Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, Spemannstr. 37-39, 72076 Tübingen, Germany
* Author for correspondence (e-mail: Andreas.Mayer{at}Tuebingen.mpg.de)
Accepted 17 December 2002
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Summary |
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Key words: Membrane fusion, NSF, Saccharomyces cerevisiae, SNARE, Vacuole, Yeast
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Introduction |
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Other studies have identified the VTC1 homologue NRF1 in
a screen for negative regulators of the Rho GTPase Cdc42p in S. pombe
(negative regulator of Cdc forty-two,
NRF1) (Murray and Johnson,
2000) or as a hypothetical polyphosphate synthase in the S.
cerevisiae vacuole (Ogawa et al.,
2000
). Cdc42p is involved in yeast vacuole fusion
(Eitzen et al., 2001
;
Muller et al., 2001
), further
supporting the role of Vtc proteins in this process.
Vtc proteins have also been suggested to be vacuolar
transporter chaperons (VTC), a novel family of chaperons
involved in the distribution of V-ATPase and other membrane proteins in S.
cerevisiae (Cohen et al.,
1999). In this study, VTC1 was found to be a
suppressor of V-ATPase function
(svf). Null mutations in genes encoding V-ATPase subunits result in a
phenotype that is unable to grow at high pH. Deletion of VTC1 could
suppress this phenotype, suggesting a relationship between Vtc1p and V-ATPase.
The other members of the VTC family, that is, VTC2, VTC3 and
VTC4, were identified by sequence similarity. Vacuoles from a
vtc1 strain showed a reduction of some V-ATPase subunits and
reduced proton uptake activity. Some proton uptake activity of the V-ATPase is
needed for establishing a membrane potential
(Stevens and Forgac, 1997
;
Wada and Anraku, 1994
;
Yabe et al., 1999
). A proton
motive force is required for proper vacuole membrane fusion, as the proton
uncoupler p-(trifluoromethoxy)-phenylhydrazone (FCCP) inhibits the in vitro
fusion reaction (Conradt et al.,
1994
; Mayer et al.,
1996
; Ungermann et al.,
1999
). Therefore, we carried out studies on the properties of Vtc
proteins with special regard to discovering the relationships between V-ATPase
activity, V-ATPase stability and membrane fusion.
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Materials and Methods |
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Strains
BJ3505, DKY6281, OMY1 through OMY13, SBY82, SBY83, SBY85 and SBY86 have
been described previously (Muller et al.,
2002). For the construction of strains with single, double and
triple knock outs of VTC1, VTC2 and VTC3 genes, strain
BY4727 (MAT
his3
200 leu2
0
lys2
0 met15
0 trp1
63
ura3
0) (Brachmann et
al., 1998
) was used as a parent. The VTC genes were
replaced by HIS3, URA3 and LEU2 markers using PCR-generated
cassettes from plasmids pRS303 (HIS3), pRS306 (URA3) and
pRS305 (LEU2), respectively
(Brachmann et al., 1998
). The
oligonucleotides used for generation of deletion cassettes and for control PCR
have been described elsewhere (Muller et
al., 2002
). The resulting strains were OMY20
(
vtc1::HIS3), OMY21 (
vtc2::HIS3), OMY22
(
vtc3::HIS3), OMY23 (
vtc1::HIS3
vtc2::URA3), OMY24 (
vtc1::HIS3
vtc3::URA3), OMY25 (
vtc2::HIS3
vtc3::URA3) and OMY26 (
vtc1::HIS3
vtc2::URA3
vtc3::LEU2).
Construction of GFP fusion proteins
The Vtc1p-GFP construct (pYER-GFP) was generated as follows: 500 bp
upstream of the last codon before the stop codon of the VTC1 ORF were
amplified from genomic DNA using the primers 5'-CGG GCG GCC GCT TCT TAT
TTC AAT CTG CAT ACT CAT TTT-3' and 5'-CCT TCT AGA GCT AAC TTA GTG
TTA GCG TCA TTG-3', which introduced a 5' NotI site and a
3' XbaI site. Using these restriction sites, the PCR fragment
was cloned into pRS416-GFP (T. Vida), resulting in C-terminal Vtc1-GFP with a
seven amino acid spacer between the last codon of VTC1 and the start
codon of GFP. The construct was verified by sequencing. The plasmid was
transformed into yeast strains BJ3505 and DKY6281 using the URA3
marker. For the construction of Vtc3-GFP, GFP was chromosomally integrated at
the 3' end of the VTC3 ORF in strain BJ3505 by homologous
recombination. A PCR product was generated with primers (forward) 5'-CA
CTA AAA CCA ATT CAA GAT TTT ATC TTC AAT TTG GTT GGG GAA
ATG TCT AAA GGT GAA GAA TTA TTC AC-3' and (reverse)
5'-GA TCT GGG TTT AAC TAT CAC ACA CAT CTT CTC ATT ATG TGC ATT GCA TAG
GCC ACT AGT GGA TCT G-3' and plasmid pUG24
(Niedenthal et al., 1996) as a
template. The last codon of VTC3 and the start codon of GFP are
underlined. Integration was verified by PCR using primer (Vtc3 con fw)
5'-GAG GCC GCT AGG AGG GAA AGA GG-3' binding inside VTC3
and primer (kan con rev) 5'-CGA TAG ATT GTC GCA CCT GAT TGC C-3'
binding inside the kanamycin resistance marker box and by western analysis.
The resulting strain was SBY593.
Assay for proton uptake activity
Proton uptake of vacuoles was measured by the method described
(Cohen et al., 1999). The
absorbance changes of acridine orange at 491-540 nm were followed by a Beckman
DU-600 spectrophotometer. The reaction mixture in a final volume of 100 µl
contained 20 µg of vacuoles (mixture of fusion tester strains) at the
fusion concentration and condition (PS buffer, 150 mM KCl, 500 µM
MnCl2, 27°C) with 15 µM acridine orange. The reaction was
started by the addition of 5 µl of an ATP regenerating system. At the end,
10 µM of FCCP were added. Proton uptake activity was defined as the
absorbance change during the first 20 seconds of the reaction.
Preparation of whole cell extracts
107 cells from a logarithmically growing culture in YPD medium
were harvested in a microfuge (6000 g, 2 minutes at 4°C),
washed with 1 ml of cold buffer C (50 mM Tris/HCl pH 7.5, 10 mM
NaN3) and resuspended in 30 µl of SDS sample buffer with
protease inhibitors [2% SDS, 60 mM Tris/HCl pH 6.8, 10% (v/v) glycerol, 5%
(v/v) ß-mercaptoethanol, 0.005% (w/v) bromphenol blue, 100 µM pefabloc
SC, 100 ng/ml leupeptin, 50 µM o-phenanthroline, 500 ng/ml pepstatin A, 1
mM PMSF]. Glass beads were added and the samples vortexed for 2 minutes.
Another 70 µl of sample buffer were added. For Vph1p-analysis, samples were
not boiled because this hydrophobic protein aggregates when heated.
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Results |
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Vtc1p is homologous to the C-termini of Vtc2p and Vtc3p, which, like Vtc1p,
contain three potential transmembrane segments
(Cohen et al., 1999). The
N-terminus of Vtc2p is homologous to Vtc4p and to the N-terminus of Vtc3p. All
are predicted to form hydrophilic domains. Vtc4p may interact with Vtc1p. This
interaction is supported by the observation that Vtc4p was completely absent
in vacuoles from vtc1 deletion mutants and that the level of Vtc1p
was significantly reduced in vtc4 deletion strains
[Fig. 6A (c.f.
Cohen et al., 1999
)]. On the
basis of the new sequence information, however, this effect can no longer be
explained by Vtc1p functioning as a transmembrane anchor for Vtc4p, as
originally proposed (Cohen et al.,
1999
).
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A previous study showed diffuse staining of the vacuolar lumen by a
Vtc3-GFP fusion protein (Ogawa et al.,
2000). This pattern is typical for soluble vacuolar proteins but
not for vacuolar membrane proteins which stain only the vacuolar rim.
In order to reanalyze Vtc localization, we constructed a Vtc1p fusion protein
carrying GFP at the C-terminus. The fusion protein behaved as an integral
membrane protein and was functional because it rescued the vacuolar fusion
defect of a vtc1 deletion mutant (data not shown). In a
protease-deficient strain (pep4), the fusion
protein stained the vacuolar rim, indicating localization at the vacuole
membrane [Fig. 1B, upper panel
(c.f. Murray and Johnson,
2001
)]. Weak fluorescence signals could also be detected around
the nucleus, in the periphery of the cell, and in dot-like structures that may
be endosomes or Golgi elements. Vacuolar membrane staining by Vtc1p-GFP was
only observed in pep4 cells, that is, in cells with
reduced vacuolar proteolytic activity (Fig.
1B, upper panels). In wild-type (PEP4+) cells,
which have a full complement of vacuolar hydrolases, GFP stained the vacuolar
lumen (Fig. 1B, lower panels).
As Vtc1p itself still behaves as an integral membrane protein in
PEP4+ cells (data not shown), a vacuolar protease probably cleaved
the fusion protein between the membrane-embedded Vtc1p C-terminus and the
hydrophilic GFP domain, releasing GFP into the vacuolar lumen. The same
clipping must have occurred in the earlier study on Vtc3p-GFP
(Ogawa et al., 2000
) in which
a PEP4+ strain had been used. The Vtc1p-GFP fusion was resistant to
proteinase K digestion from the cytosolic side
(Fig. 2A), but it was degraded
into two major fragments if proteolysis was performed in the presence of 0.5%
Triton X-100 to lyse the vacuoles. The smaller fragment corresponds to the
molecular weight of GFP alone, which, in its correctly folded form, is
protease resistant (see also Fig.
1B, lower panels). This indicates that the C-terminus of Vtc1p
faces the vacuolar lumen.
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The N-termini of the Vtc proteins are thought to face the cytosol on the basis of the following observations: an approximately 80 kDa N-terminal piece of a Vtc3p-GFP* fusion protein (total size of Vtc3p-GFP*: 125 kDa) could be digested with protease from the cytosolic side (Fig. 2A). This is almost the entire Vtc3p portion of the fusion protein (Vtc3p itself is 95 kDa). Note that, in contrast to Vtc1p-GFP, the GFP in Vtc3p-GFP* was not protease resistant (see below) and was not fluorescent (data not shown). We indicate this by the asterisk. Although Vtc3p-GFP* functionally substituted for Vtc3p (data not shown), its GFP domain could not reach the fully folded state. This is probably due to the fact that GFP was directly fused to the C-terminus of Vtc3p, whereas the Vtc1p-GFP fusion contained a seven amino acid spacer between the two parts.
We took advantage of the protease sensitivity of the C-terminal GFP domain in Vtc3p-GFP* to monitor its resistance to proteases. The C-terminal GFP in Vtc3p-GFP* was protease resistant when whole vacuoles were used, producing a 45 kDa fragment that corresponded in size to a fusion of the C-terminal three transmembrane domains of Vtc3p plus the GFP* (Fig. 2A). This fragment was digested when the vacuoles were lysed by Triton X-100, giving the protease access to the vacuolar lumen. The C-terminus of Vtc3p must therefore be exposed to the vacuolar lumen where it is protected from proteinase K.
The intactness of the vacuoles could be independently checked by
proteolytic fragmentation of pro-alkaline phosphatase (pro-Pho8p), a vacuolar
membrane protein oriented towards the vacuolar lumen
(Klionsky and Emr, 1989).
Pro-Pho8p has one transmembrane domain, a short cytosolic N-terminal tail and
a large hydrophilic domain in the vacuolar lumen that carries a
protease-sensitive pro-peptide (Fig.
2B). Proteinase K digested only the small N-terminal cytoplasmic
tail when the vacuoles were intact (Fig.
2A). The lumenal propeptide became accessible to partial
proteolysis after lysing the vacuolar membrane with Triton X-100.
Similarly to Vtc3p, Vtc4p could be degraded into fragments as small as
22 kDa by low amounts of proteinase K added to intact vacuoles
(Fig. 2A). Thus, not only the
large hydrophilic N-terminal domain of Vtc3p but also that of Vtc4p must be
exposed to the cytosol. Our data support the topology shown in
Fig. 2B, that is, an
arrangement in which the large hydrophilic parts of the Vtc complex face the
cytosol and the C-termini face the vacuolar lumen. This experimental evidence
matches previous speculations about Vtc topology
(Cohen et al., 1999
;
Nelson et al., 2000
).
Role of Vtc proteins in membrane trafficking
Since Vtc proteins are involved in vacuole fusion
(Muller et al., 2002), we also
wanted to test whether other membrane trafficking processes depended on these
factors. We assayed ER to Golgi trafficking of carboxypeptidase Y (CPY). CPY
is a vacuolar protease that is translocated into the ER as a pro-enzyme (p1
form), travels to the Golgi and becomes glycosylated (p2 form). CPY is further
transported through the prevacuolar endosomal compartment to the vacuole where
the pro-peptide is cleaved off, resulting in the active vacuolar form (m).
Cells were pulse labeled (Stack et al.,
1995
) with 35S-methionine/35S-cysteine and
chased in non-radioactive medium for different time periods before CPY was
immunoprecipitated from the cell lysates
(Fig. 3A). In wild-type cells,
CPY was rapidly transported from the ER (0 minutes) to the Golgi (5 minutes)
and finally to the vacuole (20 minutes)
(Fig. 3A). Transition from p1
to p2, as well as from p2 to m, was delayed in
vtc3 cells. By
contrast,
vtc1 [lacking also Vtc4p
(Muller et al., 2002
)] and
vtc2 cells behaved like the wildtype
(Fig. 2A). Deletion of all four
VTC genes did not result in a stronger phenotype than deletion of
VTC3 alone (data not shown). In line with ER-Golgi transport being a
process essential for growth (Novick et
al., 1980
),
vtc3 cells also showed an increased
generation time (107 minutes) when compared to wildtype (84 minutes).
vtc1,
vtc2 and
vtc4 grew like
the wildtype.
