Specific SNARE complex binding mode of the Sec1/Munc-18 protein, Sec1p

Article (PDF Available)inProceedings of the National Academy of Sciences 103(47):17730-5 · December 2006with59 Reads
DOI: 10.1073/pnas.0605448103 · Source: PubMed
The Sec1/Munc-18 (SM) family of proteins is required for vesicle fusion in eukaryotic cells and has been linked to the membrane-fusion proteins known as soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SM proteins may activate the target-membrane SNARE, syntaxin, for assembly into the fusogenic SNARE complex. In support of an activation role, SM proteins bind directly to their cognate syntaxins. An exception is the yeast Sec1p, which does not bind the yeast plasma-membrane syntaxin, Sso1p. This exception could be explained if the SM interaction motif were blocked by the highly stable closed conformation of Sso1p. We tested the possibility of a latent binding motif using sso1 mutants in yeast and reconstituted the Sec1p binding specificity observed in vivo with purified proteins in vitro. Our results indicate there is no latent binding motif in Sso1p. Instead, Sec1p binds specifically to the ternary SNARE complex, with no detectable binding to the binary t-SNARE complex or any of the three individual SNAREs in their uncomplexed forms. We propose that vesicle fusion requires a specific interaction between the SM protein and the ternary SNARE complex.
Specific SNARE complex binding mode of the
Sec1Munc-18 protein, Sec1p
John Togneri*, Yi-Shan Cheng*, Mary Munson
, Frederick M. Hughson
, and Chavela M. Carr*
*Department of Pathology and Laboratory Medicine, University of Medicine and Dentistry of New Jersey–Robert Wood Johnson Medical School,
Piscataway, NJ 08854;
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School,
Worcester, MA 01605; and
Department of Molecular Biology, Princeton University, Princeton, NJ 08544
Edited by William T. Wickner, Dartmouth Medical School, Hanover, NH, and approved October 2, 2006 (received for review June 29, 2006)
The Sec1Munc-18 (SM) family of proteins is required for vesicle
fusion in eukaryotic cells and has been linked to the membrane-
fusion proteins known as soluble N-ethylmaleimide-sensitive fac-
tor attachment protein receptors (SNAREs). SM proteins may acti-
vate the target-membrane SNARE, syntaxin, for assembly into the
fusogenic SNARE complex. In support of an activation role, SM
proteins bind directly to their cognate syntaxins. An exception is
the yeast Sec1p, which does not bind the yeast plasma-membrane
syntaxin, Sso1p. This exception could be explained if the SM
interaction motif were blocked by the highly stable closed confor-
mation of Sso1p. We tested the possibility of a latent binding motif
using sso1 mutants in yeast and reconstituted the Sec1p binding
specificity observed in vivo with purified proteins in vitro. Our
results indicate there is no latent binding motif in Sso1p. Instead,
Sec1p binds specifically to the ternary SNARE complex, with no
detectable binding to the binary t-SNARE complex or any of the
three individual SNAREs in their uncomplexed forms. We propose
that vesicle fusion requires a specific interaction between the SM
protein and the ternary SNARE complex.
membrane fusion syntaxin Sso1p Sec9p Snc2p
uk aryotic cell g rowth and organization depend on targeted
membrane-fusion reactions between vesicles and other in-
tracellular membranes. Fusion of the vesicle and t arget mem-
branes is thought to be catalyzed by a family of membrane
proteins called soluble N -ethylmaleimide-sensitive factor attach-
ment protein receptors (SNAREs; refs. 1 and 2), which assemble
bet ween membranes in a c onformation similar to the fusion-
active (fusogen ic) state of many viral membrane-fusion proteins
(3, 4). Additional conserved protein families are required for vesicle
transport and fusion throughout the cell (5, 6). For example, the
Sec1Munc-18 (SM) family of proteins is proposed to be essential
for activation of SNARE-complex assembly at the vesicle-fusion
step (7), and a general requirement of SM proteins for vesicle fusion
is supported by studies in a variety of organisms (8).
Activation of SNARE-complex assembly is a plausible mech-
an ism for SM-dependent vesicle fusion. SM proteins bind and
potentially modulate the c onformation of SNA RE proteins that
are homologous to the neuronal synaptic membrane protein,
synt axin (9). Syntaxins can adopt a fusion-inactive ‘‘closed’’
c onformation (10), in which an N-terminal three-helix bundle
motif (H
) binds the C-terminal
-helical SNARE motif (H3;
ref. 11). The structure of the neuronal SM protein, nSec1, bound
to the closed conformation of Syntaxin 1a has been interpreted
as an inter mediate required to convert syntaxin to the ‘‘open’’
c onformation, which then assembles with the other SNAREs to
for m the fusogen ic SNARE complex (12). Alternatively, the
closed syntaxin-binding mode may represent a snapshot of nSec1
acting as an inhibitor of assembly, a function that has been
attributed to SM proteins required for highly regulated exoc y-
tosis events, such as synaptic vesicle fusion (13, 14).
The un iversality of the closed-synt axin-binding mode for
vesicle fusion is called into question by the different synt axin-
binding modes that have been observed for other SM proteins (9,
15). The SM proteins, Sly1p and Vps45p, interact with an
N-ter minal peptide-finger motif of their c ognate syntaxins,
Sed5p and Tlg2p (16, 17). A crystal structure of Sly1p bound to
the peptide-finger motif of Sed5p reveals that the surface used
by Sly1p is distinct from that used by nSec1p to bind Synt axin 1A
(18). An indirect synt axin-binding mode may describe the in-
teraction of the yeast vacuolar SM protein, Vps33p, which is part
of a large protein complex that binds to the cognate syntaxin,
Vam3p (19, 20). In the most extreme case, Sec1p from yeast
extracts has no observable affinity for the yeast synt axin, Sso1p,
but instead binds, perhaps indirectly, to the assembled SNARE
c omplex (21, 22). It has been suggested that the absence of a
detect able Sec1p–Sso1p interaction in yeast may be due to the
st ability of the closed conformation of Sso1p, which could result
in an inaccessibility of Sec1p-binding sites (15).
We examined the specificity of endogenous Sec1p for the three
exocytic SNAREs in yeast and report that Sec1p binds the
SNAREs only when they are assembled as a ternary SNARE
complex. The uncomplexed SNAREs and the binary t-SNARE
complex do not coprecipitate with Sec1p from yeast lysates. When
purified from yeast, Sec1p binds specifically and directly to the
ternary SNARE complex, reconstituting the binding specificity
observed in vivo for the endogenous full-length SNAREs. Purified
Sec1p binds to the cytoplasmic domain of the exocytic SNARE
complex with a 1:1 stoichiometry, and binding requires the loop
region connecting the two SNARE motifs in the Sec9p SNARE.
This interaction represents a SNARE-complex-binding mode that
is not mediated through the peptide-finger motif or through a latent
binding site exposed in the open conformation of the syntaxin
SNARE. This particular binding mode may offer a snapshot of an
SM protein activating membrane fusion through an interaction with
the assembled SNARE complex.
Endogenous Sec1p Binds Specifically to the SNARE Complex, Not to
Individual SNAREs.
A n interaction bet ween endogenous Sec1p and
the exocytic SNARE complex was observed in yeast lysates by
using an immunoprecipitation (IP) protoc ol followed by immu-
noblot analysis (21). Recently, additional interactions between
SM proteins and unc omplexed SNA REs have been reported (23,
24), including an interaction between recombinant Sec1p and the
plasma membrane syntaxin, Sso1p (25).
To examine the binding specificity of endogenous Sec1p in
greater det ail, we used the IP protoc ol under conditions that
maint ain the indiv idual SNAREs, Sec9p, Sso1p, and Snc2p,
Author contributions: M.M., F.M.H., and C.M.C. designed research; J.T., Y.-S.C., M.M.,
F.M.H., and C.M.C. performed research; M.M. and F.M.H. contributed new reagents
analytic tools; J.T., Y.-S.C., M.M., F.M.H., and C.M.C. analyzed data; and J.T. and C.M.C.
wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
Abbreviation: IP, immunoprecipitation.
To whom correspondence should be addressed. E-mail: carrcm@umdnj.edu.
© 2006 by The National Academy of Sciences of the USA
November 21, 2006
vol. 103
no. 47 www.pnas.orgcgidoi10.1073pnas.0605448103
separated from each other. Mutations that block SNARE-
c omplex assembly, such as in the exocyst mutant sec5–24,
maint ain the steady-state level of individual SNAREs but abol-
ish the SNARE-complex interaction with Sec1p (22). At the
restrictive temperature, none of the three unc omplexed
SNA REs coprecipitates with the C-terminally triple Myc-tagged
Sec1p (Sec1p-MYC
)insec5–24 (Fig. 1a). In the wild-type
c ontrol (SEC), there is no block in SNARE-complex assembly
at the higher temperature, and the interaction with Sec1p-MYC
is preser ved, as demonstrated by persistence of all three
SNA REs in the IP.
In case binding of Sec1p to any individual SNARE requires
disassembly of SNARE complexes, we repeated the IP under
disassembly conditions. Fig. 1b shows coprecipitation of the
SNA RE complex with Sec1p-MYC
under the normal assay
c onditions (ATP), which preserve SNARE complexes. Com-
plex disassembly is activated upon addition of an ATP-
regeneration system (ATP; ref. 21), and under these condi-
tions, no coprecipitation of disassembled SNAREs is observed
for the SEC strain. To demonstrate that the ATP sensitivity is
due to SNARE-complex disassembly, we repeated the ex peri-
ment in a sec18–1 strain. The mutant gene, sec18–1, encodes an
ATPase-defective version of NSF. The synaptic protein NSF
(and its homologs) is a chaperone required for general SNARE-
c omplex disassembly. When shifted to restrictive temperature,
SNA RE complexes are preserved in the sec18–1 strain, even in
the presence of ATP. Therefore, the ATP sensitivity of SNARE
c oprecipitation w ith Sec1p-MYC
is due to disassembly by
Sec18p. Once SNARE complexes are disassembled, the sepa-
rated SNAREs do not bind to Sec1p.
Endogenous Sec1p Binds SNARE Complexes Containing Mutant Sso1p.
The IP protocol was used to test the possibility that Sec1p
interacts with a latent motif that is accessible only in the open
c onformation of Sso1p (cartoon of conformations; Fig. 5, which
is published as supporting information on the PNAS web site).
Because wild-type Sso1p is st ably folded in the closed confor-
mation, binding to a latent motif was tested by using yeast strains
in which the wild-type Sso1p (and Sso2p, the product of the
duplicate gene, SSO2) has been replaced by either a truncation
or conformational mutant of Sso1p.
Deletion of the first 30 amino acids of Sso1p removes a
potential ‘‘peptide-finger’’ motif, described as an interaction
domain between synt axins and SM proteins known to function
at other vesicle trafficking steps in the cell (16, 17). The result is
a truncated protein, Sso1p[31–290], which functionally replaces
the proteins encoded by the endogenous wild-type genes, SSO1
and the duplicate, SSO2, indicating that the putative peptide-
finger motif is not required for the essential function of these
proteins (data not shown). Fig. 1c shows ATP-sensitive c opre-
cipit ation of Sec9p, Snc2p, and the truncated Sso1p[31–290] with
. This result indicates that the first 30 amino acids
of Sso1p are not required for SNARE-c omplex assembly or for
the interaction of the SNARE complex with Sec1p.
