(Received for publication, June 9, 1995)
From the
The Saccharomyces cerevisiae vacuolar
H-ATPase (V-ATPase) is a multi-subunit complex that
can be structurally and functionally divided into peripheral
(V
) and integral membrane (V
) sectors. The vma22-1 mutation was isolated in a screen for mutants
defective in V-ATPase function. vma22
cells contain no
V-ATPase activity due to a failure to assemble the enzyme complex;
V
subunits accumulate in the cytosol, and the V
100-kDa subunit is rapidly degraded. Turnover of the 100-kDa
integral membrane protein was found to occur in the endoplasmic
reticulum (ER) of vma22
cells. The product of the VMA22 gene, Vma22p, is a 21-kDa hydrophilic protein that is
not a subunit of the V-ATPase but rather is associated with ER
membranes. The association of Vma22p with ER membranes was perturbed by
mutations in VMA12, a gene that encodes an ER membrane protein
(Vma12p) that is also required for V-ATPase assembly. These results
indicate that Vma22p, along with Vma21p and Vma12p, form a set of ER
proteins required for V-ATPase assembly.
The acidification of many organelles including the Golgi,
vacuoles, endosomes, and clathrin-coated vesicles is generated via a
vacuolar-type proton-translocating ATPase or V-ATPase. ()The
electrochemical gradient generated by V-ATPases is used in such
cellular processes as protein sorting, receptor-mediated endocytosis,
and zymogen activation (Mellman et al., 1986).
The yeast
V-ATPase is similar in structure and function to V-ATPases from other
fungi, plants, and animals (Uchida et al., 1985; Kane et
al., 1989). All are multi-subunit complexes that, in a manner
similar to the well characterized FF
-ATPase,
can be divided structurally and functionally into V
and
V
sectors. The yeast V-ATPase V
sector consists
of subunits peripherally associated with the membrane and constitutes
the catalytic and regulatory functions of the enzyme complex. The
V
portion of the enzyme contains integral membrane subunits
and forms a pore through which protons are translocated into an
organelle (Kane and Stevens, 1992).
Biochemical and genetic analyses have demonstrated that the yeast V-ATPase complex comprises at least ten polypeptides ranging in molecular mass from 100 to 14 kDa. The genes encoding the 100-kDa (VPH1, Manolson et al.(1992)), 69-kDa (VMA1/TFP1, Shih et al.(1988); Hirata et al.(1990)), 60-kDa (VMA2/VAT2, Nelson et al.(1989); Yamashiro et al.(1990)), 54-kDa (VMA13, Ho et al. (1993b)), 42-kDa (VMA5, Beltran et al.(1992); Ho et al. (1993a)), 36-kDa (VMA6, Bauerle et al. (1993)), 32-kDa (VMA8, Nelson et al.(1995); Graham et al.(1995)), 27-kDa (VMA4, Foury (1990)), 14-kDa (VMA7, Nelson et al.(1994); Graham et al.(1994)), and two hydrophobic polypeptides of 17-kDa (VMA3, Nelson and Nelson(1989); VMA11, Umemoto et al.(1991)) have been cloned and sequenced.
Disruption of genes (vma, vacuolar membrane ATPase)
encoding V-ATPase subunits (with the exception of VPH1) gives
rise to a characteristic set of phenotypes (Vma)
associated with the loss of enzyme function. These include slow growth,
an inability to grow on media buffered to a neutral pH, sensitivity to
high Ca
, and a Pet
phenotype. In
addition, vma ade2 double mutant colonies are white instead of
the red color typical of ade2 colonies. These phenotypes have
been used to design screens for mutations in genes encoding structural
subunits of the V-ATPase complex as well as factors required for the
assembly/stability but which are not subunits of the final enzyme
complex (Ho et al., 1993a; Ohya et al., 1991).