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Endocytosis was followed via the fluorescent styryl dye FM4-64. FM4-64
inserts into the plasma membrane, becomes endocytosed and then transferred to
the vacuole by vesicular transport (Vida
and Emr, 1995). After incubating cells with FM4-64 for 2 minutes,
small intracellular vesicular structures were stained in wild-type and
vtc1 mutants (Fig.
3B). During a 45 minute chase in medium without dye, staining of
the small vesicles in wild-type cells was gradually lost and the vacuolar
membrane became increasingly fluorescent. Transiently, slightly larger
punctate structures (three to five per cell) were also stained that might
correspond to endosomes. After 45 minutes, all stain had been transferred to
the vacuoles. The pulse-chase pattern was indistinguishable from wildtype and
all vtc deletion mutants, even when all VTC genes or
pairwise combinations thereof had been deleted (data not shown). This result
differs from that obtained using a similar approach in S. pombe,
where a
vtc1/nrf1 mutant was reported to have a severe
endocytosis defect (Murray and Johnson,
2000
; Murray and Johnson,
2001
). The reason for this different behaviour is unclear. In
summary, endocytic trafficking to the vacuole is independent of Vtc proteins
in S. cerevisiae. By contrast, ERGolgi transport and Golgi to vacuole
transport appears to be facilitated by Vtc3p, although it does not absolutely
depend on it.
Proton uptake activity, V-ATPase assembly and V0
conformation
Conflicting reports exist describing the effect of deletion of
VTC1 on the proton translocation activity of vacuolar membranes. In
vitro assays with isolated subvacuolar vesicles indicated a reduction in
proton translocation activity by 70% in subvacuolar vesicles prepared from
vtc1 mutants, as determined via the pH-dependent absorbance
change of acridine orange in the vesicles
(Cohen et al., 1999
;
Nelson et al., 2000
). By
contrast, qualitative in vivo assays using the DpH-dependent accumulation of
quinacrin in vacuoles detected no changes to the wildtype
(Ogawa et al., 2000
). Since a
proton motive force (pmf) across the membrane is required for vacuolar fusion
(Conradt et al., 1994
;
Mayer et al., 1996
;
Ungermann et al., 1999
), we
tested whether the fusion defects of vtc mutants
(Muller et al., 2002
) could be
explained by reduced proton translocation.
We measured the apparent proton uptake activity of vacuoles from different
vtc deletion mutants using acridine orange
(Cohen et al., 1999). In
contrast to Cohen et al., we used intact vacuoles instead of subvacuolar
vesicles. Our vacuoles are prepared by a rapid and gentle procedure that
preserves the soluble contents of this compartment. Therefore, the apparent
proton translocation activity we measure may comprise not only V-ATPase pump
activity but also H+ uptake via other mechanisms, such as import of
protons by antiporting amino acids or ions. Vacuoles also contain an
ATP-driven Ca2+ pump and a Ca2+/H+ antiporter
that may drive proton uptake and partially substitute for V-ATPase activity
(Ohsumi and Anraku, 1981
;
Ohsumi and Anraku, 1983
;
Wada et al., 1992
). Apparent
proton translocation activity is the relevant parameter for our purpose, that
is, for analyzing the correlation with vacuole fusion. Under the conditions of
our in vitro fusion assay, the apparent proton translocation activity of
vtc1 and
vtc4 vacuoles was reduced to 85% and
50% of wild-type activity, respectively
(Figs. 4A,B). Activities of
vtc2 and
vtc3 vacuoles were equal to or even
slightly higher than those of wild-type vacuoles. The apparent proton uptake
activities of vtc mutant vacuoles did not correlate with their fusion
activities (Fig. 4B). Whereas
vtc2 vacuoles were fusion competent,
vtc3
vacuoles, despite their wild-type-like apparent proton uptake activity, did
not fuse at all. Vtc3p must therefore have a direct role in fusion that is
independent of proton uptake.
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vtc4 and
vtc1 vacuoles were unable to fuse,
however, they showed significantly reduced apparent proton translocation
activities. Therefore, we determined the level of translocation activity that
would become limiting to fusion. We measured apparent proton translocation of
wild-type vacuoles with different concentrations of the H+-ATPase
inhibitor concanamycin A (Drose and
Altendorf, 1997
) and in parallel determined the fusion activities.
Concanamycin A reduced the apparent proton uptake activity in a
concentration-dependent manner (Fig.
4C), abolishing the signal in the acridine orange assay completely
at concentrations above 0.5 µM. Even at these concentrations fusion
proceeded with an efficiency of
70%. We attribute this to limitations in
the sensitivity of the proton uptake assay. Below the levels of proton uptake
detectable in this assay a basal pmf obviously remains that is sufficient to
drive fusion. The fusion signal observed with >0.5 µM of concanamycin A
was still sensitive to the proton uncoupler FCCP, demonstrating that it
depended upon a basal proton motive force (data not shown). As we worked with
intact vacuoles containing high concentrations of amino acids and other
solutes, a basal pmf (below the detection level of the acridine orange assay)
could be regenerated by efflux of these solutes via proton antiporters
(Ohsumi and Anraku, 1981
;
Ohsumi and Anraku, 1983
;
Wada et al., 1992
). This may
enable intact isolated vacuoles to retain a basal V-ATPase-independent proton
uptake activity. Subvacuolar vesicles that are commonly used for V-ATPase
assays would not show such an activity
(Cohen et al., 1999
;
Nelson et al., 2000
).
Pharmacological reduction of the apparent proton uptake activity to 85%
(the level observed with vtc1) or 50% (as observed with
vtc4; Fig. 4B)
of the control levels reduced the fusion activity of wild-type vacuoles only
moderately, to 87% and 83% of the untreated control, respectively
(Fig. 4C). This is in striking
contrast to the profound fusion defect of
vtc1 and
vtc4 vacuoles (Fig.
4B) and thus separates these two phenomena. We could test this
aspect with an independent second approach, using affinity-purified antibodies
to Vtc4p that can inactivate the protein on wild-type vacuoles
(Muller et al., 2002
). This
approach avoids potential secondary effects owing to deletion of genes.
Antibodies to Vtc4p had no effect on the proton uptake activity of wild-type
vacuoles, but they inhibited vacuole fusion
(Fig. 4D). Taken together, Vtc
proteins have a direct role in vacuolar membrane fusion
(Muller et al., 2002
) that is
separable from their potential influence on vacuolar proton translocation
activity (Cohen et al., 1999
;
Nelson et al., 2000
).
The involvement of Vtc proteins in both vacuolar proton translocation and
membrane fusion could be due to conformational changes of the V-ATPase caused
by physical interactions of Vtc proteins with this enzyme. A physical
interaction between V-ATPase and Vtc proteins was shown by cofractionation
(Cohen et al., 1999) and
coimmunoprecipitation (Muller et al.,
2002
). We tested whether Vtc mutations affect V-ATPase
conformation or stability. Differences in proteolytic susceptibility are a
well established indicator of altered conformations or associations of a
protein. We discovered that the stability of an AU1 peptide tag on the
C-terminus of Vph1p strongly depended on the presence of Vtc proteins.
Vacuoles were isolated from wild-type,
vtc1 and
vtc3 cells expressing Vph1p with chromosomally encoded tags on
the C-terminus, either a His6-HA3 tag or an AU1 tag
(Fig. 5A). The amount of Vph1p
was equal in all strains, as checked by decoration with a monoclonal antibody
to Vph1p itself. However, antibodies against the tags revealed that the AU1
tag was largely degraded in wild-type vacuoles, whereas it was stable in
vtc1 and
vtc3 vacuoles. By contrast, the
His6-HA3 tag was stable in all strains. The strains used
were depleted of vacuolar proteases (
pep4,
prb1), making post-lysis effects by altered levels of vacuolar
proteases unlikely. The picture was essentially the same in whole cell
extracts of living yeast cells (Fig.
5B), suggesting that the tag was already degraded inside the cell
and not during vacuole isolation. Therefore, the Vtc complex appears to modify
the conformation of V-ATPase so that the C-terminus of Vph1p becomes more
accessible to proteases.
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This conclusion is supported by changes in the assembly state of the
V-ATPase on isolated vtc mutant vacuoles. The fully assembled
V-ATPase consists of a membrane-integral V0 sector and a peripheral
V1 sector (Stevens and Forgac,
1997). The major peripheral subunits Vma1p and Vma2p were
significantly reduced on
vtc1 vacuoles
(Fig. 6A). By contrast, Vma4p,
which forms part of the interface to the V0 sector, was barely
affected (Fig. 6A). Vma1p and
Vma2p were also reduced on
vtc4 and on
vtc2
vacuoles (Fig. 5A). However,
vtc3 vacuoles carried more Vma1p, Vma2p and Vma4p than
wild-type vacuoles. The V0 subunits Vma6p and Vph1p, and the
vacuolar SNARE Nyv1p were present in equal amounts on the vacuoles of all
strains, indicating equal loading of the lanes
(Fig. 6A). Therefore, only some
peripheral subunits of the V1 sector differ in abundance on the
membranes of
vtc vacuoles, but not the membrane-integral
V0 sectors. Cohen et al. reported that only deletion of
VTC1 led to a reduction of V1 association
(Cohen et al., 1999
), but that
other vtc deletions had no effect (detected via the V1
subunits Vma5p and Vma8p). Integral vacuolar membrane proteins were not
included as internal reference, raising the possibility that different levels
of vacuolar membranes had been analyzed. Alternatively, this could indicate
that only some V1 subunits are affected rather than the entire
V1 sector. This notion is supported by the fact that the
differences we detected for the V1 subunit Vma4p were less
pronounced than those for Vma2p and that Cohen et al.
(Cohen et al., 1999
) also
reported less significant differences for Vma4p.
In whole cell extracts, V1 subunits of deletion mutants were as
abundant as they are in wild-type extracts
(Fig. 6B). Thus, the
association between V0 and V1 subunits appears to be
influenced by Vtc proteins. This association is labile on
vtc1,
vtc2 and
vtc4 vacuoles.
The V1 and V0 subunits can undergo regulated cycles of
dissociation and reassociation in response to depletion or replenishment of
glucose in the growth medium (Parra and
Kane, 1998
). Loss of V1 subunits from the mutant
vacuoles might therefore be caused by enhanced disassembly or by a block in
reassembly. We assayed V1/V0 dissociation and
reassociation in living cells according to published procedures
(Parra and Kane, 1998
) using
coprecipitation of V1 and V0 from whole cell lysates as
an assay. This did not reveal significant differences in any of the
vtc mutants (data not shown). Thus, we prefer the interpretation that
the stability of the V1/V0 holoenzyme is compromised in
vtc1,
vtc2 and
vtc4 mutants,
leading to partial loss of V1 subunits. In combination with the
altered proteolytic sensitivity of Vph1p-AU1, this provides in vivo evidence
for an interaction of Vtc proteins with the V-ATPase, which affects the
conformation of V0 and the stability of the holoenzyme.
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Discussion |
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A Vtc protein copurified with V-ATPase components upon chromatographic
fractionation (Cohen et al.,
1999), and we could coimmunoprecipitate V-ATPase and Vtc proteins,
indicating a physical interaction (Muller
et al., 2002
). The data presented here suggest that Vtc proteins
influence the conformation and/or molecular interactions of the V0
subunit Vph1p (Fig. 5).
Presence of the Vtc proteins alters the proteolytic sensitivity of Vph1p and
the interaction of V0 and V1 subunits. We detected
significant differences in the levels of V1 subunits Vma1p and
Vma2p on
vtc1,
vtc2 and
vtc4
vacuoles. Previously, point mutations in Vph1p were shown to influence the
assembly state and/or stability of the V1/V0 holoenzyme
(Leng et al., 1998
;
Leng et al., 1999
). These point
mutations mapped to the C-terminal 50 amino acids of Vph1p, that is, to the
same region for which our tagged version of Vph1p indicated Vtc-dependent
alterations of proteolytic sensitivity. The fact that this C-terminal stretch,
which was suggested to be at the lumenal side of the membrane
(Leng et al., 1999
), influences
the assembly of V1 onto the cytosolic side could only be explained
by significant conformational rearrangements of V0. When combined,
the results of Leng et al. and our findings suggest that the C-terminus of
Vph1p exhibits significant flexibility in its conformation and associations.
This, together with the observation of genetic and physical interactions
between V-ATPase and Vtc proteins, suggests that the VtcV-ATPase
association is chaperon-like in the genuine sense, that is, that of a protein
affecting the folding state of another polypeptide. We propose that such an
influence on V0 conformation may form one basis of Vtc protein
function in vacuole fusion. Vtc proteins affect two stages of vacuole fusion.
Vtc1p and Vtc4p regulate the activation of vacuolar SNAREs by Sec18p/NSF
(Muller et al., 2002
). Vtc3p
is involved in a later step, probably subsequent to docking and the formation
of V0 trans-complexes. Conformational changes of V0
would be strongly expected to play a role in this late stage, and it is
conceivable that Vtc3p might regulate them.
Vtc proteins were proposed to be polyphosphate synthases
(Ogawa et al., 2000) because
vtc mutations reduce the formation of vacuolar polyphosphate to
various degrees. This reduction becomes apparent only when yeast cells are
shifted from phosphate-depleted media to high phosphate media
(Ogawa et al., 2000
). It
remained unclear whether Vtc proteins play a direct role in polyphosphate
synthesis or whether polyphosphate deficiency in
vtc1 and
vtc4 mutants arises as a secondary effect, perhaps from
problems in membrane trafficking. The topology of the Vtc complex, which we
have experimentally determined now, makes the possibility of it having a
function as a polyphosphate synthase very unlikely. All parts of the Vtc
proteins except their transmembrane domains face the cytosol. An
enzyme-synthesizing polyphosphate inside the vacuole would be expected to face
the vacuolar lumen.