The conformational mutant, Sso1p-Open1, contains three
amino acid substitutions (V84E, K95E, and Y148A) in H
, the
N-ter minal autoinhibitory domain, which stabilize Sso1p in the
open conformation (26). Coprecipitation of Sec9p, Snc2p, and
Sso1p-Open1 with Sec1p-MYC
indicates that the mutations do
not interfere with the interaction bet ween Sec1p-MYC
and the
Sso1p-Open1 SNARE complex (Fig. 1c). Because Sso1p-Open1
rapidly assembles after disassembly, accumulation of Sso1p-
Open1 SNARE complexes is ex pected for this confor mational
mut ant. However, reassembly is problematic for testing whether
Sec1p can interact with the uncomplexed open conformation of
Sso1p. We address this question using purified proteins (below).
Purification of Sec1p from
Saccharomyces cerevisiae
. Previous stud-
ies using the rec ombinant cy toplasmic domains of Sso1p, Snc2p,
and the SNAP-25 homologous domain of Sec9p demonstrated
that this soluble SNA RE c omplex, and not the uncomplexed
Sso1p, binds to Sec1p from a yeast lysate (21). If the interaction
bet ween Sec1p and the SNARE complex is direct, we ex pected
to be able to reconstitute that interaction in vitro. Binding
rec onstitution was performed with recombinant SNARE pep-
tides purified from Escherichia coli and Sec1p purified from S.
cerevisiae. Attempts to purify Sec1p using either E. coli or
baculovirus systems failed due to aggregation of the Sec1p
product in physiological buffers (M.M., data not shown). When
we tried the E. coli system overexpressing GroELES and
-Sec1p (25), the purified recombinant His
-Sec1p did not
remain in solution after centrifugation for 30 min at 300,000
g (Fig. 6 Inset, which is published as supporting information on
the PNAS web site).
Soluble Sec1p was purified from S. cerevisiae by using the
pYES2 CT vector and host (Invitrogen, Carlsbad, CA). This
Fig. 1. In yeast, Sso1p, Sec9p, and Snc2p bind Sec1p as a SNARE complex, not
as individual SNAREs. (a) All three SNAREs coprecipitate from lysate of the
wild-type strain, SEC (NY1689), both at 25°C and after a shift to restrictive
temperature (37°C). In the vesicle-tethering mutant strain, sec5–24 (NY2228),
SNARE-complex assembly is blocked at 37°C, and none of the SNAREs are
detected in the IP. Sec1p-MYC
was immunoprecipitated with the 9E10 mono-
clonal antibody, and coprecipitation of Sso1p, Sec9p, and Snc2p (arrows) was
detected by using Western blot analysis. The input for each IP is shown (Left)
and represents 1.5% of the total. The positions of molecular weight standards
are indicated. (b) The interaction between Sec1p and SNAREs is abolished
when SNARE complexes are disassembled. SEC (NY1689) and the SNARE-
complex disassembly mutant strain, sec18 –1 (NY1691), were lysed either in the
presence () or absence () of ATP. Coprecipitated Sso1p, Sec9p, and Snc2p
(arrows) were detected by using Western blot analysis, as in a.(c) SNARE
complexes assembled with Sso1p[31–290] or Sso1p-Open1 coprecipitate with
. ATPase sensitivity of coprecipitation of SNAREs with Sec1p-MYC
was tested as in b. In the absence () of ATP, all three SNAREs coprecipitated
with Sec1p-MYC
in lysates from the sso1 mutants, sso1[31–290] (CCY18), and
sso1p-open1 (CCY17), as well as the isogenic wild-type control (CCY16). In the
presence () of ATP, SNAREs coprecipitated only from lysates of the sso1p-
open1 strain, presumably due to rapid reassembly of SNARE complexes.
Togneri et al. PNAS
November 21, 2006
vol. 103
no. 47
ex pression vector adds to the C terminus of the SEC1 coding
sequence a V5 epitope, plus a six-His tag (His
), for affin ity
purification. The additional sequences do not disrupt Sec1p
function in vivo, because there was no growth defect when the
endogenous essential SEC1 gene was replaced with SEC1-V5-
under the regulation of the SEC1 promoter (dat a not
We used high-speed centrifugation to separate soluble Sec1p-
f rom agg regates. Centrifugation at 300,000 g sedi-
ments particles larger than 13S (e.g., ribosomes and exocyst
c omplexes), whereas soluble proteins smaller than 13S remain in
the supernatant fraction. Sec1p-V5-His
purified from S. cerevi-
siae remained in the supernatant fraction (Fig. 5 Inset). Further-
more, Sec1p-V5-His
eluted from a Superdex 200 sizing c olumn
at the position predicted for the monomeric protein, M
of 89
kDa (Fig. 5). The M
observed by MS is 89,344 11 Da (mean
of five runs, SD; SynthesizingSequencing Facility, Depart-
ment of Molecular Biology, Princeton University, Princeton,
NJ). This mass is consistent with the calculated M
of 89,353 Da
for Sec1p-V5-His
with the N-terminal Met cleaved, and the Ser
that follows acetylated, a common cotranslational modification
in eukaryotes, including yeast (27).
