Department of Biotechnology, the University of Tokyo, Yayoi, Bunkyo-Ku,
Tokyo 113-8657, Japan
* Present address: Max-Planck-Institute of Molecular Cell Biology and Genetics,
Pfotenhauerstrasse 108, D-01307, Dresden, Germany
Author for correspondence (e-mail:
asdfg{at}mail.ecc.u-tokyo.ac.jp)
Accepted 17 June 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Sec1 family protein, SNARE complex, Vesicle fusion, Sly1 protein, Saccharomyces cerevisiae
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Members of the Sec1 family have strong physical interactions with target
membrane (t-)SNAREs. Munc18/nSec1 binds to syntaxin 1a, a neural plasma
membrane t-SNARE (Hata et al.,
1993), and Sly1 binds to Sed5, a yeast Golgi t-SNARE
(Søgarrd et al., 1994
).
Recently, the X-ray crystal structure of the Munc18-syntaxin 1a complex was
reported (Misura et al.,
2000
). However, experimental data indicate that the interaction
between Sec1 family proteins and t-SNAREs differs depending on their
localization and species. Munc18 is not included in the so-called 20S SNARE
complex (Söllner et al.,
1993
), whereas Sly1 is coprecipitated with the similar complex
from the yeast sec18-1 cells
(Søgarrd et al., 1994
).
Typical t-SNAREs have three helical regions for protein-protein interaction.
Munc18 binds to the helical regions H1 and H3 of syntaxin 1a and inhibits the
binding between syntaxin 1a and VAMP, a neural vesicular (v-)SNARE
(Pevsner et al., 1994
), as
well as the binding between syntaxin 1a and
-SNAP
(Hayashi et al., 1994
). But
Sly1 binds only to the H1 region of Sed5 and does not bind to the rest of Sed5
and therefore does not interfere with the interaction between Sed5 and the
yeast
-SNAP Sec17 (Kosodo et al.,
1998
). Moreover, Sec1 does not interact with uncomplexed Sso1, a
yeast plasma membrane t-SNARE (Carr et al.,
1999
).
Genetic studies in S. cerevisiae have indicated that a number of
ER-Golgi secretory genes have interactions with SLY1. A single copy
of the gain-of-function mutation SLY1-20 was originally identified as
a suppressor mutation that enables the ER-Golgi vesicular transport to occur
in the absence of YPT1 (Dascher et
al., 1991). Interestingly, it could also suppress the
temperature-sensitive defects caused by mutations in many other ER-Golgi
secretory genes including uso1-1 and bet1-1
(Ossig et al., 1991
;
Sapperstein et al., 1996
;
Kito et al., 1996
). The
sly1ts allele was identified in a screen for a mutant that
arrests transcription of the genes for ribosomal proteins and RNAs at the
restrictive temperature (Mizuta and
Warner, 1994
). The sly1ts mutant grows
normally at 25°C but arrests growth when the temperature is shifted to
37°C and accumulates the ER-form precursor of vacuolar enzymes. Multicopy
USO1, BET1 or HSD1 genes could suppress the temperature
sensitivity of sly1ts
(Kosodo et al., 2001
).
Barlowe and colleagues have reproduced the ER-Golgi transport in vitro by
using semi-intact cells and temperature-sensitive mutants. They proposed that
membrane fusion of the transport vesicles can be classified into tethering,
docking and fusion steps. Mutant proteins of Sly1, Bet1, Bos1, Sed5 and a
yeast NSF Sec18 do not interfere with tethering, but block docking and fusion
steps (Barlowe, 1997;
Cao et al., 1998
). They have
also showed that Sly1, Sed5 and Ypt1 are required on the acceptor Golgi
membrane (Cao and Barlowe,
2000
).
Thus the role of Sec1 family proteins, including Sly1, in membrane fusion
has not been fully understood yet (Jahn,
2000). Although the binding between t-SNAREs and Sec1 family
proteins has an important role, the function of the binding is unclear
(Carr, 2001
). From this point
of view, we focused on the interaction between Sly1 and the fusion machinery
in this report.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Plasmids
The plasmids used in this study are listed in
Table 2 and were constructed as
follows. The DNA fragments encoding various proteins were amplified either
from genomic DNA or from plasmid DNAs harboring the gene by polymerase chain
reaction (PCR) with Pfu DNA polymerase (Stratagene). The myc-encoding sequence
was subcloned from pKT10mycC (Matsui et
al., 1996), and the HA-encoding sequence was from pYN168
(Kosodo et al., 2001
). The
recombinant proteins were produced as glutathione S-transferase (GST) fusion
proteins in pGEX4T-3 (Pharmacia), as maltose-binding protein (MBP) fusion
proteins in pMAL-c2 (New England Biolabs) or as Strep-tagged fusion proteins
in pASK-IBA2 (Genosys) in E. coli. For protein production in yeast, a
low-copy vector pRS416 or multicopy vectors pRS426
(Sikorski and Hieter, 1989
)
and pYES2.0 (Invitrogen) were used.
|
Protein purification and protein-protein binding assays
Production and purification of the GST- and MBP-tagged proteins were done
as described previously (Kosodo et al.,
1998). Strep-tagged Sly1/Sly1ts was produced in E.
coli, extracted and bound to StrepTactin-Sepharose (Sigma) and then
eluted with 2.5 mM desthiobiotin after extensive washing. The concentration of
all recombinant proteins was determined by a protein assay kit (BioRad), and
proteins were stored with 40% glycerol at -20°C.
Binding assays of recombinant proteins were carried out as described
previously (Kosodo et al.,
1998).
Preparation of yeast lysate
Yeast cells were grown to an OD600 of 0.6 to 0.8 in YEPD or SD
medium. They were harvested by centrifugation, washed in distilled water and
resuspended in an ice-cold B88 buffer (150 mM KOAc, 5 mM Mg(OAc)2,
20 mM HEPES, 200 mM sorbitol, pH 6.8) with PIC (1 mM phenylmethylsulfonyl
fluoride (PMSF), 1 µg/ml 1,10-phenanthroline, 1 µg/ml pepstatinA, 1
µg/ml aprotinin, 1 µg/ml leupeptin). One-half volume of acid-washed
glass beads was added to the cell suspension in a glass tube, which was then
vortexed eight times for 30 seconds, with a 30 second incubation on ice
between each burst. The crude lysate was centrifuged at 500 g
for 3 minutes at 4°C to remove unbroken cells. The supernatant was used as
a yeast lysate in this study.
Differential centrifugation
The yeast lysate was centrifuged at 10,000 g for 10 minutes
at 4°C to generate the supernatant (S10) and pellet (P10) fractions. The
S10 was centrifuged at 100,000 g for 60 minutes at 4°C to
generate the high-speed supernatant (S100) and pellet (P100) fractions. The
same amount of each fraction was resuspended in the Laemmli sample buffer
(Laemmli, 1970) and applied to
SDS-polyacrylamide gel electrophoresis (PAGE). Immunodecoration of western
blotting was performed using indicated primary antibodies followed by
horseradish-peroxidase-coupled goat anti-mouse or anti-rabbit IgG as secondary
antibodies at a 1:5000 dilution. The bound antibodies were visualized by
enhanced chemiluminescence (Pierce).
Immunoprecipitation from yeast lysate
Affinity-purified rabbit anti-Sed5 or the monoclonal anti-myc antibody 9E10
was used for immunoprecipitation. Triton X-100 was added to the yeast lysate
to a 1% final concentration. The lysate was incubated on ice for 5 minutes to
solubilize membranes and centrifuged at 10,000 g for 5 minutes
to remove debris. After the lysate was mixed with an antibody and incubated at
4°C for 1 hour, Protein A-Sepharose beads (Amersham) were added, and the
mixture was incubated at 4°C for 2 hours. Beads were washed five times
with B88 buffer with 0.5% Tween 20 and boiled in the Laemmli sample buffer for
1 minute. The supernatants were analyzed by SDS-PAGE and western blotting.
Binding assay of SNARE proteins in yeast lysate
The sly1ts strain was transformed with pYN110
(6myc-SED5 under SED5 promoter on a CEN plasmid) or with
pKD201 (3HA-BET1 under GAL1 promoter on a 2µ plasmid).
The cells were grown to an OD600 of 0.5 in 800 ml SD medium and, as
for the sly1ts/pKD201 strain, they were further incubated
in 200 ml SG medium to produce 3HA-Bet1 protein for 2 hours. After harvesting
the cells by centrifugation, lysates were prepared by vortexing with glass
beads in 8 ml ice-cold B88 buffer. The protein concentration of both lysates
was adjusted to 8.0 mg/ml, and the aliquots (850 µl each) were stored at
-80°C. The lysates from both strains were defrosted before the reaction
and centrifuged at 5000 g for 5 minutes to remove the
cytoskeleton and aggregates before the reaction. The cleared lysates of two
strains (600 µl) were mixed and then divided into eight aliquots (150
µl, each including 1.2 mg protein) quickly on ice. The recombinant
Strep-tagged Sly1 or Sly1ts protein (2.4 µg) was added to each
mix. The reaction was started by transferring the mix to a 35°C bath and
incubated up to the indicated time. In case of the experiments in
Fig. 6, 1 mM ATP or 1 mM
AMP-PNP (final concentration) was added during the reaction. Other conditions
for each reaction are described in the figure legends. At the end of the
reaction, the mixtures were diluted by adding 850 µl of B88 buffer with 1%
Triton X-100 and subjected to immunoprecipitation by the anti-myc antibody.
Anti-Sly1, anti-myc and anti-HA antibodies were used for western blotting.
Quantification of the signals was performed by a luminoimage analyzer
(LAS-1000plus, Fuji film).
|
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To demonstrate the protein interaction directly, we adopted a
coprecipitation assay using recombinant proteins. Glutathione S-transferase
(GST) was fused at the N-terminus of Sly1 or Sly1ts protein, and
maltose-binding protein (MBP) was fused at the N-terminus of Sed5TMD
(amino acids 1-324 of Sed5), Sed5H1 (amino acids 15-39), Sed5H2 (amino acids
146-169) or Sed5H3 (amino acids 252-311), each of which carried a myc tag at
the C-terminus. The fusion proteins were produced in E. coli,
purified by affinity resins and used in the binding assay at 4°C. As shown
in Fig. 1B, a considerable
amount of GST-Sly1 was recovered in the immunoprecipitate of
MBP-Sed5
TMD-myc (lane 1), whereas much less GST-Sly1ts could
be recovered (lane 2). GST-Sly1 was coprecipitated with MBP-Sed5H1-myc, which
contains the Sly1-binding helix of Sed5 (lane 3;
Kosodo et al., 1998
), but a
detectable amount of GST-Sly1ts was not coprecipitated (lane 4).
