From the Interdisziplinäres Zentrum für
Neurowissenschaften (IZN), University of Heidelberg, Im
Neuenheimer Feld 307, 69120 Heidelberg, Germany, the
§ Biochemie-Zentrum Heidelberg (BZH), University of
Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany, and the
¶ Lehrstuhl für Chemie der Biopolymere, Technische
Universität München, Weihenstephaner Berg 3, 85345 Freising, Germany
Received for publication, September 17, 2002
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ABSTRACT |
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It is presently not clear how the function of
SNARE proteins is affected by their transmembrane domains. Here, we
analyzed the role of the transmembrane domain of the vacuolar SNARE
Vam3 by replacing it by a lipid anchor. Vacuoles with mutant Vam3 fuse poorly and have increased amounts of cis-SNARE complexes,
indicating that they are more stable. As a consequence efficient
cis-SNARE complex disassembly that occurs at priming as a
prerequisite of fusion requires addition of exogenous Sec18.
trans-SNARE complexes in this mutant accumulate up to
4-fold over wild type, suggesting that the transmembrane domain of Vam3
is required to transit through this step. Finally, palmitoylation of
Vac8, a reaction that also occurs early during priming is reduced by
almost one-half. Since palmitoylated Vac8 is required beyond
trans-SNARE complex formation, this may partially explain
the fusion deficiency.
Membrane fusion along the secretory pathway requires the
specific interaction of
SNAREs1 that are localized to
vesicles and target organelles (1). Fusion reactions are controlled by
Sec18/NSF and t-SNAREs, also termed syntaxins, consist of three domains: the
regulatory N terminus, the coiled-coil domain required for SNARE
complex formation (7-11), and the transmembrane domain (TMD), by which
most SNAREs are anchored to membranes (12). Transmembrane domains of
SNAREs are important for SNARE function. Alterations in synaptobrevin
or syntaxin TMDs in Caenorhabditis elegans cause strong
neurotransmission defects (13). C. elegans and vertebrates contain syntaxin isoforms that differ only by their TMDs (14-16). Furthermore, TMDs of syntaxin 1A and synaptobrevin II drive homo- as
well as heterodimerization (17-19). Moreover, the interaction of
syntaxin 1A with synaptobrevin, but not SNAP-25, depends on the TMD of
the t-SNARE (20). In addition, in vitro fusion of liposomes
requires anchoring of SNAREs via transmembrane domains (21) and tight
coupling between the coiled-coil and the TMD (22). Replacing the TMDs
of exocytic SNAREs in yeast arrested secretion (23). Interestingly, TMD
peptides alone are sufficient to drive fusion of liposomes (24),
suggesting that they constitute autonomous fusogenic domains. However,
the evidence suggesting that SNAREs themselves may act as catalysts
relies mostly on minimal systems. It does not exclude the possibility
that SNAREs regulate other factors that could act as fusogens on
cellular membranes or enhance SNARE-mediated fusion (25-27).
Yeast vacuole fusion occurs in a cascade of priming, docking, and
fusion (28). Five SNAREs, the t-SNARE-like proteins Vam3, Vam7, and
Vti1, and the v-SNAREs Nyv1 and Ykt6 are found in the vacuolar
cis-SNARE complex (29). Vam3, Vti1, and Nyv1 carry TMDs,
Ykt6 is anchored via a prenyl anchor, and Vam7 binds to membranes via
its PX domain (28, 30, 31). During priming, Sec18 and Sec17 disassemble
cis-SNARE complexes in an ATP-dependent manner.
Docking requires the Rab GTPase Ypt7, the HOPS tethering complex, and
two Rho GTPases, Rho1 and Cdc42 (32-34), as well as the Vtc complex
(35) and remodeling of actin (36). This is followed by the formation of
trans-SNARE complexes (26). The final step of yeast vacuole
fusion requires protein phosphatase 1, calmodulin, the proteolipid (V0
sector) of the vacuolar ATPase (37-39), and the palmitoylated fusion
factor Vac8 (40-46). Acylation of Vac8 is an essential subreaction of
vacuole fusion and occurs at priming (44). Although Vac8 functions
after trans-SNARE pairing (42), its acylation, a requirement
for the function of Vac8, occurs already at priming. Due to its
acylation Vac8 therefore connects priming and fusion.
Here, we analyzed the function of the TMD of the t-SNARE Vam3. For this
we replaced the TMD with a prenyl anchor and determined the
consequences of the TMD alteration for fusion activity and for defined
SNARE complex formation at substeps of fusion. We show that the TMD is
essential for cis-SNARE complex function and for the
transition from trans-SNARE complex formation to full fusion.
