* Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755-3844; Georg-August-Universität
Göttingen, Biochemie II, 37073 Göttingen, Germany; § Protein Research Group, Department of Molecular Biology, Odense
University, 5230 Odense M, Denmark; and
University of Oregon, Institute of Molecular Biology, Eugene, Oregon 97405
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Abstract |
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Vacuole SNAREs, including the t-SNAREs Vam3p and Vam7p and the v-SNARE Nyv1p, are found in a multisubunit "cis" complex on isolated organelles. We now identify the v-SNAREs Vti1p and Ykt6p by mass spectrometry as additional components of the immunoisolated vacuolar SNARE complex. Immunodepletion of detergent extracts with anti-Vti1p removes all the Ykt6p that is in a complex with Vam3p, immunodepletion with anti-Ykt6p removes all the Vti1p that is complexed with Vam3p, and immunodepletion with anti-Nyv1p removes all the Ykt6p in complex with other SNAREs, demonstrating that they are all together in the same cis multi-SNARE complex. After priming, which disassembles the cis-SNARE complex, antibodies to any of the five SNARE proteins still inhibit the fusion assay until the docking stage is completed, suggesting that each SNARE plays a role in docking. Furthermore, vti1 temperature-sensitive alleles cause a synthetic fusion-defective phenotype in our reaction. Our data show that vacuole-vacuole fusion requires a cis-SNARE complex of five SNAREs, the t-SNAREs Vam3p and Vam7p and the v-SNAREs Nyv1p, Vti1p, and Ykt6p.
Key words: SNAREs; membrane fusion; yeast vacuoles; NSF; ![]() |
Introduction |
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THE targeting of vesicles to their destination in the
secretory pathway depends on several layers of
specificity. The GTPases of the Rab/Ypt family are
critical for virtually every vesicle trafficking step (Novick
and Zerial, 1997). In addition, membrane proteins with cytosolic coiled-coil domains, termed SNAREs, are found
on vesicles (v-SNAREs) and organelles (t-SNAREs). It
has been proposed that SNAREs act in a lock-key mechanism to specify the docking of vesicles with their target
membrane and even to catalyze their fusion (Söllner et al.,
1993
; Ferro-Novick and Jahn, 1994
; Rothman, 1994
; Hay
and Scheller, 1997
; Weber et al., 1998
; Weis and Scheller,
1998
).
SNAREs are found in multisubunit complexes together
with two soluble proteins, the ATPase NSF and its cofactor -SNAP, on organelles and on vesicle membranes
(Walch-Solimena et al., 1995
; Otto et al., 1997
; Swanton et al.,
1998
; Ungermann et al., 1998a
; Ungermann and Wickner,
1998
). Studies of yeast vacuole fusion have shown that
ATP hydrolysis by yeast NSF (Sec18p) causes release of
yeast
-SNAP (Sec17p) from the membrane (Mayer et al.,
1996
) and disassembly of the SNARE complex (Söllner et al.,
1993
), enabling the individual SNAREs to participate in
the downstream docking reaction (Nichols et al., 1997
; Ungermann et al., 1998a
). Docking of vesicles with their target
membrane also involves tethering by velcro factors and
Rab proteins (Pfeffer, 1996
). In yeast, for example, Uso1p
and the GTPase Ypt1p tether ER-derived vesicles to the
Golgi apparatus before the action of SNAREs (Cao et al., 1998
). Likewise, the mammalian Uso1p homologue p115
interacts with GM130 to promote the fusion of Golgi vesicles after mitosis (Löwe et al., 1998
). Other possible tethering factors include rabaptin 5 (Stenmark et al., 1995
),
Vac1p (Burd et al., 1997
), and EEA1 (Mills et al., 1998
; Simonsen et al., 1998
; Christoforidis et al., 1999
). Recent
studies on the homotypic fusion of yeast vacuoles suggest
that the docking reaction can be subdivided into a reversible tethering reaction mediated by the GTPase Ypt7p and a subsequent pairing of the SNAREs in trans (Ungermann
et al., 1998b
). The mechanism of the final fusion step is still
unclear. SNAREs have been implicated as fusion catalysts
based on their ability to mediate lipid exchange in a reconstituted fusion assay (Weber et al., 1998
). However, trans-SNARE pairs can be disassembled by Sec18p without influencing the fusion rate, suggesting that SNARE pairs
may not be the proximal fusion catalysts (Ungermann et al.,
1998b
). Similarly, the fusion of cortical granules in sea urchin eggs can be preceded by a Ca2+-dependent disassembly of the SNARE complex without affecting the fusion
rate (Coorssen et al., 1998
; Tahara et al., 1998
). Furthermore, yeast vacuoles require Ca2+ and calmodulin and a
phosphatase for the fusion step per se (Conradt et al.,
1992
; Peters and Mayer, 1998
), suggesting that these proteins act after the SNAREs.
We have identified a cis-SNARE complex on the vacuole membrane which contains the t-SNAREs Vam3p
and Vam7p and the v-SNARE Nyv1p as well as Sec17p
(-SNAP), Sec18p (NSF), and LMA1 (Ungermann et al.,
1998a
; Ungermann and Wickner, 1998
; Xu et al., 1998
). We
now show that two additional SNAREs, Vti1p and Ykt6p,
are physically and functionally part of this complex.
Vti1p has been previously characterized as an essential
v-SNARE required for trafficking between the Golgi apparatus and the vacuole (Fischer von Mollard et al., 1997
;
Lupashin et al., 1997
). Ykt6p was initially identified in a
complex with the Golgi t-SNARE Sed5p (Søgaard et al.,
1994
). It does not have a transmembrane domain, but is
prenylated and may partition between the cytosol and membranes (McNew et al., 1997
). Both proteins are essential for
viability, interact genetically, and were suggested to be involved in retrograde trafficking to the cis-Golgi membrane
(Fischer von Mollard et al., 1997
; Lupashin et al., 1997
).
Our data show that these proteins are components of a
heteropentameric SNARE complex and that each of the
subunits has a vital role in homotypic vacuole fusion.
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Materials and Methods |
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Yeast Strains
Temperature-sensitive (ts)1 alleles in VTI1 were introduced into yeast
strains BJ3505 and DKY6281 by transformation and loop in-loop out of
plasmids containing the vti1 ts alleles and a URA3 marker at the VTI1 locus (Fischer von Mollard et al., 1997). Ura+ transformants were selected
and Ura
clones which were generated in a second selection with 5-fluoroorotic acid were tested for loss of the wild-type VTI1 sequences by their ts
growth and CPY-secretion phenotypes (Fischer von Mollard et al., 1997
).
Biochemical Methods
Reagents were as described by Haas (1995), Mayer et al. (1996)
, and Haas
and Wickner (1996)
. SDS-PAGE, immunoblotting using ECL (Haas et al.,
1994
), and purification of IgGs and his6-tagged Sec18p (Haas and Wickner, 1996
) were as described. Rabbit antibodies were generated against
Ni-NTA purified His6-Ykt6 protein and His6-Nyv1p that was overproduced in Escherichia coli. For coimmunoprecipitations, vacuoles were
sedimented (10 min, 8,000 g, 4°C) after any priming reaction with ATP,
washed with 500 µl PS buffer (10 mM Pipes/KOH, pH 6.8, 200 mM sorbitol), and detergent solubilized in 1 ml of buffer A (1% digitonin, 50 mM
NaCl, 20 mM Hepes/KOH, pH 7.4, 2 mM EDTA, 1× PIC [Xu and Wickner, 1996
], 1 mM PMSF, and 10 µg/ml
2-macroglobulin). The detergent
extract was placed onto a nutator for 10 min at 4°C, the insoluble material
was removed by centrifugation (10 min, 16,000 g), and the supernatant
was applied to protein A-immobilized IgGs (Harlow and Lane, 1988
; Ungermann et al., 1998a
). Incubations, washes, and elution of bound proteins
were as described (Ungermann et al., 1998a
).