Neither GST-Sly1 nor GST-Sly1ts bound to MBP-Sed5H3-myc (lanes 5
and 6). From these results, we conclude that Sly1ts has a defect in
binding to Sed5.
Binding of Sly1 proteins to the membrane fraction
The Sly1 protein is not predicted to have a transmembrane domain, and the
recombinant Sly1-fusion proteins produced in E. coli were soluble. By
contrast, nearly 60% of Sly1 localized on the Golgi membrane in yeast cells
(Lupashin et al., 1996). This
membrane localization of Sly1 has been explained by its interaction with Sed5
(Grabowski and Gallwitz, 1997
;
Cao and Barlowe, 2000
). Because
Sly1ts turned out to be defective in binding to Sed5, we examined
its intracellular localization by differential centrifugation. The lysate with
myc-tagged Sly1, Sly1-20 or Sly1ts was separated into the membrane
fractions, P10 or P100, and the soluble fraction S100. Sly1, Sed5, Scs2 [an ER
membrane protein (Kagiwada et al.,
1998
)], Van1 [a Golgi membrane protein
(Hashimoto and Yoda, 1997
)]
and Pgk1 [a soluble cytosolic protein
(Schneiter et al., 1999
)] in
these fractions were detected by western blotting. As shown in
Fig. 2, we could not find any
difference between distributions of the myc epitope in these strains. The
distributions of Sed5 and Van1 were similar. It is unlikely that this
distribution was caused simply by contamination because Pgk1 was only detected
in one fraction S100. This result suggests that the yeast membrane has
other factors than Sed5 to localize more than half of the total Sly1 protein
on the membrane.
|
Sly1 was included in the Sed5-Bet1 SNARE complex
Using an in vitro assay, it was shown that a combination of the Q-SNAREs
(Sed5, Bos1 and Sec22) and an R-SNARE (Bet1) constitute a potential minimum
fusion machinery in the ER-Golgi transport, although other combinations may
exist (McNew et al., 2000).
Thus, it was important to see whether Sly1 binds only to free Sed5 or whether
it also binds to Sed5 in the SNARE complex. We found that the
sly1ts mutation is partially suppressed by introduction of
multicopy BET1 (Kosodo et al.,
2001
). This observation suggests that Bet1 may have an important
role among v-SNAREs, in concert with Sly1. Therefore, we focused our study on
the interaction with Bet1 and Sly1. First, we examined whether Sly1 is in the
Bet1-Sed5 SNARE complex in the normal yeast cells. The 20 S complex found in
the sec18-1 cell at the nonpermissive temperature contained Sed5,
Bet1 and Sly1 proteins as well as Bos1 and Sec17. In case of exocytosis in
neuron, it was shown that v-SNARE(VAMP2) was excluded from the
n-Sec1(Munc-18)/t-SNARE(syntaxin1) complex
(Pevsner et al., 1994
).
The genomic SLY1 was replaced with SLY1-6myc in the
wild-type strain. The cells grew well without any detectable defect. The
lysate was prepared from them, with 1% Triton X-100 added to solubilize the
membrane, and then divided into three parts. Immunoprecipitation was performed
using an anti-myc monoclonal antibody, an affinity-purified anti-Sed5 antibody
or, as a control, preimmune serum from the rabbit from which we raised the
anti-Sed5 antiserum. As shown in Fig.
3, Bet1 was detected in the immunoprecipitate of anti-Sed5 (lane
2). This indicates the presence of the Bet1-Sed5 SNARE complex in the lysate.
Sly1-6myc was also detected in this immunoprecipitate. In accordance with
this, both Bet1 and Sed5 were detected in the immunoprecipitate of Sly1-myc
(lane 3). Because Sly1 does not bind to Bet1 directly, this indicates the
presence of a complex containing these three proteins. Preimmune serum
precipitated none of these proteins (lane 1). These results strongly suggest
that Sly1 still binds to the Sed5-Bet1 SNARE complex. As the H1 helix of Sed5
binds to Sly1 and the H3 helix binds to Bet1
(Kosodo et al., 1998;
Sacher et al., 1997
),
simultaneous binding of Sed5 to both Sly1 and Bet1 would be possible. This
Bet1-Sed5 interaction was suggested to be susceptible to the action of
dissociating ATPase, because Bet1 was not detected in similar experiments when
0.5 mM ATP was added to the immunoprecipitation mix (data not shown).
|
Sly1 stimulated the trans-SNARE complex formation between the
membranes
As a complex containing Sly1, Sed5 and Bet1 was detected, Sly1 may somehow
be involved in the formation of the Bet1-Sed5 SNARE complex. We constructed an
in vitro assay system to evaluate the effect of Sly1 in this reaction. The
lysate of the sly1ts mutant was selected as the basal
condition, because Sly1 is an essential protein and its depletion by other
means have obvious shortcomings. The sly1ts mutant was
transformed either with 6myc-SED5 on a CEN plasmid (strain
I) or with a GAL1p-3HA-BET1 construct on a 2µ plasmid (strain II).