Reagents and Strains--
Reagents were purchased from Sigma,
unless stated otherwise. All VAM3 constructs were generated
using site-directed oligonucleotide mutagenesis (47) on a
single-stranded template of pRS406-VAM3 (T7 Mutagene kit, Bio-Rad). All
mutations were verified by dideoxynucleotide chain-termination
sequencing. Plasmids carrying the VAM3 promotor and open
reading frame with the respective mutations and the
URA3 marker were digested with BstBI and
transformed into BJ3505vam3 Vacuole Fusion Assay--
Vacuoles are prepared from BJ3505 and
DKY6281 strains as described previously (48). Fusion is measured by a
biochemical complementation assay. BJ3505 vacuoles contain pro-alkaline
phosphatase due to a lack of the processing protease Pep4p. Vacuoles
from DKY6281 lack alkaline phosphatase. After fusion of the membranes, Pep4p matures pro-alkaline phosphatase to the catalytically active protein, which can be assayed spectrophotometrically (48).
Standard fusion reactions were performed with 3 µg of each vacuole
type in reaction buffer (5 mM MgCl2, 125 mM KCl, 20 mM PIPES/KOH, pH 6.8, and 200 mM sorbitol) supplemented with an ATP regenerating system
(0.5 mM ATP, 40 mM creatine phosphate, 0.1 mg/ml creatine kinase), 10 µM CoA, cytosol (0.5 µg/µl), His-Sec18 (200 ng/ml), and a protease inhibitor mixture
(49) in a 30-µl volume.
Coimmunoprecipitation--
After the fusion reaction, vacuoles
were pelleted (20,000 × g, 5 min, 4 °C), washed
once with 1 ml of reaction buffer containing 150 mM KCl,
and reisolated as before. Vacuoles were detergent-solubilized in 1 ml
of lysis buffer (20 mM Tris/HCl, pH 7.4, 150 mM
NaCl, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl
fluoride, 0.4 × protease inhibitor mixture, 10 µg/ml
Functional Characterization of Vam3 Mutants--
To analyze the
role of the TMD of the SNARE Vam3 in vacuole fusion, we replaced it by
the isoprenylation sequence of Ykt6 (51). As a control, we inactivated
the coiled-coil domain (231-250) by mutating five critical residues
(Fig. 1A).
We first determined whether lipid anchoring or coiled-coil mutations
affected vacuole morphology (Fig. 1B). As expected,
Vam3-(231-250) cells had fragmented vacuoles, suggesting an
inactivation of Vam3 function (Fig. 1B, panels c
and d; Ref. 52). In contrast, vacuoles with lipid-anchored
Vam3 were as wild-type vacuoles, suggesting functionality in
vivo. Second, we asked whether the lack of a transmembrane domain
would influence fusion efficiency. Interestingly, fusion of Vam3-CCIIM
vacuoles is strongly reduced in an in vitro fusion assay
(see "Experimental Procedures"; Ref. 48). Since Vam3 is essential
for homotypic vacuole fusion (53), vacuoles deleted for Vam3 or
carrying the coiled-coil mutant fused poorly (Fig.
2A). Vam3-CCIIM was expressed
and localized to vacuoles comparable with wild-type Vam3 and was not
recovered from other compartments or from the cytosol of the yeast cell
(Fig. 2, B and C). Thus, sorting and membrane
attachment did not depend on the presence of the TMD, but presumably on
the previously defined dileucine motif within the cytoplasmic domain
(54).
Analysis of cis- and trans-SNARE Complexes--
We then questioned
whether removal of the Vam3 TMD had affected the composition of
cis-SNARE complexes. Both wild-type and Vam3-CCIIM vacuoles
contained comparable amount of vacuolar SNAREs (not shown), indicating
that sorting of other membrane proteins to vacuoles with Vam3-CCIIM was
not impaired. cis-SNARE complexes were analyzed by
immunoprecipitation with antibodies to two subunits of the vacuolar
t-SNAREs Vti1 and Vam3 (55). Comparable results were obtained with
either antibody, indicating that they are indeed part of the same
complex (see below). Similar amounts of Nyv1 were isolated with Vam3 by
coimmunoprecipiation, regardless of the vacuole type (Fig.
3A, lanes 1 and
5). However, Ykt6 and Vti1 were clearly enriched in SNARE
complexes from Vam3-CCIIM vacuoles (Fig. 3A, lanes
1 and 5), suggesting that the SNARE complex becomes more stable or is altered in its composition in the absence of the
Vam3-TMD. Fewer complexes were observed for the coiled-coil mutant of
Vam3, as reported previously (not shown; Ref. 52).
To analyze the consequences of the altered SNARE complexes we
investigated Sec18/ATP-dependent priming that causes
disassembly of SNARE complexes as shown previously (50). Disassembly
was most efficient for the coiled-coil mutant (not shown). While
wild-type SNARE complexes also readily dissociated when vacuoles were
preincubated with ATP only (Fig. 3A, lanes 1 and
2), additional Sec18 was required to cause partial
disassembly of Vam3-CCIIM containing cis-SNARE complexes as
indicated by reduced coimmunoprecipitation of Vti1 and Ykt6 with Vam3
(compare lanes 6 and 7).
We therefore asked whether vacuoles carrying Vam3-CCIIM required Sec18
also for fusion. In all previous fusion experiments we routinely added
recombinant Sec18 to the reactions. When we omitted Sec18, vacuoles
carrying Vam3-CCIIM were completely inactive, but fused as soon as
recombinant Sec18 was included (Fig. 3B, black
bars). This dependence on exogenous Sec18 is not due to a lack of
endogenous Sec18 on vacuoles (Fig. 3C). Thus, the absence of
the Vam3 TMD causes enrichment of more stable cis-SNARE
complexes that require higher Sec18 concentrations for activation.
Even more striking in contrast to wild-type SNARE complexes, where all
SNAREs separated from Vam3 during priming, Nyv1 associated more tightly
with Vam3-CCIIM when vacuoles were preincubated with Sec18 and ATP. One
possibility was an association of Nyv1 with Vam3-CCIIM in
trans between vacuoles. We used inhibitors to analyze this
in more detail. The docking inhibitor Gdi1 extracts the Rab GTPase Ypt7
and blocks vacuole-vacuole contact (56, 57). If the interaction of the
t-SNARE complex (which contains Vam3 and Vti1) and the v-SNARE Nyv1
would indeed be a result of efficient trans-SNARE complex
formation, Gdi1 addition to the priming reaction should block this
association. Indeed, Nyv1 was completely displaced from Vti1 if Gdi1
had been added to the preincubation of Vam3-CCIIM vacuoles (Fig.
3D, lane 8). This suggested the poor disassembly observed for Vti1 and Vam3-CCIIM in Fig. 3A could also
include efficient reassembly of trans-SNARE complexes. In
agreement with this assumption, addition of the late fusion inhibitor
BAPTA that allows vacuole docking (37) to the CCIIM vacuoles did not
influence the association of Nyv1 with Vti1 (Fig. 3D,
lanes 7 and 8). Similar results were obtained
when SNARE complexes were isolated with anti-Vam3 antibodies (not
shown). Thus, the association of Nyv1 and Vti1 occurred after docking
and could reflect increased trans-SNARE complex formation.
To confirm that replacement of the TMD of Vam3 by a lipid anchor
supports the formation of trans-SNARE complexes, we employed an established assay to measure trans-SNARE complexes in the
fusion reaction (26). In brief, we fused vacuoles from one strain
deleted for Vam3 with vacuoles isolated from a nyv1
Replacing wild-type Vam3 by Vam3-CCIIM in the nyv1
trans-SNARE complexes were analyzed by incubating vacuoles
from vam3
Interestingly, even though trans-SNARE complexes
accumulated, we did not see an effect on the fusion kinetics (not
shown). Acquisition of resistances to priming or docking inhibitors was similar for wild-type and CCIIM mutant vacuoles, although the overall
signal of Vam3-CCIIM vacuoles is reduced 5-fold (Fig. 6, A and B, and
Fig. 2A). Thus, fusion must have been blocked at a late
stage. The palmitoylation of Vac8 occurs early during vacuole fusion
(44), but acylated Vac8 is required late in the fusion reaction,
presumably after trans-SNARE pairing (42). Since a portion
of Vac8 is associated with the SNARE complex and palmitoylation
requires Sec18 (44), a defect in Vac8 acylation could explain why
fusion is reduced and how this could be triggered by a priming defect.
When vacuoles were labeled with [3H]palmitate, Vac8,
being present equally on both vacuole types, was reproducibly labeled
almost twice as efficient on wild-type than on Vam3-CCIIM vacuoles
(Fig. 6C). Since palmitoylated Vac8 is required in a
reaction after formation of trans-SNARE pairing (42), this
45% reduction could contribute to the reduced fusion observed.
Our data shed light on the possible function of a SNARE TMD during
an authentic fusion reaction in a eukaryotic cell. Three stages of
vacuole fusion are clearly affected by alterations in the TMD: the
dissociation of cis-SNARE complexes during the priming step,
trans-SNARE pairing, and palmitoylation of the fusion factor Vac8.
cis-SNARE complexes are altered and more stable in the
absence of the Vam3 TMD. This is most obvious for the interaction of Vam3-CCIIM with Vti1 and Ykt6. Addition of exogenous Sec18 is essential
to obtain a low level of fusion of vacuoles containing Vam3-CCIIM.
In agreement with this, increased Sec18 concentrations are also
required to disassemble stable cis-SNARE complexes. Removal of the TMD of Vam3 might influence the conformation of the cytoplasmic domain and thus the interaction with Vti1 and Ykt6 (2, 20), thereby
interfering with priming. In addition, it is possible that the altered
cis-SNARE complex may lack certain factors necessary for
efficient priming. We did, however, not detect reduced levels of Sec18,
nor of the other SNAREs or of Sec17 (not shown). It is noteworthy that
a similar priming defect is also observed on vacuoles lacking subunits
of the Vtc-complex, a membrane integral protein complex that associates
with V0 and with the v-SNARE Nyv1 (35). Vacuoles from
vtc1 Vacuoles containing Vam3-CCIIM appeared intact in vivo and
had a residual fusion activity of ~20-30% in vitro. This
activity may suffice in vivo to maintain vacuolar structure.
Deletion of the vacuolar v-SNARE Nyv1 alone does not lead to vacuolar
fragmentation (53), suggesting partial redundancy with another SNARE
in vivo. However, deletion of Nyv1 in Vam3-CCIIM cells did
cause a synthetic phenotype and resulted in vacuolar fragmentation.
This demonstrates on the one hand that Nyv1 is involved in vacuolar
fusion also in vivo. On the other hand it confirms that
Vam3-CCIIM is not fully functional in vivo. It is important
to note that vacuoles contain three t-SNAREs, Vti1, Vam7 and Vam3, of
which Vti1 and Vam3 have TMDs. We thus show that a single SNARE TMD per
t-SNARE complex is not sufficient to drive homotypic vacuole fusion.
Our data confirm previous observations in several fusion systems that
SNAREs depend on an intact coiled-coil domain to function in fusion
(4). The observed fusion defect of the coiled-coil mutant in vacuole
fusion as also observed by Wang et al. (52) is a direct
result of the poor assembly of trans-SNARE complexes. This
straightforward explanation does not hold for the fusion defect
observed with lipid-anchored Vam3. Here, trans-SNARE
complexes containing Vam3-CCIIM accumulated to about 4-fold higher
levels than in the corresponding wild type, although fusion between
Vam3-CCIIM vacuoles was strongly reduced. We conclude that the
formation of the trans complex during docking does not
automatically induce fusion. trans-SNARE complexes may not
be required throughout fusion. This observation is in contrast to a
requirement of SNAREs to induce fusion of liposomes (5, 6), but agrees
with findings that trans-SNARE complexes are dispensable for
the transition from docking to fusion during homotypic vacuole fusion
and cortical vesicle exocytosis (25, 26). In fact,
trans-SNARE complexes form during the fusion reaction and
can be detected, even if fusion is blocked by fusion inhibitors (this
study; Refs. 26 and 58). In addition, the TMD may play a functional
role downstream of docking. Previously, it was reported that SNAREs
with long, but not with short, prenyl anchors induced fusion in the
reconstituted proteoliposome system (21). This prompted the suggestion
that bilayer-spanning membrane anchors of SNAREs present on both
membranes stress lipid bilayers sufficiently upon
trans-SNARE pairing to induce fusion (21). A caveat is the
instability of the proteoliposomes used as they could not sequester
5-kDa oligonucleotides (21); this indicated a severely disturbed
bilayer structure and question the validity of this approach. It is
possible that the SNARE TMD is required at the final step of membrane
fusion, i.e. at the stage of actual lipid merger. Indeed,
Grote et al. (23) observed that replacement of the TMDs of
other yeast SNAREs (Snc2p or Sso2p) by prenyl anchors also affected
membrane fusion. Lysolipid addition to cells partially rescued this
defect, possibly to overcome a stage of exocytosis arrested at
hemifusion. Structurally, TMD-TMD interactions may drive fusion by
supporting and finishing the "zippering up" of the coiled-coil
domains (59) into the lipid bilayer itself (18, 19). Alternatively,
conformational flexibility of SNARE TMDs may support fusion. In fact,
liposomes fuse efficiently when just TMD peptides corresponding to
synaptic SNAREs or viral fusion proteins were incorporated into the
bilayer (24, 60). Since sequence variants displaying more stable
Finally, the Vam3 TMD could be critical to direct or bind to other
proteins that function after docking. In fact, several factors have
been reported that act downstream of the SNAREs in yeast vacuole
fusion, including the protein phosphatase 1, calmodulin, a subunit of
the Vtc complex, the V0 proteolipid of the vacuolar ATPase (35,
37-39), and Vac8 (41-46, 61). Vac8 becomes palmitoylated coincident
with priming and requires Sec18 (44). The level of Vac8 palmitoylation
was reduced on vacuoles containing the Vam3-CCIIM mutant. The acylated
Vac8 is, however, required after trans-SNARE pairing (42),
possibly to coordinate proteins at the fusion site. It may thus couple
priming and fusion. Even though its function is poorly understood it is
clear that Vac8 function is associated with its palmitoylation state
(41, 42, 44, 46, 61, 62). It is likely that a more tightly associated
and altered SNARE complex is the main cause of the reduced Vac8
palmitoylation. The accumulation of trans-SNARE complexes
may be associated with the reduction in palmitoylation, but cannot be
the only reason, since trans-SNARE complexes did not
accumulate between vacuoles lacking Vac8 entirely (42). The
identification of a direct binding partner of the Vam3 TMD may be
informative to fully elucidate the role of SNAREs and in particular
their TMDs in fusion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-SNAP/yeast Sec17 that disassemble preexisting SNARE
complexes or activate t-SNAREs (2, 3). Upon docking of a vesicle with
its target membrane, SNAREs from opposite membranes form
trans-SNARE complexes, which are a prerequisite for complete
fusion (4). In vitro studies with recombinant SNAREs,
reconstituted into liposomes, indicated that SNAREs can drive bilayer
mixing by pairing in a cognate fashion (5, 6).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and DKY6281vam3
strains. Clones that grew on uracil-deficient plates were analyzed for
Vam3 production by immunoblotting using antibodies against Vam3.
Strains used for the trans-SNARE assay were generated as
follows: NYV1 was deleted in BJ3505vam3
and DKY6281vam3
by transformation of a PCR fragment
containing a KAN marker and distal regions of the open reading frame of
NYV1. Colonies growing on YPD (1% yeast extract, 2%
peptone, 2% glucose) + Geneticin were restreaked and analyzed for
deletion of NYV1 by immunoblotting. Then the pRS406-vectors
carrying mutations in VAM3 were genomically integrated into
the ura3 locus of the BJ3505nyv1
vam3
strain
as described above. Similarly, pRS406-NYV1 containing the promotor and
open reading frame of NYV1 was integrated into the
DKY6281nyv1
vam3
strain to generate
DKY6281vam3
.
2-macroglobulin) and incubated for 10 min on a nutator
at 4 °C. Insoluble material was removed by centrifugation
(20,000 × g, 5 min, 4 °C). A fraction (5%) of the clarified supernatant was removed, and proteins were precipitated by
the addition of trichloroacetic acid (13% v/v). The remaining detergent extract was added to protein A-Sepharose beads containing the
coupled antibodies (50) and incubated on a nutator overnight at
4 °C. The beads were reisolated by brief centrifugation and washed
three times in lysis buffer containing 0.1% Triton X-100. Elution of
the bound proteins was by addition of 1 ml of 100 mM glycine, pH 2.5, and 0.025% Triton X-100. Proteins were precipitated with trichloroacetic acid (13% v/v), washed with 1 ml of
ice-cold 100% acetone, briefly dried, and resolved in SDS sample
buffer. Protein complexes were analyzed by SDS-PAGE and Western blotting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Vam3 mutants and vacuole morphology.
A, Vam3 mutants used in this study. The coiled-coil domains
(hatched boxes; HA, HB,
HC, and H3) were predicted with the coils
program (63). Gray boxes indicate the dileucine sorting
motif (LL) and the transmembrane domain (TM). The
H3 domain was mutagenized at five positions to alanine. Vam3-CCIIM
contains the farnesylation site of yeast Ykt6 instead of the TMD.
B, vacuole morphology. Log phase grown cells from the
respective BJ strains were incubated in 50 µl of YPD with FM4-64 (30 µM, Molecular Probes, Eugene, OR) for 20 min at 30 °C
(64). Cells were reisolated by centrifugation (1 min, 5000 × g), washed twice with 1 ml of YPD medium, and chased for 15 min at 30 °C. Cells were analyzed using a standard fluorescence
microscope. Panels a and b, wild type; panels
c and d (VAM3-231-50), panels e and
f, VAM3-CCIIM.
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Fig. 2.
Fusion and localization of lipid anchored
Vam3. A, fusion of BJ and DKY strains containing the
indicated mutations. Vacuoles (3 µg of each) were incubated in a
30-µl reaction with fusion buffer in the presence of ATP, cytosol,
Sec18 (3 µg/ml), and CoA (10 µM). Fusion was assayed
after 90 min at 26 °C. Fusion of the wild-type vacuoles was set to
100%, and fusion-independent alkaline phosphatase activity was
subtracted. The results represent averages of five independent
experiments (+S.D.). B, expression and localization of Vam3
mutants. Vacuoles (10 µg) of the indicated strains were analyzed by
SDS-PAGE and Western blotting. Immunoblots were decorated with
antibodies to Vam3 and Nyv1. C, subcellular localization of
Vam3-CCIIM. Log-phase-grown yeast cells were prepared for lysis as
during the vacuole preparation. After lysis with lyticase the yeast
pellet was resuspended in 20 mM PIPES/KOH, pH 6.8, 200 mM sorbitol and 40 µg/ml DEAE-dextran were added. Cells
were briefly incubated on ice for 2 min, then heat shocked for 2 min at
30 °C. Unbroken cells and debris were removed by centrifugation
(400 × g, 5 min, 4 °C). Lysates containing 50 mg of
protein each were centrifuged for 10 min at 8000 × g,
then the supernatant was spun again at for 10 min at 16,000 × g. Proteins of the remaining supernatant were
trichloroacetic acid-precipitated. Proteins of the untreated
lysate, each pellet fraction, and the supernatant were analyzed by
SDS-PAGE and Western blotting. Vacuoles are found exclusively in the
8000 × g pellet, while Golgi and endoplasmic
reticulum are recovered in the 16,000 × g
pellet (34).
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Fig. 3.
Alterations of cis-SNARE
complex affect priming. A, altered cis-SNARE
complex composition and priming. Vacuoles (60 µg) were incubated with
or without ATP for 10 min at 26 °C in the presence of cytosol, CoA,
and Sec18, reisolated (5 min, 20,000 × g, 4 °C),
and detergent-solubilized. For comparison, a portion (1%) of the total
detergent extract was removed and precipitated with trichloroacetic
acid (13% v/v). Protein complexes were analyzed by
coimmunoprecipitation with protein A-immobilized antibodies to Vam3
(for details see "Experimental Procedures"). Proteins were released
from the beads by addition of 1 ml of 0.1 M glycine, pH
2.6, and precipitated with trichloroacetic acid, washed with
ice-cold acetone, analyzed by SDS-PAGE, and immunoblotted with the
respective antibodies. B, Sec18-dependent fusion
of Vam3-CCIIM vacuoles. Wild-type and Vam3-CCIIM vacuoles were
incubated for 90 min at 26 °C in the presence of ATP, cytosol, CoA,
and the indicated amounts of recombinant Sec18. Alkaline phosphatase
activity was determined. A representative example (n = 3) is shown. C, localization of Sec18 to vacuoles. Purified
vacuoles (10 µg) were solubilized in sample buffer, and proteins were
analyzed by SDS-PAGE. Immunoblots were decorated with the indicated
antibodies. D, accumulation of trans-complexes in
the Vam3-CCIIM mutant. Vacuoles were incubated for 30 min at 26 °C
with or without ATP in the presence of cytosol, CoA, and Sec18. Where
indicated, Gdi1 (30 µg/ml) or BAPTA (1 mM) was added.
Then vacuoles were sedimented, detergent-solubilized, and processed for
coimmunoprecipitation with antibodies to Vti1. A fraction of the
detergent extract (5%) is shown for comparison.
strain. Complexes between Nyv1 and Vam3 only form if the vacuoles dock
or fuse and can be detected by coimmunoprecipitation with antibodies to
Vam3 (26). Even though vam3
but not nyv
vacuoles are fragmented, fusion of vam3
and
nyv1
vacuoles requires the same proteins and factors like
wild-type vacuoles (26). Thus, results obtained with mutant vacuoles
can be related to the wild-type situation.
cells
dramatically changed the vacuole morphology (Fig.
4A). Vacuoles lacking Nyv1 are
enlarged (53). This suggested that another SNARE can replace Nyv1
in vivo, although Nyv1 is essential for in vitro
fusion (50). In contrast, vacuoles fragment when Vam3 is anchored via a
lipid anchor in the nyv1
background (Fig. 4A, panels g and h). On those vacuoles, only Vti1 has
a transmembrane domain within the SNARE complex, which is apparently
not sufficient to drive vacuole fusion in vivo. TMDs of Nyv1
and of Vam3 could also be critical for the fusion reaction itself,
either as catalysts of fusion or as interaction partners of other
fusion factors.
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Fig. 4.
Analysis of Nyv1 deletion strains containing
Vam3 TMD mutants. A, vacuolar morphology.
BJnyv1 vam3
with or without genomically encoded mutant
or wild-type Vam3 were labeled with 30 µM FM4-64
(Molecular Probes) and visualized by fluorescence microscopy (64).
Panels a and b, BJnyv1
vam3
;
panels c and d, BJnyv1
;
panels e and f, BJnyv1
VAM3-231-50; panels g and h, BJnyv1
VAM3CCIIM. B, accumulation of
trans-complexes in the mutants. Vacuoles from
BJnyv1
with Vam3-wt or the respective mutants and
DKYvam3
(65 µg each) were incubated together at
26 °C for 40 min in a 600-µl reaction with or without ATP in the
presence of cytosol and CoA. Protein complexes were analyzed by
coimmunoprecipiation with protein A-immobilized antibodies to Vam3 (for
details see "Experimental Procedures"). A fraction (5%) of the
detergent extract was removed and proteins were precipitated by 13%
trichloroacetic acid. C, fusion of the deletion
strains. BJnyv1
, BJnyv1
VAM3CCIIM, and
BJnyv1
VAM3-231-50 vacuoles (3 µg each) were fused
against the DKYvam3
strain in the presence of ATP,
cytosol, Sec18, and CoA for 90 min at 26 °C. Fusion of
DKYvam3
and BJnyv1
vacuoles was set to
100%. D, analysis of protein composition of vacuoles.
Isolated vacuoles (10 µg) from the respective strains were
solubilized in sample buffer and analyzed as before.
and the respective nyv1
cells in
the presence or absence of ATP. Detergent extracts were then
immunoprecipitated with anti-Vam3 antibodies. The amount of
coprecipitated Nyv1 is a measure of formed trans-SNARE
complexes (26). Lipid-anchored Vam3 formed trans-SNARE
complexes at least as efficiently as the wild-type Vam3 did, while the
coiled-coil mutant did not (Fig. 4B). However, fusion was
poor for both mutant Vam3 proteins (Fig. 4C). This was not
due to a lack of known vacuolar SNAREs on the CCIIM vacuoles (Fig.
4D). Thus, replacement of the Vam3 TMD by a lipid anchor did
permit trans-SNARE complex formation, but affected a late step in the fusion reaction. Furthermore, trans-SNARE
complexes of Vam3-CCIIM vacuoles were authentic and not a result of
in vitro assembly in detergent, since the docking inhibitor
Gdi1 did block formation of trans-SNARE complexes, while
GTP
S, a fusion inhibitor, did not (Fig.
5A). We repeatedly observed
that trans-SNARE complexes were more stable and resisted
higher salt concentrations when Vam3-CCIIM vacuoles were used.
Therefore, we compared trans-SNARE formation with CCIIM and
wild-type Vam3 over time (Fig. 5B). We also used higher
concentrations of salt for the immunoprecipitation to analyze the
stability and accumulation of trans-SNARE complexes. Low
amounts of recombinant Sec18 were included to allow efficient priming
of Vam3-CCIIM containing vacuoles. These concentrations are not
sufficient to disrupt trans-SNARE complexes, while
incubation with high amounts of Sec18 do disassemble
trans-SNARE complexes containing Vam3-CCIIM (not shown), as
shown previously for trans-SNARE complexes containing
wild-type Vam3 (26). At these low Sec18 concentrations, a 4-fold
stimulation of trans-SNARE complex formation was seen in the
absence of the Vam3 TMD compared with wild-type (Fig. 5C,
lane 5 versus lane 11, and
D). Thus, formation of trans-SNARE complexes is
strongly favored in the absence of the Vam3 TMD, but fusion is strongly
reduced. This indicates that the Vam3 TMD itself may have a direct role
in fusion or influence in addition downstream reactions.
