|Home | About | Journals | Submit | Contact Us | Français|
In this study, we have analyzed the association of the Sec1p interacting protein Mso1p with the membrane fusion machinery in yeast. We show that Mso1p is essential for vesicle fusion during prospore membrane formation. Green fluorescent protein-tagged Mso1p localizes to the sites of exocytosis and at the site of prospore membrane formation. In vivo and in vitro experiments identified a short amino-terminal sequence in Mso1p that mediates its interaction with Sec1p and is needed for vesicle fusion. A point mutation, T47A, within the Sec1p-binding domain abolishes Mso1p functionality in vivo, and mso1T47A mutant cells display specific genetic interactions with sec1 mutants. Mso1p coimmunoprecipitates with Sec1p, Sso1/2p, Snc1/2p, Sec9p, and the exocyst complex subunit Sec15p. In sec4-8 and SEC4I133 mutant cells, association of Mso1p with Sso1/2p, Snc1/2p, and Sec9p is affected, whereas interaction with Sec1p persists. Furthermore, in SEC4I133 cells the dominant negative Sec4I133p coimmunoprecipitates with Mso1p–Sec1p complex. Finally, we identify Mso1p as a homologue of the PTB binding domain of the mammalian Sec1p binding Mint proteins. These results position Mso1p in the interface of the exocyst complex, Sec4p, and the SNARE machinery, and reveal a novel layer of molecular conservation in the exocytosis machinery.
Evolutionarily conserved molecular machinery regulates transport vesicle targeting, tethering, and fusion in eukaryotic cells. In yeast Saccharomyces cerevisiae this machinery involves the activity of the eight-subunit (Sec3p, Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, Exo70p, and Exo84p) tethering complex, the exocyst, a rab family small GTPase Sec4p, and the exocytic SNARE complex (Snc1/2p, Sec9p, and Sso1/2p) thought to drive the actual lipid bilayer fusion (Jahn et al., 2003 ). The exocyst subunit Sec15p has been shown to act as an effector for Sec4p (Guo et al., 1999 ). In addition, sec4-8 mutant cells are defective in Snc1/2p–Sec9p–Sso1/2p SNARE complex assembly (Carr et al., 1999 ). Despite these and numerous other studies, the regulatory mechanisms for SNARE complex formation are still poorly understood. The Sec1/Munc18 (SM) protein family members represent central regulators of SNARE complex function. These proteins perform an essential, albeit currently poorly understood function in SNARE complex regulation (Gallwitz and Jahn, 2003 ; Toonen and Verhage, 2003 ; Kauppi et al., 2004 ).
Yeast S. cerevisiae possesses four Sec1p-family proteins: Sec1p mediates vesicle fusion at the plasma membrane (Novick et al., 1981 ; Carr et al., 1999 ), Sly1p mediates vesicle fusion at the endoplasmic reticulum–Golgi interface (Ossig et al., 1991 ), Vps33p is required for endosome to vacuole transport and vacuole maintenance (Banta et al., 1990 ), and Vps45p mediates Golgi-to-vacuole transport (Piper et al., 1994 ; Cowles et al., 1994 ). Sec1p is the closest homologue of the mammalian Munc18-1 that has been shown to associate with syntaxin 1 and to regulate the syntaxin 1–synaptobrevin–SNAP-25 SNARE complex assembly (Misura et al., 2000 ; Kauppi et al., 2004 ). Munc18-1 association with syntaxin 1 has been proposed to maintain syntaxin 1 in a closed conformation and inhibit it to enter the SNARE complex (Misura et al., 2000 ). However, overexpression of SM genes in different cell systems has both positive and negative effects on secretion (for review, see Kauppi et al., 2004 ). Due to these partially conflicting results and the lack of detailed understanding of their regulation, the function of SM proteins is still unsolved.
Yeast cells defective for Sec1p function accumulate post-Golgi vesicles, indicating a positive role for Sec1p in membrane fusion (Novick et al., 1980 , 1981 ). It seems that the binding mode of the yeast Sec1p differs from the Munc18 proteins because at least a pool of Sec1p can associate with Sso1/2p–Snc1/2p and Sec9p-containing SNARE complexes (Carr et al., 1999 ). Recently, Sec1p was shown also to interact with the exocyst complex or even its subcomplexes lacking some of the exocyst subunits (Wiederkehr et al., 2004 ). In the same study, overexpression of Sec4p or Sec1p was shown to increase the amount of SNARE complexes in exocyst-defective cells. The results suggest a role for Sec1p in the molecular interface in between the exocyst complex and the SNARE machinery. An additional layer of regulation in transport vesicle fusion is provided by phosphorylation and dephosphorylation of SNARE components. Yeast Sso1p and Sso2p proteins are phosphorylated by protein kinase A in vegetatively grown cells. This phosphorylation event inhibits SNARE complex assembly and suppresses endo- and exocytosis (Marash and Gerst, 2001 ). Dephosphorylation of the t-SNARE Sso1p by a ceramide activated type 2A protein phosphatase again promotes association of Sso1p into SNARE complexes (Marash and Gerst, 2001 ). Based on these and other results, it is evident that phosphorylation-dephosphorylation cycles are important regulators of SNARE complex function (for review, see Weinberger and Gerst, 2005 ).
Presently, only a few SM binding proteins are known. In mammalian cells, the Mint1 and 2 and the Doc2 proteins have been shown to interact with Munc18-1 (Okamoto and Sudhof, 1997 ; Verhage et al., 1997 ). In addition to Munc18-1, Doc2 interacts also with a syntaxin interacting protein Munc13 (Verhage et al., 1997 ). Mint1 and 2 are neuronally expressed proteins and Mint1 has been shown to interact with CASK and Veli proteins in metazoan cells (Borg et al., 1998 , Butz et al., 1998 ). Mint 1 was originally characterized as a protein interacting through its PTB domain with β-amyloid precursor protein (APP), which has been implicated in Alzheimer's disease development (Borg et al., 1996 ; King and Turner, 2004 ). Metazoan Mint-defective cells have reduced neurotransmission, supporting an active role for these proteins in neurotransmission (Ho et al., 2003 ). The third Mint protein Mint3, which lacks the Munc18 binding domain, is ubiquitously expressed and rather poorly characterized (Okamoto and Sudhof, 1998 ). Based on their molecular interactions, Mint proteins have been suggested to link different regulatory molecules to the neuronal exocytosis process (Okamoto and Sudhof, 1997 ; Butz et al., 1998 ; Ho et al., 2003 ; King and Turner, 2004 ).
In yeast, three SM interacting proteins, Mso1p, Sso2p, and Vac1p, have been identified. Vac1p is a phosphatidyl-inositol-3-phosphate binding protein that interacts with Vps45 and is required for proper vacuole maintenance (Weisman and Wickner, 1992 ). The t-SNARE Sso2p was shown previously to interact with Sec1p in the yeast two-hybrid assay (Brummer et al., 2001 ). The Sec1p interacting protein Mso1p was identified in a multicopy suppressor screen for sec1-1 temperature-sensitive mutant (Aalto et al., 1997 ). Deletion of MSO1 is not lethal in vegetatively growing cells, but it leads to vesicle accumulation at the site of cell growth, implicating a positive role for Mso1p in exocytosis. Previously, we observed that deletion of Mso1p completely blocked sporulation (Jantti et al., 2002 ), a process in which four new haploid cells are formed within the diploid mother cell at the end of the second meiotic division through de novo plasma membrane generation. Assembly of these so-called prospore membranes occurs at the cytoplasmic side of the spindle pole bodies (SPBs) and requires the function of a meiosis-specific structure of the SPBs, the meiotic plaque (Taxis and Knop, 2004 ). Genetic interaction of MSO1 deletion with sec1 and sec4 mutations link MSO1 functionally to the vesicle docking and SNARE complex assembly (Aalto et al., 1997 ).
To shed light on Mso1p function and regulation of exocytosis, we have analyzed in detail association of Mso1p with the molecular machinery regulating membrane fusion in yeast cells. Our study positions Mso1p functionally in close association with Sec4p and the exocytotic SNARE complex. In addition, our results suggest a novel molecular link between the exocyst complex subunit Sec15p and the exocytotic SNARE complex. We furthermore show that Mso1p displays homology with PTB domains of mammalian Sec1p binding Mint proteins, suggesting the existence of a novel layer of conservation that regulates the membrane fusion machinery.
The yeast strains used are shown in Table 1. Yeast cells were grown as described previously (Sherman, 1991 ). Disruptions of MSO1 gene in H304 and H973 yeast strains were done by PCR amplification of the hphMX4 cassette from pAG32 followed by transformation into the corresponding strains. The H2937, H2955, and H2785 strains were generated by integrating StuI cut YIpProtA-MSO1 or the YIpProtA-mso1(59-210) or pRS406, respectively, into the ura3-52 locus of H2658. To obtain strains H2820, H2821, and H2855, the MSO1 deleted strains H2658 and H2661 were transformed with Stu1-linearized plasmids YIpMSO1U, YIpMSO1-T47A, or the empty vector pRS406. Hemagglutinin (HA), and 4GFP tagging of genes (MSO1 [H2657, YJM2], SEC1 [YNR60-3, YNR53-1), SEC2 [YNR27]) at the authentic chromosomal locus was performed for each of the genes using the PCR tagging strategy (Janke et al., 2004 ). Strains expressing SEC4-N34 and SEC4-I133 mutants from GAL1 promoter were generated by integrating the ClaI linearized plasmids NRB571 and NRB598 (Collins et al., 1997 ) into the leu2 locus. H3331 (sec4-8 MSO1-3HA-kanMX) was generated by crossing H1128 and H2657 followed by tetrad dissection.
The plasmids used are listed in Table 2. To generate the constructs used in the yeast two-hybrid assay, DNA fragments encoding amino- or carboxy-terminal deletions of Mso1p were generated by PCR with oligos containing the NcoI and XhoI sites. These fragments were then ligated into NcoI/XhoI cut pEG202 (Golemis et al., 1998 ). The construct with the wild-type (wt) MSO1, YEpMSO1-bait, and YEpSEC1-fish have been described previously (Brummer et al., 2001 ). To obtain constructs for constitutive overexpression from ADH1 promoter of wt MSO1 or different MSO1 deletion mutants, DNA fragments encoding the full-length and amino- or carboxy-terminal deletions of Mso1p were generated by PCR with oligos containing the XhoI/XbaI sites and cloned into XhoI/XbaI cut pVT102U vector (Vernet et al., 1987 ). To generate YCpMSO1U and YIpMSO1U, a genomic fragment encoding MSO1 promoter, open reading frame and terminator (400 base pairs upstream of ATG, 300 base pairs downstream of stop) was amplified by PCR and cloned as an XhoI-SpeI fragment to the BluescriptSK– to yield pMSO1Gen. From this plasmid, genomic MSO1 was transferred as an XhoI/SpeI fragment to pRS406 and pRS416 (Sikorski and Hieter, 1989 ).
The point mutations T43A, T46A, and T47A were generated using the site-directed mutagenesis (QuikChange; Stratagene, La Jolla, CA) and the plasmid pMSO1-TOPO as template. For yeast-two hybrid, T43A, T46A, and T47A mutants were amplified by PCR from the mutagenized plasmids and cloned as NcoI/XhoI fragments into pEG202 to yield MSO1T43A-bait, MSO1T46A-bait, and MSO1T47A-bait. YEpmso1T43A, YEpmso1T46A, and YEpmso1T47A were generated by PCR and cloned as a XbaI/XhoI fragment into pVT102U. To obtain YIpmso1T47A, the mutagenesis was carried out by using pMSO1Gen as the template. After site-directed mutagenesis, the T47A mutant and the wt fragments were transferred to XhoI/SpeI cut pRS406 (Sikorski and Hieter, 1989 ). YIpmso1(59-210) with endogenous MSO1 promoter was generated by PCR with an 5′ oligo containing NdeI site and lacking the coding sequence for the 58 first amino acids. This oligo was used to amplify the mso1(59-210) coding region and the terminator sequence and cloned into the NdeI/SpeI cut YIpMSO1U. The pMAX54 [MBP-MSO1(38-84)] was generated by PCR and ligated as NcoI/HindIII fragment into pETM40 vector (http://www.embl-heidelberg.de/ExternalInfo/geerlof/draft_frames/index.html). This leads to the expression of an Mso1p fragment spanning amino acids 38–84 fused to the carboxy-terminus of MBP. All constructs generated by PCR were verified by sequencing. The oligonucleotides used for the generation of all constructs are available upon request. Plasmids pJM146 (from James McNew, Rice University, Houston, TX) and pT-GroE (from Shunsuke Ishii, RIKEN Tsukuba Institute, Ibaraki, Japan) for bacterial expression of His6-Sec1p and GroE, respectively, have been described previously (Yasukawa et al., 1995 ; Scott et al., 2004 ).
EGY48 strain was transformed with a dual reporter system responsive to transcriptional activation through the LEU2 gene and the lacZ fusion gene in vector pSH18-34. Plasmids pRFHM1 and pSH17-4 were used as controls for the assay. The two-hybrid assay was performed essentially as described previously (Golemis et al., 1998 ). For each interaction, at least three individual transformants were tested. The induction of LEU2-gene was assayed by growth on SCGAL-ura-trp-leu + 1% raffinose and the LacZ-genes as a blue color on SCGAL-ura-trp-leu+x-gal + 1% raffinose agar plates.
The sec1-1 (H305) and sec1-11 (H306) mutants were transformed with plasmids overexpressing different mutant versions of MSO1 from pVT102U. The suppression test was done by spotting 105 cells and three 10-fold dilutions thereof on selective plates and incubating the plates at different temperatures for 3 d. To test synthetic lethal interactions between mso1-T47A and sec1-1, sec1-11, sec2-48, and sec4-8 mutants, the strains H2784, H2855, and H2786 were crossed with H305 and H306 and strains H2820, H2821 and H2785 with H1127 and H1128. After tetrad dissection, the genotypic combinations of the resulting spores were analyzed.
The HA (12CA5) and the myc-tag antibodies (9E10) were purchased from Roche Diagnostics (Indianapolis, IN). The proteins were separated either in SDS-PAGE using the buffer system of Laemmli (Laemmli, 1970 ) with acrylamide concentrations from 7.5–15% or by using NU-PAGE 4–20% gradient gels (Invitrogen, Carlsbad, CA) for Coomassie staining as appropriate for the proteins analyzed. For Western analysis, the protein were transferred onto nitrocellulose membranes. Bound antibodies were visualized with the ECL detection system (GE Healthcare, Piscataway, NJ). Anti-Sec1p antibodies used were either the one described previously (K6) (Aalto et al., 1997 ) or anti-Sec1p (#57) (from James McNew, Rice University; Scott et al., 2004 ). Anti-Sec9p-CT antibodies have been described previously (Brennwald et al., 1994 ). Anti-Sec4p (R232), Sec15p (R233), Sso1/2p (K8), Sso1p (K6916), Sso2p (K6906), Mso1p (R285), tubulin, and Ady3p antibodies have been described previously (Aalto et al., 1997 ; Moreno-Borchart et al., 2001 ; Jantti et al., 2002 ; Toikkanen et al., 2003 ). Anti-Sec8p antibody was obtained from Wei Guo (University of Pennsylvania, Philadelphia, PA). Anti-Snc1p and Snc2p specific antibodies were generated in rabbits by using cytosolic domains (GST-Snc1-TMD and His6-Snc2-TMD) of the corresponding proteins as antigens.
Immunofluorescence and electron microscopy of sporulating yeast cells have been described previously (Moreno-Borchart et al., 2001 ).
His6-Sec1p Pull-Downs. The expression of soluble His6-Sec1p in Escherichia coli was obtained by coexpression of pT-GroE and pJM146 as reported previously (Yasukawa et al., 1995 ; Scott et al., 2004 ). The same growth conditions were used for the control with the empty plasmid. Cells were harvested, lysed by sonication followed by centrifugation at 100,000 × g for 60 min at 4°C. To produce MBP-Mso1p(38-84) or MBP alone, BL21DE3 pRIL E. coli cells were transformed either with pMAX54 [MBP-Mso1p(38-84)] or pETM40 vector. Cells were grown at 25°C to OD600 0.8 and induced with 0.2 mM isopropyl β-d-thiogalactoside at the same temperature for 15 h. Cells were harvested and lysed by sonication. The lysates were then centrifuged at 15,000 × g. Equal amounts of His6-Sec1p and MBP-Mso1p(38-84) lysates were mixed and incubated for 5 h with mild agitation at 4°C. Amylose (E8021L; New England Biolabs, Beverly, MA) or Ni-NTA resin (30210; QIAGEN, Valencia, CA) was added and incubated overnight at 4°C. After centrifugation at 2000 rpm, resins were washed three times with buffer A (20 mM Tris, pH 7.2, 150 mM NaCl, 10 mM imidazole). Proteins were eluted with 20 mM maltose in 10 mM Tris, pH 7.2, or 300 mM imidazole in buffer A, respectively. Loading buffer was added to the samples and SDS-PAGE followed by Western blot detection for Mso1p and Sec1p was performed. The same procedures were applied to all controls.
Protein A Pull-Downs. Strains H2937 (ProtA-MSO1), H2955 [ProtA-mso1(59-210)], and H2785 (Δmso1) were grown on liquid YPD medium to a density of OD600 1–2. The cells were centrifuged at 3000 × g for 10 min at 4°C and washed once with water. The cells were then resuspended in 10% volume in water and dropped into the liquid nitrogen to create small pellets. The pellets were kept at –70°C until further use. For cell lysis, 5 g of each pellet was resuspended at a ratio of 1:1 (wt/vol) in ice-cold TEA-buffer (10 mM tetraethylammonium [TEA], pH 7.5, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 50 mM NaF) supplemented with protease and phosphatase inhibitors (Complete Protease inhibition cocktail [Roche Diagnostics], 4 mM 4-(2-aminoethyl)benzenesulfonyl fluoride [AEBSF; Roche Diagnostics], 40 mM β-glycerolphosphate, 5 mM benzamidine, 5 mM Pefablock). The cells were broken by vortexing in the presence of glass beads with repeated intervals of 20–30 s until ~80% of the cells were broken. After lysis the cell suspension was adjusted to 1% (vol/vol) NP-40, kept on ice for 30–45 min, centrifuged at 3000 × g for 10 min at 4°C, where after the supernatants were transferred to new tubes. The supernatants were then centrifuged at 10,000 × g for 10 min at 4°C and used for pull-downs.