Studies of the assembly of the V-ATPase in cells mutant in either
V or V
subunit genes have indicated that
assembly requires the coordination of integral membrane subunits and
peripheral subunits. Disruption of a gene encoding any single V
peripheral subunit of the V-ATPase complex leads to a failure of
the V
sector to assemble on the vacuolar membrane and the
stable accumulation of remaining V
subunits in the cytosol.
The V
sector of the enzyme, however, is transported to and
stable in the vacuolar membrane in the absence of an assembled V
sector (Doherty and Kane, 1993; Ho et al., 1993a). The
absence of a V
sector subunit leads to the
destabilization/degradation of the remaining V
polypeptides
as well as a failure of V
subunits to associate with the
vacuolar membrane (Kane et al., 1992; Bauerle et al.,
1993).
Recent studies have characterized the VMA12 and VMA21 genes, which are involved in V-ATPase assembly but whose
products (Vma12p and Vma21p) are not subunits of the final vacuolar
enzyme complex (Hirata et al., 1993; Hill and Stevens, 1994).
Vma21p is a membrane protein of the endoplasmic reticulum (ER), and
mutations in VMA21 lead to a rapid degradation of the 100-kDa
V polypeptide. A model has been proposed in which assembly
of the V-ATPase complex is initiated in the ER, and failure to do so
results in the rapid turnover of the 100-kDa integral membrane
polypeptide within the ER (Hill and Stevens, 1994).
In this work, we
describe the characterization of the VMA22 gene and its
product, Vma22p. Similar to Vma21p, Vma22p is a non-subunit assembly
factor that is associated with the ER. Loss of Vma22p function leads to
a rapid and specifically ER-based degradation of the 100-kDa V polypeptide and failure of the V-ATPase complex to assemble.
The
structure of the vma22 disruption in KHY38 and KHY39 was
confirmed by PCR amplification using oligonucleotide AUX1 as described
above and oligonucleotide STHN2 (5`-ATCACATGGTGTTTTATGGG-3`) to amplify
a 1.4-kb DNA fragment specific for the vma22
::URA3 disruption. The vma22
disruption in KHY13 and KHY34
cells was also confirmed by PCR amplification. Oligonucleotides STHN2
as above and oligonucleotide LEU5OUT (5`-CAAGTATTGTGATGCAAGC-3`)
corresponding to sequences 3` to the LEU2 open reading frame
were used to amplify a 380-bp DNA fragment specific for the vma22
::LEU2 disruption.
Figure 1:
Schematic diagram of VMA22 (A) restriction map of the 1-kb SnaBI-SpeI DNA fragment contained within pKH2211. A, blackarrow represents the VMA22 open reading frame, and flanking DNA is represented by a solidline. The vma22::URA3 and vma22
::LEU2 disruption constructs are outlined.
The region of pKH2211 used to generate anti-Vma22p antibodies is also
shown. Restriction endonucleases are B*, BglII (*
indicates this site was introduced by in vitro mutagenesis); S, StyI; SnSnaBI; Sp, SpeI; X, XmnI. B, nucleotide
sequence of VMA22 and predicted amino acid sequence of Vma22p.
The nucleotide sequence of the SnaBI-SpeI fragment
contained in pKH2211 is shown (Genbank accession no. U24501). The
initiation and termination codons of VMA22 are in bold. Vma22p amino acid residues are given in the single
letter code and are numbered in bold. C,
immunoblot analysis to detect Vma22p. Whole cell protein extracts were
prepared from SNY28 (wild type), KHY38 (vma22
::URA3), and
KHY13 (vma22
::LEU2) cells and 15 µg of protein loaded
onto SDS-PAGE gels (15% acrylamide). The resulting immunoblots were
probed with a dilution of affinity-purified anti-Vma22p antiserum and
visualized by chemiluminescent detection. ORF, open reading
frame; WT, wild type.
Whole cell protein extracts and vacuolar membrane fractions were prepared as described (Hill and Stevens, 1994). Vacuolar membrane fractions were extracted with chloroform/methanol as described by Ho et al. (1993b). Monoclonal antibodies recognizing the 100- (10D7, 7B1), 69- (8B1), 60- (13D11), and 42-kDa (7A2) V-ATPase subunits were used as described by Kane et al.(1992). Monoclonal antibodies specific for the 100-, 69-, and 60-kDa V-ATPase subunits and Dpm1p were from Molecular Probes Inc. Polyclonal antibodies directed against the 100-, 54-, and 27-kDa V-ATPase subunits were purified and used as described by Hill and Stevens(1994). Polyclonal antibody directed against phosphoglycerol kinase was used as described by Nothwehr et al.(1995). Monoclonal antibodies specific for the ER membrane protein, Dpm1p, were used as described by Piper et al.(1994). Secondary antibodies conjugated to horseradish peroxidase were purchased from Amersham. Immunoblots were detected using chemiluminescent detection (DuPont NEN).
VMA22 was
cloned by complementation of the pH and color defects of vma22-1 cells. Strain KHY28 was transformed with a
plasmid library (Rose et al., 1987). 1 of 2500 Ura transformants was red, able to grown on media buffered to pH 7.5,
and complementation was plasmid dependent. Plasmid pKH2201 was
recovered and found to contain a yeast genomic DNA insert of 8.5 kb.
Various subclones of pKH2201 (pKH2203, pKH2204, pKH2206, pKH2207,
pKH2208, pKH2209, and pKH2211) demonstrated that a 1-kb SnaBI-SpeI fragment (pKH2211, Fig. 1A) was sufficient to fully complement vma22-1.
The nucleotide sequence of the 1-kb
complementing fragment in pKH2211 was determined for both strands
(Genbank accession number U24501). One long open reading frame (VMA22) of 543 bp was found predicting a protein (Vma22p) of
181 amino acids with a molecular mass of 21 kDa (Fig. 1B). Data base searches showed that VMA22 was identical to the sequence designated YHR060w in the CPR2-ERG7 intergenic region of chromosome VIII
recently identified by the yeast genome project. No significant
homology to other sequences in the data base was found. Rabbit
antiserum generated using an E. coli expressed Vma22p fusion
protein (codons 16-181 of Vma22p) was used to detect Vma22p in
yeast cells. Anti-Vma22p antibodies recognized a single band of 21 kDa
in wild-type cells, and this 21-kDa protein was absent in vma22 strains (KHY38 and KHY13) (Fig. 1C).
Strains carrying a null allele of vma22 (Fig. 1A) were constructed by
transforming SF838-1D and SNY28 with either the vma22
::LEU2 fragment contained within pKH2213 (KHY34 and
KHY13) or the vma22
::URA3 fragment (KHY38 and KHY34). The
structure of the disruption was confirmed by PCR amplification of the
disrupted VMA22 open reading frame using oligonucleotides
complementary to sequences flanking VMA22 (see
``Experimental Procedures''). A strain carrying a marked
allele of VMA22 (VMA22::URA3) was crossed with KHY28 (vma22-1), the diploid sporulated, dissected, and the
tetrad progeny examined. All tetrads examined showed 2 Vma
Ura
:2 Vma
Ura
segregation indicating close linkage and, taken together with
complementation analysis, demonstrating that the cloned gene was VMA22.
To address the basis of the
lack of V-ATPase activity seen in vma22 cells, we
examined the levels of various subunits of the enzyme complex. Western
blot analyses of the V-ATPase subunits in wild-type (SNY28) and vma22
(KHY38) cells are shown in Fig. 2A.
Immunoblots of whole cell protein extracts indicated that the level of
the 100-kDa V-ATPase subunit (Vph1p) was substantially lowered in vma22
cells as compared to wild-type cells. However, the
steady state levels of other subunits (69, 60, 54, 42, 36, and 27 kDa)
appeared comparable between wild-type and vma22
cells.
