From the Department of Biochemistry and Molecular Biology, State University of New York Health Science Center, Syracuse, New York 13210
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ABSTRACT |
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Vacuolar proton-translocating ATPases are
composed of a complex of integral membrane proteins, the
Vo sector, attached to a complex of peripheral
membrane proteins, the V1 sector. We have examined the
early steps in biosynthesis of the yeast vacuolar ATPase by
biosynthetically labeling wild-type and mutant cells for varied pulse
and chase times and immunoprecipitating fully and partially assembled
complexes under nondenaturing conditions. In wild-type cells, several
V1 subunits and the 100-kDa Vo subunit associate within 3-5 min, followed by addition of other Vo
subunits with time. Deletion mutants lacking single subunits of the
enzyme show a variety of partial complexes, including both complexes that resemble intermediates in the assembly pathway of wild-type cells
and independent V1 and Vo sectors that form
without any apparent V1Vo subunit interaction.
Two yeast sec mutants that show a temperature-conditional
block in export from the endoplasmic reticulum accumulate a complex
containing several V1 subunits and the 100-kDa
Vo subunit during incubation at elevated temperature. This
complex can assemble with the 17-kDa Vo subunit when the temperature block is reversed. We propose that assembly of the yeast
V-ATPase can occur by two different pathways: a concerted assembly
pathway involving early interactions between V1 and
Vo subunits and an independent assembly pathway requiring
full assembly of V1 and Vo sectors before
combination of the two sectors. The data suggest that in wild-type
cells, assembly occurs predominantly by the concerted assembly pathway,
and V-ATPase complexes acquire the full complement of Vo
subunits during or after exit from the endoplasmic reticulum.
Vacuolar proton-translocating ATPases
(V-ATPases)1 are highly
conserved proton pumps found in all eukaryotic cells (reviewed in Ref.
1). V-type ATPases couple hydrolysis of cytoplasmic ATP to transport of
protons from the cytosol into internal organelles or, in certain cells,
across the plasma membrane. The catalytic sites for ATP hydrolysis
reside in a peripheral complex of subunits called the V1
sector of the enzyme, and the proton pore appears to be contained
within a complex of integral membrane and tightly bound peripheral
subunits called the Vo sector. V-ATPases have been
implicated in constitutive physiological processes ranging from protein
sorting to pH and calcium homeostasis to activation of lysosomal
proteases (reviewed in Refs. 1-3).
The yeast V-type ATPase is composed of at least 13 different subunits,
which have been identified by a combination of genetic and biochemical
techniques (1, 4, 5). The V1 sector of the yeast vacuolar
ATPase is composed of a 69-kDa catalytic subunit, a 60-kDa subunit that
appears to play a regulatory role, and six other peripheral subunits of
relative molecular masses 54, 42, 32, 27, 14, and 13 kDa. The
Vo sector of the yeast enzyme consists of a 100-kDa
integral membrane subunit, a tightly associated peripheral subunit of
36 kDa, and a trio of proteolipid subunits of 23, 17, and 16 kDa. The
genes for all of these subunits have been cloned, and the subunit size,
letter designation (for comparison to V-ATPases from other organisms),
and gene names are listed in Table I. Deletion mutants lacking each of these subunits have been constructed. Deletion of any of these genes, except for the individual
STV1 and VPH1 genes (6), results in a well
defined set of Vma
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
growth phenotypes, including failure
to grow in medium containing high concentrations of calcium, pH
conditional growth (the cells grow at pH 5, but fail to grow in medium
buffered to above pH 7), and failure to grow on nonfermentable carbon
sources (7, 8). The STV1 and VPH1 genes appear to
encode functionally similar 100-kDa subunits present in different
cellular locations; deletion of both genes is necessary to generate a
full Vma
phenotype (6).
Subunit composition of the yeast vacuolar H+-ATPase
The assembly of V-ATPases presents a fascinating set of problems. These enzymes are composed of a combination of membrane proteins that are believed to traverse at least portions of the secretory pathway and peripheral proteins that appear to be synthesized as cytoplasmic proteins and never enter the secretory pathway. V-ATPases reside in multiple organelles of eukaryotic cells, but correct targeting of these complexes is critical because functional assembly of a V-ATPase in an inappropriate location could fundamentally change the characteristics of that organelle by changing its internal pH. In addition, the assembly state of V-ATPases has been shown to be modified in response to extracellular conditions (9-11), indicating that V-ATPases are dynamic structures and assembly may be linked to regulation of enzyme activity.
The physical requirements for enzyme assembly have been probed by a
number of different methods. The V1 sector of the bovine clathrin-coated vesicle enzyme has been assembled from individual subunits derived from either heterologous expression systems or biochemical purification (12). The V1, Vo, and
V1Vo complexes of the bovine enzyme have been
reassembled from partial complexes obtained by dissociation of the
enzyme in vitro (13-16). The yeast V-ATPase has also been
reassembled from partial complexes in vitro (17, 18).