Sec1p Binds Directly to the Cytoplasmic SNARE Complex. The cy to-
plasmic domain of the exoc ytic SNARE complex, which includes
Sso1p[1–265] and Snc2p[1–93] (without transmembrane do-
mains), plus the SNAP-25 domain, Sec9p[416651], was previ-
ously shown to bind Sec1p from a yeast lysate (21). Therefore,
we used this cy toplasmic SNA RE complex to test for a direct
interaction with Sec1p-V5-His
in a pulldown assay (Fig. 2; see
Mater ials and Methods). Bound proteins and peptides were
quantit ated by densitometry by using stocks of the proteins and
peptides as c oncentration standards (Supporting Text, which is
published as supporting information on the PNAS web site). The
c ytoplasmic SNARE complex bound directly to Sec1p-V5-His
(Fig. 3a). By incubating increasing concentrations of SNARE
c omplexes with a constant amount of Sec1p-V5-His
, we found
that SNARE-c omplex binding saturated at 1
M, with a stoi-
chiometry of one cytoplasmic SNARE complex bound to each
(Fig. 7, which is published as supporting infor-
mation on the PNAS web site). We detected no binding to the
individual SNAREs, or the t-SNA RE complex, Sec9p[416651]
Fig. 2. SNARE peptides used in this study. (a) SNARE peptides were mixed
stoichiometrically to define the three types of SNARE complexes used for the
binding studies. Lane 1: The cytoplasmic SNARE complex, Sso1p[1–265]:
Sec9p[416 651]:Snc2p[1–93]. Lane 2: The core complex, Sso1p[179
265]:Sec9p[416 651]:Snc2p[1–93]. Lane 3: The four-helix bundle, Sso1p
[179–265]:Sec9p[416 –504]:Sec9p[571– 651]:Snc2p[1–93]. Peptides are ob-
served as Coomassie-stained bands, separated on a 16.5% Tris-tricine gel.
Molecular weight markers are indicated. (b) A schematic of each of the parent
proteins with SNARE peptides defined by amino-acid positions. H
: the
N-terminal inhibitory domain of Sso1p. H
: the SNARE motif helix. Sec9p H
and H
: the two SNARE-motif helices of the SNAP-25 domain of Sec9p. TM,
transmembrane domains of Sso1p and Snc2p.
Fig. 3. In vitro reconstitution of Sec1p-SNARE-complex binding. (a) Purified
binds to the cytoplasmic SNARE complex but not to individual
SNAREs or the t-SNARE complex. For each binding reaction, SNAREs were
added to a final concentration of 1
M. Lane 1, Control IP: the cytoplasmic
SNARE complex was added to V5 antibody immobilized on Protein G Sepha-
rose in the absence of Sec1p-V5-His
. Lanes 2– 6: Assembled SNARE complexes
or individual SNARE peptides were added, as indicated (), to 0.4
immobilized on V5-Protein G Sepharose. Lane 7: The same
t-SNARE complex used in lane 6, but with Snc2p added to the binding reaction
). Arrows indicate Coomassie-stained bands of the proteins and peptides,
which were separated on a 16.5% Tris-tricine gel. Molecular weight markers,
V5 antibody heavy chain (H
) and light chain (L
) are indicated. (b) The Sec9p
loop region is required for interaction with Sec1p-V5-His
. Binding of SNARE
peptides to immobilized Sec1p-V5-His
was performed as in a. Lane 1: Control
IP. Lane 2: The cytoplasmic SNARE complex. Lane 3: The cytoplasmic SNARE
complex assembled with H
and H
of Sec9p, in the absence of the loop region,
Sso1p[1–265]:Sec9p[416–504]:Sec9p[571– 651]:Snc2p[1–93]. Lane 4: The four-
helix bundle, Sso1p[179–265]:Sec9p[416 –504]:Sec9p[571– 651]: Snc2p[1–93].
www.pnas.orgcgidoi10.1073pnas.0605448103 Togneri et al.
plus Sso1p[1–265] (Fig. 3a, lanes 3–6) up to 10
M (Fig. 7a).
However, when 1
M Snc2p[1–93] was added to the sample
c ontain ing 1
M t-SNARE c omplex (Fig. 3a,
), ternary com-
plexes rapidly assembled and bound to Sec1p-V5-His
(c ompare
lanes 2 and 7).
A Sec9p Loop Region Is Required for SNARE-Complex Binding to Sec1p.
To determine the SNARE domains necessary for interaction
with Sec1p-V5-His
, the binding assay was performed with
SNA RE complexes assembled from shortened versions of the
SNA RE peptides (Fig. 2). In the core c omplex, Sec9p[416651]
and Snc2p[1–93] are assembled with Sso1p[179–265], which is
tr uncated to remove the H
domain. In the four-helix bundle,
Sso1p[179–265] and Snc2p[1–93] are assembled with
Sec9p[416–504] and Sec9p[571–651], which represent Helix
) and Helix
), without the intervening loop region. For
each combination of SNARE peptides, heterologously tagged
SNA RE c onstructs were used in a separate pulldown assay to
c onfirm SNARE-c omplex assembly (Fig. 8, which is published as
supporting information on the PNAS web site). The cytoplasmic
SNA RE complex, the t-SNARE complex, the four-helix bundle
and each of the individual peptides behaved as soluble mono-
mers, remaining in the supernatant fraction after centrifugation
at 300,000 g (Fig. 9, which is published as supporting infor-
mation on the PNAS web site).
A lthough the cy toplasmic SNARE complex binds Sec1p-V5-
, the four-helix bundle does not (Fig. 3b), suggesting that
either or both of the domains H
of Sso1p or the loop region
Sec9p are required for binding. Two lines of evidence suggest
that the H
domain is not sufficient for binding. First, the
binary t-SNA RE complex, which includes H
as part of
Sso1p[1–265], does not bind Sec1p-V5-His
(Fig. 3a, lane 6).
Sec ond, the core complex, which retains the Sec9p loop region
but lacks H
, binds to Sec1p-V5-His
(Fig. 4, lane 6). The core c omplex behaves as a particle,
sedimenting at 300,000 g (Fig. 9, lane 4). Rotary-shadowing
electron microscopy revealed oligomers c ontaining six to eight
c ore SNARE complexes (Fig. 10, which is published as support-
ing information on the PNAS web site). The dimensions of the
individual components of the oligomer are in agreement with
similar studies of yeast exoc ytic SNA RE complexes (28). The
oligomeric nature of the core complex complicates interpreta-
tion of the requirement of Sso1p H
for Sec1p-V5-His
Nonetheless, we could test whether the Sec9p loop region is
required by using a combination of SNA RE peptides that lacks
the Sec9p loop region, but retains H
: Sso1p[1–265], Snc2p[1–
93], Sec9p[416–504] and Sec9p[571–651]. The resulting SNARE
c omplex assembles (Fig. 8), yet it does not bind Sec1p-V5-His
(Fig. 3b, lane 3; Fig. 7a), suggesting that the Sec9p loop region
is an important binding determinant.
Sec1p Does Not Bind to the Open Conformation of Sso1p. To test for
a latent Sec1p-interaction site, Sso1p mut ants were tested for
their abilit y to interact with purified Sec1p-V5-His
. Wild-t ype
Sso1p[1–265] is stably folded in the closed conformation (Fig. 5),
and it does not bind Sec1p-V5-His
(Fig. 4, lane 1). The
c onformational mutant, Sso1p[1–265]-Open1, favors the open
c onformation, yet uncomplexed Sso1p[1–265]-Open1 does not
interact with Sec1p-V5-His
(Fig. 4, lane 3). Complete removal
of the H
domain, as in Sso1p[179–265], exposes the Sso1p
SNA RE motif (H
), but Sso1p[179–265] does not bind to
(Fig. 4, lane 5). All three Sso1p peptides were
tested up to 10
M (Fig. 7a). SNA RE complexes made with each
of these Sso1p peptides do interact with Sec1p-V5-His
(Fig. 4,
lanes 2, 4, and 6). Note that the suprastoichiometric binding of
SNA RE complexes made with Sso1p[179–265] is likely to be a
c onsequence of the oligomeric nature of the core c omplex
(described above and in Figs. 9 and 10). With that caveat, it
appears that the H
tr uncation and mutations in Sso1p[1–265]-
Open1 do not disrupt the Sec1p-binding site. Taken together,
these results suggest that, rather than a latent binding motif on
Sso1p, Sec1p binds specifically to a site on the assembled
SNA RE complex, a site that includes the Sec9p loop region.
Direct Binding of Yeast Sec1p to the Exocytic SNARE Complex. The
results of these studies demonstrate that Sec1p purified from
yeast binds specifically and directly to the exocytic SNARE
c omplex. SNARE-complex binding was reconstituted in vitro
with the purified cytoplasmic domain of the ternary SNARE
c omplex, which consists of Sso1p[1–265], Sec9p[416651], and
Snc2p[1–93]. All three components of the SNARE complex are
necessary to reconstitute the interaction, because Sec1p-V5-His
binds to none of the individual SNA REs or to the binary
t-SNA RE complex, in contrast to a recent observation (25).
Binding studies with truncated SNARE domains revealed that
the Sec9p H
loop region is required to support an interaction
with Sec1p.
A specific interaction between Sec1p and the ternary SNARE
complex is consistent with what is observed in yeast. Using an IP
protocol with lysates from mutants defective in SNARE-complex
assembly, we observed none of the individual SNAREs or the
t-SNARE complex coprecipitated with Sec1p. Using the same
protocol with wild-type yeast, we found that disassembly of
SNARE complexes abolishes Sec1p binding to any of the SNAREs,
as expected if binding requires the fully assembled SNARE com-
plex. Likewise, Sec1p concentrate s at sites of SNARE-complex
assembly, where secretory vesicle s fuse with the plasma membrane
(22, 26, 29). Furthermore, Sec1p is mislocalized in sec mutants
defective for SNARE-complex assembly and shows an increase in
polarized localization in mutants that accumulate SNARE com-
Fig. 4. Sec1p does not bind to the open conformation of Sso1p. SNARE-
complex binding to purified Sec1p-V5-His
was performed as in Fig. 3. For each
binding reaction, SNAREs were added to a final concentration of 1
M. Lane
1: Uncomplexed Sso1p[1–265]. Lane 2: cytoplasmic SNARE complex (SC), as-
sembled with Sso1p[1–265]. Lane 3: Uncomplexed Sso1p[1–265]-Open1. Lane
4: Cytoplasmic SC assembled with Sso1p[1–265]-Open1. Lane 5: Uncomplexed
SNARE motif, Sso1p[179 –265]. Lane 6: Cytoplasmic SC assembled with
Sso1p[179–265], also called the core complex (Fig. 2). Arrows indicate Coo-
massie-stained bands of the proteins and peptides, which were separated on
a 16.5% Tris-tricine gel. Molecular weight markers, the V5 antibody heavy
chain (H
) and light chain (L
), are indicated.
Togneri et al. PNAS
November 21, 2006
vol. 103
no. 47
plexes (21, 22, 26), supporting the conclusion that Sec1p binds to
SNARE complexes. Our ability to reconstitute the specific inter-
action between Sec1p and the SNARE complex not only supports
these in vivo observations, it demonstrates that this interaction does
not require other factors, such as additional proteins (30, 31) or
Another Way for SM Proteins to Interact with SNAREs. Using wild-
t ype and mutant Sso1p constructs, our binding studies with yeast
Sec1p indicate the following: (i) Sec1p has no observable affinity
for Sso1p in the closed conformation, r uling out the closed-
synt axin-binding mode characterized for nSec1Munc-18 (12,
32); (ii) the N-terminal 30 amino acids of Sso1p are not required
for Sec1p binding or SNARE function, ruling out the syntaxin
peptide-finger-binding mode characterized for Sly1p and
Vps45p (16, 17); (iii) Sec1p purified from yeast binds directly and
stoichiometrically to the purified SNARE complex, ruling out an
indirect binding mode, as suggested for Vps33p (19, 20); and (iv)
there is no evidence for an interaction between Sec1p and
individual nonsyntaxin SNAREs, as has been observed for Sly1p
(23, 33). We also ex plicitly tested the proposal that Sec1p can
bind to the t-SNA RE complex (25) and found no evidence, in
vivo or in vitro, for an interaction between Sec1p and the
t-SNA RE complex formed by Sec9p and Sso1p. Not ably, we did
observe nonspecific interactions between Sec1p-V5-His
fied from yeast and recombinant SNARE peptides that were
t agged with heterologous sequences (data not shown). We
c onclude that the interaction observed bet ween Sec1p and the
exoc ytic SNARE complex represents a specific mode of SNARE
interactions: the SNARE-complex-binding mode.
Evidence for a SNARE-Complex-Binding Mode Raises New Questions.
The finding that at least one SM protein binds specifically to the
assembled SNA RE c omplex raises new questions about the role
of SM proteins in vesicle traf ficking and membrane fusion. One
suggested function is protection of SNARE complexes from
disassembly before membrane fusion (21). However, we see no
evidence for protection from disassembly when over-expressing
Sec1p in yeast (Fig. 11a, which is published as supporting
infor mation on the PNAS web site), or when using an in vitro
disassembly assay with purified components (Fig. 11b). Alter-
natively, Sec1p may activate membrane fusion by zippering
together the membrane proximal ends of the SNAREs (21, 25,
34, 35). This possibility remains to be tested.
Does the SNARE-complex interaction observed in yeast repre-
sent an intermediate common to all SM proteins? If so, we expect
to find that other SM proteins use a similar SNARE-complex-
binding mode to interact with their cognate SNARE complexes.
Indeed, recent findings indicate that the SNARE-complex inter-
actions observed for Sly1p and Vps45p are not simply the result of
the SM protein binding to the syntaxin peptide-finger motif, as
previously believed (23, 33).
In the cell, SM proteins may function with protein complexes
that link vesicle tethering and SNARE-complex assembly. The
vacuolar SM protein, Vps33p, is part of a large protein complex
k nown as HOPS (19, 36). HOPS interacts with assembled
SNA RE complexes and is required for vacuole docking and
fusion (37, 38). HOPS binds Vam7p, the vacuole-specific
SNA RE homologous to neuronal SNAP-25 (37). We find that
the Sec1p interaction with SNARE complexes requires a binding
site on the plasma membrane-specific SNAP-25 homolog, Sec9p,
suggesting that interaction with this t-SNA RE may be a common
requirement for SM protein function. Like HOPS, the plasma
membrane-specific vesicle tethering complex, known as the
exoc yst, is both a Rab effector (39) and required for SNA RE-
c omplex assembly (22). Although Sec1p is not a stable compo-
nent of the exocyst, it may associate with low af finit y to promote
SNA RE-c omplex assembly (30). Although we observe no acti-
vation of assembly with Sec1p alone (Fig. 12, which is published
as supporting information on the PNAS web site), one exocyst
protein, Sec6p, binds directly to Sec9p and inhibits assembly of
SNA RE complexes (40). If the Sec6p–Sec9p interaction repre-
sents a regulatory intermediate in SNARE-c omplex assembly,
perhaps association of Sec1p with Sec9p and the exocyst is
required for a subsequent step to activate SNAREs for mem-
brane fusion.
Whether SM proteins share common functions will require a
c ombination of mechanistic studies using purified proteins in
SNA RE complex-assembly and membrane-fusion reactions,
plus introduction of mutations designed to test the predictions of
a model for SM-dependent SNARE-complex assembly and
membrane fusion.
Materials and Methods
Plasmid Construction. For c onstruction of ex pression vectors
pK LN9 Sso1p[179–265], pKLN10 Sec9p[416–504] and pKLN11
Sec9p[571–651] (K. N. Tomishima and F.M.H., unpublished
results), BamHI and EcoRI restriction sites were added to the
ends of the SSO1 and SEC9 sequences by PCR, and the products
were ligated into the pGEX4T-1 vector (GE Healthcare, Pisca-
t away, NJ). For construction of pYES2CT Sec1p, BamHI and
HindIII restriction sites were added to the ends of the SEC1
sequence by PCR using pNB680 (21) as template. The PCR
product was then ligated into the pYES2CT vector (Invitrogen)
and confirmed by sequencing (IDT, Piscat away, NJ).
Immunoprecipitation Protocol. Seven S. cerevisiae strains were used
for the IP experiments (Table 1). For CCY16, CCY17, and
CCY18, FHY102 (26) was modified by integration of a triple-
myc tag at the C terminus of SEC1, as described (21) and
transfor med with the plasmids indicated. Cell lysis and IP were
performed as described, with at least 50% recovery of Sec1p-
using the 9E10 antibody (21). The proteins that co-
precipit ated with Sec1p-MYC
were detected by Western blot
analysis. Blots were probed with primary antibodies against
Sso1p (16,371; ref. 26); Sec9p (CUMC938) and Snc2p (CUMC6;
ref. 41) and with the secondary antibody, peroxidase-conjugated
anti-rabbit IgG (Sigma, St. Louis, MO). Antibody recognition
Table 1. Yeast strains used in this study
Strain Genotype Source
CCY16 MATa his31 leu20 ura30 sec1::SEC1MYC
URA3 sso1::kanMX4 sso2::kanMX4 pMM250 (CEN LEU2 SSO1) This study
CCY17 MATa his31 leu20 ura30 sec1::SEC1MYC
URA3 sso1::kanMX4 sso2::kanMX4 pMM266 (CEN LEU2 sso1-open1) This study
CCY18 MATa his31 leu20 ura30 sec1::SEC1MYC
URA3 sso1::kanMX4 sso2::kanMX4 pFH24 (CEN LEU2 sso131–290) This study
FHY102 MATa his31 leu20 ura30 sso1::kanMX4 sso2::kanMX4 pMM250 (CEN LEU2 SSO1) M. Munson
his31/ his31 leu2/leu2 trp1–289/trp1–289 ura3–52/ura3–52 Invitrogen
NY1689 MAT
his3-200 leu2–3, 112 trp1 ura3–52 sec1::SEC1MYC
URA3 P. Novick
NY1691 MAT
sec18–1 his3-200 leu2–3, 112 trp1 ura3–52 sec1::SEC1MYC
URA3 P. Novick
NY2228 MAT
sec5–24 ura3–52 sec1::SEC1MYC
URA3 P. Novick
www.pnas.orgcgidoi10.1073pnas.0605448103 Togneri et al.
was detected by using a chemiluminescence reagent (Pierce,
Rockford, IL).
Protein Purification. Recombinant SNARE peptides Sso1p[1–
265], Sso1p[1–265]-Open1 Snc2p[1–93] and Sec9p[416651]
were ex pressed and purified as described (10, 21, 26).
Sso1p[179–265], Sec9p[416–504] and Sec9p[571–651] were ex-
pressed in E. coli BL21 cells as GST fusion proteins in a
pGEX4T-1 expression vector as described by the manufacturer
(GE Healthcare). After removal of GST, Sec9p[416–504] and
Sec9p[571–651] were further purified by using Benzamadine
Sepharose (GE Healthcare) and Sso1p[179–265] by anion ex-
change (MonoQ, GE Healthcare). Purified peptides were con-
centrated by ultrafiltration (Millipore Amicon, Billerica, MA;
Ultra-15) and stored at 80°C.
His-t agged Sec1p (Sec1p-V5-His
) was ex pressed in yeast by
using the pYES2CT Sec1p plasmid transformed into the S.
cerevisiae strain, INVSc1, as described by the manufacturer
(Invitrogen). Af ter a 14-h induction in galactose, cells were
harvested and resuspended in wash buffer (50 mM Hepes, pH
7.4 20 mM NaF20 mM NaN
). Washed cells were lysed in
buf fer A (50 mM Hepes, pH 7.4150 mM KCl10% glycerol30
mM imidazole) plus 1 mM PMSF and yeast protease inhibitor
mixture (21), using a microfluidizer, model M-110Y (Microflu-
idics, Newton, MA). The lysate was clarified by centrifugation;
af ter 10,000 g for 30 min (Sorvall, Guelph, ON, Canada; rotor
SS-34), the supernatant f raction (S10) was centrifuged for
another 45 min at 100,000 g (Beckman, Fullerton, CA; rotor
Ti-70). Sec1p-V5-His
f rom the S100 was purified on a nickel-
charged chelating column (HiTrap, GE Healthcare), with a
gradient of 10–100% buffer B (50 mM Hepes, pH 7.4150 mM
KCl250 mM imidazole10% glycerol). For storage, 2 mM DTT
and 1 mM EDTA were added and the aliquots were frozen at
SNARE Binding with Immobilized Sec1p-V5-His
. To standardize the
amount of Sec1p-V5-His
in the binding reaction, 0.4
n ickel purified Sec1p-V5-His
was immunoaffinity-purified by
using 1.8
g of V5 monoclonal antibody (Invitrogen). Excess
was washed from the resin with SNARE-binding
buf fer (50 mM Hepes, pH 7.4100 mM NaCl0.5% IGEPAL
0.5 mM DTT1 mM EDTA). Before addition to the binding
reaction, SNA RE c omplexes were assembled, as described (10).
The time required for completion of SNARE-complex assembly
was measured separately by using a SNARE-assembly assay
(Supporting Text, which is published as supporting infor mation
on the PNAS web site). One micromolar SNARE or SNARE
c omplex was added to immobilized Sec1p-V5-His
and incu-
bated for2hat4°C. The reactions were separated by SDS
PAGE and stained with Coomassie blue R250.
We are g rateful to Arati Tripathi for helpful discussions and mass
spectrometry analysis; Karin Nicholson Tomishima for construction of
pKLN9, -10, and –11; Jenn ifer Streltsova for c onstruction of pYESC2
Sec1p; Jenna Hutton for comments and construction of MYC-tagged
SEC1 in the sso1 mutants; Donald Winkelmann for electron microscopy
of core SNARE c omplexes; Barbara Siminovich-Blok for the replace-
ment of SEC1 with the SEC1-V5-His
construct in yeast; Hays Rye
(Princeton University) for helpful comments and the E. coli system
overexpressing GroELES; Bill Wickner (Dartmouth Medical School,
Hanover, NH) for the generous gifts of purified Sec17p and Sec18p; and
Patrick Brennwald (University of North Carolina School of Medicine,
Chapel Hill, NC) for providing
Sec9p and
Snc2p antibodies. This work
was supported by National Institutes of Health Grants R01GM066291
(to C.M.C.), R01GM071574 (to F.M.H.), R01GM068803 (to M.M.), and
5T32GM08319-14 (to J.T.), and the Pew Scholars Program for Biomed-
ical Sciences (to C.M.C.).
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Togneri et al. PNAS
November 21, 2006
vol. 103
no. 47
    • "The role of the Sec1/Munc18 (SM) family of proteins in SNARE mediated fusion has been long recognized. SM proteins are known to interact predominantly with the syntaxin (Stx) family of SNAREs (Misura et al., 2000), but SM protein binding to the whole SNARE complex has also been observed (Carr et al., 1999; Togneri et al., 2006; Lobingier and Merz, 2012). For the Golgi localized SM protein, Sly1, the main type of reported binding was to the N-terminus of Stx5 (Yamaguchi et al., 2002), a binding mode that is consistent with a role in SNARE complex formation (Kosodo et al., 2002; Peng and Gallwitz, 2002). "
    [Show abstract] [Hide abstract] ABSTRACT: Glycosylation is recognised as a vitally important posttranslational modification. The structure of glycans that decorate proteins and lipids is largely dictated by biosynthetic reactions occurring in the Golgi apparatus. This biosynthesis relies on the relative distribution of glycosyltransferases and glycosidases, which is maintained by retrograde vesicle traffic between Golgi cisternae. Tethering of vesicles at the Golgi apparatus prior to fusion is regulated by Rab GTPases, coiled-coil tethers termed golgins and the multisubunit tethering complex known as the conserved oligomeric Golgi (COG) complex. In this review we discuss the mechanisms involved in vesicle tethering at the Golgi apparatus and highlight the importance of tethering in the context of glycan biosynthesis and a set of diseases known as congenital disorders of glycosylation.
    Full-text · Article · Mar 2016
    • "In this study, Munc18-1 expression was found to be upregulated in the active zone of unsusceptible rats, thereby directly promoting syntaxin-1 stability and regulating the formation of vesicle priming. Thus, elevated Munc18-1 may act as a compensatory regulator of accelerated syntaxin-1 (Zilly et al., 2006), binding simultaneously to the SNARE complex to control the assembly of the Munc18-1/SNARE membrane fusion complex (Togneri et al., 2006; Zilly et al., 2006; Khvotchev et al., 2007; Rathore et al., 2010; Lim et al., 2013). Finally, to address whether additional core components of the SNARE complex were affected, SNAP25 and VAMP2 expression in the synaptic junction was analyzed. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: While stressful events are recognized as an important cause of major depressive disorder, some individuals exposed to life stressors maintain normal psychological functioning. The molecular mechanism(s) underlying this phenomenon remain unclear. Abnormal transmission and plasticity of hippocampal synapses have been implied to play a key role in the pathoetiology of major depressive disorder. Methods: A chronic mild stress protocol was applied to separate susceptible and unsusceptible rat subpopulations. Proteomic analysis using an isobaric tag for relative and absolute quantitation coupled with tandem mass spectrometry was performed to identify differential proteins in enriched hippocampal synaptic junction preparations. Results: A total of 4318 proteins were quantified, and 89 membrane proteins were present in differential amounts. Of these, SynaptomeDB identified 81 (91%) having a synapse-specific localization. The unbiased profiles identified several candidate proteins within the synaptic junction that may be associated with stress vulnerability or insusceptibility. Subsequent functional categorization revealed that protein systems particularly involved in membrane trafficking at the synaptic active zone exhibited a positive strain as potential molecular adaptations in the unsusceptible rats. Moreover, through STRING and immunoblotting analysis, membrane-associated GTP-bound Rab3a and Munc18-1 appear to coregulate syntaxin-1/SNAP25/VAMP2 assembly at the hippocampal presynaptic active zone of unsusceptible rats, facilitating SNARE-mediated membrane fusion and neurotransmitter release, and may be part of a stress-protection mechanism in actively maintaining an emotional homeostasis. Conclusions: The present results support the concept that there is a range of potential protein adaptations in the hippocampal synaptic active zone of unsusceptible rats, revealing new investigative targets that may contribute to a better understanding of stress insusceptibility.
    Full-text · Article · Sep 2015
    • "Sro7 interacts with both Sec9 and Exo84, a component of the exocyst, and functions in the docking and fusion of post-Golgi vesicles with the plasma membrane [32,33]. Sec1 binds to the assembled SNARE complex and is involved in the activation of membrane fusion [34,35] . The observation that these genes could suppress the lethality caused by Rga1-C538 overexpression suggests that excess Rga1-C538 may impair exocytosis. "
    [Show abstract] [Hide abstract] ABSTRACT: In budding yeast, Rga1 negatively regulates the Rho GTPase Cdc42 by acting as a GTPase-activating protein (GAP) for Cdc42. To gain insight into the function and regulation of Rga1, we overexpressed Rga1 and an N-terminally truncated Rga1-C538 (a.a. 538-1007) segment. Overexpression of Rga1-C538 but not full-length Rga1 severely impaired growth and cell morphology in wild-type cells. We show that Rga1 is phosphorylated during the cell cycle. The lack of phenotype for full-length Rga1 upon overexpression may result from a negative regulation by G1-specific Pho85, a cyclin-dependent kinase (CDK). From a high-copy suppressor screen, we isolated RHO3, SEC9, SEC1, SSO1, SSO2, and SRO7, genes involved in exocytosis, as suppressors of the growth defect caused by Rga1-C538 overexpression. Moreover, we detected that Rga1 interacts with Rho3 in two-hybrid and bimolecular fluorescence complementation (BiFC) assays. Rga1 preferentially interacts with the GTP-bound form of Rho3 and the interaction requires the GAP domain and additional sequence upstream of the GAP domain. Our data suggest that the interaction of Rga1 with Rho3 may regulate Rho3's function in polarized bud growth.
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