For pull-downs 300 μl of IgG-coated Dynabeads was used. Incubation with the lysate was done at 4°C by end-over rotation for 2–4 h where after the tubes were fixed to a magnetic collector and the supernatants were discarded. The IgG-coated beads were washed five times at 4°C for 5–10 min: once with TEA-buffer with protease inhibitors and 1% NP-40, one to two times with TEA without inhibitors in a presence of NP-40, one to two times with wash buffer:TEA buffer supplemented with 5 mM ammonium acetate, 5 mM MgCl2. The beads were collected and eluted twice with 50 μl with 1% SDS at 65°C for 5 min. The eluted fractions were collected and concentrated in the vacuum to a final volume of 20–30 μl followed by mixing with 30 μl of urea-SDS sample buffer (HU-buffer (Knop et al., 1999 ), heating at 65°C for 10 min, and centrifugation for 5 min before loading onto the SDS-PAGE gels. For small-scale ProtA pull-downs, cells were treated as in immunoprecipitations except that IgG-coupled beads were used for precipitation (see below)
Immunoprecipitations. Cells were grown to early logarithmic growth phase and adjusted to 20 mM NaF. Thereafter, 300–400 OD600 of cells were collected immediately by centrifugation and washed once with ice-cold water supplemented with 20 mM NaF followed by centrifugation and a wash with ice-cold lysis buffer (50 mM HEPES, pH 7.5, 150 mM KCl, 5 mM EDTA, 5 mM EGTA, 20 mM NaF, 2 mM NaVaO4). Cells were then harvested at 3000 rpm/4°C and resuspended in 4 ml of lysis buffer supplemented with the Complete Protease inhibition cocktail (Roche Diagnostics) and 4 mM AEBSF (Roche Diagnostics). The cells were lysed by vortexing together with acid-washed glass beads six to 10 times for 15 s with a 30-s pause on ice. Then, 0.5% NP-40 was added to the lysates followed by a 30-min incubation on ice. The lysates were centrifuged 10,000 × g 10 min at 4°C, and protein concentration of the supernatant was measured using the Bradford reagent (Bio-Rad, Hercules, CA). Equal amounts of protein (6–12 mg) were used for immunoprecipitations.
The lysates were precleared by incubation with magnetic protein G-coupled beads (Dynal Biotech, Oslo, Norway) for 30 min at 4°C followed by centrifugation at 10, 000 × g for 10 min. Precleared lysate were subjected to immunoprecipitations for 1 h at 4°C with identical amounts of either anti-HA (12CA5) or anti-myc (9E10) monoclonal antibodies covalently coupled to protein G magnetic beads. Antibody coupling was performed according to the manufacturer's instructions (Dynal Biotech). The beads were washed three times with 1 ml of lysis buffer. The precipitated proteins were released into 60 μl of elution buffer (HU buffer without dithiothreitol [DTT]), heated for 5 min at 95°C. The elutes were adjusted to 100 mM DTT, heated as described above, and subjected to SDS-PAGE and Western blotting.
Deletion of MSO1 is not lethal, but results in a moderate vesicle accumulation phenotype in vegetatively grown yeast cells (Aalto et al., 1997 ). Our previous observation that deletion of Mso1p completely inhibited sporulation of diploid yeast cells (Jantti et al., 2002 ) prompted us to study in detail this sporulation defect by immunofluorescence and electron microscopy. As controls, we used wild-type diploid cells and cells deleted for Mcp54p and Mcp70p, two components of the meiotic spindle pole body that are necessary for the initiation of prospore membrane (PSM) formation, which occurs at the cytoplasmic side of the SPB early during meiosis II (Taxis and Knop, 2004 ). Based on the 4,6-diamidino-2-phenylindole (DAPI) labeling, MSO1 deletion did not impair entry into and progression through the meiotic divisions (our unpublished data). However, analysis of different stages of PSM assembly, by using Ady3p as a marker for prospore membrane (Moreno-Borchart et al., 2001 ), revealed that prospore membranes did not assemble in the Δmso1 mutant and that the Ady3p marker localized to clusters at the meiotic SPBs (Figure 1A). The phenotype was reminiscent to the one observed in the Δmpc54 Δmpc70 mutant. However, in the Δmso1 mutant a larger fraction of the precursor structures was detected at the SPBs compared with the Δmpc-control, suggesting that the failure of PSM assembly was due to a block slightly later in the process. This result suggests that Mso1p is required for prospore membrane formation in sporulating cells.
To study the defect in the Δmso1 cells in meiosis in more detail, the formation of the prospore membrane was examined by electron microscopy in wt, Δmpc54 Δmpc70, and the Δmso1 mutants. In wild-type cells, the prospore membrane surrounds the newly formed haploid nucleus (Figure 1B). In MSO1-deleted cells (Δmso1), no prospore formation occurred, but instead numerous 60- to 70-nm vesicles accumulated at the SPB (Figure 1B). These vesicle were frequently seen lined along the cytoplasmic side of the SPB, which at this stage of meiosis is modified with the meiotic plaque composed of Mpc54p, Mpc70p, and Spo74p (Knop and Strasser, 2000 ; Nickas and Neiman, 2002 ). This indicates that these vesicles have been docked to the scaffold of the meiotic SPB, but they seem to have failed to fuse and form a prospore membrane. This phenotype was observed at several SPBs (n > 10). In Δmpc54 Δmpc70 cells devoid of the meiotic plaque, only a few vesicles were detected adjacent to SPB (Figure 1B). These results show that Mso1p is essential for membrane fusion in sporulating cells and support the previously proposed role for Mso1p in regulation of membrane fusion. In addition, these results suggest that prospore membrane biogenesis is initiated by formation of a homogenously sized population of vesicles and their subsequent homotypic fusion.
Morphological data indicated that Mso1p acts in membrane fusion at the site of exocytosis and PSM formation. To analyze Mso1p localization, a cassette encoding four copies of GFP was inserted in to the MSO1 locus. This resulted in carboxy-terminal tagging of Mso1p with GFP (Mso1–4GFP) and enabled expression of Mso1-GFP from endogenous MSO1 promoter. Mso1p is a low abundance protein in yeast cells (our unpublished data), and therefore multiple GFP tags were required to visualize the protein in living cells. The fusion proteins were functional in vivo, because they did not impair spore formation (our unpublished data). In vegetatively grown haploid cells, Mso1-GFP localized to sites of vesicle fusion at the plasma membrane, at the bud-tip and bud-neck, but was not detectable on migrating vesicles (Figure 2A). In sporulating diploid cells, Mso1-GFP was found to localize to areas around the SPBs in early stages of meiosis II (Figure 2B). Faint staining was visible all along the extended prospore membranes throughout the growth of the membranes during anaphase of meiosis II (Figure 2B, top, metaphase; bottom, anaphase). Ady3p was visualized by immunofluorescence to detect the leading edge of the growing prospore membranes (Moreno-Borchart et al., 2001 ). Previously, Sec1p has been localized by GFP tagging to a restricted area in the bud and in the neck region of dividing vegetatively grown mitotic cells (Carr et al., 1999 ). In addition to this localization, Sec1p also has been localized by indirect immunofluorescence with anti-Sec1p antibodies to other areas of plasma membrane in the mother cell (Scott et al., 2004 ). The Mso1-GFP localization data are in agreement with the proposed role for Mso1p in Sec1p binding.
Previously, we showed that the full-length Mso1p interacts with Sec1p (Aalto et al., 1997 ). To map in more detail the Sec1p-binding domain in Mso1p, we prepared amino- and carboxy-terminal deletion series of Mso1p and investigated their interaction with full-length Sec1p in the yeast two-hybrid assay. Deletion of Mso1p from the carboxy terminus up to amino acid E58 gave similar growth on Leu– plates and generated β-galactosidase activity comparable with that of wt Mso1p (Figure 3A). When Mso1p was reduced to only the 37 amino-terminal amino acids, no Sec1p interaction was detected. The role of the amino terminal Mso1p fragment in Sec1p binding was further mapped by deletions of the amino terminal part of Mso1p. Amino acids 1–38 could be deleted without any significant decrease in Sec1p binding. However, when the first 58 amino terminal amino acids were deleted, Sec1p interaction was lost (Figure 3A). This assay localizes the minimal Sec1p binding domain between amino acids E38 and E58. All the constructs used resulted in similar expression of the encoded wt and mutant proteins (our unpublished data).
To verify the role of the amino-terminal part of the Mso1p in Sec1p binding, yeast strains were prepared in which the endogenous MSO1 was replaced by an amino-terminally protein A-tagged full-length Mso1p or a mutant version lacking the first 58 amino acids [ProtA-Mso1(59-210)]. When lysates prepared from these cells were subjected to precipitation experiments with IgG-conjugated magnetic beads followed by SDS-PAGE and Western detection with anti-Sec1p and Mso1p antibodies, it was evident that the ProtA-Mso1p efficiently brought down Sec1p, whereas ProtA-Mso1p(59-210) did not bind Sec1p (Figure 3B).
To verify that the interaction of Sec1p with Mso1p is direct, the putative Sec1p-binding domain fused to MBP, MBP-Mso1p(38-84), and His6-tagged Sec1p were produced in E. coli. Clarified lysates were mixed together and incubated for 5 h at 4°C followed by binding to either Nickel-NTA or amylose resins. In pull-downs with Ni-NTA resin, specific binding between His6-Sec1p and MBP-Mso1p(38-84) was observed, whereas no binding was detected in samples containing His6-Sec1p and MBP or MBP-Mso1p(38-84) and the empty plasmid control (Figure 3C). Pull-downs performed with amylose resin revealed the same specific interactions between His6-Sec1p and MBP-Mso1p(38-84) (our unpublished data).
MSO1 was identified in a genetic screen for multicopy suppressors of sec1-1 temperature-sensitive mutant (Aalto et al., 1997 ). We made use of this genetic interaction to test the role of the observed Sec1p binding domain for in vivo Mso1p function. Multicopy plasmids expressing wt MSO1, different mutant version of the gene, or the empty vector were transformed to sec1-1 and sec1-11 temperature-sensitive mutants, and the growth of the transformants was tested at different temperatures. The wt MSO1 was able to efficiently rescue the growth of sec1-1 and sec1-11 mutant cells at 37°C (Figure 4A). The mutant version of MSO1 encoding amino acids 1–132 was still able to rescue the growth of both sec1-1 and sec1-11 mutants. Additional deletion of MSO1 resulted in decreased suppression capacity, and an Mso1p fragment spanning amino acids 1–94 no longer was able to rescue the growth of sec1-1 at 37°C and displayed also reduced rescue of the less severe sec1-11 mutant at this temperature. This fragment, however, was still able to rescue the growth at a lower, but still restrictive temperature for both mutants. Additional deletion of Mso1p beyond amino acid N78 caused loss of growth rescue in both mutants even at 35°C.
The in vivo function of the amino terminal part of Mso1p was further mapped by testing the capability of the amino terminal deletion constructs of Mso1p to rescue the growth of sec1-1 and sec1-11 at restrictive temperature. Mutants lacking amino acids 1–37 were still fully active in vivo (Figure 4B). However, when a segment containing the Sec1p binding domain (amino acids 1–58) was deleted, the growth rescue phenotype was completely lost in both sec1 mutants. The mutant Mso1p containing only the amino-terminal part of the protein (amino acids 1–58), including the Sec1p binding domain, could not suppress sec1 mutants (Figure 4B). Similar expression of the encoded mutant protein was obtained for all constructs used (our unpublished data). The essential role of the Sec1p binding domain for vesicle fusion was further verified in sporulating diploid cells expressing solely the mso1 mutant deleted for the 57 amino-terminal amino acids. In these cells, no prospore membrane formation was observed, and the sporulating cells displayed a very similar vesicle accumulation phenotype as seen in MSO1-deleted cells (our unpublished data; Figure 1B). This suggests that interaction of Mso1p with Sec1p is required for prospore membrane precursor vesicle fusion and prospore membrane formation. Together, these results show that the Sec1p binding domain is necessary, but not sufficient, for full Mso1p in vivo function.
We next proceeded to further characterize the Sec1p binding domain. Threonine residues were selected for the initial targets in mutagenesis. The wt MSO1 was mutagenized to yield three different mutant versions of MSO1, mso1T43A, mso1T46A, and mso1T47A. These mutants and the wt MSO1 were overexpressed in sec1-1 and sec1-11 cells and their capability to suppress the ts phenotype was scored. In contrast to wt and the T43A and T46A mutants, the T47A was unable to rescue the growth at the restrictive temperature (Figure 5A). The role of T47 in Sec1p binding was tested by the yeast two-hybrid system. The T47A mutation reduced, but did not inhibit, Sec1p binding when compared with T43A and T46A mutants (our unpublished data). For all constructs used, similar expression of the encoded mutant protein was verified by Western blotting (our unpublished data).
MSO1 deletion in combination with sec1-1, sec1-11, sec2-41, or sec4-8 mutation is lethal (Aalto et al., 1997 ). This suggests that Mso1p function may be functionally closely associated not only with Sec1p, but also Sec2p and Sec4p. We therefore tested synthetic interactions of the Sec1p binding-deficient mutant form of mso1T47A in combination with sec1 mutants or with sec2-41 or sec4-8. For this, the haploid mso1T47A mutant was crossed with the corresponding haploid sec mutants. The diploids were sporulated, and after tetrad dissection the genotypes of the viable spores were scored (Figure 5B). The mso1T47A showed synthetic lethality with both sec1 alleles. Interestingly, mso1T47A was not synthetically lethal with sec2-41 or sec4-8 mutants. This suggests that in contrast to the complete loss of Mso1p, the Sec1p-binding deficient form of Mso1p is still capable of supporting cell growth in cells compromised for the Sec2p or Sec4p function. This indicates that the carboxy-terminal part of Mso1p may contain determinants functionally associated with Sec2p and Sec4p.
To study whether Mso1p could be found in association with other proteins than Sec1p, we performed immunoprecipitations in a yeast strain in which triple HA-tagged Mso1p (Mso1p-3HA) expression (the sole copy of MSO1 in these cells) was under control of the endogenous MSO1 promoter to retain wild-type expression levels of the protein. Immunoprecipitates were analyzed by immunoblotting using antibodies specific for Sec1p, Sso1/2p, Sec9p, Snc1/2p, Sec15p, and HA. Anti-HA antibody coupled magnetic beads efficiently precipitated Mso1p-HA, in contrast to the negative control beads in which identical amount of anti-myc antibody was coupled to (Figure 6A). In addition to Mso1p-HA, Sec1p, Sso1/2p, Sec9p, Snc1/2p, and Sec15p coimmunoprecipitated reproducibly with Mso1p-HA (Figure 6A). No coprecipitation of Sec4p or the exocyst subunit Sec8p was observed with Mso1-HA under the experimental conditions used (our unpublished data).
We next asked whether Mso1p is necessary for the observed coimmunoprecipitation of Sec15p, Sec1p, and the SNARE complex components. For this, anti-HA and anti-myc control immunoprecipitations were performed in two strains in which the sole genomic copy of SEC1 contained a carboxy-terminal triple HA tag and MSO1 was either deleted or not. Immunoprecipitates were analyzed by immunoblotting using antibodies specific for Sso1/2p, Sec9p, Snc1/2, Sec15p, and HA. In both strains, efficient coimmunoprecipitation of the same proteins was observed with Sec1p-HA (Figure 6B). These results show that deletion of Mso1p does not significantly affect the coimmunoprecipitation of Sec1p-HA with Sso1/2–Sec9–Snc1/2 SNARE complex components or Sec15p in vegetatively grown cells. It is thus likely that Mso1p performs a nonessential, regulatory function in association with Sec15p, Sec1p, and the Sso1/2–Sec9–Snc1/2 complex in these cells.
To address the stoichiometry of the interaction between Mso1p and Sec1p, we purified Mso1p using protein A tagging. This revealed that protein A-Mso1p and Sec1p were present in a complex in almost stoichiometric amounts, whereas no SNARE or Exocyst components could be detected under these rather stringent purification conditions, either by mass spectroscopy or Western blotting. This suggests that Mso1p can form a stable stoichiometric complex with Sec1p (Figure 6C).
MSO1 deleted cells display synthetic genetic interactions with sec4-8 mutation (Aalto et al., 1997 ) (Figure 5B). To evaluate the effect of sec4 mutations on molecular interactions of Mso1p, we performed anti-HA immunoprecipitations from vegetatively grown cells that express Mso1p-HA or Sec2p-HA from their endogenous promoters. In addition, these cells expressed dominant negative mutant forms of Sec4p under the inducible GAL1 promoter. The Sec4p-S34N mutant is analogous to Ras N17 mutation and presumably results in Sec4p arrested in an intermediate state between GDP and GTP bound forms, whereas the N133I mutation results in impaired guanine nucleotide binding of Sec4p (Walch-Solimena et al., 1997 ; Collins et al., 1997 ). The expression of sec4 mutants was induced by shifting the cells to a galactose containing growth medium for 2 h. The cells were then lysed and processed for immunoprecipitations. Immunoprecipitates were analyzed by immunoblotting using antibodies specific for HA, Sec1p, Sec4p, Sso1p, and Sso2p. In cells expressing wt Sec4p, coimmunoprecipitation of Sec1p and Sso1p and Sso2p proteins with Mso1p-HA was observed (Figure 7A). These proteins did not coprecipitate with Sec2p-HA. Both mutant forms of Sec4p coprecipitated with Sec2p-HA, supporting the previously detected in vitro interaction (Walch-Solimena et al., 1997 ). In addition, small amounts of the endogenous wild-type Sec4p were detected. No Mso1p was found to precipitate together with Sec2p-HA. Interestingly, however, Sec4p-N133I, but not Sec4p-S34N, mutant coprecipitated weakly, but specifically, with Mso1p (Figure 7A). In addition, the presence of Sec4p-N133I, and to a lower extent the Sec4p-S34N, mutant specifically reduced the amount of the coprecipitated Sso1p and Sso2p.
To further investigate the role of Mso1p in association with Sec4p, we carried out immunoprecipitations of Mso1p-HA from extracts of the temperature-sensitive sec4-8 mutant cells. These cells were grown either at 24°C or shifted to 37°C for 15 or 30 min before harvesting, where after the cells were lysed and subjected for immunoprecipitations with anti-HA-coupled or with anti-myc-coupled negative control beads. Immunoprecipitates were analyzed by Western blotting with antibodies to Sec1p, Snc1/2, Sso1/2p, Sec9p, Sec4p, Sec15p, and HA. In cells incubated at the restrictive temperature, Sec1p still efficiently coprecipitated with Mso1-HA (Figure 7B), whereas coimmunoprecipitation of Snc1/2, Sso1/2p, or Sec9p was abolished. In these immunoprecipitations, Sec15p still coprecipitated with Mso1 and Sec1p, whereas no Sec4-8p was detected. Interestingly, this was also true for the immunoprecipitation at permissive temperature (Figure 7B). This suggests that association of Mso1p with SNARE complexes is compromised in the sec4-8 mutant. Furthermore, these results show that association of Mso1p with Sec1p and Sec15p is not affected by sec4-8 mutation.
The molecular machinery regulating exocytosis is conserved from yeast to mammalian cells (Jahn et al., 2003 ). However, for Mso1p no mammalian homologue has been identified so far. In different yeast species close homologues for Mso1p can be found (Figure 8A). Database searches for mammalian homologues were hampered due to the high serine content (17% of all amino acids) of S. cerevisiae Mso1p. Searches to a small database containing patented protein sequences without functional information, identified a homologous protein sequence in which the homology was not restricted to serine residues. When this sequence was used to search GenBank sequences, the PTB domains of the mammalian Sec1p (Munc18) binding proteins (Okamoto and Sudhof, 1997 ), the Mint proteins, were identified as homologues of this sequence. Direct alignment of yeast Mso1 proteins verified the homology. The Kluyveromyces lactis Mso1p (KlMso1p) showed the highest homology with the PTB domains of Mint proteins of all known hemiascomycete yeast Mso1p homologues (Figure 8B). The homology is 46% and the identity 12%. Significantly, the similarity is not restricted to serine residues but is distributed throughout the length of Mso1p. The homology between KlMso1p with Mint PTB domains is higher than the homology between known PTB domains of human Shc, Mint1, and Numb proteins (30% similarity, 6% identity), underlining the fact that known PTB domains may share very low sequence homology but still retain a PTB-like fold (Yan et al., 2002 ). The phylogenetic tree shows the relationship between fungal Mso1p homologues and the Mint proteins (Figure 8C). Notably, the homology between the ascomycete Schizosaccharamyces pombe Mso1p and the Mints is higher than the hemiascomycete Mso1p.
Sec1p protein family members play an essential but poorly understood role in regulation of membrane fusion in eukaryotic cells (Gallwitz and Jahn, 2003 ; Toonen and Verhage, 2003 ; Kauppi et al., 2004 ). Numerous studies in different model systems have resulted in partly contradictory conclusions on their function. Here, we studied the Sec1p binding protein Mso1p, to understand its functional link to Sec1p and regulation of membrane fusion. We previously noticed that no sporulation occurred in diploid yeast cells deleted for MSO1 (Jantti et al., 2002 ). This prompted us to investigate in more detail the role of Mso1p in this process. In meiosis, formation of the spores occurs inside the mother cell. Thereby, the PSM, which are the precursors of the spore walls and the plasma membranes of the spores, become assembled de novo, discontinuously from the plasma membrane of the mother cell. The meiotic spindle pole bodies provide on their cytoplasmic side a specific scaffold, called the meiotic plaque, on top of which the earliest structures of the prospore membranes can be found (Moreno-Borchart and Knop, 2003 ). Up to now, it had been enigmatic how the initial steps of PSM assembly occur. Our observation of a class of small and homogenous vesicles in the Δmso1 mutant that seem to have docked to the SPB, but not fused, suggest that PSM formation is mediated by homotypic vesicle fusion that most likely involves secretory vesicles. This is in agreement with the previous finding that sec1, sec4, or sec8 mutants also block prospore membrane formation, although in these cases no detailed analysis of the phenotype with regard to the processes at the SPB has been carried out (Neiman, 1998 ). This suggests that core exocytic machinery is involved in all steps that govern the PSM assembly pathway, including the very early assembly processes. However, during PSM initiation, the membrane fusion machinery seems to be more stringently regulated compared with constitutive exocytosis in vegetatively grown cells. This is exemplified by the fact that PSM formation depends on the presence of Mso1p, whereas in Mso1p-deleted vegetatively grown cells only a moderate accumulation of vesicles at the sites of membrane growth is visible (Aalto et al., 1997 ). This essential function of Mso1p in PSM formation seems to be mediated through its interaction with Sec1p as mutant cells expressing a Sec1p binding defective form of Mso1p had a very similar phenotype compared with cells where the whole Mso1p was deleted.
The findings that Mso1p is essential for membrane fusion, most likely in association with Sec1p, prompted us to study in more detail the molecular interactions of Mso1p with Sec1p. Mapping the Sec1p interaction domain of Mso1p by the yeast two-hybrid system revealed an amino-terminal peptide (aa 37–59) to be necessary and sufficient for this protein–protein interaction. This interaction is direct because it could be reproduced in vitro with bacterially expressed components. Mso1p seems to form a rather stabile complex with Sec1p as Sec1p copurified in stoichiometric amounts with ProtA-Mso1p from yeast lysates under stringent conditions and in all experiments performed, an Mso1p–Sec1p complex was always observed. Within the Sec1p interacting peptide, T47 turned out to be a critical amino acid for Mso1p in vivo function and Sec1p–Mso1p interaction. The in vivo effect of the T47A mutation is presently unclear. Based on the in vitro binding results with bacterially expressed proteins it is evident that phosphorylation of T47 is not an absolute requirement for Sec1p binding. However, we cannot presently exclude the possibility that, phosphorylation of this residue may still serve as a means to regulate the Mso1p–Sec1p interaction in vivo. By using the mso1T47A mutant, we could show that cells expressing mso1T47A in combination with sec2 or sec4 mutations were viable, although this mutation formed a synthetic lethal combination with sec1 mutants. This is in contrast to the previous findings where complete loss of Mso1p formed a lethal combination also with sec2 and sec4 mutations. This underlines the importance of the Sec1p binding domain for Mso1p in vivo function and suggests that the carboxy-terminal part of Mso1p functionally interacts with the GTPase Sec4p and its exchange factor Sec2p.
Sec4p has been shown to interact with Sec15p, a subunit of the exocyst tethering complex (Guo et al., 1999 ). Furthermore, in sec2 and sec4 mutants no assembly of plasma membrane Sso1/2–Sec9–Scn1/2 SNARE complexes takes place and localization of Sec1p to the bud site is lost (Carr et al., 1999 ). These results functionally link Sec4p with Sec1p and SNARE complex function. However, no physical interactions between Sec4p and the SNARE complex or the SNARE complex interacting Sec1p has been reported so far. Our immunoprecipitation experiments carried out both with Mso1p and Sec1p identified coprecipitation of Mso1p, Sec1p, the Ssop–Sec9p–Sncp SNARE complex subunits, and the exocyst subunit Sec15p. Under the conditions used, we did not detect Sec8p in the Mso1p–Sec1p-containing complexes. This suggests that only Sec15p and possibly some other subunits of the exocyst complex associate directly or indirectly with Mso1p. The coimmunoprecipitation of Mso1p and Sec1p with Sec15p is supported by the recent findings showing that Sec1p coimmunoprecipitates with the exocyst complex or even its subcomplexes lacking some of the subunits (Wiederkehr et al., 2004 ). Previously, we showed that MSO1 deletion in combination with conditional mutations in exocyst subunits (sec3-2, sec5-24, sec6-4, sec8-9, sec10-2, and sec15-1) and the t-SNARE component sec9-4 formed a harmful combination for the cells (Aalto et al., 1997 ). At the same time, no synthetic interaction was detected when combining MSO1 deletion and sec18-1, a mutation affecting the disassembly of the SNARE complexes. These results support the close functional association of Mso1p with vesicle tethering and SNARE complex assembly. It has previously been proposed that exocyst complex assembly takes place during vesicle docking at the plasma membrane (Wiederkehr et al., 2004 ). Although Sec8p was not detected in our immunoprecipitations, it is possible that Mso1p interacts with the vesicle associated exocyst complex at the early stages of vesicle tethering. Exocyst is a large protein complex (Hsu et al., 1998 ), and it is likely that the complex has to be at least partially disassembled or displaced before SNARE assembly. Therefore, Mso1p also could facilitate formation of the t-SNARE complexes in a timely manner with the exocyst complex disassembly. The timing of these associations could be regulated through the association of Mso1p with Sec4p.
A functional association of Mso1p with Sec4p is supported by the findings that the combined effect of MSO1 deletion and sec4-8 mutation is lethal even at the permissive temperature. This lethality is not observed when only the Sec1p binding domain of Mso1p is mutated indicating that the Sec4p interaction is undisturbed by this mutation. We observed coimmunoprecipitation of Sec4I133 protein, a dominant negative mutant of Sec4p, with Mso1p, whereas under the conditions used, we could not observe coimmunoprecipitation with wt Sec4p, sec4-8p, or Sec4N34p. In sec4-8, SEC4N34 and SEC4I133 mutant cells association of Mso1p with Sec1p persisted, whereas at the same time in sec4-8 and SEC4I133 cells, interaction with the SNARE complex subcomponents was impaired. Our results thus suggest that the association of Mso1p with Sec1p is not regulated by the GTPase activity of Sec4p. Sec4-8p binds poorly GTP, Sec4N34 binds preferentially GDP, whereas Sec4I133p is presumably defective both in GDP and GTP binding (Walworth et al., 1989 ). Although, Sec4N34p and I133 mutants bind similarly the Sec4p function regulating proteins Sec2p and Dss1p (Collins et al., 1997 ; Walch-Solimena et al., 1997 ), these mutants are likely to posses slightly different conformations due to their different nucleotide binding properties. This could affect their binding to Mso1p or to possible, presently unknown proteins that may mediate this interaction. We speculate that GTPase cycle defective Sec4I133 protein is locked into a Sec1p and Mso1p containing complex and that this interaction impairs Mso1p–Sec1p association with the SNARE machinery and thus assembly of functional SNARE complexes. On GTP hydrolysis, Sec4p may normally be rapidly released from Sec1p and Mso1p and thus allow assembly of the t-SNARE complexes. A role for Mso1p as a protein facilitating Sec1p and Sec4p interaction with the SNARE machinery could explain the apparently positive role for Mso1p in membrane fusion as deletion of it or inhibition of its interaction with Sec1p impairs membrane fusion. Localization of Mso1-GFP to the bud tip and the site of PSM generation suggest that Mso1p–Sec1p–Sso1/2p interactions take place at sites of active membrane fusion. Because of the nature of immunoprecipitation experiments, we do not know whether all proteins coimmunoprecipitated here are part of a large protein complex or whether several smaller Mso1p containing complexes with different compositions have been retrieved. Clearly, to fully understand Mso1p function and the detailed composition of the protein complexes it associates with, further biochemical and genetic studies are needed. These results nevertheless for the first time position Mso1p to the molecular interface of the putative vesicle tethering machinery and the SNARE machinery.
In analogy to the exocyst, the HOPS protein complex is required for vesicle tethering during homotypic vacuole fusion. Within this complex, both the SM protein Vps33p and the Rab-family small GTPase Ypt7p were found in association with the SNARE machinery (Wurmser et al., 2000 ; Price et al., 2000 ). Our results together with the previous results with the HOPS complex imply that in different membrane fusion events, there exists a molecular machinery that links the vesicle tethering machinery with the SNARE machinery, possibly through a small GTP binding protein. In exocytosis, Mso1p and Sec1p seem to possess molecular interactions to fulfill such a task.
Database searches identified Mso1p as a homologue of the PTB domains of mammalian Sec1p binding Mint proteins. In mammalian cells, three Mint proteins exist. Mint1 and Mint2 proteins bind to Munc18 (Okamoto and Sudhof, 1997 ), the neuronal Sec1p homologue, whereas the ubiquitously expressed Mint3 has not been shown to interact with Munc18 (Okamoto and Sudhof, 1998 ). Mint proteins are multidomain proteins, which contain, in addition to the PTB domain, two PDZ domains and a separate Munc18 interaction domain (Okamoto and Sudhof, 1997 ). The PTB domain of Mint proteins has been reported to bind to the YENPTY peptide present in Alzheimer's disease-linked APP and to regulate cellular APP levels (Biederer and Sudhof, 2000 ; King and Turner, 2004 ). In addition to its association with Munc18, Mint1 protein has been shown to be part of a tripartite protein complex containing Mint1 (LIN-10), CASK (LIN-2), and Veli (LIN-7) and also to bind presenilins that are components of the APP processing enzyme γ-secretase (Borg et al., 1998 ; Butz et al., 1998 ; Lau et al., 2000 ). Based on a mouse knockout study, Mint1 and 2 may display partially redundant neuronal functions (Ho et al., 2003 ). Importantly, this study showed a clear role for Mint1 in neurotransmitter release. Although the detailed cellular function of Mint proteins remains obscure, the existing data support their role as adapter proteins that may form protein scaffolds with diverse downstream effectors in neuronal exocytosis. Although the expression of the Mint PTB domains could not rescue the loss of Mso1p in yeast cells (our unpublished data), the analogy of Sec1p binding and the sequence homology support the idea that Mso1p and Mint proteins are functional homologues. The interaction of Mso1p with Sec1p is confined to a short amino-terminal region of Mso1p, which makes it likely that the remaining part of the Mso1p has additional interaction partners. The Mint PTB domain also has been shown to bind phosphoinositol-4,5-bisphosphate and other phosphoinositol phosphates that are required for exocytosis (Okamoto and Sudhof, 1997 ; Odorizzi et al., 2000 ; Cockcroft and De Matteis, 2001 ). Our attempts to detect a similar interaction of Mso1p with PIPs were not successful. This leaves the identification of the ligand for the putative PTB-pocket of Mso1p to future work.
Together, our results demonstrate that vesicle fusion at the SPB in meiosis is a highly regulated membrane fusion process that bears evolutionarily conserved features with regulated vesicle fusion processes in higher eukaryotes. The functional importance of Mso1p for membrane fusion in yeast meiosis indicates that tight control of Sec1p via Mso1p has evolved as a distinct way how to mediate timed vesicle fusion. This unexpected novel layer of functional conservation in exocytosis regulation in yeast indicates an important additional role for the PTB-domain like protein Mso1p in conjunction with Sec1p, the exocyst subunit Sec15p, and the small GTPase Sec4p.
We acknowledge Pat Brennwald, Jeffrey Gerst, Wei Guo, Shunsuke Ishii, James McNew, and Peter Novick for generously providing strains, plasmids, and/or antibodies. We also thank Jeffrey Gerst for sharing unpublished results. Per-Harald Jonson and Marko Hyvönen helped with sequence alignments and Johanna Pispa provided comments on the manuscript. We thank Outi Könönen for excellent technical assistance. This work was financially supported by the Academy of Finland Grant Nos. 8244, 42160, 52096, 209120, and 211171 and the Institute of Biotechnology. This work is part of the research program “VTT Industrial Biotechnology” (Academy of Finland; Finnish Center of Excellence program, 2000–2005, Project No. 64330).
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05–03–0243) on July 19, 2005.