These strains were grown at 25°C, and the expression of 3HA-BET1
in strain II was induced in SG medium for 2 hours. The cells were harvested,
disrupted by vortexing with glass beads, centrifuged at 4000 g
for 5 minutes and the supernatants (S4) were stocked at -80°C. Almost all
endogenous Sly1ts protein was recovered in S4 (data not shown).
Sly1 or Sly1ts protein with Strep tag at the C-terminus was
produced in E. coli and purified by affinity resin to a single band
in SDS-PAGE. The lysates of strains I and II were mixed and incubated with or
without adding a purified recombinant protein. Then, the mixture was diluted
6.7-fold by B88 buffer on ice, and Triton X-100 was added to 1% of the final
concentration to dissolve the membranes. The monoclonal anti-myc antibody was
used to precipitate 6myc-Sed5, and 3HA-Bet1, exogenous
Sly1/Sly1ts-Strep and endogenous Sly1ts in the
precipitate were detected by western blotting.
First, we measured the effect of temperature on the cell-free system.
0°C serves as a control for basal reactions. At 15°C, both wild-type
and sly1ts strains grew slowly without apparent difference
(data not shown). At 25°C, the wildtype grew slightly faster than the
sly1ts mutant (data not shown). 35°C is a restrictive
temperature for the sly1ts strain and the vesicle
transport between ER-Golgi stops at this temperature
(Mizuta and Warner, 1994). The
reaction mix was kept at these temperatures for 20 minutes. At 0°C, no
increase of the amount of Bet1 was found when either Sly1-Strep or
Sly1ts-Strep was added (Fig.
4, lanes 1 and 2). At 15°C, a slight increase in the amount of
3HA-Bet1 was detected in the precipitates by adding either protein
(Fig. 4, lanes 3 and 4). At
25°C, either protein gave a considerable increase, but Sly1-Strep had a
stronger effect (Fig. 4, lanes
5 and 6). At 35°C, Sly1ts-Strep had little effect
(Fig. 4, lanes 7 and 8). The
activity of recombinant proteins in the assay system may have a good
accordance with yeast growth.
The temperature dependence of the binding of 3HA-Bet1 to 6myc-Sed5 (Fig. 4) also indicates that the Bet1-Sed5 complex was formed during the incubation at each temperature and not in the later process after the dilution and addition of Triton X-100. When Triton X-100 was added before mixing the lysates, we could not find any increase in the amount of 3HA-Bet1 (data not shown).
We further evaluated the time course of the change in the amount of coprecipitated proteins after the temperature shift from 0°C to 35°C. During the incubation at 35°C, the amount of coprecipitated 3HA-Bet1 clearly increased by adding Sly1-Strep protein to the mixture (Fig. 5A, lanes 1-3). Addition of the same amount of Sly1ts-Strep did not give such an increase (Fig. 5A, lanes 4-6). The exogenous Sly1-Strep protein was found in the precipitates, but Sly1ts-Strep was hardly detected in them (lanes 1-6). We detected a faint band of endogenous Sly1ts protein in all lanes (compare with Fig. 4). Most of the endogenous Sly1ts protein bound to 6myc-Sed5 before cell disruption was not released in the present condition and might prevent exogenous Sly1ts-Strep from replacing its position.
|
The amount of protein in the immunoprecipitate was measured using a
luminoimage analyzer. At 0°C, no difference was found in the amount of
3HA-Bet1 irrespective of whether Sly1-Strep or Sly1ts-Strep was
added. The SNARE-mediated membrane fusion was temperature dependent and did
not occur at 0°C (Weber et al.,
1998). At 35°C, the increased amount of 3HA-Bet1 with
Sly1-Strep was 17 times of that with Sly1ts-Strep
(Fig. 5B). These indicate that
Sly1-Strep stimulated formation of the Bet1-Sed5 trans-SNARE complex in a
time-and temperature-dependent manner.
Next, we addressed the question of whether the trans-SNARE complex is formed from the cis-SNARE complex or whether its dissociation by NSF/Sec 18 ATPase is required to prime complex formation. If 1 mM ATP was added into the reaction mix, the amount of coprecipitable 3HA-Bet1 decreased (Fig. 6, lanes 2 and 6), and it was almost under the limit of detection in the case of Sly1ts-Strep. This indicates that ATPase Sec18 that dissociates the cis-SNARE complex was active in our reaction mix. If the unhydrolyzable analog AMPPNP was added instead of ATP, the result was the same as the result without its addition (compare lane 1 with 3, and lane 5 with 7). This indicates that trans-SNARE complex formation in our system did not require ATP hydrolysis. However, if the lysate was incubated in the presence of ATP with Sly1-Strep or Sly1ts-Strep and then AMPPNP was added, the amount of 3HA-Bet1 significantly increased (lane 4 or 8). This result indicates that the dissociated SNAREs present at the time of lysate preparation participated in the formation of the trans-SNARE complex under the standard conditions. Probably ATP in the lysate was soon exhausted by reactions that consumed ATP. We guess that the pre-incubation of lysate with ATP increased the amount of dissociated SNAREs by the action of the NSF/Sec18 ATPase and thus the amount of the newly formed SNARE complex increased. AMPPNP probably inhibited further action of Sec18 ATPase, which would also dissociate the newly formed SNARE complex after vesicle fusion. We conclude that dissociation of pre-formed SNARE complex is also a prerequisite for trans-SNARE complex formation in the presence of Sly1 protein.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sly1 and the other membrane fusion machinery
GLUT4 vesicle trafficking is abolished at the restrictive temperature in a
3T3L1 mutant cell that has the same point mutation in Munc18c as the yeast
sly1ts allele
(Thurmond and Pessin, 2000).