Purification of the Vam3p Complex
His6-Vam3p was immobilized on Aminolink resin (Pierce) and used as an
affinity matrix to purify antibodies to Vam3p. Affinity-purified antibodies
(200 ng) and an equal amount of nonimmune rabbit IgGs were covalently
linked to 1 ml protein A-Sepharose (Amersham-Pharmacia; Harlow and
Lane, 1988). Vacuoles were prepared by a batch purification. Cells from
6-liter overnight cultures were lysed with oxalyticase and DEAE dextran
as described (Haas, 1995
). After heat shock, cell lysates were chilled on
ice, diluted with 15% Ficoll in PS buffer (200 mM sorbitol, 10 mM Pipes/
KOH, pH 6.8) to 4% Ficoll (final concentration), and transferred to 60Ti
tubes (Beckman). Lysates were centrifuged (50,000 rpm, 4°C, 60 min, 60Ti
rotor) and vacuoles harvested from the top, diluted 20-fold with cold PS
buffer, and centrifuged (JA20, 10,000 rpm, 10 min, 4°C). The vacuole pellet was resuspended in PS buffer.
For purification of the SNARE complex, 26 mg of vacuoles was lysed in
10 ml of 1.5% Triton X-100, PBS (Harlow and Lane, 1988), pH 7.4, 2 mM
EDTA, 1× PIC (Xu and Wickner, 1996
), and 1 mM PMSF (lysis buffer).
After 30 min at 4°C on a nutator, the detergent extract was centrifuged for
30 min in a 60Ti rotor at 4°C, 35,000 rpm. The supernatant was collected
and incubated on a nutator for 1.5 h at 4°C with 3 ml of a protein A resin
bearing nonimmune IgGs. The flow through was collected, reapplied to
fresh resin, and incubated as before. Three such sequential preadsorption
steps were performed. The sample was then halved. One half was applied to a control resin, the other to the immobilized affinity-purified antibodies
to Vam3p. The detergent extracts were incubated with the resins for 18 h
on a nutator at 4°C. The flow throughs were collected and the resins were
washed with 50 ml of 150 mM, 350 mM, and 500 mM NaCl in lysis buffer.
Bound proteins were eluted with 4 ml 0.1 M glycine/HCl, pH 2.6, 0.025%
Triton X-100, precipitated by TCA, washed with 1 ml of ice-cold acetone,
and dried at 56°C for 5 min. Aliquots were analyzed by SDS-PAGE and
Coomassie blue-stained or transferred to nitrocellulose for immunoblotting.
Proteins were identified by comparing their tryptic peptide mass maps
to the Saccharomyces cerevisiae sequence database (Jensen et al., 1998).
Protein bands were excised from the gel, rinsed, and the protein samples
were digested with trypsin in the gel matrix (Shevchenko et al., 1996
). Extracted peptide mixtures were analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (REFLEX;
Bruker Daltonics). The peptide mass maps were used to query a comprehensive sequence database for unambiguous protein identification (PeptideSearch software, provided by M. Mann and P. Mortensen, EMBL)
(Jensen et al., 1996
, 1997
).
Vacuole Fusion
Vacuole fusion is measured by a biochemical complementation assay
(Conradt et al., 1992; Haas et al., 1994
). Vacuoles from DKY6821 have
normal proteases but lack the membrane protein alkaline phosphatase.
Vacuoles from BJ3505 accumulate alkaline phosphatase in the unprocessed and catalytically inactive "pro" form due to the deletion of the
gene encoding the protease Pep4p. Incubation of a mixture of these vacuoles in reaction buffer at 27°C in the presence of cytosol and ATP leads to
fusion, content mixing, and processing of pro-alkaline phosphatase by
Pep4p. The active alkaline phosphatase is measured by a colorimetric assay at the end of the fusion reaction.
Vacuoles (Haas, 1995) were used immediately after isolation. The standard fusion reaction (30 µl) contained 3 µg of each vacuole type (BJ3505
and DKY6281) in reaction buffer (10 mM Pipes/KOH, pH 6.8, 200 mM
sorbitol, 150 mM KCl, 0.5 mM MgCl2, 0.5 mM MnCl2), 0.5 mM ATP, 3 µg/ml
cytosol, 3.5 U/ml creatine kinase, 20 mM creatine phosphate, and a protease inhibitor cocktail (PIC; Xu and Wickner, 1996
) containing 7.5 µM
pefabloc SC, 7.5 ng/ml leupeptin, 3.75 µM o-phenanthroline, and 37.5 ng/ml
pepstatin. To reduce proteolysis in the coimmunoprecipitation experiments, only the protease A-deficient BJ3505 vacuoles were analyzed. One
unit of fusion activity is defined as 1 µmol p-nitrophenol phosphate hydrolyzed per minute and milligram of BJ3505.
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Results |
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Identification of Vti1p and Ykt6p as Part of the Vacuolar SNARE Complex
To identify proteins that interact with the vacuolar t-SNARE
Vam3p, a detergent extract of vacuoles was incubated with
immobilized affinity-purified antibodies to Vam3p or with
a control IgG resin. Retained proteins were eluted from
each column and analyzed by SDS-PAGE and immunoblotting (Fig. 1 A) or Coomassie staining (Fig. 1 B). Vam3p
was solubilized completely under the experimental conditions and was retained specifically on the anti-Vam3p-affinity column (compare flow through and eluate in Fig. 1
A). The Coomassie-stained protein bands (Fig. 1 B) were
identified by peptide mapping by MALDI mass spectrometry combined with sequence database searching (Jensen
et al., 1998). In addition to Vam3p, Vam7p, and Sec17p, two additional SNARE proteins were specifically eluted
from the anti-Vam3p column and identified by MALDI
mass spectrometry: Vti1p (Fig. 1 C; Fischer von Mollard et
al., 1997
), an essential v-SNARE implicated in Golgi to
vacuole trafficking, and Ykt6p (Fig. 1 D), a v-SNARE previously implicated in trafficking through the Golgi (Søgaard et al., 1994
; McNew et al., 1997
). The vacuole cis-SNARE complex is neither SDS resistant nor as stable as
the exocytic complex in neurons (Hayashi et al., 1994
,
1995
; Otto et al., 1997
). Thus, washing the column in high
salt removed a substantial amount of Nyv1p, though its
presence in the cis-SNARE complex was confirmed by immunoblotting (data not shown). Both Vti1p and Ykt6p
were found in substoichiometric amounts, consistent with
their association to Vam3p being salt-sensitive (data not shown). Because of the lability of this cis-SNARE complex during immunoisolation, we cannot be sure that we
have identified all its constituents, and the isolation of
functional cis-SNARE complex for analysis in a reconstituted fusion assay (Sato and Wickner, 1998
) may reveal
additional components.
|
Though Vti1p was found previously in a complex with
Vam3p (Holthuis et al., 1998), Ykt6p has so far only been
described as a Golgi-specific SNARE (Søgaard et al.,
1994
; Lupashin et al., 1997
; McNew et al., 1997
). We therefore used coimmunoprecipitation to test whether antibodies to each protein would immunoprecipitate the vacuolar
cis-SNARE complex in a manner similar to antibodies to
Vam3p. This was indeed the case (Fig. 2 A, lanes 1, 3, and 5). Upon ATP/Sec18p/Sec17p-dependent priming, the cis-SNARE complex disassembled (lanes 2, 4, and 6) and
cross-immunoprecipitation was lost. While each of the
three antibodies immunoprecipitated a disproportionate
amount of its cognate protein, reflecting partial SNARE
complex disassembly or in vitro lability, each also precipitated the other SNARE complex components (Nyv1p,
Vam7p, and Sec17p) in a similar proportion. The composition of the SNARE complex was also examined by an independent approach. All members of the SNARE complex cosediment in a glycerol velocity gradient, suggesting that they are largely in a complex with each other (Fig. 2
B). Vti1p and Ykt6p do not completely copurify with
Vam3p, in agreement with their role in other trafficking
reactions (Fig. 2 C; Fischer von Mollard et al., 1997
; Lupashin et al., 1997
). They are both present in a complex with
Vam3p on the vacuole membrane and behave similarly to
previously identified members of the vacuolar SNARE complex. The localization of Vti1p and Ykt6p to the vacuole does not depend on previously characterized members
of the SNARE complex (Fig. 2 C).
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To determine directly whether Vti1p, Nyv1p, and Ykt6p are in the same SNARE complex with each other and with the other SNAREs, successive immunoprecipitations were performed (Fig. 3). The same SNAREs were recovered in immunoprecipitates with antibodies to Vam3p (Fig. 3, lane 1), Vti1p (lane 2), Ykt6p (lane 4), or Nyv1p (lane 7). Some Ykt6p remained in the supernatant after immunoprecipitation with antibody to Vti1p, but it was not in complex with Vam3p or Vam7p (lane 3). Similarly, some Vti1p remained in the supernatant after immunoprecipitation with antibody to Ykt6p, but it was not in complex with Vam3p or Vam7p (lane 5). Immunoprecipitation with antibody to Nyv1p (lane 6) removed almost all the Ykt6p, leaving only a small amount that is not in complex with other SNAREs (lane 7). We conclude that the cis-SNARE complex contains Vam3p, Vam7p, Vti1p, Nyv1p, and Ykt6p and is at least pentameric for SNAREs.
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Vti1p and Ykt6p Are Required for Homotypic Vacuole Fusion
The above physical data establish that Vtilp and Ykt6p are
present in a cis-complex with Vam3p, Nyvlp, and Vam7p.
However, we have noted (Ungermann et al., 1998b) that
only a small percentage of the SNAREs enters the trans-complex, and thus functional criteria are needed to establish that each SNARE is part of a functional complex.
Such experiments address the possibility that a small percentage of the SNAREs in a tetrameric complex might be
the active complex for fusion, while the fifth SNARE was
present but irrelevant for function. Three lines of evidence
show that Vti1p is directly involved in the fusion reaction.
First, antibodies to Vti1p inhibit fusion to a similar extent
as Vam3p antibodies when added at different times to an
ongoing reaction (Fig. 4 A). Antibodies to Vti1p, Sec17p,
or Vam3p were added to aliquots from a fusion reaction at
several times and fusion was continued for a total of 90 min at 27°C (Fig. 4 A). All antibodies inhibited the reaction thoroughly when added at the beginning. Whereas
Sec17p completes its action during the early priming step
(Mayer et al., 1996
), Vam3p acts at a later docking stage
and thus antibodies to Vam3p inhibit at later times (Nichols et al., 1997
; Ungermann et al., 1998a
). Antibodies to
Vti1p inhibit the reaction in a kinetic fashion similar to
Vam3p antibodies, suggesting that they may act at the
same reaction. Second, Vti1p function depends on vacuole
mixing. Vacuoles from the two tester strains were incubated in the presence of ATP in separate tubes. At the indicated times, aliquots from each tube were mixed in the
absence or presence of antibodies to Vam3p, Vti1p, or
Sec17p. Of these three proteins, only Sec17p function can
be fulfilled early in the reaction (Fig. 4 B; Mayer et al.,
1996
). The reaction remains sensitive to antibodies to
Vam3p and Vti1p, showing that the completion of the
function of these proteins depends on vacuole contact.
Third, previously characterized vti1 ts alleles (Fischer von
Mollard et al., 1997
) were introduced into the tester strains
and analyzed in the fusion reaction. Vacuoles were purified from all six wild-type and vti1 ts mutant strains and
tested in all combinations. To induce the phenotype of the
ts allele, vacuoles were mixed and preincubated at the indicated temperatures without ATP for the times shown. The ts alleles are much more thermolabile in the protease-plus DKY background than in the protease-minus BJ vacuoles, perhaps because partially thermally altered mutant
Vtilp is more susceptible to proteolysis. When combined
with a wild-type partner, only the ts alleles in the DKY
background show a ts phenotype which is strongly induced
at elevated temperatures. Strikingly, combination of vacuoles with vti1 ts alleles leads to a synthetic fusion phenotype, as even vacuoles that were only preincubated on ice
retained only 5-10% fusion activity (DKY vti1-1/BJ vti1-2
and DKY vti1-2/BJ vti1-2). However, both vacuole partners show up to 60% fusion with the wild-type partner.
