both of these studies suggests a subunit
stoichiometry for the V-ATPase proteolipid ring of
c4-5c'1c"1, although other studies
have suggested that there may be two copies of subunit c" (46).
Assuming the former subunit stoichiometry, the proteolipid ring of
V0 would contain 25-29 transmembrane helices in comparison
to the 20 transmembrane helices for F0. Because there is
only one critical glutamate residue present in each of the V-ATPase
proteolipid subunits (19, 37), the total number of such residues per
V0 domain (6-7) is smaller than that for F0
(10). This difference has been suggested as an explanation for the
lower H+-ATP stoichiometry of the V-ATPases relative to the
F-ATPases (47).
Why the V-ATPases require three distinct proteolipid subunits is
unclear, but previous results (19) have indicated (and it is confirmed
in the current study) that they are not able to complement each
other's loss. Because of the unique structure predicted for subunit
c", we were interested in determining whether TM1 played an important
functional role in activity or assembly of the V-ATPase. It has been
suggested, for example, that the fifth transmembrane segment might
serve as a "plug" in the center of the proteolipid ring of
V0 (45). It also appeared possible that removal of TM1
might allow the truncated Vma16p to substitute for one of the other
proteolipid subunits in supporting activity. It was therefore
surprising that although the Vma16p- Because TM1 of Vma16p did not appear to serve a crucial functional role
in V-ATPase activity, we wished to determine whether this region of the
protein actually corresponds to a transmembrane segment. To address
this question, a series of mutants of Vma16p were constructed
containing unique cysteine residues in the N and C terminus of the
protein as well as in three of the predicted loop regions. The
reactivity of these cysteine residues toward the membrane-impermeant
maleimide AMS and the membrane-permeant maleimide MPB was then
determined in intact vacuolar membrane vesicles. This same approach was
used to address the membrane topology of subunit a of the V-ATPase (27)
as well as subunit a of the F-ATPase (35, 36). The results presented in
the current study show that the N terminus of subunit c" is present on
the cytoplasmic side of the membrane, whereas the loop between TM4 and
TM5 containing Cys-178 is present on the lumenal side of the membrane.
Previous work (21) from our laboratory on the mammalian homologue of
Vma16p indicates that the C terminus of the mouse subunit c", in
contrast to the C terminus of subunit c, is present on the cytoplasmic
side of the membrane. The results presented in Fig. 3 of the present
paper confirm that the C terminus of the yeast Vma16p is also exposed
on the cytoplasmic side of the membrane. Interestingly, the least
conserved part of subunit c" is located in the putative TM1 region
(21). Because both the N and C terminus of subunit c" appear to be
exposed to the cytoplasmic side of the membrane, the polypeptide chain
likely crosses the membrane an even number of times. This suggests that
subunit c" contains four rather than five transmembrane segments and is
consistent with the lumenal labeling observed for the S178C mutant. A
model depicting the folding of subunit c" is shown in Fig.
6. The failure of the remaining 6 introduced cysteine residues to react with MPB likely reflects the
inability of this reagent to access these sites. This limited access
may be due to shielding of these parts of the polypeptide chain by
other portions of Vma16p itself or by other subunits in the
V0 domain. In particular, both subunit d (a peripheral
protein that remains tightly bound to V0 upon dissociation
of V1) and the N-terminal domain of subunit a (which is
both cytoplasmic (27) and appears from electron microscopy to fold down
on the membrane (49)) are likely candidates for this inter-subunit
shielding effect. Because immunoprecipitation of the labeled complexes
was performed using a monoclonal antibody against subunit a that only
recognizes its epitope in the free V0 domain, the observed
shielding of these sites is most likely not due to interference by the
V1 domain.
TM1). Although this construct does not complement the
loss of Vma3p or Vma11p, it does complement the loss of full-length Vma16p. Vacuoles isolated from the strain expressing Vma16p-
TM1 showed V-ATPase activity and proton transport greater than 80% relative to wild type and displayed wild type levels of subunits A and
a, suggesting normal assembly of the V-ATPase complex. These results
suggest that TM1 of Vma16p is dispensable for both activity and
assembly of the V-ATPase. To obtain information about the topology of
Vma16p, labeling of single cysteine-containing mutants using the
membrane-permeable reagent
3-(N-maleimidylpropionyl)biocytin (MPB) and the
-impermeable reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS) was tested. Both the Cys-less form of Vma16p and eight single cysteine-containing mutants retained greater than 80% of wild
type levels of activity. Of the eight mutants tested, two (S5C and
S178C) were labeled by MPB. MPB-labeling of S5C was blocked by AMS in
intact vacuoles, whereas S178C was blocked by AMS only in the presence
of permeabilizing concentrations of detergent. In addition, a
hemagglutinin epitope tag introduced into the C terminus of Vma16p was
recognized by an anti-hemagglutinin antibody in intact vacuolar
membranes, suggesting a cytoplasmic orientation for the C terminus.
These results suggest that subunit c" contains four rather than five
transmembrane segments with both the N and C terminus on the
cytoplasmic side of the membrane.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), TNY102 (vma11
), and TNY103 (vma16
), were
constructed by replacing the entire coding region of each gene
(VMA3, VMA11, or VMA16) with the
TRP gene in the YPH500 strain. YEPD buffered to pH
5.5 or pH 7.5 was used for selection of strains showing a vma
phenotype.
) plates, and growth phenotypes of the mutants were assessed on
YEPD plates buffered with 50 mM
KH2PO4 or 50 mM succinic acid to
either pH 7.5 or pH 5.5.
TM1 was detected by Western blotting using the horseradish
peroxidase-conjugated monoclonal antibody 3F10 against HA, whereas
Vph1p or Vma1p was detected by monoclonal antibody 10D7 and 8B1-F3
(Molecular Probes, OR), respectively, followed by a horseradish
peroxidase-conjugated secondary antibody (Bio-Rad). Assembly of the
V-ATPase was assessed by measurement of the amount of subunit A present
on isolated vacuolar membranes (23, 26). Blots were developed using a chemiluminescent detection method obtained from Kirkegaard & Perry Laboratories (Gaithersburg, MD).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
TM1) lacking amino acid residues 2-41 that
contain TM1 (Fig. 1). We first tested the ability of this construct to
complement the phenotype of yeast strains disrupted in each of the
proteolipid genes. It has been shown previously that yeast lacking any
of the V-ATPase genes (or the pair of genes encoding subunit a) display
a conditional lethal phenotype (vma
) characterized by an inability to
grow at pH 7.5 but retaining the ability to grow at pH 5.5 (31, 32). As
can be seen in Fig. 2b, the full-length genes are only able to complement their own disruption and not that of the other
proteolipid genes. Moreover, Vma16p-
TM1 does not complement the loss
of either Vma3p or Vma11p. Surprisingly, however, Vma16p-
TM1 does
complement the loss of the full-length Vma16p protein. This result
suggests that TM1 is not essential for V-ATPase function.
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Fig. 1.
Protein sequence alignment of the three
proteolipid subunits Vma3p (c), Vma11p (c'), and Vma16p (c") of the
yeast V-ATPase. Residues that are identical in at least two of the
sequences are indicated by shaded boxes, and the
essential glutamate residues in each subunit are indicated with
asterisks. The putative transmembrane segments of Vma16p
(TM1-TM5) are indicated with black bars.
TM2-TM5 of Vma16p correspond to TM1-TM4 of Vma3p and Vma11p. The
region of Vma16p removed in the Vma16p- TM1 construct (residues
2-41) is shown with a double-headed arrow. Residues mutated
to cysteine in Vma16p are shown as white letters in
black boxes.
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Fig. 2.
Deletion of the first transmembrane region of
Vma16p does not alter V-ATPase function. a,
schematic illustration of the domain structure of Vma3p, Vma11p, and
Vma16p. Putative transmembrane regions are indicated as
boxes, and the positions of the essential glutamate
residues in each subunit are indicated as open circles. The
glutamate residue shown in the shaded circle in TM5 of
Vma16p has been shown not to be essential for function (19).
b, growth phenotype at pH 7.5 of yeast strains
disrupted in the indicated proteolipid genes (host cell) upon
introduction of the indicated full-length genes or the Vma16p- TM1
construct. c, Western blot analysis of vacuoles
isolated from a vma16
strain expressing HA-tagged forms
of Vma16p or the Vma16p-
TM1 construct. Western blotting was
performed using antibodies against the HA epitope, the V1
subunit Vma1p, or the V0 subunit Vph1p as described under
"Experimental Procedures." d,
concanamycin-sensitive ATPase activity (hatched bars) or
ATP-dependent proton transport (open bars) was
measured for vacuoles isolated from the vma16
strain
expressing the HA-tagged forms of Vma16p or Vma16p-
TM1. The specific
activity of the ATPase in the vacuoles isolated from the strain
expressing the full-length HA-tagged Vma16p was 0.49 µmol of
ATP/min/mg of protein at 30 °C and 0.5 mM ATP, which
corresponds to ~70% of the activity measured for the full-length
untagged Vma16p.
TM1 construct in order to determine
quantitatively the effect of removal of TM1 of Vma16p on V-ATPase
function. We first wished to test the stability of the Vma16p-
TM1
protein and its ability to assemble with other V-ATPase subunits. To
accomplish this, an HA epitope tag was inserted at the C-terminal end
of both the full-length Vma16p and the Vma16p-
TM1. This was
necessary because of the lack of available antibodies against the
native Vma16p protein. As can be seen in Fig. 2c, although
Western blots of vacuoles isolated from the strain expressing
Vma16p-
TM1 showed somewhat reduced reactivity with the anti-HA
antibody relative to vacuoles isolated from the strain expressing the
full-length Vma16p, both Vma1p (subunit A of the V1 domain)
and Vph1p (subunit a of the V0 domain) were present at
normal levels on the vacuolar membrane. It has been shown previously
(19, 23) that the absence of any of the proteolipid subunits results in
the failure of V1 to assemble onto the vacuolar membrane
and aberrant assembly and targeting of the V0 domain. These
results thus suggest that TM1 of subunit c" is dispensable for normal
assembly of the V-ATPase complex. The lower level of antibody staining
of the Vma16p-
TM1 construct in isolated vacuoles may reflect partial
proteolytic removal of the HA tag or altered reactivity of the HA
epitope in this construct. Finally, measurement of both
concanamycin-sensitive ATPase activity and ATP-dependent
proton transport (as assessed by quenching of ACMA fluorescence) in
isolated vacuoles (Fig. 2d) indicates that V-ATPase
complexes containing Vma16p-
TM1 have nearly the same activity and
coupling as complexes containing the full-length Vma16p. These results
confirm that TM1 of Vma16p is not necessary for V-ATPase function.
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Fig. 3.
Antibody binding to the C terminus of
Vma16p::HA in intact and detergent-solubilized vacuolar
membrane vesicles. Left lane, vacuolar membrane
vesicles (100 µg of protein) isolated from the vma16
strain expressing the Cys-less form of HA-tagged Vma16p were incubated
with anti-HA antibody, washed to remove unbound antibody, solubilized
with C12E9, and immunoprecipitated with protein
G-Sepharose followed by separation on SDS-PAGE (15% acrylamide gels),
transfer to nitrocellulose, and Western blotting using the anti-HA
antibody as described under "Experimental Procedures." Right
lane, vacuolar membrane vesicles (described above) were first
solubilized with C12E9 followed by addition of
anti-HA antibody, immunoprecipitation, separation by SDS-PAGE, and
Western blotting as described above.
View larger version (57K):
[in a new window]
Fig. 4.
Effect of cysteine substitutions in Vma16p on
assembly and activity of the V-ATPase. a, to assess the
effects of removing endogenous cysteine residues and introduction of
unique cysteine residues in Vma16p on stability of Vma16p and assembly
of the V-ATPase, vacuoles were isolated from the vma16
strain expressing the wild type, HA-tagged Vma16p
(Wild::HA), the vector alone (Vector),
the Cys-less form of Vma16p (Cys-less::HA) or
each of the indicated single cysteine-containing mutants of Vma16p.
SDS-PAGE and Western blot analysis was then performed on 1 µg of
protein (for Vph1p and Vma1p) or 10 µg of protein (for Vma16p) using
antibodies against Vph1p, Vma1p, or HA as described under
"Experimental Procedures." b, concanamycin-sensitive
ATPase activity (hatched bars) or ATP-dependent
proton transport (open bars) was measured for vacuoles
isolated from the vma16
strain expressing the indicated
constructs as described under "Experimental Procedures." Values are
expressed relative to wild type, with each value corresponding to the
average of the three measurements on two or three independent vacuole
preparations (error bars correspond to the S.D.).
View larger version (33K):
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Fig. 5.
Labeling of single cysteine-containing
mutants of Vma16p by MBP and protection by AMS. a,
vacuolar membrane vesicles (100 µg of protein) isolated from the
vma16 strain expressing the Cys-less form of HA-tagged
Vma16p or the indicated single cysteine-containing mutants were
incubated with 250 µM MPB for 15 min at 23 °C. The
membranes were then solubilized with C12E9, and
the V0 complexes were immunoprecipitated with the
monoclonal antibody 10D7 against subunit a and protein A-Sepharose. The
samples were separated by SDS-PAGE and transferred to nitrocellulose,
and the biotinylated bands were identified using horseradish
peroxidase-conjugated NeutrAvidin and the Supersignal Western blotting
system (Pierce). The exposure time is the same for all samples in this
experiment. The asterisks mark the position of Vma16p.
b, vacuolar membrane vesicles isolated from the S5C and
S178C mutants of Vma16p were incubated in the presence or absence of
100 µM AMS for 5 min at 15 °C followed by 5-fold
dilution and labeling with MPB, SDS-PAGE, and Western blot analysis as
described above. Where indicated (ZW+), 0.25% Zwittergent
3-14 was included during treatment with AMS.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
TM1 was not able to substitute
for either subunits c or c', it was able to replace the full-length
subunit c" in supporting both assembly and activity. This result is in
conflict with that reported by Gibson et al. (46) who found
that removal of TM1 of subunit c" led to a vma
phenotype. Although
the basis for this difference in results is not certain, it should be
noted that in their study the mutant and wild type forms of Vma16p were
overexpressed in a vma16-deficient strain using a galactose-inducible
promoter (46). Because replacement of glucose in the medium with
galactose causes partial dissociation of the V-ATPase complex (48), it is possible that the vma
phenotype observed for the strain expressing the Vma16p-
TM1 construct is the result of somewhat greater
dissociation of the V-ATPase complex containing the deleted form of
Vma16p relative to the full-length protein upon glucose substitution. It should also be noted that the region removed in the Gibson et
al. (46) study was from residue Leu-12 to Ser-55 (inclusive), whereas the region removed in the present study extended from residue
Asn-2 to His-41. It is thus possible that removing the residues Gly-42
to Ser-55 (which is outside the region predicted for TM1) accounts for
the observed vma
phenotype. It is in any case clear from the present
study that TM1 of subunit c" is dispensable for both assembly and
activity of the V-ATPase complex.
View larger version (15K):
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Fig. 6.
Proposed membrane topology of Vma16p.
TM1-TM4 in the current model correspond to TM2-TM5 of the
original model and are indicated by open boxes spanning the
membrane, and TM1 (original model) is now shown as a separate domain on
the cytoplasmic side of the membrane. Ser-5, which upon substitution
with cysteine showed a cytoplasmic labeling pattern, is indicated as a
shaded circle, whereas Ser-178, which upon mutation to
cysteine showed a lumenal pattern, is indicated by an open
circle. Amino acid residues which when changed to cysteine were
not reactive with MPB are indicated by solid dots.
Mutagenesis studies (19) indicate that Glu-108 in TM2 (shown as an
open box in the current model) is essential for proton
transport activity, and Glu-188 in TM4 (shaded box) is
not.
These results suggest that the TM1 region of subunit c" does not correspond to an actual membrane spanning segment but instead represents a somewhat hydrophobic domain located on the cytoplasmic side of the membrane. What might the function of this region be? As mentioned above, this region is not well conserved between species, with only 8 of 55 residues identical between the yeast and mouse sequences (21). Because of its location on the cytoplasmic side of the membrane, it is oriented to participate in the interaction between the V1 and V0 domains, although no obvious perturbation in the stability of the V-ATPase complex is observed upon its removal. Alternatively, it may function in the regulated interaction between V1 and V0, which has been shown to play an important role in controlling V-ATPase activity in vivo (48). Additional studies will be required to elucidate the role of this region of subunit c".
The model shown in Fig. 6 for the topology of subunit c" suggests that,
like subunit c (and presumably subunit c'), subunit c" also contains
four transmembrane helices, giving a V0 structure having a
subunit stoichiometry of c4c'1c"1 a
total of 24 transmembrane helices. Because the critical glutamate
residue in subunit c" is present in TM2 (new model) rather than in TM4
of subunit c and c' (19), there is an asymmetry to the V0
proteolipid ring even beyond the presence of three different
proteolipids. Based upon current models of the arrangement of c
subunits in the proteolipid ring of F0 (50), accommodating
such an asymmetric placement of the critical carboxyl in the ring of
proteolipid subunits should not be problematic. It should also be
noted, however, that the orientation of the transmembrane segment
containing the critical glutamate residue (that is the N- to C-terminal
direction) is opposite for subunit c" relative to subunits c and
c' (21). Whether these structural asymmetries are important for the
functional properties of the V0 domain, such as the
inability of native V0 (in contrast to F0) to
conduct protons (51), remains to be determined.
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ACKNOWLEDGEMENT |
---|
E. coli strains were provided by National Institutes of Health Grant DK34928.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM34478 (to M. F.).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 Physiology,
Tufts University School of Medicine, 136 Harrison Ave., Boston, MA
02111. Tel.: 617-636-6939; Fax: 617-636-0445; E-mail:
michael.forgac@tufts.edu.
Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M209875200
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ABBREVIATIONS |
---|
The abbreviations used are:
V-ATPase, vacuolar
proton-translocating adenosine triphosphatase;
F-ATPase, F1F0-ATP synthase;
HA, influenza hemagglutinin;
TM, transmembrane segment;
Vma16p-TM1, the Vma16p protein lacking
putative transmembrane segment 1;
Me2SO, dimethyl
sulfoxide;
MES, 2-(N-morpholino)ethanesulfonic acid;
YEPD, yeast extract peptone dextrose;
MPB, 3-(N-maleimidylpropionyl) biocytin;
AMS, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid.
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