Several observations indicate that Vtc proteins control membrane fusion
independently of polyphosphate levels
(Muller et al., 2002): first,
vtc1 and
vtc4 mutants have no polyphosphates
(Ogawa et al., 2000
) and are
deficient in priming of SNARE proteins. SNARE priming and fusion can partially
be rescued by exogenous Sec18p in vitro
(Muller et al., 2002
), where
regeneration of vacuolar polyphosphates should not be possible. Second,
antibodies to Vtc4p blocked SNARE priming and fusion on wild-type vacuoles
which should have polyphosphates. Third,
vtc3 mutants
show a less severe reduction in polyphosphates than
vtc1 and
vtc4 cells do (Ogawa et
al., 2000
). They do not fuse and cannot be rescued by Sec18p
(Muller et al., 2002
). Thus,
the fusion activity of vacuoles does not correlate with polyphosphate levels.
This demonstrates that Vtc proteins do not influence fusion via polyphosphate
but perform a direct role in vacuole fusion.
Vacuoles from some of the vtc deletion mutants show altered proton
uptake activity. The effects seen in our study are qualitatively similar to
those observed in the previous studies
(Cohen et al., 1999;
Nelson et al., 2000
). Cohen et
al. reported that V-ATPase activity of
vtc1 vacuoles was
reduced to
10-30% of the wild-type signal
(Cohen et al., 1999
). We
observed only a minor reduction in apparent proton translocation activity to
85%, which is consistent with the observations by Ogawa et al.; they
observed no vacuolar acidification defects in a qualitative in vivo assay
(Ogawa et al., 2000
). The
difference in our results and those of Cohen et al. might be due to the use of
different strains, incubation conditions and methods for vacuole isolation.
For example, Cohen et al. used protease-competent cells and a slow method of
membrane isolation that produces subvacuolar vesicles and should release
vacuolar hydrolases (Cohen et al.,
1999
). This might explain the significant proteolytic degradation
of the V-ATPase subunit Vma5p, which is visible in this study, and the
stronger reduction of proton translocation activity observed. A further
important aspect to be considered has already been outlined above: the
apparent proton-translocation activity of intact vacuoles that we assay may
comprise several different H+-translocating processes, whereas
H+ translocation in subvacuolar vesicles depends solely on
V-ATPase. For example, proton translocating antiporters could drive proton
uptake by efflux of amino acids or ions from intact vacuoles but not from
subvacuolar vesicles that have lost their soluble contents in the course of
preparation.
We assume that the influence of Vtc proteins on V-ATPase conformation and stability is central to the effects of these proteins in vacuole fusion and possibly also in proton translocation. A major task in the functional analysis of these proteins will therefore be to characterize this interaction and its dynamics, particularly in the course of vacuole fusion.
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References |
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Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J., Hieter, P. and Boeke, J. D. (1998). Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14,115 -132.[CrossRef][Medline]
Cohen, A., Perzov, N., Nelson, H. and Nelson, N.
(1999). A novel family of yeast chaperons involved in the
distribution of V-ATPase and other membrane proteins. J. Biol.
Chem. 274,26885
-26893.
Conradt, B., Haas, A. and Wickner, W. (1994). Determination of four biochemically distinct, sequential stages during vacuole inheritance in vitro. J. Cell Biol. 126,99 -110.[Abstract]
Drose, S. and Altendorf, K. (1997).
Bafilomycins and concanamycins as inhibitors of V-ATPases and P-ATPases.
J. Exp. Biol. 200,1
-8.
Eitzen, G., Thorngren, N. and Wickner, W.
(2001). Rho1p and Cdc42p act after Ypt7p to regulate vacuole
docking. EMBO J. 20,5650
-5656.
Klionsky, D. J. and Emr, S. D. (1989). Membrane protein sorting: biosynthesis, transport and processing of yeast vacuolar alkaline phosphatase. EMBO J. 8,2241 -2250.[Abstract]
Leng, X. H., Manolson, M. F. and Forgac, M.
(1998). Function of the COOH-terminal domain of Vph1p in activity
and assembly of the yeast VATPase. J. Biol. Chem.
273,6717
-6723.
Leng, X. H., Nishi, T. and Forgac, M. (1999).
Transmembrane topography of the 100-kDa a subunit (Vph1p) of the yeast
vacuolar proton-translocating ATPase. J. Biol. Chem.
274,14655
-14661.
Mayer, A., Wickner, W. and Haas, A. (1996). Sec18p (NSF)-driven release of Sec17p (alpha-SNAP) can precede docking and fusion of yeast vacuoles. Cell 85, 83-94.[Medline]
Muller, O., Bayer, M. J., Peters, C., Andersen, J. S., Mann, M.
and Mayer, A. (2002). The Vtc proteins in vacuole fusion:
coupling NSF activity to V0 trans-complex formation. EMBO
J. 21,259
-269.