Though some of the effects observed may be due to enhanced protease sensitivity of the ts alleles of Vti1p, the
synthetic fusion phenotype strongly implies that Vti1p is
directly involved in the reaction, as priming (as judged by
Sec17p release and SNARE complex disassembly) is not
altered after induction of the ts phenotype (not shown).
Finally, Ykt6p antibodies inhibit with similar kinetics as
Vti1p antibodies when added to a fusion reaction (Fig. 4
D) and acquisition of resistance to antibodies to Ykt6p requires docking (Fig. 4 E).
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Thus, three v-SNAREs, Nyv1p, Vti1p, and Ykt6p, are required for the docking stage of vacuole-vacuole fusion. Since they are dissociated from the cis-SNARE complex during priming, these data suggest that inhibition by antibody to each SNARE is not due to steric hindrance of access to another SNARE. Rather, these data indicate that each SNARE has a functional role in the reaction. The complete sensitivity of fusion to antibodies to Vti1p and Ykt6p, or to ts alleles in Vti1p, suggests that each of these proteins is fully involved in the reaction rather than being in redundant tetrameric complexes of Vam3p/Vam7p/ Nyv1p/Vti1p and Vam3p/Vam7p/Nyv1p/Ykt6p.
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Discussion |
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Vti1p and Ykt6p are components of the vacuolar SNARE
complex, as both proteins copurify with the SNARE
complex and antibodies to both Vti1p and Ykt6p precipitate all the previously identified members of this complex.
This does not simply reflect an exchange of SNAREs
from other contaminating organelles into association with
Vam3p in detergent extracts, as inhibition studies with antibodies to Vti1p and Ykt6p and the synthetic fusion phenotype of ts alleles in Vti1p in our tester vacuoles indicate
that both proteins are part of a SNARE complex with a
functional role in the fusion reaction. Kinetic inhibition
curves with antibodies to Vti1p and Ykt6p are indistinguishable from those reported for antibodies to the previously identified vacuolar SNAREs Vam3p, Vam7p, and
Nyv1p (Fig. 3; Nichols et al., 1997; Ungermann et al.,
1998a
; Ungermann and Wickner, 1998
).
Our data establish that each of these SNAREs
Vam7p, Nyv1p, Vtilp, and Ykt6p
has a role in the reaction, though these roles need not be unique. Before priming, the effects of ts mutants (Fig. 4) or of deleting
SNAREs (Nichols et al., 1997
; Ungermann and Wickner,
1998
) could be due to allosteric effects on neighboring SNARE complex subunits. Similarly, antibodies which
bind to one SNARE could inactivate the function of a
pentameric cis-SNARE complex by obstructing access of a
crucial protein or ligand to another SNARE. However,
these concerns are vitiated by the observation that all
SNAREs are disassembled from the complex during ATP-dependent priming (Fig. 2 A) and thus are not associated
during docking while the reaction remains sensitive to
each anti-SNARE antibody during docking (Nichols et al.,
1997
; Ungermann et al., 1998a
; Ungermann and Wickner,
1998
; Fig. 4, A and D). The sensitivities to each of these
antibodies is a strong argument that each subunit of the
pentameric cis-SNARE complex has some role in the
overall reaction.
Our data suggest that three v-SNAREs, Nyv1p, Vti1p,
and Ykt6p, participate in the fusion reaction. This is not
without precedent as Vti1p has been recovered in a complex with Vam3p (Holthuis et al., 1998) and Ykt6p has
been shown to be a weak multicopy suppressor of Vti1p
(Lupashin et al., 1997
). What could be the role of three
v-SNAREs in the vacuole fusion reaction? The resolution of the crystal structure of the neuronal SNARE complex
(Sutton et al., 1998
) and the analysis of the exocytic
SNARE complex in yeast (Katz et al., 1998
) and neurons
(Poirier et al., 1998
) have led to the proposal that the core
of a SNARE complex consists of four parallel coiled-coil
domains provided by three proteins: syntaxin, synaptobrevin, and SNAP-25 and their homologues. The alignment of
all SNAREs at their coiled-coil domains identifies a conserved glutamine (Q) in one set of SNAREs (mainly
t-SNAREs and some v-SNAREs like Bet1p and Vti1p)
and a conserved arginine (R) in another set (most v-SNAREs
including Nyv1p and Ykt6p; Fasshauer et al., 1998
). Based
on these findings, Fasshauer et al. (1998)
propose that
each SNARE complex consists of three Q-SNARE coiled-coils (e.g., one from syntaxin, and two from SNAP25) and
one R-SNARE coiled-coil (e.g., one from synaptobrevin;
Sutton et al., 1998
). How does this compare to data for
the vacuolar SNARE complex? We already know of
five SNAREs in our complex, the t-SNARE Vam3p (or
Q-SNARE), the SNAP-25/23 homologue Vam7p (Q), and
the v-SNAREs Vti1p (Q), Nyv1p (R), and Ykt6p (R).
Vam3p and Vam7p are found in a tight complex on the
vacuole (Sato et al., 1998
; Ungermann and Wickner, 1998
).
Whereas SNAP-25 provides two coiled-coil domains to
the neuronal SNARE complex, Vam7p provides only one
(Weimbs et al., 1997
). The third Q-SNARE coiled-coil
could therefore come from Vti1p, which has been previously considered a v-SNARE. Either Nyv1p or Ykt6p
would then be the required R-SNARE. However, both proteins are part of the same cis-SNARE complex (Fig. 3)
and antibodies to either protein inhibit the fusion reaction
(Fig. 4, D and E; Ungermann et al., 1998a
). Furthermore,
vacuoles lacking Nyv1p fuse only poorly, if at all, with
each other (Nichols et al., 1997
), suggesting an essential
role of Nyv1p in the fusion reaction. In fact, Nyv1p is not
required for any of the trafficking reactions to the vacuole
(Fischer von Mollard and Stevens, 1999
), but appears to
be exclusively reserved for vacuole fusion. Thus, at least portions of the intracellular pool of all five of these
SNAREs are in a complex with each other, which may define a new, five coiled-coil core of a SNARE complex.
Not all of the vacuolar SNAREs are recovered in a cis
complex. The proportion is highest with salt-washed vacuoles, possibly due to removal of Sec18p (not shown). This
might reflect the lability of the complex or, alternatively,
that only some of the SNAREs are complexed and a second population may exist in an uncomplexed form or in
a complex with unidentified proteins. Vacuoles without Vam3p or Vam7p have no cis-SNARE complex and yet
are still capable of fusion at a measurable rate (Ungermann et al., 1998a; Ungermann and Wickner, 1998
). Furthermore, vacuoles without Vam3p do not need priming
by Sec17p/Sec18p/ATP (Ungermann et al., 1998a
), which suggests that SNAREs that are not in a cis complex can
also participate in the homotypic fusion reaction. We have
shown by deletion analysis, antibody inhibition, and the
generation of ts alleles that each of the subunits has a critical role for the fusion reaction and that a complex of all
SNAREs exists on the vacuole (Nichols et al., 1997
; Ungermann et al., 1998a
; Ungermann and Wickner, 1998
; this
study). However, we do not know whether the separate
SNAREs or the cis-SNARE complex have distinct roles or
specific activities. Previous work has shown that a detergent extract which was immunodepleted of SNAREs
can be reactivated by addition of a 200-fold purified v-t-SNARE complex (Sato and Wickner, 1998
). Future
work will be necessary to establish the stoichiometry and
functional roles of the five SNAREs during this reconstitution reaction.
Finding a role for Vti1p and Ykt6p in the vacuole-vacuole fusion reaction adds to a long list of trafficking reactions in which these proteins have been implicated (Fischer von Mollard et al., 1997; Lupashin et al., 1997
;
McNew et al., 1997
; Holthuis et al., 1998
). Ykt6p is unusual as a v-SNARE in that it is prenylated and appears to
partition between cytosol and membranes (McNew et al.,
1997
). Subcellular localization of Ykt6p has therefore been difficult. Although Ykt6p was initially identified in a
complex with the Golgi t-SNARE Sed5p (Søgaard et al.,
1994
), and may participate in trafficking between the ER
and Golgi membranes (McNew et al., 1997
), we find a significant portion of Ykt6p on the vacuole, suggesting a vital
role for this protein in vacuole function. Vti1p has been recovered in complexes with organellar t-SNAREs along the
secretory pathway: with Sed5p, the Golgi t-SNARE, with Pep12p, the endosomal t-SNARE, and with Vam3p (Fischer von Mollard et al., 1997
; Holthuis et al., 1998
;
this study). Because of their interactions with multiple
t-SNAREs, Vti1p, and Ykt6p cannot be the sole determinants of specificity in vesicular traffic. These proteins are
likely to be involved in a retrieval and recycling of trafficking factors from late organelles to, for example, the Golgi
apparatus (Fischer von Mollard et al., 1997
; Lupashin et al., 1997
; Bryant et al., 1998
). Two other v-SNAREs have
been implicated in retrograde trafficking reactions in
yeast: Sft1p in retrograde transport within the Golgi stack
(Banfield et al., 1995
), and Sec22p for the trafficking of
vesicles from the Golgi apparatus back to the ER (Spang
and Scheckman, 1998
).
The vacuolar t-SNARE Vam3p has a fundamental role
in several trafficking reactions. It has been implicated in
trafficking from the endosome to the vacuole (Darsow et al.,
1997; Götte and Gallwitz, 1997
), in the trafficking of AP3-dependent Golgi-derived vesicles to the vacuole (Cowles
et al., 1997
; Piper et al., 1997
), in aminopeptidase I transport to the vacuole and autophagocytosis (Darsow et al.,
1997
), and in homotypic vacuole fusion as the final step of
the inheritance of this organelle (Nichols et al., 1997
). Deletion of Vam3p results in a clear delay of protein trafficking to the vacuole (Darsow et al., 1997
; Nichols et al., 1997
;
Piper et al., 1997
; Wada et al., 1997
; Srivastava and Jones,
1998
). However, vam3
vacuoles can be purified by the
same floatation protocol as for wild-type vacuoles, albeit
at somewhat lower yield. These vacuoles contain all vacuolar marker proteins at the same steady-state concentration (Nichols et al., 1997
; Ungermann et al., 1998a
, Ungermann and Wickner, 1998
; Stefan and Blumer, 1999
), they
fuse with wild-type vacuoles with similar kinetics, and they show the same sensitivities to inhibitors of fusion as wild-type vacuoles (Nichols et al., 1997
; Ungermann et al.,
1998b
). Though vam3
vacuoles are fragmented and of
much smaller size (Darsow et al., 1997
; Nichols et al., 1997
;
Wada et al., 1997
), their normal protein content and behavior in the vacuole fusion reaction classifies them as vacuoles. This suggests that delivery of proteins to the vacuole, even if slow or of limited efficiency, can occur in a
Vam3p-independent fashion and raises the question of
how the t-SNARE requirement can be bypassed. The requirement for the vacuole SNARE complex in several reactions implies that other factors are required to add specificity to these trafficking reactions. Defining these factors
and their functions may contribute to the understanding of
how trafficking to and from this organelle is specified.
![]() |
Footnotes |
---|
Address correspondence to William Wickner, Department of Biochemistry, Dartmouth Medical School, 7200 Vail Building, Hanover, NH 03755-3844. Tel.: (603) 650-1701. Fax: (603) 650-1353. E-mail: william. wickner{at}dartmouth.edu
Received for publication 12 March 1999 and in revised form 3 May 1999.
C. Ungermann's present address is Biochemie Zentrum Heidelberg
(BZH), Universität Heidelberg, Im Neuenheimer Feld 326, 69120 Heidelberg, Germany.
We thank Drs. James McNew and James Rothman for providing an antiserum to Ykt6p, used in initial studies, Søren Andersen for technical assistance with sample preparation for mass spectrometry; and G. Eitzen, K. Sato, A. Price, and Z. Xu for advice and critical comments on the manuscript.
This work was supported by grants from the National Institute of General Medical Sciences to the labs of W. Wickner (GM23377) and T. Stevens (GM32448), and from the Deutsche Forschungsgemeinschaft to C. Ungermann.
![]() |
Abbreviations used in this paper |
---|
MALDI, matrix-assisted laser desorption/ionization; ts, temperature-sensitive.
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References |
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