Partial complexes formed in yeast mutants lacking single subunits of
the enzyme have provided information about the extent of assembly
possible in the absence of individual subunits, and results obtained
with the yeast deletion mutants indicate that the V1 and
Vo sectors can assemble independently in vivo (19). Although these assembly studies provide important information about subunit interactions, none of them directly addresses how V-ATPase complexes are formed biosynthetically. Myers and Forgac (20)
examined the initial stages of biosynthesis of the V-ATPase in bovine
kidney cells by performing pulse-chase studies followed by
immunoprecipitation of V-ATPase complexes from soluble and membrane
fractions using an antibody against the catalytic subunit. They
observed parallel synthesis of soluble V1 sectors and
membrane-bound V1Vo sectors at early times but
did not see recruitment of the soluble V1 sectors to newly
synthesized Vo sectors over time. In this work, we have
addressed the early steps of assembly of the yeast V-ATPase both in
wild-type cells containing a full complement of subunits and in mutant
cells lacking a single subunit. Our results suggest that assembly of
the V-ATPase may occur by two pathways, one involving very early
associations between V1 and Vo subunits and a
second involving independent synthesis of V1 and
Vo sectors. We also provide preliminary evidence suggesting that full assembly of the V-ATPase is completed during or after exit of
the membrane subunits from the endoplasmic reticulum (ER).
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EXPERIMENTAL PROCEDURES |
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Materials-- Zymolyase 100T and Tran[35S] label were purchased from ICN. Dithiobis(succinimidyl propionate) was obtained from Pierce. Molecular mass markers (high range) were obtained from Life Technologies, Inc. Restriction enzymes and other enzymes for molecular biology were purchased from New England Biolabs or Roche Molecular Biochemicals. The TA cloning system and pCRII plasmid were obtained from Invitrogen. Oligonucleotides were purchased from Genosys. All other reagents were purchased from Sigma.
Strain and Plasmid Constructions--
Yeast strains used in this
study and their genotypes are listed in Table
II. When possible, isogenic mutant and
wild-type strains were compared in the immunoprecipitations. The
SF838-5A and SF838-1D
wild-type strains are closely related and
were shown to give virtually identical results in immunoprecipitations.
The vma6
mutant strain was constructed by first
amplifying the VMA6 gene from yeast genomic DNA by
PCR using oligonucleotides 5'-CGGAAGCGCACGCTAGAAGG-3' and
5'-CGCTGCAATAGCCACCTAC-3' and Taq polymerase and inserting into the pCRII vector as directed by the manufacturer. Nucleotides
330 to +966, relative to the VMA6 open reading frame, were
then replaced with the LEU2 marker by cleaving the
VMA6-containing plasmid gene with SspI and
ligating to an HpaI fragment containing the entire
LEU2 gene. The disrupted vma6
::LEU2
fragment was released from the vector by digestion with
XbaI and BamHI. The
vma6
::LEU2 mutant strain was generated by the
one-step gene replacement technique (21). Wild-type yeast cells were
transformed with the linear vma6
::LEU2 fragment
by the lithium acetate method (22), and transformants, initially
identified by growth on minimal medium lacking leucine, were further
screened for pH-sensitive growth to identify vma mutants.
Disruption of the VMA6 gene was confirmed by PCR using
genomic DNA from strains exhibiting a Vma
phenotype amd
the same oligonucleotides used for the original amplification of the
VMA6 gene. Construction of the vma7
and vma10
strains was performed by similar methods. The
VMA7 gene was amplified using oligonucleotides
5'-GGATCCGTAATACCGTTGCCT-3' and 5'-CGAGGCCGTGTCACGTTCC-3'. The
URA3 gene was used to replace nucleotides 19-360 of the
open reading frame by fusion PCR, cloned into the pCRII vector, and the
disrupted fragment was released from the pCRII vector by digestion with
BamHI and XhoI. The VMA10 gene was
amplified using oligonucleotides 5'-AGCCTTGTAATGCCTATCAG-3' and
5'-GATAGTTGTAGTCCCTCTGG-3'. The URA3 gene was used to
replace nucleotides 2-336 of the open reading frame by fusion PCR, and the disrupted fragment was amplified and used directly for
transformation. Disruption of the VMA7 and VMA10
genes in yeast was confirmed by PCR using genomic DNA as a
template. For generation of the vma13
and
vma12
strains, the VMA13 and VMA12
genes in plasmids pNUVA4540 and pNUVA410, respectively (a generous gift
from Yasuhiro Anraku and Ryogo Hirata, University of Tokyo) were
transferred to pBluescript KS+. The 1085-base pair BglII
fragment of VMA13 was replaced with an HpaI
fragment containing the LEU2 gene to form plasmid pYO6. A
linear fragment containing the disrupted VMA13 gene was
released by digestion with BamHI and SacII. The VMA12 gene was disrupted by digestion with EcoRI,
treatment of the resulting fragment with T4 DNA polymerase to generate
blunt ends, and ligation with an HpaI fragment containing
the LEU2 gene to form plasmid pYO4. The
vma12
::LEU2 disruption fragment was released by
digestion with XhoI and SacII. Absence of the
VMA13- and VMA12-encoded subunits in the deletion
strains was confirmed by Western blot. A single Myc epitope tag was
added to the C terminus of the VMA3 gene, immediately before
the stop codon, by fusion PCR. Oligonucleotides
5'-TCTTCAGAAATAAGCTTTTGTTCACAGACAACATCTTGAGTAGC-3' and
5'-AGCTTATTTCTGAAGAAGACTTGTAAGGCAGCTTCTGAATCACT were used for
incorporation of the Myc tag, and oligonucleotides
5'-GCAACAATAACACAGATCGCA-3' and 5'-TAGAAGTTGATAAGGTTGGG-3' were
used to amplify the VMA10 gene. All molecular biology manipulations
were carried out as described (23).
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Media for growth of yeast strains was prepared as described by Sherman et al. (24). For immunoprecipitations, yeast strains were grown overnight in supplemented minimal medium lacking methionine that contained 2% dextrose. sec mutants were maintained at 25 °C.
Immunoprecipitations--
Immunoprecipitations were carried out
as described (9). For pulse-chase studies, Tran[35S]
label was added for the indicated pulse time, and the chase was
initiated by addition of unlabeled methionine and cysteine to a final
concentration of 0.33 mg/ml each. For experiments with the
sec mutants, cells were maintained and converted to
spheroplasts at 25 °C. Spheroplasts from the sec1-1
strain were preincubated at 25 or 38 °C for 5 min before addition of
Tran[35S] label and then labeled for 60 min at the same
temperature. sec18-1 spheroplasts were preincubated for 5 min at 25 or 37 °C, labeled for 30 min at the indicated temperature,
and then chased in the presence of excess methionine and cysteine.
sec12-4 spheroplasts were preincubated for 5 min at 25 or
35 °C and then labeled for 30 min at the same temperature and
chased. Immunoprecipitations were carried out using either 5 µl of
purified 8B1 monoclonal antibody, 250 µl of 8B1 cultured supernatant,
or 250 µl of 13D11 cultured supernatant, each brought to a final
volume of 500 µl with phosphate-buffered saline containing 5% bovine
serum albumin, or 500 µl of 10D7 cultured supernatant.
Carboxypeptidase Y (CPY) and the Myc epitope-tagged VMA3
were immunoprecipitated from labeled spheroplasts under denaturing
conditions as described (47) using polyclonal antisera against CPY
(provided by Dr. Tom Stevens) and anti-Myc monoclonal antibody 9E10
(Santa Cruz Biotechnology), respectively. Immunoprecipitated samples
were analyzed by SDS-polyacrylamide gel electrophoresis and
autoradiography as described (19).
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RESULTS |
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Kinetics of Assembly of the Wild-type Yeast V-ATPase--
We first
addressed the early steps in assembly of the yeast V-ATPase by
immunoprecipitating partial complexes of the ATPase following a brief
pulse with Tran[35S] label and varied times of chase in
the presence of excess unlabeled methionine and cysteine. Wild-type
yeast cells were converted to spheroplasts, biosynthetically labeled
for 3 min, and then solubilized under nondenaturing conditions in the
presence of cross-linker as described previously (9). Partially or
fully assembled complexes of the ATPase were then immunoprecipitated with monoclonal antibodies 8B1, 13D11, or 10D7, which recognize the
69-, 60-, and 100-kDa subunits, respectively (19). Previous experiments
have demonstrated that the 8B1 and 13D11 monoclonal antibodies can
recognize the individual subunits, partially assembled complexes, and
the fully assembled V-ATPase (19). In contrast, the 10D7 monoclonal
antibody recognizes a cryptic epitope that is exposed on the 100-kDa
subunit alone or assembled as part of Vo complexes but is
hidden when V1 subunits are bound to the Vo subunits (25). The results of the immunoprecipitations with monoclonal
antibodies 8B1 and 13D11 are shown in Fig.
1. The amount of 69-kDa subunit and the
amount of 60-kDa subunit immunoprecipitated by each of the specific
antibodies appeared to be fairly constant during the 2-90-min chase
times. During the chase time, however, the collection of V-ATPase
subunits co-precipitated by the antibodies changes as subunits labeled
during the 3-min pulse were incorporated into complexes with the 69-kDa
(Fig. 1A) and 60-kDa (Fig. 1B) subunits.
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Several features of the results shown in Fig. 1 are notable. First, labeled V1 and certain Vo subunits, specifically the 100-kDa subunit, are co-precipitated to a large extent at very early times of chase, whereas the 17-kDa subunit, another Vo subunit, appears at later chase times. (The 100-kDa subunit appears as a doublet in these immunoprecipitations. We have observed this frequently, and we believe that both bands represent the 100-kDa subunit because both are also immunoprecipitated by the anti-100-kDa antibody and are missing in Vo mutants lacking the 100-kDa subunit.) This result suggests that the V1 and Vo sectors may not be assembling independently and that assembly of the V1 sector does not necessarily precede attachment of the V1 sector to portions of the Vo sector. Second, certain bands, specifically the band at approximately 19 kDa, are present in complexes immunoprecipitated at very early times and actually disappear as the chase time increases.
We used the 10D7 monoclonal antibody in immunoprecipitations to monitor
the assembly state of the free Vo sector of the yeast vacuolar H+-ATPase, and the results are shown in Fig.
2. The 17- and 36-kDa subunits were
co-precipitated with the 100-kDa subunit with increasing chase time,
and a protein of approximately 19 kDa again appeared at the earliest
chase times and then disappeared, as shown in Fig. 1. A number of other
proteins are also co-precipitated by this antibody, but at present, we
have focused on the subunits that are found as part of the intact
V-ATPase or also co-precipitated by the anti-V1 subunit
antibodies. The 10D7 antibody has been used in conjunction with the
anti-V1 subunit antibodies to reveal the reversible
disassembly of the yeast V-ATPase in response to changes in carbon
source. In these experiments, assembled Vo sectors were
co-precipitated by the antibody under conditions where the V1 and Vo sectors were disassembled, but the
Vo sectors disappeared into the intact complex under
conditions where the enzyme was fully assembled (9, 48). Significantly,
we saw no disappearance of the Vo sectors
immunoprecipitated by the 10D7 antibody during the 90-min chase time,
suggesting that these Vo sectors are not being chased into
V1Vo complexes, but instead are forming an
independent pool of Vo sectors.
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The data in Fig. 2A suggest that the 17- and 36-kDa subunits may appear at somewhat earlier chase times in the Vo complexes immunoprecipitated by the 10D7 antibody than in the V1Vo complexes immunoprecipitated by the 8B1 and 13D11 antibodies. We addressed this question directly for the 17-kDa subunit by comparing complexes immunoprecipitated from identical quantities of wild-type cells with the three antibodies after a 5-min pulse and 0- and 5-min chases. As shown in Fig. 2B, the anti-V1 subunit antibodies coprecipitated the 69-, 60-, 32-, and 27-kDa V1 subunits, the 100-kDa Vo subunit, and the 19-kDa protein after a 5-min pulse. The 17-kDa subunit appeared only after the subsequent 5-min chase. In contrast, the 10D7 antibody coprecipitated the 17-kDa subunit with the 100-kDa subunit at both chase times. These results indicate that the labeled 17-kDa subunit is assembling more rapidly with the free Vo sectors immunoprecipitated by the 10D7 antibody than with the V1Vo complexes. Equally important, these results emphasize that the Vo subunits present in V1Vo complexes and the Vo subunits in "free Vo sectors" immunoprecipitated by the 10D7 antibody are distinct subpopulations; the free Vo complexes do not necessarily arise by disassembly of the V1Vo complexes during isolation.
In order to have a second probe of Vo subunit assembly, we
constructed an epitope-tagged version of the VMA3 gene. This
gene encodes the most abundant of three proteolipid subunits that are part of the yeast vacuolar H+-ATPase (45). Placement of the
Myc epitope tag just before the stop codon yielded a
Myc-VMA3 construct that was able to fully complement the
growth defects of a vma3 mutant strain (data not shown).
We were also able to immunoprecipitate a protein of slightly greater
than 17 kDa, reflecting the size of the tag, using the Myc antibody
under denaturing conditions (Fig. 2C). The Myc antibody did
not immunoprecipitate this protein or any other V-ATPase subunits under
nondenaturing conditions, however. This limited the utility of this
construct for assembly studies, but it did allow us to identify
unambiguously the 17-kDa band seen in the nondenaturing immunoprecipitations as the VMA3 gene product. As shown in
Fig. 2C, a protein of the same size as the
Myc-VMA3 immunoprecipitated by the anti-Myc antibody under
denaturing conditions was coprecipitated by both anti-V1
and anti-Vo subunit antibodies under nondenaturing conditions, and the 17-kDa band disappeared under these conditions.
Assembly of Partial Complexes in Mutants Lacking One
Subunit--
We previously examined the extent of assembly of the
yeast vacuolar H+-ATPase in a number of mutants lacking one
subunit of the enzyme (19). The results showed that the V1
and Vo sectors could assemble separately, leading us to
speculate that assembly of the enzyme in wild-type cells might occur by
combination of preassembled V1 and Vo sectors.
At the time those experiments were done, a rather limited collection of
deletion mutants was available, and the immunoprecipitations were
performed in the absence of cross-linker, so unstable complexes
containing both V1 and Vo subunits may not have
been identified. In light of the kinetic data shown in Figs. 1 and 2,
we examined whether the deletion mutants might contain partial
V1Vo complexes that were not detected
previously. The extent of assembly was measured in a more complete set
of deletion mutants using the conditions of the experiments shown in
Fig. 1, including cross-linker to stabilize weakly bound complexes. The
results of immunoprecipitation following a 60-min pulse with Tran[35S] label and no chase are shown in Fig.
3. Panels A and B
of Fig. 3 show the complexes immunoprecipitated by the 8B1 (anti-69-kDa subunit) and 13D11 (anti-60-kDa subunit) antibodies, respectively. Assembled V-ATPase complexes, containing both V1 and
Vo sector subunits, were immunoprecipitated by both
antibodies from wild-type cells after the 60-min pulse. The
vph1stv1
, vma3
, vma6
, vma7
, and
vma12
mutants appear to contain core V1
complexes, containing minimally the 69-, 60-, 32-, and 27-kDa
subunits, without attached Vo subunits. The
vma10
mutant, which lacks the 13-kDa V1
subunit and as a result has lowered levels of the 27-kDa subunit (27), contains a partial complex consisting of the 100-, 69-, 60-, and possibly the 32-kDa subunits (the 32-kDa subunit appears to be coprecipitated only by the 8B1 antibody). The vma8
mutant, which lacks the 32-kDa V1 subunit, may contain a
similar complex containing the 27-kDa subunit (again immunoprecipitated
by only one of the anti-V1 subunit antibodies) instead of
the 32-kDa subunit. The vma4
mutant, which lacks the
27-kDa V1 subunit, contains an even smaller complex that
appears to include the 69- and 60-kDa subunits but lacks the 100- and
32-kDa subunits. The vma5
and vma13
mutants, which lack the 42- and 54-kDa V1 subunits,
respectively, show some level of assembly of V1 with all of
the Vo subunits, as indicated by co-precipitation of the
100-, 36-, and 17-kDa subunits with the V1 subunits. These
complexes may be rather unstable because the Vo subunits
appear to be at low levels in the immunoprecipitations from
vma13
cells, and only the 13D11 antibody could
co-precipitate the Vo subunits from vma5
cells. These results are consistent with previous experiments (18, 19,
26, 27) in that a number of the mutants can assemble a core
V1 complex, containing at least the 69-, 60-, 32-, and
27-kDa V1 subunits. However, they also indicate a range of
potential partial complexes and assembly intermediates that was not
appreciated earlier.
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Vo complexes were immunoprecipitated by the 10D7 antibody
from the same set of mutants (data not shown). The results were entirely consistent with previous results. Wild-type cells and all of
the mutants lacking V1 subunits, except the
vma7 mutant, clearly assembled free Vo
complexes, as indicated by coprecipitation of the 36- and 17-kDa
subunits with the 100-kDa subunit. All of the mutants lacking a
Vo subunit abolished Vo sector assembly, as did
the vma7
and vma12
mutations. Vma12p is an
assembly factor for the Vo subunits (28, 29), and Vma7p is
unique among the V1 subunits because its absence has been
shown to prevent localization and assembly of the Vo
subunits at the vacuole (30, 31). Taken together, these results
indicate that V1 and Vo sectors can assemble independently in mutants lacking one subunit, even though assembly does
not appear to occur via independent assembly of the two sectors when
all of the subunits are present (Figs. 1 and 2).
Figs. 1 and 2 show interactions between V1 and
Vo sector subunits at very early time points. We reasoned
that some of the mutant strains might form similar complexes at early
stages of assembly but later dissociate these complexes because absence of a subunit prevents assembly from proceeding to formation of a
stable, fully assembled complex. This question was addressed by
examining the complexes formed in the deletion mutants under conditions
similar to those used for wild-type cells in Fig. 2B (5-min
pulse and a 0- or 5-min chase). The results of immunoprecipitation with
the 13D11 antibody are shown in Fig. 4.
There is evidence of assembly of the V1 subunits in almost
all of the mutant strains, and the vma4, vma5
, vma8
,
vma10
, and even the vma1
mutants show some level
of co-precipitation of the 100-kDa Vo subunit. The
vma5
, vma8
, and vma10
mutants show some
assembly of the V1 and Vo sectors after a
60-min pulse, so it was not surprising that some interactions could be
detected at earlier times. The vma4
mutant did not show
interactions between V1 and Vo sectors after a
60-min pulse (Fig. 3), but did appear to show some interaction at these
early times of assembly. The 100-kDa subunit was present at levels in
the complexes from vma4
cells comparable to wild-type after the 5-min chase, and the 17-kDa subunit was also visible at this
time. These results suggest that the vma4
mutant starts to assemble the V-ATPase by a pathway similar to that seen in wild-type
cells, but loses interaction between the V1 and
Vo sectors at later times of assembly. In contrast, the
vma3
mutant showed no evidence of early interactions
between V1 and Vo sectors, but was still able
to proceed with assembly of the V1 sector. These results
indicate that early interactions between the 100-kDa subunit and
V1 subunits are not essential for V1 assembly,
and there must, therefore, be multiple potential routes for
V1 sector assembly.
