Yeast Sec1p Functions before and after Vesicle Docking

Abstract

Sec1/Munc18 (SM) proteins bind cognate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and stimulate vesicle membrane fusion. Before fusion, vesicles are docked to specific target membranes. Regulation of vesicle docking is attributed to some but not all SM proteins, suggesting specialization of this earlier function. Yeast Sec1p seems to function only after vesicles are docked and SNARE complexes are assembled. Here, we show that yeast Sec1p is required before and after SNARE complex assembly, in support of general requirements for SM proteins in both vesicle docking and fusion. Two classes of sec1 mutants were isolated. Class A mutants are tightly blocked in cell growth and secretion at a step before SNARE complex assembly. Class B mutants have a SNARE complex binding defect, with a range in severity of cell growth and secretion defects. Mapping the mutations onto an SM protein structure implicates a peripheral bundle of helices for the early, docking function and a deep groove, opposite the syntaxin-binding cleft on nSec1/Munc-18, for the interaction between Sec1p and the exocytic SNARE complex.

INTRODUCTION

Neurotransmitter secretion, organelle function, and eukaryotic cell growth depend on vesicle fusion reactions regulated by the dynamic interplay between soluble and membrane-anchored protein complexes. Vesicle-anchored (v) and target-membrane–anchored (t) soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) assemble to pin together and catalyze the fusion of vesicles with their target membranes (Weber et al., 1998). Before fusion, soluble Rab GTPases and multicomponent vesicle tethering complexes are required to organize vesicle docking and the subsequent assembly of cognate v-t SNARE complexes (Cai et al., 2007). Sec1/Munc18 (SM) proteins are soluble factors that have been implicated in regulation of vesicle docking, SNARE complex assembly, and vesicle membrane fusion (Jahn, 2000; Burgoyne et al., 2009).

Due to the apparent multiplicity of roles, a coherent model for SM protein function has been difficult to envision. Dramatic vesicle docking defects are observed in the neurons of Caenorhabditis elegans unc-18 mutants (Weimer et al., 2003), neurons of Drosophila rop mutants (Schulze et al., 1994), chromaffin cells of Munc18-1 mutants (Voets et al., 2001; Ciufo et al., 2005; de Wit et al., 2006), and vacuoles of yeast vps33 mutants (Rieder and Emr, 1997). So far, a requirement for yeast Sec1p before vesicle docking at the plasma membrane has not been demonstrated, suggesting specialization of different SM proteins for regulatory functions before SNARE complex assembly.

Only recently has a consensus emerged (Sudhof and Rothman, 2009), confirming the observation in yeast (Carr et al., 1999; Togneri et al., 2006) that SM proteins function as part of the core vesicle fusion machinery, promoting fusion when bound to the assembled SNARE complex (Scott et al., 2004; Dulubova et al., 2007; Shen et al., 2007; Mima et al., 2008). However, no SM protein–SNARE complex structure has been reported, and the mechanism of fusion activation remains to be discovered. The absence of evidence for Sec1p function before vesicle docking may mean the only essential function for SM proteins is stimulation of membrane fusion through interactions with assembled SNARE complexes. Alternatively, there is a general requirement of SM proteins for both vesicle docking and fusion, but docking mutants have not yet been isolated for yeast Sec1p.

Defects in vesicle tethering, docking, and fusion at the yeast plasma membrane are indistinguishable by electron microscopy (Novick and Schekman, 1979; Novick et al., 1980); yet, successful vesicle docking can be determined using an assay for assembled SNARE complexes. With this assay, exocyst mutants, with low or undetectable SNARE complex abundance, were determined to block exocytosis before vesicle docking (Grote et al., 2000). Here, we use random and site-directed mutagenesis to isolate a diverse panel of yeast sec1 mutants and identify defects in cell growth, SNARE complex assembly, and secretion of cargo carried by dense and light secretory vesicles. Based on these phenotypes, the sec1 mutants separate into two classes. Class A mutants exhibit a tight block in fusion of both dense and light vesicles and a defect in SNARE complex assembly, suggesting a block before vesicle docking. Class B mutants have SNARE complexes in wild-type abundance, but they are defective in SNARE complex binding. The two classes of mutants cluster in different regions of the SM protein structure, suggesting a separation of vesicle docking and membrane fusion functions. Thus, like other SM proteins, Sec1p is required both before and after docking, in support of the view that SM proteins use both functions to regulate vesicle membrane fusion.

MATERIALS AND METHODS

Yeast Strains and Media

Strains and plasmids used in this study are listed in Table 1. Unless otherwise indicated, yeast cells were grown on yeast peptone dextrose (YPD) media at 25°C (permissive temperature) or 38°C (restrictive temperature). Yeast transformations were performed according to the lithium acetate protocol, as described previously (Gietz and Schiestl, 2007). Sporulation, dissection, and tetrad analysis were performed as described previously (Guthrie and Fink, 1991), by using an Axiophot 20 dissection microscope (Carl Zeiss, Thornwood, NY). Selection was performed on 5-fluoroorotic acid (5FOA) (US Biological, Swapscott, MA) or Synthetic Complete (SC) media lacking leucine (SC-leu) or uracil (SC-ura; MP Biomedicals, Santa Ana, CA), as indicated.

Table 1.

Yeast strains

Strain	Genotype	Source
CCY6	BY4743, MATa/α his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 lys2Δ0/LYS2 MET15/met15Δ0 ura3Δ0/ura3Δ0 YDR164c::kanMx4/YDR164c (SEC1)	EUROSCARF
CCY32	MATa leu2Δ0 his3Δ1 ura3Δ0 met15Δ0 YDR164c::KANMX pRS416 SEC1	This study
CCY19	CCY32, but pRS416 SEC1 is replaced with pCC64 SEC1	This study
CCY28	CCY32, but pRS416 SEC1 is replaced with pCC117 sec1-32	This study
CCY29	CCY32, but pRS416 SEC1 is replaced with pCC89 sec1-34	This study
CCY44	CCY32, but pRS416 SEC1 is replaced with pCC137 sec1-50	This study
CCY45	CCY32, but pRS416 SEC1 is replaced with pCC131 sec1-51	This study
CCY46	CCY32, but pRS416 SEC1 is replaced with pCC82 sec1-52	This study
CCY50	CCY32, but pRS416 SEC1 is replaced with pCC84 sec1-65	This study
CCY51	CCY32, but pRS416 SEC1 is replaced with pCC103 sec1-66	This study
CCY52	CCY32, but pRS416 SEC1 is replaced with pCC134 sec1-67	This study
CCY53	CCY32, but pRS416 SEC1 is replaced with pCC90 sec1-68	This study
CCY55	CCY32, but pRS416 SEC1 is replaced with pCC140 sec1-55	This study
CCY57	CCY32, but pRS416 SEC1 is replaced with pCC104 sec1-80	This study
CCY58	CCY32, but pRS416 SEC1 is replaced with pCC74 sec1-83	This study
CCY59	CCY32, but pRS416 SEC1 is replaced with pCC161 sec1-84	This study
CCY64	CCY32, but pRS416 SEC1 is replaced with pCC130 sec1-56	This study
CCY65	CCY32, but pRS416 SEC1 is replaced with pCC155 sec1-57	This study
CCY66	CCY32, but pRS416 SEC1 is replaced with pCC128 sec1-58	This study
CCY67	CCY32, but pRS416 SEC1 is replaced with pCC133 sec1-59	This study
CCY71	CCY32, but pRS416 SEC1 is replaced with pCC129 sec1-81	This study
CCY91	CCY32, but pRS416 SEC1 is replaced with pCC156 sec1-35	This study
CCY92	CCY32, but pRS416 SEC1 is replaced with pCC157 sec1-36	This study
CCY94	CCY32, but pRS416 SEC1 is replaced with pCC159 sec1-38	This study
CCY95	CCY32, but pRS416 SEC1 is replaced with pCC160 sec1-39	This study
CCY100	CCY32, but pRS416 SEC1 is replaced with pCC154 sec1-44	This study
CCY102	CCY32, but pRS416 SEC1 is replaced with pCC92 sec1-46	This study
CCY104	CCY32, but pRS416 SEC1 is replaced with pCC94 sec1-48	This study
CCY105	CCY32, but pRS416 SEC1 is replaced with pCC95 sec1-49	This study
CCY106	CCY32, but pRS416 SEC1 is replaced with pCC68 sec1-75	This study
CCY107	CCY32, but pRS416 SEC1 is replaced with pCC69 sec1-76	This study
CCY108	CCY32, but pRS416 SEC1 is replaced with pCC70 sec1-77	This study
CCY109	CCY32, but pRS416 SEC1 is replaced with pCC71 sec1-78	This study
CCY110	CCY32, but pRS416 SEC1 is replaced with pCC73 sec1-79	This study
CCY162	CCY32, but pRS416 SEC1 is replaced with pCC96 sec1-82	This study
CCY42	CCY32 sec18-1, product of a cross with NY432 (P. Novick)	This study
CCY43	CCY42, but pRS416 SEC1 is replaced with pCC89 sec1-34	This study
GY3323	MATa leu2-3,112 ura3-52 trp1 his3Δ-200 exo70::HIS3 (CEN TRP1 exo70-38) L-A-	W. Guo
NY778	MATα leu2-3,112 ura3-52 sec6-4	P. Novick
NY784	MATa, leu2-3,112 ura3-52 sec10-2	P. Novick