View larger version (40K):
[in a new window]
Fig. 5.
Accumulation of trans-SNARE
complexes in the absence of the Vam3 TMD. A, authentic
trans-SNARE complexes of Vam3-CCIIM vacuoles. Vacuoles (65 µg each) from BJnyv1 containing Vam3-wt or Vam3-CCIIM
were incubated with DKYvam3
at 26 °C in a 600-µl
reaction with or without ATP in the presence of cytosol, CoA, Sec18,
and the indicated docking inhibitor Gdi1 (30 µg/ml) or fusion
inhibitor GTP
S (2 mM) for 40 min. Protein complexes were
analyzed by coimmunoprecipitations with antibodies to Vam3 as described
under "Experimental Procedures." B, accumulation of
trans-SNARE complexes of Vam3-CCIIM triggered by Sec18
addition. The experiment was done as described in the legend to
B. Sec18 (3 µg/ml) was added where indicated. At the times
indicated samples were set on ice before processing for
coimmunoprecipitation. C, quantification of
trans-SNARE complexes. trans-SNARE complex
accumulating in the presence of Sec18 after 40 min were quantified by
laser densitometry (n = 8). Ratios of Nyv1 and Vam3
signals of each precipitation with Vam3-CCIIM were set to 100%.
View larger version (18K):
[in a new window]
Fig. 6.
A late block in fusion of Vam3-CCIIM vacuoles
caused by Vac8 acylation defect. A and B,
time of addition experiment. A 30× scale fusion reaction was started
in the presence of ATP, Sec18, CoA (10 µM), and cytosol
at 26 °C. Aliquots (30 µl) were removed at the indicated times,
and antibodies (200 ng/µl) to Sec17 or Vam3 were added or left on
ice. Then samples were incubated at 26 °C for a total of 90 min
before being assayed for fusion. To allow comparison of wild-type and
Vam3-CCIIM vacuoles the final fusion was set to 100% in each graph.
C, reduced palmitoylation of Vac8 on Vam3-CCIIM vacuoles.
Vacuoles from both strains were incubated for 10 min at 26 °C with
ATP, CoA (10 µM), Sec18, and [3H]palmitate.
Vacuoles were recovered by centrifugation (5 min, 8000 × g) and solubilized in sample buffer. Proteins were analyzed
by SDS-PAGE and fluorography as described previously (44). Pal-Vac8 on
films was quantified by laser densitometry. An average of 10 experiments is shown (±S.E.).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and vtc4
cells also require Sec18 addition to overcome their deficiency in priming and to partially rescue fusion. Vtc4 is present on CCIIM and wild-type vacuoles in equal
amounts.2 Our data therefore
suggest a more tightly associated SNARE complex due to a lack of the
Vam3 TMD rather than an effect caused by a lack of Vtc proteins.
-helical conformations in isotropic solution were less fusogenic,
fusogenicity may depend on the conformational flexibility of the
-helical SNARE TMDs in the membrane.
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ACKNOWLEDGEMENTS |
---|
We thank Rico Laage for his help during the initial phase of this project, members of the Ungermann and Langosch group for critical assessment of the manuscript, and Gabriela Müller and Ruth Jelinek for expert technical assistance. We are grateful to Andreas Mayer for helpful comments on the manuscript.
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FOOTNOTES |
---|
* This work was supported by a grant from the Deutsche Forschungsgemeinschaft (LA 699/8-1 (to D. L. and C. U.) and by Boehringer Ingelheim Fonds (to L. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence may be addressed: Lehrstuhl für
Chemie der Biopolymere, Technische Universität München,
Weihenstephaner Berg 3, 85345 Freising, Germany. Tel.: 49-8161-713500;
Fax: 49-8161-4404; E-mail: biopolymere@bl.tum.de (for D. L.) or
Biochemie Zentrum Heidelberg, University of Heidelberg, Im Neuenheimer
Feld 328, 69120 Heidelberg, Germany. Tel.: 49-6221-544180; Fax:
49-6221-544366; E-mail: cu2@ix.urz.uni-heidelberg.de (for C. U.).
Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M209522200
2 J. Rohde, L. Dietrich, D. Langosch, and C. Ungermann, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
SNARE, soluble NSF attachment protein receptor (where NSF is
N-ethylmaleimide-sensitive factor);
SNAP, soluble NSF
attachment protein;
TMD, transmembrane domain;
PIPES, 1,4-piperazinediethanesulfonic acid;
BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid;
GTPS, guanosine 5'-O-(thiotriphosphate).
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