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Assembly of the Yeast Vacuolar H+-ATPase in sec
Mutants--
The pulse-chase studies described above provide an
initial picture of the steps in V-ATPase assembly but do not provide
any information about where in the cell the various stages of assembly might occur. Previous studies have demonstrated that three resident proteins of the ER, Vma12p, Vma21p, and Vma22p, are essential for
assembly of the yeast V-ATPase (28, 32, 33) and that at least the
100-kDa Vo subunit must pass through the ER (29) and a
post-Golgi, prevacuolar compartment (34) en route to the vacuole.
Recent results indicate that Vo assembly occurs in the ER
(49), but do not address where subunits of the V1 and
Vo sectors of the V-ATPase begin to interact or the
possibility of distinct free Vo and
V1Vo subpopulations raised by the results described above. We attempted to obtain a preliminary correlation of
assembly and transport of the V-ATPase to the vacuole by examining the
extent of assembly of the V-ATPase in several yeast sec
mutants blocked at various steps in transport through the secretory
pathway (35, 36). The yeast sec mutants are all
temperature-sensitive mutants that allow normal transport through the
secretory pathway at the permissive temperature (usually 25 °C) but
exhibit an arrest in transport at elevated temperature. Epistasis
studies of these mutants and their subsequent molecular
characterization indicate that they arrest transport at different
stages in the secretory pathway (35, 36). sec1-1 is a
late-acting sec mutant that arrests vesicle fusion with the
plasma membrane but does not affect the transport of soluble or
membrane proteins to the vacuole (36-38). sec12-4 is an
early acting sec mutant that prevents exit of newly synthesized secreted and vacuolar proteins from the ER (35, 38).
sec18-1 acts at multiple places in the secretory pathway but
exhibits an early block (similar to that seen in sec12-4
cells) in exit of newly synthesized secreted and vacuolar proteins from the ER (36-40). As shown in Fig.
5A, the sec1-1
mutant assembled both fully assembled V-ATPase complexes,
immunoprecipitated by the 8B1 and 13D11 antibodies, and free
Vo complexes, immunoprecipitated by the 10D7 antibody, at
both the permissive and nonpermissive temperatures. These complexes
were comparable to those formed in wild-type cells (Fig.
5A). sec18-1 mutant cells also assembled an
apparently wild-type V-ATPase when incubated at 25 °C for 30 min
(Fig. 5B, 25 °C samples). However, labeling at the
nonpermissive temperature for 30 min resulted in accumulation of
complexes that contained the 100-kDa subunit and V1
subunits but no 17-kDa subunit. There was relatively little difference
in the Vo complexes formed at the permissive or
nonpermissive temperature. Very similar results were observed in a
sec12-4 mutant (data not shown).
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Some of the sec mutants have been shown to be reversible; transport intermediates formed at elevated temperature can be chased through to their normal location when the mutants are returned to the permissive temperature (35). In order to distinguish whether the complexes formed at elevated temperature in the sec18-1 mutants were true assembly intermediates or "dead-end" complexes resulting from a compromised secretory pathway, we determined whether these complexes could go on to assemble with the 17-kDa subunit during a subsequent chase period at 25 °C. As shown in Fig. 5B, the V1 subunit-containing complexes formed at elevated temperature in sec18-1 mutants can combine with the labeled 17-kDa subunit during a subsequent 30-min chase at 25 °C but do not assemble with the 17-kDa subunit if the cells are chased at 37 °C. A similar pattern was seen in the sec12-4 mutant (data not shown). Both the arrest of transport from the ER and the reversal of the arrest with the return to the permissive temperature were confirmed by immunoprecipitating the soluble vacuolar protein carboxypeptidase Y from the same cells shown in Fig. 5, A and B. CPY exhibits easily distinguishable forms, characteristic of organelle-specific posttranslational modifications, as it transits from the ER (p1 form) to the Golgi apparatus (p2 form) to the vacuole (mature form) (38, 47). As expected, sec1 mutants exhibit all three forms of CPY, representing newly synthesized CPY at various stages in transport, at both the permissive and nonpermissive temperatures (Fig. 5C). sec18 mutant cells exhibit all three forms after a 30-min labeling at the permissive temperature but accumulate the p1 form, indicating an ER block, at the nonpermissive temperature. Significantly, the accumulated p1 form can be converted to the p2 and m forms during a chase at the permissive, but not the nonpermissive, temperature. The results suggest that during the 30-60-min incubation at elevated temperature, we stabilized a genuine assembly intermediate in the sec18 mutant, similar to intermediate complexes seen at very early (0-2 min) chase times in wild-type cells. Furthermore, this intermediate appears to be competent for full assembly of the V-ATPase when the sec block is released.
There is a potential question of whether wild-type complexes lacking
the labeled 17-kDa subunit at early time points (Figs. 1 and
2B) are true assembly intermediates or merely complexes containing unlabeled 17-kDa subunit. The results with the
sec mutants argue that these complexes are true
intermediates that become competent for assembly with the 17-kDa
subunit at a well defined stage of transport. These results do not
support an exchange of the 17-kDa subunit from the Vo
complexes formed at elevated temperature to the
V1Vo complexes but instead suggest that full assembly of the free Vo sectors can occur before these
early sec blocks.
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DISCUSSION |
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Multiple Assembly Pathways for the Yeast Vacuolar
H+-ATPase--
The methods used here have allowed us to
identify new features of the assembly of the yeast V-ATPase. Their
greatest strength is the ability to follow the kinetics of biosynthesis
in vivo. A number of potential assembly intermediates
present at steady state have been identified and demonstrated to form
interactions necessary for assembly in vitro, but these
experiments do not directly address the early steps of biosynthesis. We
have temporally resolved the incorporation of two V1 and
one Vo subunit into complexes and achieved a picture of
both V1Vo and Vo sector assembly
in vivo. Based on the results of these experiments, in
combination with previous experiments, we propose that assembly of the
yeast vacuolar H+-ATPase, and probably other V-type
ATPases, may proceed through at least two pathways, shown schematically
in Fig. 6. The central feature of the
model in Fig. 6 is that cells have the capacity to assemble the
vacuolar H+-ATPase either by forming early interactions
between V1 and Vo subunits and then adding to
both sectors (concerted V1Vo assembly) or by
combining preassembled V1 and Vo sectors,
possibly with addition of one or more subunits that are not initially
present in either sector (independent assembly of V1 and
Vo).
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The results presented here argue that in wild-type cells, in which all
of the subunits of the enzyme are present, biosynthetic assembly of the
V1Vo complex occurs predominantly by the
concerted pathway, with early interactions between V1
subunits and at least the 100-kDa subunit of the Vo sector,
followed by a gradual addition of the 17- and 36-kDa subunits to the
Vo sector. Labeled 17- and 36-kDa subunits are incorporated
at earlier times into the free Vo complexes (Fig.
2B), suggesting that the free Vo complexes are
being formed shortly after synthesis of the individual subunits. It is
possible that the 17- and 36-kDa subunits are incorporated at later
times in assembly of V1Vo complexes or that an
unlabeled pool of the 17- and 36-kDa subunits is preferentially
recruited to V1Vo complexes with the newly
synthesized 100-kDa subunit. However, the second possibility was
eliminated by the experiments shown in Fig. 5, which strongly support
the concerted model of assembly. The one piece of data that initially
appears to be inconsistent with this model is the failure of the
100-kDa subunit to bind to the V1 subunits at early times
in vma3 mutant. It has been shown, however, that loss of
one Vo subunit destabilizes the other Vo
subunits (25, 41) and could potentially generate other long term
changes in the cell, for example, loss of an assembly factor that
affects the behavior of the 100-kDa subunit in assembly.
We also have evidence of assembly of a pool of free Vo
sectors, consistent with the independent assembly pathway in Fig. 6, but we have no evidence that these sectors go on to form fully assembled V1Vo complexes in wild-type cells. As
described above, we saw a pool of free Vo sectors that can
be immunoprecipitated by the 10D7 antibodies and that is not chased
into intact V-ATPase complexes with increasing time. Although we cannot
readily address whether there are free V1 sectors being
formed simultaneously by the methods used here, there is ample evidence
of a pool of free V1 sectors in the cytosol of wild-type
yeast cells (18, 19, 26). The assembly pathway of these free
V1 sectors has been examined in detail by Tomashek et
al. (18). In certain deletion mutants, independent assembly of
V1 and Vo sectors appears to become the
predominant assembly pathway. Depending on the subunit that is missing,
cells can proceed to assemble either V1 or Vo sectors with no evidence of early interaction between subunits of the
two sectors. Under certain circumstances, for example, in the
vma4 mutant, the initial steps of concerted
V1Vo assembly appear to occur, but the
complexes formed are then lost, apparently because a subunit missing in
the mutant is essential for stabilization of the complexes.
Although we did not see intact V-ATPase complexes forming by the
independent assembly pathway in our studies, there is substantial evidence that the free V1 and Vo sectors
contain the structural information necessary for assembly. Using two
different in vitro reconstitution systems, Vo
sectors assembled at the vacuole in vma2 cells were shown
to be competent for structural interaction with V1 sectors
(17, 18) and activation of ATPase activity in the V1
sectors (17). In addition, free V1 sectors formed biosynthetically in vma3
cells have been demonstrated to
interact with Vo sectors in vitro (18). Finally,
it is clear that V1 and Vo sectors separated by
disassembly of the enzyme in response to glucose deprivation can later
be recombined to form an active V1Vo complex
(9, 11). Although we have not yet established whether V1
and Vo complexes formed by disassembly of the V-ATPase are
identical to those formed biosynthetically, the disassembly and
reassembly data strongly suggest the existence of an independent assembly pathway.
The suggestion of multiple, potentially competing, assembly pathways for a multisubunit complex is rather novel. Nevertheless, the model we propose is consistent with available data on V-ATPases from other systems. Myers and Forgac (20) observed parallel assembly of V1Vo and free V1 complexes in bovine kidney cells and showed that these complexes formed over a similar time course and did not exchange readily. Bovine clathrin-coated vesicles have also been shown to contain a pool of free Vo (42). It is possible that there are physiological advantages to having a means for assembling both intact V1Vo sectors and free V1 and Vo sectors. We have observed that in addition to the "acute" changes in assembly state that occur with abrupt changes in carbon source, carbon source also has a "long term" impact on assembly of the yeast vacuolar H+-ATPase (9, 48). Yeast cells grown overnight in a poor carbon source contain a higher percentage of free V1 and Vo sectors than cells grown in glucose. Significantly, these free V1 and Vo sectors can be mobilized for assembly into intact V1Vo complexes in response to an improvement in carbon source. It is intriguing to speculate that there might be an "assembly switch" that regulates the extent or rate at which each of the assembly pathways shown in Fig. 6 operates. Recent work of Tomashek et al. (18) suggests a potential assembly switch by demonstrating that ATP binding to the catalytic subunit can influence assembly.
In addition to providing new insights into how assembly of vacuolar H+-ATPases occurs, these studies contribute to an initial picture of where assembly occurs. Coupling of assembly of the ATPase to transport through a specific compartment is a potential means of regulating which compartments of the vacuolar network are acidified. The identification of ER resident V-ATPase assembly factors essential for biogenesis of the 100-kDa subunit led to the suggestion that assembly of the yeast Vo sector occurred in the ER (29, 32, 33, 49). Myers and Forgac (20) suggested that association of V1 and Vo sectors occurred in the ER, based on recruitment of newly synthesized V1 subunits to a membrane fraction in the presence of brefeldin A or a 15 °C block to transport. Li et al. (46) have observed assembled V1Vo complexes in a microsomal fraction, suggesting interaction between V1 and Vo complexes as early as the ER in plants. The assembly and transport of Vo subunits to the vacuolar membrane in mutants lacking V1 subunits (17, 25, 43, 44) implies that assembly of V1 and Vo sectors is not essential for triggering Vo subunit transport to the vacuole. Our results with the sec mutants suggest that Vo sectors can assemble in the ER but argue that full assembly of V1 and Vo subunits occurs during exit from the ER or in a subsequent compartment in wild-type cells. In the sec12-4 and sec18-1 mutants at the nonpermissive temperature, the assembly intermediate that accumulated after 30 min resembled the complexes present in wild-type cells at the 2-5-min chase times (Fig. 1). Based on this evidence, we would suggest that attachment of the 100-kDa subunit to V1 subunits in the concerted pathway takes place before the sec18-1 block but that acquisition of the 17-kDa subunit takes place later in transport to the vacuole.
In the course of these experiments, we have also biochemically
identified a new protein, with a molecular mass of approximately 19 kDa, that exhibits properties expected of an assembly factor (specifically, transient appearance at early stages of both
V1Vo and Vo sector assembly). We do
not yet know the identity of this protein. Polyclonal antibodies
against Vma12p, a previously identified assembly factor (28), did not
immunoprecipitate the 19-kDa protein. The 19-kDa protein seems to
disappear as the 17-kDa protein appears in the
V1Vo and Vo complexes, but it is
not an unprocessed form of the 17-kDa subunit because it does not
undergo a molecular mass shift when Vma3p is epitope-tagged (data not
shown). It is still possible that the 19-kDa protein represents one of
the other proteolipid subunits (Vma11p or Vma16p) (45), but the best
evidence available suggests that both of these protein are part of the final V-ATPase complex. Further experiments will be necessary to
identify this protein.
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ACKNOWLEDGEMENTS |
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We thank Tom Stevens, Morris Manolson, Ryogo Hirata, and Yasuhiro Anraku for providing strains and antibodies used in this work; Yemisi Oluwatosin for construction of the pYO4 and pYO6 plasmids; and Chris Tachibana for helpful advice on CPY immunoprecipitations.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant R01-GM50322 (to P. M. K.).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.
An American Heart Association Established Investigator. To whom
correspondence should be addressed: Dept. of Biochemistry and Molecular
Biology, State University of New York Health Science Center at
Syracuse, 750 E. Adams St., Syracuse, NY 13210. Tel.: 315-464-8742;
Fax: 315-464-8750; E-mail: kanepm{at}hscsyr.edu.
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ABBREVIATIONS |
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The abbreviations used are: V-ATPase, vacuolar proton-translocating ATPase; H+-ATPase, proton-translocating ATPase; V1, peripheral sector of V-ATPase; Vo, membrane sector of V-ATPase; ER, endoplasmic reticulum; CPY, carboxypeptidase Y; PCR, polymerase chain reaction.
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REFERENCES |
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