Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

Scott, Brenton L.; