Specific SNARE complex binding mode of the
Sec1?Munc-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 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 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
tracellular membranes. Fusion of the vesicle and target 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
between membranes in a conformation similar to the fusion-
active (fusogenic) state of many viral membrane-fusion proteins
transport and fusion throughout the cell (5, 6). For example, the
Sec1?Munc-18 (SM) family of proteins is proposed to be essential
for activation of SNARE-complex assembly at the vesicle-fusion
is supported by studies in a variety of organisms (8).
Activation of SNARE-complex assembly is a plausible mech-
anism for SM-dependent vesicle fusion. SM proteins bind and
potentially modulate the conformation of SNARE proteins that
are homologous to the neuronal synaptic membrane protein,
syntaxin (9). Syntaxins can adopt a fusion-inactive ‘‘closed’’
conformation (10), in which an N-terminal three-helix bundle
motif (Habc) 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 intermediate required to convert syntaxin to the ‘‘open’’
conformation, which then assembles with the other SNAREs to
form the fusogenic 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 exocy-
tosis events, such as synaptic vesicle fusion (13, 14).
The universality of the closed-syntaxin-binding mode for
vesicle fusion is called into question by the different syntaxin-
binding modes that have been observed for other SM proteins (9,
ukaryotic cell growth and organization depend on targeted
membrane-fusion reactions between vesicles and other in-
15). The SM proteins, Sly1p and Vps45p, interact with an
N-terminal peptide-finger motif of their cognate 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 Syntaxin 1A
(18). An indirect syntaxin-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 syntaxin, Sso1p,
but instead binds, perhaps indirectly, to the assembled SNARE
complex (21, 22). It has been suggested that the absence of a
detectable Sec1p–Sso1p interaction in yeast may be due to the
stability 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
binding site exposed in the open conformation of the syntaxin
SNARE. This particular binding mode may offer a snapshot of an
the assembled SNARE complex.
Endogenous Sec1p Binds Specifically to the SNARE Complex, Not to
the exocytic SNARE complex was observed in yeast lysates by
using an immunoprecipitation (IP) protocol followed by immu-
noblot analysis (21). Recently, additional interactions between
plasma membrane syntaxin, Sso1p (25).
To examine the binding specificity of endogenous Sec1p in
greater detail, we used the IP protocol under conditions that
maintain the individual 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: firstname.lastname@example.org.
© 2006 by The National Academy of Sciences of the USA
November 21, 2006 ?
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no. 47 www.pnas.org?cgi?doi?10.1073?pnas.0605448103
separated from each other. Mutations that block SNARE-
complex assembly, such as in the exocyst mutant sec5–24,
maintain 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 uncomplexed
SNAREs coprecipitates with the C-terminally triple Myc-tagged
Sec1p (Sec1p-MYC3) in sec5–24 (Fig. 1a). In the wild-type
control (SEC?), there is no block in SNARE-complex assembly
at the higher temperature, and the interaction with Sec1p-MYC3
is preserved, as demonstrated by persistence of all three
SNAREs 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
SNARE complex with Sec1p-MYC3 under the normal assay
conditions (?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 experi-
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-
complex disassembly. When shifted to restrictive temperature,
SNARE complexes are preserved in the sec18–1 strain, even in
the presence of ATP. Therefore, the ATP sensitivity of SNARE
coprecipitation with Sec1p-MYC3 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
conformation of Sso1p (cartoon of conformations; Fig. 5, which
is published as supporting information on the PNAS web site).
Because wild-type Sso1p is stably 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 syntaxins 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 copre-
Sec1p-MYC3. This result indicates that the first 30 amino acids
of Sso1p are not required for SNARE-complex 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 Habc, the
N-terminal autoinhibitory domain, which stabilize Sso1p in the
open conformation (26). Coprecipitation of Sec9p, Snc2p, and
Sso1p-Open1 with Sec1p-MYC3indicates that the mutations do
not interfere with the interaction between Sec1p-MYC3and the
Sso1p-Open1 SNARE complex (Fig. 1c). Because Sso1p-Open1
rapidly assembles after disassembly, accumulation of Sso1p-
Open1 SNARE complexes is expected for this conformational
mutant. 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 recombinant cytoplasmic domains of Sso1p, Snc2p,
and the SNAP-25 homologous domain of Sec9p demonstrated
that this soluble SNARE complex, and not the uncomplexed
Sso1p, binds to Sec1p from a yeast lysate (21). If the interaction
between Sec1p and the SNARE complex is direct, we expected
to be able to reconstitute that interaction in vitro. Binding
reconstitution 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 GroEL?ES and
His6-Sec1p (25), the purified recombinant His6-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
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
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)
are indicated. (b) The interaction between Sec1p and SNAREs is abolished
when SNARE complexes are disassembled. SEC? (NY1689) and the SNARE-
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
was tested as in b. In the absence (?) of ATP, all three SNAREs coprecipitated
with Sec1p-MYC3in 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 ?
expression vector adds to the C terminus of the SEC1 coding
sequence a V5 epitope, plus a six-His tag (His6), for affinity
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-
His6 under the regulation of the SEC1 promoter (data not
We used high-speed centrifugation to separate soluble Sec1p-
V5-His6from aggregates. Centrifugation at 300,000 ? g sedi-
ments particles larger than 13S (e.g., ribosomes and exocyst
complexes), whereas soluble proteins smaller than 13S remain in
the supernatant fraction. Sec1p-V5-His6purified from S. cerevi-
siae remained in the supernatant fraction (Fig. 5 Inset). Further-
more, Sec1p-V5-His6eluted from a Superdex 200 sizing column
at the position predicted for the monomeric protein, Mrof 89
kDa (Fig. 5). The Mrobserved by MS is 89,344 ? 11 Da (mean
of five runs, ?SD; Synthesizing?Sequencing Facility, Depart-
ment of Molecular Biology, Princeton University, Princeton,
NJ). This mass is consistent with the calculated Mrof 89,353 Da
for Sec1p-V5-His6with 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 cyto-
plasmic domain of the exocytic SNARE complex, which includes
Sso1p[1–265] and Snc2p[1–93] (without transmembrane do-
mains), plus the SNAP-25 domain, Sec9p[416–651], was previ-
ously shown to bind Sec1p from a yeast lysate (21). Therefore,
we used this cytoplasmic SNARE complex to test for a direct
interaction with Sec1p-V5-His6in a pulldown assay (Fig. 2; see
Materials and Methods). Bound proteins and peptides were
quantitated by densitometry by using stocks of the proteins and
peptides as concentration standards (Supporting Text, which is
published as supporting information on the PNAS web site). The
cytoplasmic SNARE complex bound directly to Sec1p-V5-His6
(Fig. 3a). By incubating increasing concentrations of SNARE
complexes with a constant amount of Sec1p-V5-His6, we found
that SNARE-complex binding saturated at 1 ?M, with a stoi-
chiometry of one cytoplasmic SNARE complex bound to each
Sec1p-V5-His6(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-SNARE complex, Sec9p[416–651]
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.
proteins with SNARE peptides defined by amino-acid positions. Habc: the
N-terminal inhibitory domain of Sso1p. H3: the SNARE motif helix. Sec9p Ha
and Hb: the two SNARE-motif helices of the SNAP-25 domain of Sec9p. TM,
transmembrane domains of Sso1p and Snc2p.
SNARE peptides used in this study. (a) SNARE peptides were mixed
Sec1p-V5-His6binds 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-His6. Lanes 2–6: Assembled SNARE complexes
or individual SNARE peptides were added, as indicated (?), to 0.4 ?g of
Sec1p-V5-His6 immobilized on V5-Protein G Sepharose. Lane 7: The same
(*). 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 (Hc) and light chain (Lc) are indicated. (b) The Sec9p
loop region is required for interaction with Sec1p-V5-His6. Binding of SNARE
peptides to immobilized Sec1p-V5-His6was performed as in a. Lane 1: Control
IP. Lane 2: The cytoplasmic SNARE complex. Lane 3: The cytoplasmic SNARE
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].
In vitro reconstitution of Sec1p-SNARE-complex binding. (a) Purified
www.pnas.org?cgi?doi?10.1073?pnas.0605448103Togneri 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
containing 1 ?M t-SNARE complex (Fig. 3a,*), ternary com-
plexes rapidly assembled and bound to Sec1p-V5-His6(compare
lanes 2 and 7).
To determine the SNARE domains necessary for interaction
with Sec1p-V5-His6, the binding assay was performed with
SNARE complexes assembled from shortened versions of the
SNARE peptides (Fig. 2). In the core complex, Sec9p[416–651]
and Snc2p[1–93] are assembled with Sso1p[179–265], which is
truncated to remove the Habcdomain. In the four-helix bundle,
Sso1p[179–265] and Snc2p[1–93] are assembled with
Sec9p[416–504] and Sec9p[571–651], which represent Helixa
(Ha) and Helixb(Hb), without the intervening loop region. For
each combination of SNARE peptides, heterologously tagged
SNARE constructs were used in a separate pulldown assay to
supporting information on the PNAS web site). The cytoplasmic
SNARE 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).