Thurmond and Pessin showed the mutant Munc18c had a reduced affinity for the
t-SNARE syntaxin 4. On the basis of our results and their report, we conclude
that a single amino-acid substitution R266K in Sly1 renders its binding
activity to t-SNARE much lower and temperature labile, and consequently
vesicle transport is abolished at a nonpermissive temperature. Therefore, the
binding of the Sec1 family protein to t-SNARE would be necessary to form the
correct SNARE complex.
Sly1 is one of the Sec1 family proteins in S. cerevisiae. The Sec1
family proteins have long been implied as regulators of the membrane fusion
process in vesicular transport because of their high affinity for t-SNAREs
that trigger membrane fusion (Halachmi and
Lev, 1996; Nichols and Pelham,
1998
). The Munc 18/n-Sec1 protein was first identified and
purified from mammalian brain owing to its high binding affinity to the
t-SNARE syntaxin 1 (Hata et al.,
1993
). Munc18 was thought to be a negative regulator of membrane
fusion, since it prevented synaptobrevin and SNAP from binding to syntaxin 1
(Pevsner et al., 1994
).
However, the negative regulator model does not fully explain why the protein
is essential for neurotransmitter release and intracellular vesicle transport
(Ossig et al., 1991
;
Verhage et al., 2000
). By
contrast, it has also been proposed that the Sec1 family proteins have a
positive or chaperone-like role rather than a negative role
(Grabowski and Gallwitz, 1997
;
Yang et al., 2000
;
Jahn, 2000
). Our results
strongly suggest that Sly1, at least, has a positive role in SNARE complex
formation, which is followed by membrane fusion.
We have previously reported that Sly1 had no effect on binding of Bet1 to
Sed5 using recombinant protein without a transmembrane domain
(Kosodo et al., 1998) (K.Y.,
N.Y., A.H. and Y.K., unpublished). Recently, it has been reported that the
transmembrane domain of syntaxin 1 is critical for its proper interaction with
other proteins (Lewis et al.,
2001
). Actually, Sly1 did not have an effect on the binding after
membrane solubilization by Triton X-100 in our cell free system (data not
shown). The lipid bilayer and transmembrane domain of SNAREs therefore are
probably indispensable for the correct SNARE complex formation and functional
role of Sly1. To confirm this, we have shown for the first time the direct
role of Sec1/Munc18 family in trans-SNARE complex assembly by a novel in vitro
assay system using recombinant Sly1 proteins and intact SNAREs with a
transmembrane domain embedded in the membrane in the yeast lysate. This method
will be applied to address the role of other fusion-mediating proteins in
yeast ER-Golgi vesicle transport such as Uso1, Ypt1, Yip1, Yif1, Sec22, Bos1
or Ykt6 in SNARE complex formation with Sly1 function.
The complex containing Sed5, Bet1, Sly1 as well as Bos1 and Sec17 was
discovered in the detergent extract of sec18-1 mutant cells at the
nonpermissive temperature (Søgarrd
et al., 1994). Although this 20 S complex was originally regarded
as a docking complex, it is now considered as a remnant of the fusion
machinery. Dissociation of this cis-SNARE complex by the ATPase Sec18 is a
pre-requisite for activation and priming of SNAREs to be used in the next
round of membrane fusion. Our present data confirmed that a complex containing
Sed5, Bet1 and Sly1 exists in the normally growing wild-type yeast cell
(Fig. 3), which is also
consistent with our previous results using a binding experiment with
recombinant proteins (Kosodo et al.,
1998
). After submission of our manuscript, Peng and Gallwitz
reported the formation of a complex containing the Sec22/Sed5/Bos1/Bet1 SNARE
complex, Sly1 and Sec17 by mixing recombinant proteins
(Peng and Gallwitz, 2002
).
They suggested that the specificity of the SNARE complex is determined by
protein-protein interaction of the components. However, the SNARE complex
assembly occurred extremely slowly as it took hours, and importantly the
kinetics were not influenced by the presence or absence of Sly1. These
apparent contradictions with our present data are probably caused by the
absence of membrane in their system. The kinetics of our system are closer to
the process occurring in the cell, and the trans-SNARE complex assembly did
not occur in our system after the addition of Triton X-100. It is likely that
Sly1 helps to assemble the trans-SNARE complex when the SNAREs have
transmembrane domains and are anchored to the membrane.
The Sec-1 family may function at two different stages. The Sec1 family
protein may act upon t-SNAREs before SNARE complex formation followed by the
interaction of v-SNARE and t-SNARE. In this case, the Sec1 family protein
would change or fix t-SNAREs into an effective conformation. The other is that
the Sec1 family protein may bind to t-SNARE after SNARE complex formation, as
is the case in yeast exocytosis. It has been reported that yeast Sec1 does not
bind to a single Sso1 molecule, a t-SNARE of plasma membrane, but binds to the
v-t-SNARE complex (Grote et al.,
2000). We suggest that Sly1 acts before SNARE complex formation
because the amount of Sly1 binding to Sed5 is almost the same before and after
SNARE complex formation in our cell-free assay system
(Fig. 5A, lane 1 or 3).
Probably, Sly1 binds to Sed5 before SNARE complex formation, which would,
consequently, induce Sed5 into an efficient binding state for Bet1.
SNAREs themselves are the minimal fusion machinery, and trans-SNARE complex formation drives membrane fusion. Thus formed cis-SNARE complexs must be dissociated by the action of NSF/Sec18 ATPase in combination with SNAP/Sec17 before they are used in the next round of membrane fusion. Our data (Fig. 6) implies that the dissociation of cis-SNARE complex is a pre-requisite for stimulation of trans-SNARE complex assembly by Sly1.
SNAREs are considered to be in a cycle of assembly and disassembly in the cell. Solubilization of membrane by detergent stopped trans-SNARE complex assembly, although disassembly could occur if ATP was supplied. The amount of Bet1-Sed5 complex reflects the amount of SNARE complex present at the time of the detergent extraction. During this study, we found that the amount of Bet1 in the Sed5 immunoprecipitate was significantly less when Sly1 is fully active than when it is less active [the sly1ts mutant was compared with the parent, and the sly1ts/vector was compared with the sly1ts/SLY1 plasmid (K.Y., N.Y., A.H. and Y.K., unpublished)]. Apparently, the amount of Bet1-Sed5 complex in detergent extract decreases when Sly1 is more active. So, Sly1 may stimulate disassembly of the cis-SNARE complex by the action of NSF/Sec18 ATPase as well as the assembly of trans-SNARE complex in the presence of the membrane. To examine this biochemically, it will be necessary to develop a cell-free system in which SNAREs are at a specific stage of the assembly/disassembly cycle so that the reaction can be started simultaneously. Genetic data may support the idea that Sly1 may be involved in both assembly and disassembly of SNARE complex. Synthetic lethality of mutations generally suggests that processes that the mutant gene products concern are closely related. When the sec18-1 and sly1ts mutants were crossed and the progeny was processed for tetrad analysis, the cells that have both mutant alleles did not grow even at the permissive temperature for each single mutation (K.Y., N.Y., A.H. and Y.K.). Thus, the mutant alleles sec18-1 and sly1ts were synthetic lethal.
Role of Sec1 family proteins at the membrane fusion step
Bryant and James recently reported that Vps45, one of the yeast Sec1-family
proteins, is essential for ternary SNARE complex formation
(Bryant and James, 2001).
During vesicle transport between the late Golgi and the endosome, the t-SNARE
Tlg2 and its binding partners Tlg1 and Vti1 form a ternary complex. In the
absence of Vps45, which binds to Tlg2, Tlg2 suffers rapid degradation, and
even if the degradation is prevented through abolition of proteasome activity
Tlg2 cannot form a ternary complex. Bryant and James suggest that Sec1 family
proteins have chaperone-like activity on t-SNAREs and play an essential role
in the activation process that allows t-SNARE to participate in ternary
complex formation. Although their observations are mainly made on the basis of
immunoprecipitation from cell lysates of null mutants, they are essentially
consistent with our results in that Sec1 family proteins enhance ternary SNARE
complex formation.
There are four Sec1 family genes in S. cerevisiae
(Aalto et al., 1992). Sec1 acts
in vesicular transport to the plasma membrane, Vps33 or Vps45 to the vacuole
or endosome and Sly1 to the Golgi apparatus
(Halachmi and Lev, 1996
).
Although Sec1 family proteins have no transmembrane domain, a part of them
localizes on the organelle membranes as a part of protein complexes. Vps33 is
a component of `C-Vps complex' on the vacuolar membrane with Vps11, Vps16,
Vps18, Vps39 and Vps41 (Wurmser et al.,
2000
). Vps45 interacts with Vps21 and Vac1 and localizes on the
endosomal membrane (Tall et al.,
1999
). As shown in Fig.
2, in spite of different binding affinities for Sed5, both Sly1
and Sly1ts, Sec1 proteins similarly localize on the membrane. This
suggests that Sly1 localization is not only dependent on Sed5, but also that
other factors have a crucial role. Sly1 might be a part of a certain protein
complex, as other Sec1 family proteins are, and might settle on Golgi membrane
through this complex.
Our findings with Sly1 and observations of other investigators suggest that
Sec1 family proteins generally localize on the target membranes independently
from the SNARE proteins that they bind to. SNARE proteins cycle between
organelle membranes. For example, Sed5 mainly localizes on the Golgi membrane,
but it cycles between the ER and Golgi membranes (Wooding et al., 1998;
Cho et al., 2000). There are
probably some mechanisms to induce SNARE-dependent membrane fusion and to mix
the content of cargo exactly on the Golgi membrane. When Sly1 regulates
trans-SNARE complex formation as we have described, Sly1 should localize on
the area where membrane fusion is expected to occur. In other words, the area
of membrane fusion would not be determined without the correct localization of
Sec1 family proteins on the proper membranes.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aalto, M. K., Keranen, S. and Ronne, H. (1992). A family of proteins involved in intracellular transport. Cell 68,181 -182.[Medline]
Barlowe, C. (1997). Coupled ER to Golgi
transport reconstituted with purified cytosolic proteins. J. Cell
Biol. 139,1097
-1108.