Plasmid and DNA Manipulation

Mutagenesis studies required two centromeric plasmids: a counterselectable URA3-marked centromeric (CEN) plasmid, pRS416, to create a balancer for the sec1Δ null haploid strain; and a LEU2-marked CEN plasmid, pRS315, to transform the sec1 mutants into the balanced null strain. To make the balancer and mutant plasmids, the SEC1 gene (open reading frame [ORF] plus 745 base pairs upstream and 547 base pairs downstream) was amplified by polymerase chain reaction (PCR) by using YEp24SEC1 (pCC112; pNB680, Carr et al., 1999) as template. Primers for amplification were BSsec15′G (with BamHI) and BSsec13′J (with XbaI). The amplified 3.4-kb BamHI-SEC1-XbaI product was ligated into the two centromeric plasmids (Sikorski and Hieter, 1989; ATCC 67521; American Type culture Collection, Manassas, VA), creating pRS416SEC1 (pCC150) and pRS315SEC1 (pCC64).

To introduce wild-type SEC1 or sec1 mutants into the sec1Δ genetic background, a heterozygous sec1Δ diploid (CCY6; BY4743; EUROSCARF, Frankfurt, Germany) was transformed with pCC150. Transformed diploids were sporulated, and the resulting tetrads were dissected and analyzed on SC-ura plates. The balanced null sec1Δ URA3 strain (CCY32) was identified by sensitivity to 5FOA, indicating presence of the balancer pCC150. To replace pCC150 with wild-type SEC1 or sec1 mutants on a LEU2 plasmid, CCY32 was transformed with pCC64 or pRS315 containing the sec1mutant alleles (for plasmid names, see Table 1). The transformants were selected on SC-leu, and pCC150 was counterselected on 5FOA. The resulting LEU2 ura3 sec1 strains are listed in Table 1.

Site-directed point mutations were introduced using QuikChange II site-directed mutagenesis kit (Stratagene, Cedar Creek, TX) with pCC64 as a template and polyacrylamide gel electrophoresis (PAGE)-purified oligonucleotide primers (JLH1-10; IDT, Piscataway, NJ), designed with nonoverlapping ends as described previously (Zheng et al., 2004). Conserved sites, identified using the hmmbuild-f algorithm of the HMMER suite of programs (hmmer2.3.2, http://hmmer.janelia.org; Hannenhalli and Russell, 2000), were selected for mutagenesis based on amino acid identity in each of the four SM protein families: Sec1, Sly1, Vps33, and Vps45 (Figure 1). “Cleft” mutations were designed based on syntaxin contact sites on the nSec1/Syntaxin 1a structure (Misura et al., 2000). The “furrow” mutant was designed based on clusters of same-charge amino acids that mapped to the surface of a conserved furrow in domain 3 of nSec1(PDB ID 3C98; Burkhardt et al., 2008), Sly1p (PDB ID 1MQS; Bracher and Weissenhorn, 2002); sSec1 (PDB ID 1EPU, 1FVH; Bracher et al., 2000), and Munc18c (PDB ID 2PJX; Hu et al., 2007). The “groove” mutant was designed to alter the charge property flanking the conserved gap, or groove, between domains 1 and 2. Charged residues were mutated to reverse the charge, unless otherwise indicated (Table 2).

Figure 1.

Positions of sec1 mutations. (A) Sequence conservation among the four SM protein families: Sec1, Vps33, Vps45, and Sly1. Segments of conserved amino acid sequence from S. cerevisiae Sec1p (Sce Sec1p) are aligned with the homologous sequence segments from H. sapiens Munc-18/nSec1 (Hsa nSec1), Vps33a (Hsa Vps33), Vps45 (Hsa Vps45), and Sly (Hsa Sly1). A capital letter in the consensus sequence (top line) indicates >50% identity. A lowercase letter indicates the highest probability amino acid at that position, and “x” indicates no conservation identified. The asterisks above the consensus sequence indicate conserved sites chosen for mutagenesis. SM protein sequences were separated by phylogenetics (Supplemental Figure S1). Sequences were grouped into four families (Supplemental Table 1), and each group was aligned using the CLUSTALW algorithm in the software package MacVector version 8.1.1. Alignments were evaluated to determine the consensus sequence for all four families (top line), by using the hmmbuild-f algorithm of the HMMER suite of programs (hmmer2.3.2, http://hmmer.janelia.org; Hannenhalli and Russell, 2000). (B) Conserved sites chosen for mutagenesis map to buried positions on an SM protein structure. Sites marked by asterisks in A are modeled as spheres (aspartate, red; histidine and arginine, blue) on a ribbon representation of Munc18-1/nSec1: domain 1 (aa 4-134, black), domain 2 (aa 135-245 and 480-592, light gray), and domain 3 (aa 246-479, dark gray). For labeling, amino acid numbers are based on the Munc18-1/nSec1 sequence, with the corresponding yeast numbers in parentheses. Images of nSec1 were created using Pymol (DeLano Scientific, Palo Alto, CA) and Protein Data Bank file 3C98 (Burkhardt et al., 2008), with the syntaxin 1a chain omitted. (C) class A mutations (orange spheres) map to a helical cluster in domain 3b. The positions marked are altered in the randomly generated mutants sec1-34 [G443E, K494T], sec1-38 [S391Y, D429G, E446K, I544F], sec1-39 [M402T, Y437N, I478T], and sec1-49 [Y437N, I260T] and in conserved-site mutants sec1-51 [R252A, D255A], sec1-65 [R252A, E604R], and sec1-68 [R252A, D307H, H371D]. For labeling, the amino acid numbers are based on the Munc18-1/nSec1 sequence, with the corresponding yeast numbers and mutations in parentheses. Images of nSec1 were created as described in B. (D) Many of the class B mutations (blue spheres) map to a groove between domains 1 and 2. The positions marked are altered in the randomly generated mutants sec1-32 [L417S, L639S], sec1-35 [N167I, F221Y, F235L], sec1-36 [I249N], sec1-44 [F620S, R643I, L633V, G696S], sec1-46 [F156S, I187N], sec1-48 [N81S, R638P]; in conserved-site mutants sec1-50 [E604A], sec1-52 [D255A], sec1-55 [R252L, E604L], sec1-66 [R252A, H371D], sec1-67 [D255N]; and in the groove surface mutant sec1-58 [K79E, Y80E, R169E, K170E]. Images are labeled as described in C. Where the same position has been mutated to more than one amino acid, a comma is used to separate the mutations (i.e., D255A,N). Images were created as described in B.

Table 2.

Mutagenesis of yeast SEC1

Mutagenesisa	Mutation in Sec1p	Growth defectb (°C)	Allele
Random	N81S, R638P	38	sec1-48
Random	F156S, I187N	38	sec1-46
Random	N167I, F221Y, F235L	38	sec1-35
Random	I249N	38	sec1-36
Random	S391Y, D429G, E446K, I544F	36	sec1-38
Random	M402T, Y437N, I478T	31	sec1-39
Random	L417S, L639S	38	sec1-32
Random	Y437N, I260T	35	sec1-49
Random	G443E, K494T	31	sec1-34
Random	F620S, R643I, L633V, G696S	38	sec1-44
Conserved	D251A	None	sec1-83
Conserved	D251A, R252A	None	sec1-84
Conserved	R252A	None	sec1-78
Conserved	R252A, D255A	28	sec1-51
Conserved	R252L, D255V, E604L	Lethal
Conserved	R252A, D307H	None	sec1-80
Conserved	R252A, D307H, H371D	36	sec1-68
Conserved	R252A, H371D	36	sec1-66
Conserved	R252A, E604A	None	sec1-81
Conserved	R252A, E604R	32	sec1-65
Conserved	R252E, E604R	Lethal
Conserved	R252L, E604L	38	sec1-55
Conserved	E604A	36	sec1-50
Conserved	D255A	37	sec1-52
Conserved	D255N	35	sec1-67
Conserved	D307H	None	sec1-75
Conserved	D307H, H371D	None	sec1-77
Conserved	H371D	None	sec1-76
Groove surface	K79E, Y80E, R169E, K170E	38	sec1-58
Furrow surface	G380R, E381K, D429K	None	sec1-59
Cleft surface	R63D	None	sec1-56
Cleft surface	K64E	None	sec1-82
Cleft surface	R63A, K64A	None	sec1-57
Cleft surface	R63D, K64E	Lethal
Cleft surface	E291K, E297R, D360R, D362K	None	sec1-79

^aRationale for mutagenesis.

^bTemperature at which growth defect is observed.

Random mutations were introduced into the SEC1 open reading frame by using Gene Morph II EZ clone domain mutagenesis kit (Stratagene) with pCC64 as a template. Five pairs of PAGE-purified oligonucleotide primers (JLH1-10; IDT) were designed with nonoverlapping ends. Ten sec1 mutant plasmid libraries were generated by PCR mutagenesis: two from the entire SEC1 ORF (PCR 1 and 2), plus two for each of four shorter sequences (PCR 3 and 4, aa 3-132; PCR 5 and 6, aa 133-262; PCR 7 and 8, aa 513-645; and PCR 9 and 10, aa 263-512). The resulting sec1 mutant plasmid libraries were transformed into CCY32, and transformants were selected on SC-leu. We isolated 2250 colonies that were struck onto 5FOA plates to counterselect the balancer pCC150, and the resulting ura3-LEU2 colonies were screened for temperature-sensitive (ts) or cold-sensitive (cs) growth defects at 38 and 19°C, respectively. Many of the mutants contained multiple lesions; however, when we attempted to isolate individual point mutations, the ts growth defect was lost. Mutations in the SEC1 open reading frame were mapped by DNA sequencing (IDT; Table 2).

Immunoprecipitation Assays for SNARE Complex Assembly and Sec1p Binding

Assembled SNARE complexes were assayed by coimmunoprecipitation (IP) of exocytic SNAREs. Wild-type (SEC1) and sec1 mutants were lysed after a 20-min incubation at permissive (25°C) or restrictive (38°C) temperature and analyzed for assembled SNARE complexes, as described (Grote et al., 2000), with the following modifications. An α-Sec9p antibody (Sec9-228; raised against Sec9p, aa 416-651; Covance, Denver, PA) was used to IP Sec9p, an α-Sso1p antibody (Sso1-16,371; gift from M. Munson, University of Massachusetts Medical School, Worcester, MA) was used to detect coprecipitation of Sso1p and an α-Snc2p antibody (Snc2-213, raised against GST-Snc2p, aa 1-93; Covance) to detect Snc2p by Western blot analysis.

Sec1p-binding to SNARE complexes was assayed by co-IP of SNARE complexes with Sec1p. Wild-type (SEC1) and sec1 mutants were lysed after a 20-min incubation at 25 or 38°C, and an α-Sec1p antibody (Sec1-229; raised against Sec1pV5His₆; Covance,) was used to IP wild-type and mutant Sec1p. Western blot analysis was used to detect co-IP of Sso1p, which reflects the abundance of assembled exocytic SNARE complexes present in the cell before lysis (Carr et al., 1999). It should be noted that yeast Sec1p does not bind to unassembled Sso1p or the t-SNARE complex of Sec9p and Sso1p (Togneri et al., 2006). In both IP assays, the abundance of Sso1p in the input lysates was detected by Western blot analysis by using α-Sso1p (Sso1-16,371).

Invertase and Bgl2p Secretion Assay

Invertase assays were performed as described previously (Wiederkehr et al., 2003). In brief, SEC1 and sec1 mutants were shifted to 38°C for 1 h in low glucose medium (0.1%). Total and external invertase activities were measured using an enzyme assay (Goldstein and Lampen, 1975). Internal invertase was determined by subtracting the external invertase activity from the total. The average percentage of internal invertase was determined from four independent experiments, unless otherwise indicated.

Internal Bgl2p accumulation was determined as described previously (Harsay and Schekman, 2007). In brief, yeast strains were grown at 25 or 38°C for 1 h. Cells were spheroplasted and internal Bgl2p was assayed by Western blot, by using an α-Bgl2p antibody (gift from Edina Harsay, University of Kansas, Lawrence, KS). An α-alcohol dehydrogenase (ADH) antibody (Abcam, Cambridge, MA) was used to detect ADH, which was used as an internal loading control. The intensity of the Bgl2p bands was normalized with the ADH band intensity and quantified by ChemiDocXRS and Quantity One, version 4.4.1 (Bio-Rad Laboratories, Hercules, CA). The average internal Bgl2p intensity was determined from three independent experiments, unless otherwise indicated.

Bud Emergence Assay

Cultures of early log phase yeast cells were arrested at G1 by addition of 6 μM α-factor (Sigma-Aldrich, St. Louis, MO) for 2 h at 25°C. The cells were washed with fresh YPD medium to remove the α-factor and incubated for 60 min at the indicated restrictive temperatures. For each strain studied, the bud size was measured using IP Lab Image Analysis software (BioVision Technologies, Exton, PA), with images captured by differential interference contrast (DIC) microscopy. For each strain, 200 cells in total were counted. Daughter cells ≤2 μm in diameter were counted as small buds, and daughter cells >2 μm were counted as large buds.

SNARE Complex Binding by Immobilized Sec1p-V5-His₆

His-tagged Sec1p (Sec1p-V5-His₆), Sec1-34p, or Sec1-58p was expressed in yeast by using pYES2/CT plasmid transformed into the Saccharomyces cerevisiae strain INVSc1, as described by the manufacturer (Invitrogen, Carlsbad, CA). After induction with 2% galactose, cells were harvested and resuspended in wash buffer (50 mM HEPES, pH 7.4, 20 mM NaF, and 20 mM NaN₃). Washed cells were lysed in binding buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.5% Igepal, 0.5 mM dithiothreitol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and yeast protease inhibitor mixture [Complete; Roche Applied Science, Indianapolis, IN]). The lysate was clarified by sequential centrifugation at 15,000 × g and 100,000 × g for 30 min each. To standardize the amount of Sec1p-V5-His₆ used for the binding reactions, 2 μg of V5 monoclonal antibody (Invitrogen) was used to purify and immobilize Sec1p-V5-His₆ on protein G-Sepharose resin (Invitrogen). Excess Sec1p-V5-His₆ and cellular proteins were washed away with binding buffer. Before addition to the binding reaction, SNARE complexes or Sso1p/Sec9CT binary complexes were assembled as described previously (Nicholson et al., 1998). Binary or ternary SNARE complexes (0.3 or 1 μM) were added to immobilized Sec1p-V5-His₆, or mutant proteins, and incubated for 2 h at 4°C. After three washes with binding buffer, bound proteins were detected by SDS-PAGE analysis and stained with Coomassie Blue R250.

RESULTS

Sec1p interacts specifically with the exocytic SNARE complex (Carr et al., 1999; Togneri et al., 2006), and the SM protein–SNARE complex interaction has been shown to stimulate vesicle fusion (Scott et al., 2004; Dulubova et al., 2007; Shen et al., 2007; Mima et al., 2008). There is also evidence that SM proteins may function upstream of vesicle fusion, through interactions with vesicle tethering machinery (Sato et al., 2000; Seals et al., 2000; Wiederkehr et al., 2004; Wagner et al., 2006; Peplowska et al., 2007). However, it is unknown whether these interactions represent a general function of SM proteins. To better clarify the role or roles of Sec1p, we used site-directed and random mutagenesis to generate a large panel of sec1 mutants.

Isolation of sec1 Alleles

Sites with a high percentage of amino-acid identity among SM protein sequences were targeted for mutagenesis of the yeast SEC1 gene. From 37 taxa, 164 unique sequences were identified with similarity to Sec1, Slyl, Vps33, or Vps45 from yeast (S. cerevisiae) and human (Homo sapiens). Sequences were sorted into four families (Secl, Slyl, Vps33, and Vps45; Supplemental Table S1) by using a phylogenetic approach similar to those used in other studies of SM protein family evolution (Supplemental Figure S1; Arac et al., 2005; Koumandou et al., 2007). Based on these results, a separate multiple sequence alignment was made for each family, by using the CLUSTALW algorithm in the software package MacVector version 8.1.1 under default settings. The four alignments were used as input for Protein Family Alignment Annotation Tool (Johnson et al., 2003), to determine percentage of identity at each amino acid position for each family. The four alignments were also used as input for the HMMER algorithm hmmbuild-f (Hannenhalli and Russell, 2000), to determine a consensus sequence for each family. The four consensus sequences were aligned and hmmbuild-f was used a second time to determine a consensus among all four families (Figure 1A).

Charged residues occupy many of the most highly conserved sites. Therefore, sites that were >95% conserved by charge were chosen for mutagenesis. Mapping the conserved sites onto the nSecl/Muncl8 structure revealed two charge pairs (Figure 1B): the R235/E549 (R252/E604 in yeast Sec1p) salt bridge buried at the protein core, and D285/H347 (D307/H371) located in domain 3a. D234 (D251) seems to be involved in backbone hydrogen bonds in the core of domain 2. Another conserved aspartate, D238 (D255), forms hydrogen bonds with a short helical turn (D238-V243 in nSec1/Munc18), and with R235, interactions observed in all SM protein crystal structures resolved to date (Bracher et al., 2000; Misura et al., 2000; Bracher and Weissenhorn, 2002; Hu et al., 2007). Chosen sites were substituted with alanine or leucine, to replace the charges with neutral or hydrophobic residues (Table 2). Pairs of charges were also reversed, to determine the effect of conserving the bond, but with reversed topology. Aspartate, D238 (D255) was substituted with alanine, or asparagine, a favored N-cap residue (Aurora and Rose, 1998), to test its possible function as a helix-capping residue. Of the 18 conserved-site mutants studied, eight exhibited a ts growth defect, two were lethal and eight showed no growth defect at either 38 or 19°C (Table 2).

Random mutations were introduced into the SEC1 coding sequence by PCR mutagenesis (see Materials and Methods). From 10 sec1 mutant plasmid libraries, each created by an independent PCR reaction, 2250 transformants in total were screened. Of these, 10 exhibited ts growth defects upon loss of the SEC1 balancer plasmid. The fact that none of the conserved-site mutations was isolated from this screen indicates that the random mutagenesis approach we used was not saturating.

In total 19 mutants with a recessive, ts growth defect were isolated from the combined random and site-directed mutagenesis approaches, including an additional surface mutation, described below. The positions of the mutations were determined by DNA sequencing of the SEC1 open reading frame (IDT; Table 2) and mapped on the nSecl/Muncl8 structure (Figure 1). Although we also screened for conditional viability at 19°C, no cold-sensitive mutants were isolated. Because SEC1 is an essential gene, mutations that disrupt folding or abolish an essential binding interaction are expected to be lethal at all temperatures and therefore missed by the random mutagenesis screen. However, three site-directed mutants were determined to be lethal upon replacement of the wild-type gene with the sec1 mutant allele (Table 2).

Ts mutants could be defective due to a weakened binding interaction, or a temperature-sensitive folding defect. A previous study found increased degradation at restrictive temperature (Brummer et al., 2001) in the sec1-11 mutant (Novick and Schekman, 1979), suggesting a folding defect in that ts sec1 allele. In another study, we showed that recombinant, wild-type Sec1p produced in Escherichia coli was insoluble, with no detectable Sec1p in the supernatant fraction after centrifugation at 300,000 × g (Togneri et al., 2006), suggesting aggregation due to misfolding. Wild-type Sec1p in yeast lysates separates into a particulate and a soluble fraction: the soluble form is monomeric and binds specifically to SNARE complexes (Togneri et al., 2006). Although it is possible to have local folding defects that do not exhibit increased degredation or aggregation, we used these criteria to test for a temperature-sensitive, global folding defect that might explain a loss of Sec1p function in sec1 mutants. The stability and solubility of the mutant proteins were determined to be comparable with Sec1p from an isogenic wild-type strain (Supplemental Figure S2).

Two Classes of sec1 Mutants

Based on the reported functions of other SM proteins for both vesicle docking and fusion, we expected that the isolation of a large panel of sec1 mutants would reveal 1) whether there is a docking role for Sec1p and 2) the surface used by Sec1p to bind the exocytic SNARE complex for activation of vesicle membrane fusion. To determine whether we had isolated separate functions for Sec1p, we screened the 20 loss-of-function sec1 mutants for SNARE complex assembly (an indication of vesicle docking) and SNARE complex binding, and we found that the mutants fall into two functionally distinct classes: class A and class B (Figure 2).

Figure 2.

Phenotypes of class A and class B sec1 mutants. (A) SNARE complex abundance is diminished in class A mutants, whereas SNARE complex binding to Sec1p is disrupted in class B mutants. An α-Sec9p antibody (Sec9-288) was used to IP Sec9p in an assay for assembled SNARE complexes (top). An α-Sec1p antibody (Sec1-229) was used to IP Sec1p in an assay for Sec1p-binding to SNARE complexes (middle). Coprecipitation of Sso1p with either Sec9p or Sec1p was detected by Western blot analysis by using an α-Sso1p antibody (Sso1-16,371). Wild-type (SEC1) and the indicated sec1, sec6, and exo70 mutant yeast strains were lysed after a 20-min shift from permissive (25°C) to restrictive (38°C) temperature. Protein concentration in the lysates was determined using the Bradford assay (Bio-Rad Laboratories). Bound proteins were separated by SDS PAGE, and 0.75% of the lysates used for the IPs is shown (Input, bottom). (B) Both invertase and Bgl2p secretion are tightly blocked in class A mutants and blocked with a range of severity in class B mutants. Accumulation of invertase and Bgl2p was measured in wild-type (SEC1) and the indicated mutant yeast strains after a 60-min shift to the restrictive temperature (38°C). Invertase activity was measured using an enzyme assay, and the average percentage of internal invertase (±SEM; n = 4) was plotted on a bar graph (black; see also Supplemental Figure S2). The average internal Bgl2p was measured by Western blot analysis by using an α-Bgl2p antibody (gray; for Westerns, see Supplemental Figure S2). The averaged units of intensity plotted (±SEM; n = 3) represent the optical density per square millimeter of each Bgl2p band, normalized to the intensity of internal loading control (ADH).

Class A mutants are defective in SNARE complex assembly. Assembled SNARE complexes were detected using a co-IP assay (Togneri et al., 2006). A Sec9p IP was used to screen the sec1 mutants for coprecipitation of Sso1p (Figure 2A, top). Yeast exocytic SNARE complexes assemble in vivo and do not continue to assemble or exchange in lysates prepared under ATP-depleting conditions (Carr et al., 1999). Thus, the intensity of the Sso1p band detected by Western blot analysis reflects the abundance of assembled SNARE complexes in the cell at the time of lysis. In class A mutants, such as sec1-34 and sec1-68, the abundance of SNARE complexes was significantly diminished after a 20-min shift to the restrictive temperature. Diminished levels of assembled SNARE complexes in the class A mutants suggests a block before vesicle docking, a phenotype observed previously for mutants of the exocyst tethering complex, such as sec6-4 (Figure 2A; Grote et al., 2000).

Class B mutants have a SNARE-complex binding defect, despite wild-type levels of SNARE complexes. The Sec1p–SNARE complex interaction is detected in a binding assay (Figure 2A, middle), by using an α-Sec1p antibody (Sec1-229) to IP wild-type or mutant Sec1p (Supplemental Figure S3). Yeast Sec1p binds specifically to the assembled exocytic SNARE complex, composed of Sso1p, Sec9p, and Snc2p, and not to the t-SNARE complex (Sso1p and Sec9p), or the individual SNAREs (Togneri et al., 2006). Therefore, the Sec1p IP was used to screen the sec1 mutants for SNARE complex binding. In this assay, the Sso1p band on the Western blot reflects the fraction of SNARE complexes coprecipitating with Sec1p (Carr et al., 1999; Togneri et al., 2006). SNARE complexes coprecipitating with Sec1p from mutant strains were significantly diminished, compared with an isogenic wild-type control (Figure 2A, middle). Reduced Sso1p coprecipitation with Sec1p from class A mutants is probably due to the diminished levels of assembled SNARE complexes in those strains (Figure 2A, top). For class B mutants, SNARE complexes are abundant (Figure 2A, top; coprecipitation of Snc2p and Sso1p with Sec9p, Supplemental Figure S3), suggesting diminished coprecipitation of Sso1p is due to a SNARE-complex binding defect in mutant Sec1p protein. By contrast, neither SNARE complex assembly nor Sec1p binding was defective in the exocyst mutant exo70-38 (Figure 2A), suggesting the ts defect in this exocytosis mutant is independent of SNARE complex assembly or Sec1p binding.

Secretory Phenotypes of Class A and B Mutants

Electron micrographs of thin sections of yeast exocytosis mutants reveal gross accumulation of secretory vesicles after exposure to the restrictive temperature (Novick and Schekman, 1979; Novick et al., 1980). The secretory vesicles that accumulate in exocytosis mutants, such as sec6-4, separate into two peaks on density gradients (Harsay and Bretscher, 1995): dense vesicles, which carry the enzyme invertase to the plasma membrane via the endosome, and the more abundant light vesicles, which carry cell wall components, such as the endoglucanase Bgl2p.

To determine the types of secretory vesicles that accumulate in the class A and class B sec1 mutants, the intracellular levels of invertase and Bgl2p were monitored using established assays (see Materials and Methods). In general, both classes of mutants exhibited higher intracellular Bgl2p levels compared with the isogenic wild-type control strain and resembled the exocyst mutant sec6-4 (Figure 2B, gray bars; for Bgl2p Western blots of all mutants, see Supplemental Figure S4A; Harsay and Bretscher, 1995; He et al., 2007). In contrast to the Bgl2p phenotype, a strong block in invertase secretion was found to be a hallmark of class A mutants (Figure 2B, black bars; all mutants, Supplemental Figure S4B). Class A mutants uniformly retained >90% of the invertase enzyme, similar to that observed for the exocyst mutants, sec6-4 (Novick et al., 1980). However, in class B mutants, the invertase phenotypes ranged from a strong intracellular accumulation, as in sec1-67 (>90%), to very weak or no significant increase in invertase accumulation, as in sec1-50 (<25%; Figure 2B and Supplemental Figure S4B).

Bud Emergence Is Blocked in a Subset of sec1 Mutants

Studies of exo70-35 and exo70-38, and the secretion-specific allele, cdc42-6, draw a connection between a block in Bgl2p secretion (without an invertase secretion defect) and delayed bud emergence, suggesting a link between these genes and the cell cycle (He et al., 2007; Adamo et al., 2001). Given that a subset of class B mutants exhibited a similar secretion phenotype, sec1 mutants were examined for a possible delay in bud emergence (Figure 3). MATa strains of sec1 mutants were arrested in G1 by exposure to α-factor and then washed with fresh media to initiate growth. After 60 min at the restrictive temperature, the majority of wild-type cells had produced large buds (>65%), with few unbudded cells (10%). Class A mutants, such as sec1-68, exhibited very few cells with buds (3%), whereas most cells remained in the unbudded stage (97%), resembling the exocyst mutant sec10-2 (>96% unbudded). Class B mutants exhibited a range of budding defects. Mutants tightly blocked in both Bgl2p and invertase secretion, such as sec1-67, exhibited a severe budding defect (99% unbudded). In sec1-58, an allele with a partial block in invertase secretion (Supplemental Figure S4B), ∼50% of the cells remained unbudded. Surprisingly, no discernible budding defect was observed for sec1-50, an allele that resembles exo70-35, exo70-38, and cdc42-6 in its secretion phenotype. Using growth curves, we determined that the onset of the temperature-sensitive growth defect observed for sec1-50 occurs after the first doubling (data not shown); thus, in this case, growth arrest is not due to a bud emergence defect. In general, there seems to be a correlation between the severity of the invertase secretion block and the inability of sec1 mutants to produce buds at restrictive temperatures.

Figure 3.

Growth of the daughter cell is tightly blocked in class A mutants. Wild-type (SEC1) and the indicated sec1 and sec10 mutant cells were synchronized using α-factor. Growth of the daughter cell (bud) was initiated at restrictive temperature (38°C) by washing out the α-factor with fresh media. Images were taken at 0 and 60 min, by using DIC microscopy and analyzed using IP Lab Image Analysis software (BioVision Technologies). The percentage of small-budded cells (arrowheads; <2 μm; gray bars), large-budded cells (arrows; >2 μm; white bars), and unbudded cells (black bars) were quantified for the 60-min time point and plotted as a bar graph (n = 200).

A Distinct Function for Sec1p Upstream of SNARE Complexes

The differences observed between sec1 mutants could be explained trivially, if a common function were disrupted, but more severely in class A than in class B mutants. Like class B mutants, coprecipitation of SNARE complexes with the mutant Sec1p is strongly diminished in class A mutants (Sec1p IP, Figure 4A). SNARE complex abundance is also severely reduced in class A mutants, with little Sso1p coprecipitation in the Sec9p IP assay (Sec9p IP, Figure 4A). By contrast, the assay for assembled SNARE complexes revealed wild-type coprecipitation of Sso1p from class B mutants (Sec9p IP, Figure 2A), including sec1-67, with a invertase secretion defect as strong as the class A mutants (Figure 2B and Supplemental Figure S4).

Figure 4.

A function distinct and before the SNARE complex binding activity of Sec1p is defective in class A mutants. (A) Reduced abundance of SNARE complexes is observed in all class A sec1 mutants. Representative Sso1p Western blots indicate the results of the assays for assembled SNARE complexes (αSec9p; top) and for Sec1p-binding to SNARE complexes (αSec1p; middle), as described in Figure 2A. Bound proteins were separated by SDS PAGE, and 0.75% of the lysates used for the IPs are shown (Input). (B) Sec9p, Sso1p, and Snc2p are present at wild-type abundance in class A sec1 mutants. Wild-type (SEC1) and sec1 mutants were lysed after 20-min incubation at the restrictive (38°C) temperature. For each lysate, 10 μg of total protein was loaded and separated by SDS-PAGE. SNARES were detected by Western blot analysis by using α-Sec9p, α-Sso1p, and α-Snc2p (Snc2-213). The sec1-65 sample was run on a separate gel, which did not resolve the doublet characteristic of Sec9p. (C) Rapid accumulation of SNARE complexes in sec18-1 increases the abundance of and binding to SNARE complexes by Sec1-34p. The results of the SNARE complex assembly (αSec9p) and binding (αSec1p) IP assays are shown for wild-type (SEC1 SEC18), sec18-1 (SEC1 sec18-1), sec1-34 (sec1-34 SEC18), and the double mutant sec1-34 sec18-1. Cells were lysed after 20-min incubation at the restrictive (38°C) temperature. The Western blot also indicates the relative abundance of Sso1p and Snc2p in 0.75% of the lysates used for the IPs, as shown in Figure 2A. (D) High-copy expression of SEC9 restores growth and partially restores SNARE complex assembly and mutant Sec1p binding to SNARE complexes in the class A mutant sec1-34. On an SC-leu-ura plate incubated at restrictive temperature (32°C), growth of the following transformants is shown in triplicate: wild-type (SEC1) with 2μ SEC9, sec1-34 with a control vector and sec1-34 with 2μ SEC9 (top). Bottom, results of the SNARE complex assembly (αSec9p) and binding (αSec1p) IP assays, performed as described in Figure 2A. Also indicated on the Western blot is the abundance of Sec9p (overexpressed in the strains transformed with 2μ SEC9), Sso1p and Sncp in 0.75% of the lysates used as inputs for the IP assays.

We considered the possibility that lower SNARE complex abundance in class A mutants could be due to a decrease in the overall abundance of one or all of the exocytic SNAREs: Sec9p, Sso1p, or Snc2p. Decreased SNARE abundance has been observed previously for other SM protein mutants such as vps45Δ, which has diminished levels of the syntaxin SNARE, Tlg2p in the absence of Vps45p (Bryant and James, 2001). Western blot analysis of Sec9p, Sso1p and Snc2p indicated that all three exocytic SNAREs are present in wild-type abundance after a shift to the restrictive temperature (Figure 4B), indicating decreased SNARE protein concentration is not the cause of the low abundance of SNARE complexes in the class A mutants.

We reasoned that if the function lost in class A mutants is distinct from class B mutants, Sec1p from a class A mutant should have detectable SNARE complex binding activity. If SNARE complexes could be restored in a class A sec1 mutant, the mutant Sec1p could be tested for the SNARE complex binding function using the Sec1p IP assay. By crossing class A mutant sec1-34 with the SNARE complex disassembly mutant sec18-1, we isolated a double mutant, sec1-34 sec18-1. With this mutant, we could take advantage of the rapid onset of SNARE complex accumulation in sec18-1 (Grote et al., 2000) in the sec1-34 background at restrictive temperature (38°C; Sec9p IP, Figure 4C). Although diminished relative to the sec18-1 single mutant, SNARE complexes are detectable at much greater abundance in the sec1-34 sec18-1 double mutant than in the sec1-34 single mutant. Furthermore, a small fraction of the SNARE complexes that persist in sec1-34 sec18-1 at restrictive temperature, although nonfunctional for restoring growth, bind to and coprecipitate with the mutant Sec1-34p protein (Sec1p IP, Figure 4C).

SNARE complex abundance and Sec1p binding are also restored in sec1-34 by an excess of Sec9p. The sec1-34 ts growth phenotype is suppressed by high-copy expression of Sec9p, by using a 2μ SEC9 plasmid, and increased SNARE complex abundance and binding is observed using the IP assays (Figure 4D). Together with the sec1-34 sec18-1 double mutant results, these results suggest that class A mutants have a severe block in secretion, not because the mutant Sec1p protein is unfolded, nor due to a defect in SNARE-complex binding, but probably due to a deficiency in a function required upstream of vesicle docking and SNARE complex assembly.

Conserved sites are buried and may play a role in SM protein structure and dynamics that is required for both vesicle docking and SNARE-complex binding functions. In contrast to the class A and class B mutants isolated from the random mutagenesis screen, which map to distinct regions of the SM protein structure (Figure 1), conserved-site mutations are found in both class A and class B, as might be expected if the two essential functions are conserved. Charge pairs, such as those in the conserved salt bridge formed by R235 and E549, are not necessarily classified together (also true for Sly1p, Li et al., 2005), as would be expected if a single function were disrupted by a point mutation in either one of the pair. The salt bridge per se is not essential: changing both R235 and E549 simultaneously to the charge-neutral amino acid alanine (sec1-51, R252A, E604A; Table 2) resulted in wild-type growth. However, changing them instead to leucines (sec1-55) resulted in a class B phenotype (Supplemental Figure S4), suggesting packing and/or flexibility at this position is important for a SNARE complex interaction.

Our analysis of conserved sites has also uncovered a role for an invariant aspartate, D238 in nSec1/Munc18-1. Unlike a nearby aspartate, D234 (D251 in yeast), which tolerates alanine substitution (i.e., sec1-83 and sec1-84), D238 (D255) is sensitive to mutation. D238 appears in the structures to be acting as an N-cap, stabilizing a short helical turn (residues 238–243) through H-bonds with the side chain carboxylate. Replacement of D238 with alanine (sec1-52, D255A), which cannot make the capping H-bonds, resulted in a temperature-sensitive growth defect and a class B secretory phenotype (Supplemental Figure S4). However, despite its favorability to form an N-cap (Aurora and Rose, 1998), asparagine at that position has an even more dramatic secretory block (sec1-67, D255N), indicating that an N-cap cannot be the sole conserved function. This invariant aspartate may also be required as a charge partner for R235 during SM protein folding or for conformational changes required for the vesicle docking and/or membrane fusion mechanisms.

Mapping the SNARE Complex Binding Surface on Sec1p

Mutations that affect the Sec1p-SNARE complex interaction are expected to map to binding surfaces or to buried core positions that stabilize the SNARE-complex binding conformation of Sec1p. The crystal structures of nSec1/Munc18 (Misura et al., 2000), sSec1 (Bracher et al., 2000), Sly1p (Bracher and Weissenhorn, 2002), and Munc18c (Hu et al., 2007) have three defined channels that are connected to each other and are large enough to accommodate part or all of a SNARE complex (Figure 5A). An area described as the cleft is formed between domains 1 and 3 where closed conformation of Syntaxin 1a binds to nSec1/Munc18 (Misura et al., 2000). The groove describes the channel formed between domains 1 and 2, opposite of the syntaxin-binding cleft. Close to the base of the molecule is the furrow, flanked by domain 3a and 3b.

Figure 5.

Mutations in a conserved groove disrupt Sec1p binding to the SNARE complex. (A) The positions of surface-directed mutations are mapped on nSec1. Mutations were introduced at three surfaces, indicated with dashed lines: the cleft, sec1-79 [E291K, E297R, D360R, D362K] (green spheres) and pCC72 [R63D, K64E] (black spheres); the furrow, sec1-59[G380R, E381K, D429K] (yellow spheres); and the groove sec1-58[K79E, Y80E, R169E, K170E] (blue spheres). The box (left; ribbon representation) highlights the groove region, where amino acids (blue spheres) are altered in class B mutants sec1-35 [N167I, F221Y, F235L], sec1-36 [I249N], sec1-46 [F156S, I187N], and sec1-48 [N81S, R638P] (see Table 2) and the site-directed groove surface mutant sec1-58[K79E, Y80E, R169E, K170E] (bold type). For labeling, amino acid numbers are based on the Munc18-1/nSec1 sequence, with the corresponding yeast numbers and mutations in parentheses. Images were created as described in Figure 1. (B) Groove mutants have a ts growth defect. Wild-type (SEC1) and sec1 mutants of the cleft (sec1-79), furrow (sec1-59), and groove (sec1-35, sec1-36, sec1-46, sec1-48, and sec1-58) were tested for growth at 25 and 38°C. A yeast strain bearing the plasmid pCC72 [R63D, K64E] in place of SEC1 was inviable. (C) Sec1p from the groove mutants binds weakly to assembled SNARE complexes. Wild-type (SEC1) and ts sec1 mutants (sec1-35, sec1-36, sec1-46, sec1-48, and sec1-58) were assayed for assembled SNARE complexes (top) and SNARE complex binding (middle), as described in Figure 2A. Input (bottom) represents 0.75% of the lysate used for the IPs.

Using the nSec1/Munc18 crystal structure as a guide, mutations were designed and introduced onto the surface of the cleft, groove and furrow of Sec1p, in an attempt to disrupt interactions with the exocytic SNARE complex. Clusters of same-charged surface residues in yeast Sec1p were reversed to the opposite charge by site-directed mutagenesis (Figure 5A). No growth defect was observed for mutations introduced in the furrow (sec1-59) or cleft (sec1-79; Figure 5B), with the exception of the cleft mutations at R65/E66 (yeast Sec1p R63D, K64E), which is a recessive lethal mutation. By contrast, directed mutagenesis in the groove resulted in a mutant, sec1-58, with a temperature-sensitive growth defect (Figure 5B). The mutant exhibited wild-type abundance of SNARE complex, with a strong defect in Sec1p binding to SNARE complexes (Figure 5C). The binding defect observed in sec1-58 was similar to that observed for other class B mutants, sec1-35, sec1-36, sec1-46, and sec1-48, which cluster in the groove area (Figure 5, A and C). Thus, the results of both site-directed and random mutagenesis converge on a groove between domains 1 and 2 of Sec1p as a surface that, although not necessary for SNARE complex assembly, seems to be important for SNARE complex binding to Sec1p.

An in vitro binding assay with purified, soluble SNAREs (Togneri et al., 2006) was used to further examine the SNARE-complex binding defect in the designed groove mutant sec1-58. Cytoplasmic SNARE complexes were incubated with Sec1p modified at the C terminus with a V5 epitope and a six-histidine tag for purification (Figure 6). Wild-type Sec1p-V5-His₆ binds ternary, cytoplasmic SNARE complexes with an apparent binding constant of 300 nM (Togneri et al., 2006). At this concentration of cytoplasmic SNARE complexes, we detected weak binding to immobilized Sec1-58p-V5-His₆, compared with either wild-type Sec1p-V5-His₆, or the class A mutant, Sec1-34p-V5-His₆. SNARE complex binding is specific, with no detectable binding to binary t-SNARE complexes (first three lanes, Figure 6), and requires the SNARE-motif linker region of Sec9p (Supplemental Figure S5), as has been demonstrated previously for wild-type Sec1p (Togneri et al., 2006).

Figure 6.

In vitro binding between the SNARE complex and mutant Sec1p. Sec1p-V5-His₆ protein from wild-type (SEC1), class A mutant sec1-34, and class B mutant sec1-58 was affinity purified from yeast lysates by using an α-V5 antibody and immobilized on protein G-Sepharose resin for binding studies. The cytoplasmic domains of binary (Sso1p[1-265] and Sec9[416-651]) or ternary (Sso1p[1-265], Sec9p[416-651], and Snc2p[1-93]) SNARE complexes were incubated at 300 nM with immobilized mutant and wild-type Sec1p, as described previously (Togneri et al., 2006). Bound Sec1p-V5-His₆ and SNAREs were resolved by SDS-PAGE and stained using Coomassie Blue R250. Sec1-58p-V5-His₆ migrates slightly slower than Sec1p-V5-His₆ from the other strains. An asterisk marks a band likely to be a degradation product of Sec1p-V5-His₆ that copurify with the protein from the mutant strains. At higher SNARE complex concentrations, but not observed in this gel, the Snc2p band is detected at the position marked (Snc2p[1-93]; see Supplemental Figure S5). A diffuse band detected slightly above the Snc2p[1-93] position is a contaminant. V5 antibody heavy chain (Hc) and light (Lc) are also indicated. The positions of molecular weight standards are indicated in kilodaltons.

The Sec1p from class A mutant sec1-34 binds to SNARE complexes accumulated in vivo (Figure 4, C and D) and Sec1-34p-V5-His₆ binds to purified cytoplasmic SNARE complexes, in vitro (Figure 6), albeit with slightly weaker affinity than the wild-type protein. Thus, the severe secretion and growth phenotypes observed for class A mutants are unlikely to be attributed to a severe SNARE complex binding defect. By contrast, the binding defect observed in vivo for class B mutants, such as sec1-58, is also observed in vitro, with purified soluble proteins, implicating the groove surface as an important interaction site between Sec1p and exocytic SNARE complexes.

DISCUSSION

Using random and site-directed mutagenesis, we have identified two classes of sec1 mutants that suggest 1) a requirement for SM proteins before vesicle docking extends to yeast Sec1p and 2) a groove opposite the syntaxin-binding cleft is necessary for Sec1p interaction with the exocytic SNARE complex. These findings help to bridge a gap between Sec1p, which specifically binds to assembled SNARE complexes (Togneri et al., 2006), after vesicle docking (Grote et al., 2000), and other SM proteins, which have a high affinity for the unassembled syntaxin SNARE and function before vesicle docking (Munson and Bryant, 2009). Despite different modes of SNARE interactions (Gallwitz and Jahn, 2003), there is a growing consensus that SM proteins are required to activate SNARE complexes for membrane fusion (Scott et al., 2004; Dulubova et al., 2007; Shen et al., 2007; Starai et al., 2008). It is less clear whether every vesicle docking reaction requires an SM protein and what function the SM protein provides to ensure specific vesicle attachment and SNARE complex assembly.

The mechanism used by SM proteins to regulate vesicle docking may have evolved to match the different requirements of regulated vesicle fusion, for example, exocytosis in neuroendocrine cells versus the more constitutive vesicle fusion required for yeast cell budding. In chromaffin cells, Munc18-1 binds the syntaxin N-peptide domain to inhibit synaptic SNARE complex assembly until the N-peptide domain is displaced (Burkhardt et al., 2008). The syntaxin N-peptide binding mode is widespread among the SM proteins, including yeast Sly1p and mammalian Sly1 (Yamaguchi et al., 2002), yeast Vps45p and mammalian Vps45 (Dulubova et al., 2002; Carpp et al., 2006), worm unc-18 (Johnson et al., 2009), Munc18-1 (Dulubova et al., 2007; Khvotchev et al., 2007; Burkhardt et al., 2008), and Munc18c (Latham et al., 2006), and it has been proposed as part of a general mechanism for regulation of vesicle docking (Shen et al., 2007; Burkhardt et al., 2008; Johnson et al., 2009). However, the syntaxin N-peptide interaction is dispensible for the function of Sly1p (Peng and Gallwitz, 2004), Vps45p (Carpp et al., 2006), and Vps33p (Starai et al., 2008). Moreover, Sec1p does not bind to the N-peptide or any other domain of its cognate syntaxin Sso1p (Togneri et al., 2006), bolstering the argument that this binding mode is not required for general SM protein function. Instead, we favor the view that the essential role that SM proteins play in vesicle docking involves interactions with other regulatory proteins, such as Rabs and tethering complexes.

SM proteins have been functionally, and in some cases physically, linked to vesicle tethering complexes (Sato et al., 2000; Seals et al., 2000; Wiederkehr et al., 2004; Wagner et al., 2006; Peplowska et al., 2007; Laufman et al., 2009). Tethering precedes docking: tethering complexes reversibly attach vesicles to target membranes by using a mechanism intimately connected to the GTPase cycle of Rabs (Cai et al., 2007). The yeast vacuolar SM protein Vps33p copurifies as a component of the homotypic vacuolar tethering complex HOPS (Sato et al., 2000; Seals et al., 2000), which interacts with the Rab Ypt7p (Mayer and Wickner, 1997; Ungermann et al., 1998) to tether and dock vacuoles. The defect in class A sec1 mutants is consistent with a block in secretion before SNARE complex assembly, possibly affecting the vesicle tethering step. Genetic studies also provide evidence for functional interaction between Sec1p and the exocyst tethering complex. A block in secretion caused by mutation or deletion of components of the exocyst tethering complex is suppressed by overexpression of Sec1p (Wiederkehr et al., 2004). Furthermore, severe growth defects caused by combined lesions in exocyst component Sec3p (Finger and Novick, 2000; Roumanie et al., 2005) and sec1-1 (Novick and Schekman, 1979) suggest that Sec1p participates with or even in parallel to the exocyst to facilitate vesicle tethering and SNARE complex assembly. However, a robust physical interaction between Sec1p and tethering factors has been difficult to demonstrate (Wiederkehr et al., 2004).

A link between SM proteins and tethering complexes indirectly connects SM protein function to Rab GTP activation of tethering; however, SM protein function may be more directly linked to Rabs. In chromaffin cells, Rab3A has been shown to interact with Munc18-1 at a loop region, near a peripheral helical cluster in domain 3b (Graham et al., 2008). Similarly, yeast mutants SLY1-20[E532K], SLY1-15[T531I] and SLY1-10[ΔE532], which map to a short helical insertion in domain 3b, allow Sly1p to function independently of the yeast Rabs Ypt1p and Ypt6p (Dascher et al., 1991; Li et al., 2007). Strikingly, most of the class A sec1 mutations map to the peripheral cluster of alpha helices in domain 3b, implicating this region for the essential function of Sec1p before vesicle docking and SNARE complex assembly. A Sec1p interaction with the Rab Sec4p could explain genetic evidence for a functional interaction. The original sec1-1[G443E] allele, which also maps to domain 3b, is suppressed by multiple copies of SEC4 (Salminen and Novick, 1987) and displays a severe growth defect in combination with loss-of-function mutation sec4-8 (Finger and Novick, 2000). However, in contrast to the Rab3A findings, studies of Sec4p have not yet physically linked it to Sec1p.

After docking, SM proteins bind to SNARE complexes and stimulate membrane fusion (Scott et al., 2004; Dulubova et al., 2007; Shen et al., 2007; Mima et al., 2008), by using a mechanism that remains to be discovered. Although no SM protein–SNARE complex structure has been resolved, the syntaxin-binding cleft has been proposed as the likely binding site for the four-helix bundle of the SNARE complex (Sudhof and Rothman, 2009). Two separate mutations introduced at a Munc18-1/syntaxin 1a contact surface of domain 1, E59K and E66A, were reported to interfere with the interaction between Munc18-1 and the assembled synaptic SNARE complex (Deák et al., 2009). We found that two simultaneous charge reversals at the analogous Munc18-1/syntaxin 1a contact site in domain 1 of Sec1p resulted in lethality (R63D, K64E; Table 2), although with no effect of alanine substitutions (sec1-57[R63A, K64A]), suggesting that an essential interaction with Sec1p is repelled by the acidic residues at this surface.

Surprisingly, isolation of class B mutants implicates another surface as critical for SNARE complex binding: a conserved groove opposite the syntaxin binding cleft. Alternatively, it is not the groove, per se, but a structural change induced by mutations in the groove that alter the SNARE complex-binding conformation. The conformation of SM proteins is expected to be altered by mutations at buried positions (Li et al., 2005; Carpp et al., 2006), a likely explanation for the defects we observe for the conserved site sec1 mutants. However, we expect that charge alterations at the protein surface, such as those designed for groove mutant sec1-58, would disrupt a local interaction without disturbing the overall protein conformation. In support of this idea, Sec1p encoded by mutant sec1-58 was expressed, purified and found to recapitulate the binding defect we observed in lysates. How the groove region is used to recognize and bind cognate SNARE complexes for activation of vesicle fusion awaits high-resolution structural analysis.

Assigning a regulatory role for yeast Sec1p in vesicle docking will require purification and reconstitution of exocytic docking intermediates, in vitro. It is likely that SM proteins are one component of a vesicle tethering/SNARE complex assembly/fusion activation machine, as has been demonstrated for Vps33p/HOPS in the highly coupled vesicle fusion reaction at the vacuole (Mima et al., 2008; Starai et al., 2008). Could SM protein–SNARE interactions form a scaffold around a fusion pore (Rizo et al., 2006) and/or play a role in changing membrane curvature to resolve hemifusion, as has been proposed for the role of synaptotagmin and SNAREs in regulated exocytosis (Martens et al., 2007)? Testing models for the mechanism of fusion activation will require high-resolution imaging of fusion intermediates, such as an SM protein-bound SNARE complex trapped between apposing membranes.

Abbreviations used:

IP

immunoprecipitation

SM

Sec1/Munc18

SNARE

soluble N-ethylmaleimide-sensitive factor attachment protein receptor.