Muller, O., Johnson, D. I. and Mayer, A.
(2001). Cdc42p functions at the docking stage of yeast vacuole
membrane fusion. EMBO J.
20,5657
-5665.
Murray, J. M. and Johnson, D. I. (2000).
Isolation and characterization of Nrf1p, a novel negative regulator of the
Cdc42p GTPase in Schizosaccharomyces pombe. Genetics
154,155
-165.
Murray, J. M. and Johnson, D. I. (2001). The
Cdc42p GTPase and its regulators Nrf1p and Scd1p are involved in endocytic
trafficking in the fission yeast Schizosaccharomyces pombe. J.
Biol. Chem. 276,3004
-3009.
Nelson, N., Perzov, N., Cohen, A., Hagai, K., Padler, V. and Nelson, H. (2000). The cellular biology of proton-motive force generation by VATPases. J. Exp. Biol. 203, 89-95.[Abstract]
Niedenthal, R. K., Riles, L., Johnston, M. and Hegemann, J. H. (1996). Green fluorescent protein as a marker for gene expression and subcellular localization in budding yeast. Yeast 12,773 -786.[CrossRef][Medline]
Novick, P., Field, C. and Schekman, R. (1980). Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell 21,205 -215.[Medline]
Ogawa, N., DeRisi, J. and Brown, P. O. (2000).
New components of a system for phosphate accumulation and polyphosphate
metabolism in Saccharomyces cerevisiae revealed by genomic expression
analysis. Mol. Biol. Cell
11,4309
-4321.
Ohsumi, Y. and Anraku, Y. (1981). Active
transport of basic amino acids driven by a proton motive force in vacuolar
membrane vesicles of Saccharomyces cerevisiae. J. Biol.
Chem. 256,2079
-2082.
Ohsumi, Y. and Anraku, Y. (1983). Calcium
transport driven by a proton motive force in vacuolar membrane vesicles of
Saccharomyces cerevisiae. J. Biol. Chem.
258,5614
-5617.
Parra, K. J. and Kane, P. M. (1998). Reversible
association between the V1 and V0 domains of yeast vacuolar H+-ATPase is an
unconventional glucose-induced effect. Mol. Cell.
Biol. 18,7064
-7074.
Peters, C., Andrews, P. D., Stark, M. J., Cesaro-Tadic, S.,
Glatz, A., Podtelejnikov, A., Mann, M. and Mayer, A. (1999).
Control of the terminal step of intracellular membrane fusion by protein
phosphatase 1. Science
285,1084
-1087.
Peters, C., Bayer, M. J., Buhler, S., Andersen, J. S., Mann, M. and Mayer, A. (2001). Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion. Nature 409,581 -588.[CrossRef][Medline]
Stack, J. H., Horazdovsky, B. and Emr, S. D. (1995). Receptor-mediated protein sorting to the vacuole in yeast: roles for a protein kinase, a lipid kinase and GTP-binding proteins. Annu. Rev. Cell Dev. Biol. 11, 1-33.[CrossRef][Medline]
Stevens, T. H. and Forgac, M. (1997). Structure, function and regulation of the vacuolar (H+)-ATPase. Annu. Rev. Cell Dev. Biol. 13,779 -808.[CrossRef][Medline]
Ungermann, C., Wickner, W. and Xu, Z. (1999).
Vacuole acidification is required for trans-SNARE pairing, LMA1 release, and
homotypic fusion. Proc. Natl. Acad. Sci. USA
96,11194
-11199.
Vida, T. A. and Emr, S. D. (1995). A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J. Cell Biol. 128,779 -792.[Abstract]
Wada, Y. and Anraku, Y. (1994). Chemiosmotic coupling of ion transport in the yeast vacuole: its role in acidification inside organelles. J. Bioenerg. Biomembr. 26,631 -637.[Medline]
Wada, Y., Ohsumi, Y. and Anraku, Y. (1992). Chloride transport of yeast vacuolar membrane vesicles: a study of in vitro vacuolar acidification. Biochim. Biophys. Acta 1101,296 -302.[Medline]
Wickner, W. (2002). New EMBO member's review:
Yeast vacuoles and membrane fusion pathways. EMBO J.
21,1241
-1247.
Yabe, I., Horiuchi, K., Nakahara, K., Hiyama, T., Yamanaka, T.,
Wang, P. C., Toda, K., Hirata, A., Ohsumi, Y., Hirata, R. et al.
(1999). Patch clamp studies on V-type ATPase of vacuolar
membrane of haploid Saccharomyces cerevisiae. Preparation and
utilization of a giant cell containing a giant vacuole. J. Biol.
Chem. 274,34903
-34910.