Bryant, N. J. and James, D. E. (2001). Vps45p
stabilizes the syntaxin homologue Tlg2p and positively regulates SNARE complex
formation. EMBO J. 20,3380
-3388.
Cao, X. and Barlowe, C. (2000). Asymmetric
requirements for a rab GTPase and SNARE proteins in fusion of COPII vesicles
with acceptor membranes. J. Cell Biol.
149, 55-65.
Cao, X., Ballew, N. and Barlowe, C. (1998).
Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is
independent of SNARE proteins. EMBO J.
17,2156
-2165.
Carr, C. M. (2001). The taming of the SNARE. Nature Struct. Biol. 8,186 -188.[CrossRef][Medline]
Carr, C. M., Grote, E., Munson, M., Hughson, F. M. and Novick,
P. J. (1999). Sec1p binds to SNARE complexes and concentrates
at sites of secretion. J. Cell Biol.
146,333
-344.
Cho, J.-H., Noda, Y. and Yoda, K. (2000). Proteins in the early Golgi compartment of Saccharomyces cerevisiae immunoisolated by Sed5p. FEBS Lett. 469,151 -154.[CrossRef][Medline]
Dascher, C., Ossig, R., Gallwitz, D. and Schmitt, H. D. (1991). Identification and structure of four yeast genes (SLY) that are able to suppress the functional loss of YPT1, a member of the RAS superfamily. Mol. Cell. Biol. 11,872 -885.[Medline]
Grabowski, G. and Gallwitz, D. (1997). High-affinity binding of the yeast cis-Golgi t-SNARE, Sed5p, to wild-type and mutant Sly1p, a modulator of transport vesicle docking. FEBS Lett. 411,169 -172.[CrossRef][Medline]
Grote, E., Carr, C. M. and Novick, P. J.
(2000). Ordering the final events in yeast exocytosis.
J. Cell Biol. 151,439
-451.
Halachmi, N. and Lev, Z. (1996). The Sec1 family: A novel family of proteins involved in synaptic transmission and general secretion. J. Neurochem. 66,889 -897.[Medline]
Hashimoto, H. and Yoda, K. (1997). Novel membrane protein complexes for protein glycosylation in the yeast Golgi apparatus. Biochem. Biophys. Res. Commun. 241,682 -686.[CrossRef][Medline]
Hata, Y., Slaughter, C. A. and Sudhof, T. C. (1993). Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin. Nature 366,347 -351.[CrossRef][Medline]
Hayashi, T., McMahon, H., Yamasaki, S., Binz, T., Hata, Y., Sudhof, T. C. and Niemann, H. (1994). Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J. 13,5051 -5061.[Abstract]
Jahn, R. (2000). Sec1/Munc18 proteins: Mediators of membrane fusion moving to center stage. Cell 27,201 -204.
Kagiwada, S., Hosaka, K., Murata, M., Nikawa, J. and Takatsuki,
A. (1998). The Saccharomyces cerevisiae SCS2 gene
product, a homolog of a synaptobrevin-associated protein, is an integral
membrane protein of the endoplasmic reticulum and is required for inositol
metabolism. J. Bacteriol.
180,1700
-1708.
Kito, M., Seog, D.-H., Igarashi, K., Kambe-Honjo, H., Yoda, K. and Yamasaki, M. (1996). Calcium and SLY genes suppress the temperature-sensitive secretion defect of Saccharomyces cerevisiae uso1 mutant. Biochem. Biophys. Res. Commun. 220,653 -657.[Medline]
Kosodo, Y., Noda, Y. and Yoda, K. (1998). Protein-Protein interactions of the yeast Golgi t-SNARE Sed5 protein distinct from its neural plasma membrane cognate Syntaxin 1. Biochem. Biophys. Res. Commun. 250,212 -216.[CrossRef][Medline]
Kosodo, Y., Imai, K., Hirata, A., Noda, Y., Takatsuki, A., Adachi, H. and Yoda, K. (2001). Multicopy suppressors of the sly1 temperature-sensitive mutation in the ER-Golgi vesicular transport in Saccharomyces cerevisiae. Yeast 18, 1-13.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227,680 -685.[Medline]
Lewis, J. L., Dong, M., Earles, C. A. and Chapman, E. R.
(2001). The transmembrane domain of syntaxin 1A is critical for
cytoplasmic domain protein-protein interactions. J. Biol.
Chem. 276,15458
-15465.