Although the cytoplasmic SNARE complex binds Sec1p-V5-
His6, the four-helix bundle does not (Fig. 3b), suggesting that
either or both of the domains Habcof Sso1p or the loop region
Sec9p are required for binding. Two lines of evidence suggest
that the Habc domain is not sufficient for binding. First, the
binary t-SNARE complex, which includes Habc as part of
Sso1p[1–265], does not bind Sec1p-V5-His6 (Fig. 3a, lane 6).
Second, the core complex, which retains the Sec9p loop region
but lacks Habc, binds to Sec1p-V5-His6suprastoichiometrically
(Fig. 4, lane 6). The core complex behaves as a particle,
sedimenting at 300,000 ? g (Fig. 9, lane 4). Rotary-shadowing
electron microscopy revealed oligomers containing six to eight
core 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 exocytic SNARE complexes (28). The
oligomeric nature of the core complex complicates interpreta-
Nonetheless, we could test whether the Sec9p loop region is
required by using a combination of SNARE peptides that lacks
the Sec9p loop region, but retains Habc: Sso1p[1–265], Snc2p[1–
93], Sec9p[416–504] and Sec9p[571–651]. The resulting SNARE
complex assembles (Fig. 8), yet it does not bind Sec1p-V5-His6
(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 mutants were tested for
their ability to interact with purified Sec1p-V5-His6. Wild-type
Sso1p[1–265] is stably folded in the closed conformation (Fig. 5),
and it does not bind Sec1p-V5-His6 (Fig. 4, lane 1). The
conformational mutant, Sso1p[1–265]-Open1, favors the open
conformation, yet uncomplexed Sso1p[1–265]-Open1 does not
interact with Sec1p-V5-His6(Fig. 4, lane 3). Complete removal
of the Habcdomain, as in Sso1p[179–265], exposes the Sso1p
SNARE motif (H3), but Sso1p[179–265] does not bind to
Sec1p-V5-His6(Fig. 4, lane 5). All three Sso1p peptides were
tested up to 10 ?M (Fig. 7a). SNARE complexes made with each
of these Sso1p peptides do interact with Sec1p-V5-His6(Fig. 4,
lanes 2, 4, and 6). Note that the suprastoichiometric binding of
SNARE complexes made with Sso1p[179–265] is likely to be a
consequence of the oligomeric nature of the core complex
(described above and in Figs. 9 and 10). With that caveat, it
appears that the Habctruncation 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
SNARE 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
complex. SNARE-complex binding was reconstituted in vitro
with the purified cytoplasmic domain of the ternary SNARE
complex, which consists of Sso1p[1–265], Sec9p[416–651], and
Snc2p[1–93]. All three components of the SNARE complex are
necessary to reconstitute the interaction, because Sec1p-V5-His6
binds to none of the individual SNAREs or to the binary
t-SNARE complex, in contrast to a recent observation (25).
Binding studies with truncated SNARE domains revealed that
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
as expected if binding requires the fully assembled SNARE com-
plex. Likewise, Sec1p concentrates at sites of SNARE-complex
assembly, where secretory vesicles 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-
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 (Hc) and light chain (Lc), are indicated.
Sec1p does not bind to the open conformation of Sso1p. SNARE-
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
not require other factors, such as additional proteins (30, 31) or
Another Way for SM Proteins to Interact with SNAREs. Using wild-
type 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, ruling out the closed-
syntaxin-binding mode characterized for nSec1?Munc-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
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 explicitly tested the proposal that Sec1p can
bind to the t-SNARE complex (25) and found no evidence, in
vivo or in vitro, for an interaction between Sec1p and the
t-SNARE complex formed by Sec9p and Sso1p. Notably, we did
observe nonspecific interactions between Sec1p-V5-His6puri-
fied from yeast and recombinant SNARE peptides that were
tagged with heterologous sequences (data not shown). We
conclude that the interaction observed between Sec1p and the
exocytic 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 SNARE complex raises new questions about the role
of SM proteins in vesicle trafficking 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
information 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
known as HOPS (19, 36). HOPS interacts with assembled
SNARE complexes and is required for vacuole docking and
fusion (37, 38). HOPS binds Vam7p, the vacuole-specific
SNARE homologous to neuronal SNAP-25 (37). We find that
the Sec1p interaction with SNARE complexes requires a binding
suggesting that interaction with this t-SNARE may be a common
requirement for SM protein function. Like HOPS, the plasma
membrane-specific vesicle tethering complex, known as the
exocyst, is both a Rab effector (39) and required for SNARE-
complex assembly (22). Although Sec1p is not a stable compo-
nent of the exocyst, it may associate with low affinity to promote
SNARE-complex 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
SNARE complexes (40). If the Sec6p–Sec9p interaction repre-
sents a regulatory intermediate in SNARE-complex assembly,
perhaps association of Sec1p with Sec9p and the exocyst is
required for a subsequent step to activate SNAREs for mem-
Whether SM proteins share common functions will require a
combination of mechanistic studies using purified proteins in
SNARE 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
Materials and Methods
Plasmid Construction. For construction of expression vectors
pKLN9 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-
taway, NJ). For construction of pYES2?CT 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 pYES2?CT vector (Invitrogen)
and confirmed by sequencing (IDT, Piscataway, NJ).
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
transformed with the plasmids indicated. Cell lysis and IP were
performed as described, with at least 50% recovery of Sec1p-
MYC3 using the 9E10 antibody (21). The proteins that co-
precipitated with Sec1p-MYC3were 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
MATa his3?1 leu2?0 ura3?0 sec1?::SEC1MYC3URA3 sso1?::kanMX4 sso2?::kanMX4 pMM250 (CEN LEU2 SSO1)
MATa his3?1 leu2?0 ura3?0 sec1?::SEC1MYC3URA3 sso1?::kanMX4 sso2?::kanMX4 pMM266 (CEN LEU2 sso1-open1)
MATa his3?1 leu2?0 ura3?0 sec1?::SEC1MYC3URA3 sso1?::kanMX4 sso2?::kanMX4 pFH24 (CEN LEU2 sso1?31–290?)
MATa his3?1 leu2?0 ura3?0 sso1?::kanMX4 sso2?::kanMX4 pMM250 (CEN LEU2 SSO1)
MATa/? his3?1/ his3?1 leu2/leu2 trp1–289/trp1–289 ura3–52/ura3–52
MAT? his3-?200 leu2–3, 112 trp1 ura3–52 sec1?::SEC1MYC3URA3
MAT? sec18–1 his3-?200 leu2–3, 112 trp1 ura3–52 sec1?::SEC1MYC3URA3
MAT? sec5–24 ura3–52 sec1?::SEC1MYC3URA3
www.pnas.org?cgi?doi?10.1073?pnas.0605448103Togneri et al.
was detected by using a chemiluminescence reagent (Pierce,
Protein Purification. Recombinant SNARE peptides Sso1p[1–
265], Sso1p[1–265]-Open1 Snc2p[1–93] and Sec9p[416–651]
were expressed 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-tagged Sec1p (Sec1p-V5-His6) was expressed in yeast by
using the pYES2?CT Sec1p plasmid transformed into the S.
cerevisiae strain, INVSc1, as described by the manufacturer
(Invitrogen). After a 14-h induction in galactose, cells were
harvested and resuspended in wash buffer (50 mM Hepes, pH
7.4?20 mM NaF?20 mM NaN3). Washed cells were lysed in
buffer A (50 mM Hepes, pH 7.4?150 mM KCl?10% glycerol?30
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;
after 10,000 ? g for 30 min (Sorvall, Guelph, ON, Canada; rotor
SS-34), the supernatant fraction (S10) was centrifuged for
another 45 min at 100,000 ? g (Beckman, Fullerton, CA; rotor
Ti-70). Sec1p-V5-His6from 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 7.4?150 mM
KCl?250 mM imidazole?10% glycerol). For storage, 2 mM DTT
and 1 mM EDTA were added and the aliquots were frozen at
SNARE Binding with Immobilized Sec1p-V5-His6. To standardize the
amount of Sec1p-V5-His6in the binding reaction, ?0.4 ?g of
nickel purified Sec1p-V5-His6was immunoaffinity-purified by
using 1.8 ?g of V5 monoclonal antibody (Invitrogen). Excess
Sec1p-V5-His6was washed from the resin with SNARE-binding
buffer (50 mM Hepes, pH 7.4?100 mM NaCl?0.5% IGEPAL?
0.5 mM DTT?1 mM EDTA). Before addition to the binding
reaction, SNARE complexes 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 information
on the PNAS web site). One micromolar SNARE or SNARE
complex was added to immobilized Sec1p-V5-His6 and incu-
bated for 2 h at 4°C. The reactions were separated by SDS?
PAGE and stained with Coomassie blue R250.
We are grateful to Arati Tripathi for helpful discussions and mass
spectrometry analysis; Karin Nicholson Tomishima for construction of
pKLN9, -10, and –11; Jennifer Streltsova for construction of pYES?C2
Sec1p; Jenna Hutton for comments and construction of MYC-tagged
SEC1 in the sso1 mutants; Donald Winkelmann for electron microscopy
of core SNARE complexes; Barbara Siminovich-Blok for the replace-
ment of SEC1 with the SEC1-V5-His6 construct in yeast; Hays Rye
(Princeton University) for helpful comments and the E. coli system
overexpressing GroEL?ES; 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,
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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vol. 103 ?
no. 47 ?