Figure 2:
Assembly of the V-ATPase in wild-type (WT) and vma22 cells. A, whole cell
protein extracts were prepared from wild-type (SNY28) and vma22
(KHY38) cells. Samples (20 µg) were loaded onto
SDS-PAGE gels, and the resulting immunoblots were probed to detect the
100-, 69-, 60-, 54-, 42-, 36-, and 27-kDa V-ATPase subunits. Whole cell
protein immunoblots were compared with those prepared from 4 µg of
vacuolar membrane extracts from both wild-type and vma22
cells. B, vacuolar membrane fractions from wild-type and vma22
cells were treated with chloroform/methanol (see
``Experimental Procedures'') and loaded onto SDS-PAGE gels;
the V-ATPase 17-kDa proteolipid was detected by silver
staining.
Western blot analyses of vacuolar membrane fractions prepared from
wild-type and vma22 cells indicated that although V
subunits appeared to be produced at wild-type levels in vma22
cells, they failed to associate with the vacuolar
membrane (Fig. 2A). In addition, indirect
immunofluorescence of the 60-kDa subunit of the V
sector in vma22
cells showed a cytosolic pattern instead of the
vacuolar membrane-staining pattern seen in wild-type cells (data not
shown). Immunoblots also indicated that while a low level of the
100-kDa V-ATPase subunit was present in vma22
cells, no
100-kDa polypeptide was present on the vacuolar membrane (Fig. 2A). Chloroform/methanol treatment of vacuolar
membranes has been shown to extract the 17-kDa proteolipid
(Vma3p/Vma11p) component of the V
sector of the V-ATPase,
which can then be detected by SDS-PAGE and silver staining (Ho et
al., 1993b). The 17-kDa proteolipid was substantially reduced in vma22
cells in contrast with the proteolipid extracted
from wild-type vacuolar membranes (Fig. 2B). In
summary, immunoblot results indicate that Vph1p (100 kDa) and
Vma3p/Vma11p (17 kDa), constituents of the V
integral
membrane sector of the V-ATPase enzyme, are destabilized in vma22
cells. Subunits that comprise the V
sector of the enzyme (69, 60, 54, 42, 36, and 27 kDa) appeared to
be present at wild-type levels and were stable in vma22
cells but were unable to associate with the vacuolar membrane.
These results fully accounted for the lack of V-ATPase activity seen in vma22
cells, since this enzyme complex was unassembled.
The low level of the 100-kDa subunit (Vph1p) seen in immunoblots
from vma22 cells led us to investigate further the nature
of this defect. We performed a kinetic analysis of the synthesis and
turnover of Vph1p in wild-type and vma22
cells. We also
investigated the effect of the PEP4 gene status on the
turnover and stability of Vph1p. Pep4p is a critical protease in the
activation cascade of vacuolar hydrolases (Ammerer et al.,
1986; Woolford et al., 1986). Any effect of a mutation in the PEP4 gene (pep4-3) on the half-life of Vph1p
would implicate vacuolar proteases in the turnover of this protein.
Wild-type and vma22 cells were radiolabeled with
methionine and chased by the addition of
non-radiolabeled methionine. At various chase times, samples were
immunoprecipitated with anti-Vph1p antibodies and analyzed by SDS-PAGE
and fluorography. In wild-type cells (SNY28), Vph1p was very stable
with a half-life of
500 min (Fig. 3, lanes1-4). The stability of Vph1p was unaffected by
introduction of the pep4-3 mutation (SF838-1D,
half-life
460 min; Fig. 3, lanes9-12), indicating that vacuolar hydrolases have
little effect on the turnover of Vph1p in wild-type cells. In vma22
cells (KHY13), Vph1p was rapidly degraded with a
half-life of only 36 min (Fig. 3, lanes5-8), and introduction of the pep4-3 mutation into vma22
cells (KHY34) did little to
stabilize the protein (half-life 40 min; Fig. 3, lanes13-16). These results suggested that the deceased
level of Vph1p seen in vma22
cells was due to its rapid
turnover and that this degradation likely occurred outside of the
vacuole.
Figure 3:
Immunoprecipitation of Vph1p from
wild-type and vma22 cells and the effects of PEP4. Wild-type and vma22
cells were
radiolabeled with [
S]methionine for 10 min and
chased for various times prior to immunoprecipitation of Vph1p and
analysis by SDS-PAGE and fluorography. Lanes1-4, Vph1p from SNY28 cells (VMA22 PEP4); lanes5-8, Vph1p from KHY13 cells (vma22
PEP4); lanes9-12, Vph1p
from SF838-1D cells (VMA22 pep4-3); lanes13-16, Vph1p from KHY34 cells (vma22
pep4-3).
To establish where in the secretory pathway Vph1p was being
degraded in vma22 cells, we made use of a
temperature-sensitive sec mutation. At the non-permissive
temperature (36 °C), sec12-4 cells are blocked in
vesicle budding from the ER (Nakano et al., 1988), and in a sec12-4 vma22
double mutant newly synthesized Vph1p
should remain in the ER. If Vph1p is being degraded in a post-ER
compartment in vma22
cells, the protein should be
stabilized in sec12-4 vma22
cells shifted to 36
°C. Alternatively, if Vph1p is being degraded in the ER of vma22
cells, then blocking membrane traffic out of the ER
will not influence its half-life.
Strains carrying sec12-4 (MBY10-7A), vma22 (KHY13), or both sec12-4 and vma22
(KHY9) alleles were
grown at the permissive temperature (22 °C), shifted to 36 °C
for 15 min prior to addition of radiolabel, and subjected to
immunoprecipitation of Vph1p as previously described. Arrest of protein
exit from the ER was monitored by immunoprecipitation of CPY. CPY is
found in the ER as the p1CPY precursor (67 kDa), is modified to form
the p2CPY precursor in the Golgi (69 kDa), and processed to the mature
enzyme (mCPY) in the vacuole (Stevens et al., 1982).
Accumulation of p1CPY served to confirm that protein traffic out of the
ER was blocked in sec12-4 and sec12-4
vma22
cells (Fig. 4C, lanes1-6). Additional controls also ensured that
degradation of Vph1p was not merely the result of higher temperature or
strain background differences (Fig. 4B).
Figure 4:
Immunoprecipitation of Vph1p from
wild-type and vma22 cells and the effects of sec12-4. A, sec12-4 (MBY10-7A), vma22
(KHY13), and vma22
sec12-4 (KHY9) cells were incubated for 15 min at 36 °C
before cells were labeled, and Vph1p was immunoprecipitated at various
times and analyzed by SDS-PAGE and fluorography. Lanes1-5, Vph1p from sec12-4 cells at 36
°C; lanes6-10, Vph1p from vma22
cells at 36 °C; lanes11-15, Vph1p
from sec12-4 vma22
cells at 36 °C. B,
graphic representation of the stability of Vph1p at 36 °C in
various strains. The radioactivity of Vph1p samples visualized by
immunoprecipitation was quantified (AMBIS Radioanalytic Imaging
System); the zero minute chase time quantity was designated as 100% and
the percentage of Vph1p remaining was plotted against time. C,
immunoprecipitation of CPY. At zero and 40 min chase time points in the
above experiment (A), samples were also harvested for
immunoprecipitation with anti-CPY antibodies. Lanes1 and 2, sec12-4 cells at 36 °C; lanes3 and 4, vma22
cells at
36 °C; lanes5 and 6, sec12-4
vma22
cells. p1CPY refers to the ER precursor form of CPY,
p2CPY refers to the Golgi precursor form of CPY, and mCPY refers to the
mature vacuolar form of CPY.
In kinetic
analyses, Vph1p was stable in the ER of sec12-4 cells at
the non-permissive temperature (Fig. 4A, lanes1-5). The half-life of Vph1p in sec12-4 cells (>400 min, Table 3) was similar to that found in
wild-type cells (half-life >400 min; Table 3). In contrast, vma22 cells showed a rapid turnover of Vph1p at the
non-permissive temperature (Fig. 4A, lanes6-10), with a half-life of
23 min (Table 3). The rapid degradation of Vph1p seen in vma22
cells was not stabilized by introduction of the sec12-4 temperature-sensitive allele (Fig. 4A, lanes11-15), with the half-life of Vph1p in these double
mutant cells still only
30 min. Together, these data clearly
indicate that the degradation of Vph1p in vma22
cells is
occurring in the ER compartment of the secretory pathway.
We had
previously determined that Vph1p was rapidly degraded in another vma mutant, vma21 (Hill and Stevens, 1994). We have
also found that the degradation of Vph1p in vma21 cells
(Hill and Stevens, 1994) occurs in the ER. KHY22 cells (vma21
, half-life
28 min) and KHY21 cells (vma21
sec12-4, half-life
20 min) showed
approximately the same rate of degradation of Vph1p at the restrictive
temperature (Fig. 4B and Table 3).
Figure 5:
Localization of Pma1p and processing of
alkaline phosphatase in wild-type and vma22 cells. A, Wild-type (SNY28) and vma22
(KHY38) cells
were fixed, spheroplasted, and stained with anti-Pma1p antibodies.
Cells were viewed by Nomarski optics to observe cell morphology and
epifluorescence microscopy using filter sets specific for DAPI to
observe nuclear staining and fluorescein to observe anti-Pma1p
staining. B, Wild-type (SNY28) and vma22
(KHY38)
cells were radiolabeled with [
S]methionine for
10 min and chased for 60 min before alkaline phosphatase was
immunoprecipitated and analyzed by SDS-PAGE and fluorography. pALP
refers to the precursor form of alkaline phosphatase, and mALP refers
to the mature vacuolar form of alkaline
phosphatase.
Figure 6: Immunolocalization of Vma22p. Wild-type cells (SNY28) carrying pKH2211 were fixed, spheroplasted, and stained with anti-Vma22p antibodies. Cells were viewed with Nomarski optics to observe cell morphology and epifluorescence microscopy using filter sets specific for DAPI to observe nuclear staining and fluorescein to observe anti-Vma22p staining.
Association of Vma22p with the ER membrane might
result from an interaction between Vma22p and an ER membrane protein.
Two intriguing candidate proteins are Vma12p and Vma21p. Yeast cells
lacking either of these two proteins (vma21 (Hill and Stevens,
1994) or vma12()) have been found to rapidly
degrade Vph1p. Vma21p (Hill and Stevens, 1994) and Vma12p (Hirata et al., 1993)
are ER membrane proteins necessary
for V-ATPase assembly and activity. We investigated whether the
subcellular fractionation profile of Vma22p could be altered by
mutations in either the VMA21 or VMA12 gene. SNY28
(wild-type), KHY4 (vma21
), and DJY63 (vma12
) cells were spheroplasted and lysed, and the
lysates were subjected to differential centrifugation to generate
lysate, P13, S100, and P100 fractions. Horazdovsky and Emr(1993) have
demonstrated that fractions generated by this procedure separate
soluble cytosolic proteins (S100) from Golgi (P100) and plasma
membrane/ER/vacuolar (P13) proteins. Lysate, P13, S100, and P100
fractions were separated by SDS-PAGE, and immunoblots were probed to
detect phosphoglycerol kinase, a soluble cytosolic protein, Dpm1p, an
ER transmembrane protein (Orlean et al., 1988), and Vma22p. In
wild-type cells, Vma22p was detected in the P13 membrane fraction (Fig. 7), consistent with an ER localization as shown by
indirect immunofluorescence experiments. While the P13 fraction
contains both ER and vacuolar membranes, additional experiments with
purified vacuolar membranes revealed that Vma22p is not associated with
this organelle (data not shown). As expected, phosphoglycerol kinase
and Dpm1p fractionated with the cytosol and P13 membranes,
respectively, in these preparations. The fractionation profile of
Vma22p was unaltered in vma21
cells (data not shown).
However, when fractions prepared from vma12
cells were
probed with anti-Vma22p antibodies, Vma22p was now detected in the S100
fraction with little detectable signal in the P13 membrane fraction.
The fractionation of phosphoglycerol kinase and Dpm1p in vma12
cells was identical to that seen in wild-type cells. These results
suggest that the association of Vma22p with the ER membrane is mediated
through an interaction with another ER membrane protein, Vma12p.
Figure 7:
Membrane association and subcellular
fractionation of Vma22p. Wild-type (SNY28) and vma12 (DJY63) cells were fractionated into lysate, P13 (vacuolar, ER,
and plasma membrane), S100 (cytosolic), and P100 (Golgi and endosomes)
fractions. Equal proportions of each fraction were loaded on SDS-PAGE
gels, and the resulting immunoblots were probed to detect Vma22p, the
cytosolic protein phosphoglycerol kinase (PGK), and the ER
membrane protein, Dpm1p.
In this work, we report the isolation and characterization of
the yeast VMA22 gene, whose product Vma22p is required for the
expression of a functional V-ATPase enzyme. Yeast cells lacking Vma22p
display phenotypes that are characteristic of other vma mutants including pH and Ca-sensitive growth.
Vacuolar membranes from vma22
cells contain no V-ATPase
activity, and correspondingly their vacuoles are not acidified.
The
specific defect in vma22 cells appears to be at the level
of assembly of the V-ATPase complex. Peripherally associated V
subunits were stable in vma22
cells but were unable
to associate with the vacuolar membrane and instead accumulated in the
cytosol. V
subunits appeared to be destabilized in vma22
cells, and the 100-kDa subunit, Vph1p, was not
found on the vacuolar membrane. The small amount of proteolipid found
in chloroform/methanol extracts from vma22
vacuolar
membranes could reflect a stable but unassembled vacuolar portion of
the V
sector or merely represent the contamination of
vacuolar membrane preparations with membranes from other organelles.
The low level of Vph1p in vma22 cells was the result
of destabilization of the protein. In kinetic analyses, Vph1p was
rapidly degraded in vma22
cells with a half-life of
40 min, approximately 8% of that observed in wild-type cells.
Degradation of Vph1p was independent of the vacuolar hydrolase, Pep4p,
indicating that the increased turnover of Vph1p seen in vma22
cells was likely occurring in an organelle other than the vacuole.
To define the subcellular organelle in which Vph1p was degraded in vma22 cells, we performed our kinetic analyses in sec12-4 vma22
double mutant cells, which accumulate
the ER compartment at the non-permissive temperature. The continued
degradation of Vph1p in these cells at the permissive or non-permissive
temperature showed clearly that degradation of Vph1p occurred in the
ER. We also confirmed that the rapid turnover of Vph1p seen in vma21
cells (Hill and Stevens, 1994) also occurred
specifically in the ER.
The fate of the proteolipid component of the
V-ATPase V sector in vma22
cells remains
uncertain due to a lack of antibodies to perform a similar kinetic
analysis as done here for Vph1p. At present, we are constructing
epitope-tagged versions of Vma3p and Vma11p to address these questions
and also determine the location of the small amount of proteolipid
present in vma22
cells.
Other membrane proteins of the
secretory pathway appeared to be unaffected by the vma22 mutation. Pma1p was present at the plasma membrane of both
wild-type and vma22
cells, as revealed by
immunofluorescence experiments. Also, the vacuolar membrane protein,
alkaline phosphatase, was transported to and matured in the vacuole
similarly in wild-type and vma22
cells. These results
suggest that the vma22
mutation may be specific in its
effects on the assembly of the V-ATPase complex.
Although vma22 cells displayed phenotypes typical of other vma mutations, including those in subunit structural genes, Vma22p was
found not to be a subunit of the V-ATPase complex. Vma22p was
de-enriched in vacuolar membrane fractions and did not co-purify with
the V-ATPase. The immunofluorescent labeling pattern of Vma22p showed
both an ER and some cytosolic localization. The ER membrane
localization of Vma22p is similar to that for Vma21p (Hill and Stevens,
1994) and is suggestive of an ER-based set of proteins required for
V-ATPase expression. Although Vma22p is predicted to be a hydrophilic
protein, both indirect immunofluorescence and subcellular fractionation
experiments indicate that Vma22p is associated with the ER membrane.
The effects of the vma22 mutation on the V-ATPase
complex described here are similar to those reported for vma21
(Hill and Stevens, 1994) and vma12
(Hirata et
al., 1993)
mutants. The 100-kDa polypeptide is rapidly
degraded in vma21
cells, and in this work we have shown
that, as in vma22
cells, Vph1p turnover occurs in the ER.
Like Vma22p, Vma21p is also an ER protein, and its ER localization is
determined by a C-terminal di-lysine motif (Hill and Stevens, 1994).
Vma12p, a 25-kDa membrane protein, also resides in the ER. (
)In vma12
cells, Vma22p did not fractionate
with membranes as in wild-type cells but rather with soluble cytosolic
proteins such as phosphoglycerol kinase. It therefore appears likely
that ER membrane association of Vma22p is mediated through an
interaction with Vma12p. Although we did not find any perturbation of
Vma22p localization in vma21
cells, it will be necessary
to extend our analysis of potential interactions between all
combinations of Vma21p, Vma12p, and Vma22p. Our results suggest that
this set of proteins is necessary for the assembly of the V
sector of the V-ATPase complex in the ER. In the future, we will
attempt to establish whether Vma21p/Vma22p/Vma12p exist as a protein
complex and also if there are other proteins associated with this
complex. Preliminary cross-linking experiments indicate that at least
Vma22p and Vma12p physically interact. (
)Because we have a
large number of other vma mutants to screen for phenotypes
similar to those of vma21/vma22/vma12, we may yet identify
additional proteins required for V-ATPase assembly.
What could the
function be of Vma22p/Vma12p/Vma21p in V-ATPase assembly? We have not
ruled out a role for these proteins in the correct insertion of the
100-kDa integral membrane protein into the ER. As yet we have no data
relating to the topology of the V sector polypeptides in
either wild-type or vma mutant cells. While it is too early to
be confident, we favor an alternative model in which the
Vma22/Vma21/Vma12 proteins act as chaperones, allowing the proper
assembly of the V
subunits after their insertion into the
ER membrane.
The requirement for additional ER proteins for folding, insertion into the ER, or oligomerization has been observed for membrane proteins other that the 100-kDa V-ATPase subunit. Shr3p, an ER protein, is required for the exit of amino acid permeases from the ER in yeast (Ljungdahl et al., 1992). Shr3p is proposed to act either in a chaperone-like role in the folding of permeases or possibly as a permease-specific component of the ER translocation machinery. In Drosophila, mutations in the cyclophilin homologue ninaA result in a failure to transport two related opsin proteins from the ER of photoreceptor cells (Colley et al., 1991). Finally, the influenza virus HA membrane protein requires BiP for trimerization (Gething et al., 1986), and this oligomerization is required for exit from the ER (Copeland et al., 1988).
In summary,
we have identified a protein, Vma22p, which is required for the ER
assembly of the V sector of the V-ATPase complex. Failure
to assemble the V
sector leads to rapid degradation of the
100-kDa polypeptide in the ER and failure to assemble the V-ATPase
enzyme complex. Vma22p, along with Vma21p and Vma12p, may form an ER
assembly complex specific for the V-ATPase enzyme.