Lupashin, V. V., Hamamoto, S. and Schekman, R. W. (1996). Biochemical requirements for the targeting and fusion of ER-derived transport vesicles with purified yeast Golgi membranes. J. Cell Biol. 132,277 -289.[Abstract]
Matsui, Y., Matsui, R., Akada, R. and Toh-e, A. (1996). Yeast src homology region 3 domain-binding proteins involved in bud formation. J. Cell Biol. 133,865 -878.[Abstract]
McNew, J. A., Parlati, F., Fukuda, R., Johnston, R. J., Paz, K., Paumet, F., Söllner, T. H. and Rothman, J. E. (2000). Compartmental specificity of cellular membrane fusion encode in SNARE proteins. Nature 407,153 -159.[CrossRef][Medline]
Misura, K. M. S., Scheller, R. H. and Weis, W. I. (2000). Three-dimensional structure of the neuronal-Sec1-syntaxin 1a complex. Nature 404,355 -362.[CrossRef][Medline]
Mizuta, K. and Warner, J. R. (1994). Continued functioning of the secretory pathway is essential for ribosome synthesis. Mol. Cell. Biol. 14,2493 -2502.[Abstract]
Nichols, B. J. and Pelham, H. R. B. (1998). SNAREs and membrane fusion in the Golgi apparatus. Biochim. Biophys. Acta 1404,9 -31.[Medline]
Ossig, R., Dascher, C., Trepte, H. H., Schmitt, H. D. and Gallwitz, D. (1991). The yeast SLY gene products, suppressors of defects in the essential GTP-binding Ypt1 protein, may act in endoplasmic reticulum-to-Golgi transport. Mol. Cell. Biol. 11,2980 -2993.[Medline]
Peng, R. and Gallwitz, D. (2002). Sly1 protein
bound to Golgi syntaxin Sed5p allows assembly and contributes to specificity
of SNARE fusion complexes. J. Cell Biol.
157,645
-655.
Pevsner, J., Hsu, S. C., Braun, J. E. A., Calakos, N., Ting, A. E., Bennet, M. K. and Scheller, R. H. (1994). Specificity and regulation of a synaptic vesicle docking complex. Neuron 13,353 -361.[Medline]
Rothman, J. E. (1994). Mechanism of intracellular protein transport. Nature 372, 55-63.[CrossRef][Medline]
Sacher, M., Stone, S. and Ferro-Novick, S.
(1997). The synaptobrevin-related domains of Bos1p and Sec22p
bind to the syntaxin-like region of Sed5p. J. Biol.
Chem. 272,17134
-17138.
Sapperstein, S. K., Lupashin, V. V., Schmitt, H. D. and Waters, M. G. (1996). Assembly of the ER to Golgi SNARE complex requires Uso1p. J. Cell Biol. 132,755 -767.[Abstract]
Schneiter, R., Brugger, B., Sandhoff, R., Zellnig, G., Leber,
A., Lampl, M., Athenstaedt, K., Hrastnik, C., Eder, S., Daum, G. et al.
(1999). Electrospray ionization tandem mass spectrometry
(ESI-MS/MS) analysis of the lipid molecular species composition of yeast
subcellular membranes reveals acyl chain-based sorting/remodeling of distinct
molecular species en route to the plasma membrane. J. Cell
Biol. 146,741
-754.
Sikorski, R. S. and Hieter, P. (1989). A system
of shuttle vectors and yeast host strains designed for efficient manipulation
of DNA in Saccharomyces cerevisiae. Genetics
122, 19-27.
Söllner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P. and Rothman, J. E. (1993). SNAP receptors implicated in vesicle targeting and fusion. Nature 362,318 -324.[CrossRef][Medline]
Søgaard, M., Tani, K., Ye, R. R., Geromanos, S., Tempst, P., Kirchhausen, T., Rothman, J. E. and Söllner, T. (1994). A Rab protein is required for the assembly of SNARE complex in the docking of transport vesicles. Cell 78,937 -948.[Medline]
Tall, G. G., Hiroko, H., DeWald, D. B. and Horazdovsky, B.
F. (1999). The phosphatidylinositol 3-phosphate binding
protein Vac1p interacts with a rab GTPase and a Sec1p homologue to facilitate
vesicle-mediated vacuolar protein sorting. Mol. Biol.
Cell 10,1873
-1889.
Thurmond, D. C. and Pessin, J. E. (2000).
Discrimination of GLUT4 vesicle trafficking from fusion using a
temperature-sensitive Munc18c mutant. EMBO J.
19,3565
-3575.
Verhage, M., Maia, A. S., Plomp, J. J., Brussaard, A. B.,
Heeroma, J. H., Vermeer, H., Toonen, R. F., Hammer, R. E., van den Berg, T.
K., Missler, M. et al. (2000). Synaptic assembly of the brain
in the absence of neurotransmitter secretion. Science
287,864
-869.
Weber, T., Zemelman, B. V., McNew, J. A., Westermann, B., Gmachl, M., Parlati, F., Söllner, T. and Rothman, J. E. (1998). SNAREpins: Minimal machinery for membrane fusion. Cell 92,759 -772.[Medline]
Wooding, S. and Pelham, H. (1998). The dynamics
of Golgi protein traffic visualized in living yeast cells. Mol.
Biol. Cell 9,2667
-2680.
Wurmser, A. E., Sato, T. K. and Emr, S. D.
(2000). New component of the vacuolar class C-Vps complex couples
nucleotide exchange on the Ypt7 GTPase to SNARE-dependent docking and fusion.
J. Cell Biol. 151,551
-562.
Yang, B., Steegmaier, M., Gonzalez, L. C., Jr and Scheller, R.
H. (2000). nSec1 binds a closed conformation of syntaxin1A.
J. Cell Biol. 148,247
-52.
Yoda, K. and Noda, Y. (2001). Vesicular transport and the Golgi apparatus in yeast. J. Biosci. Bioeng. 91,1 -11.
Related articles in JCS: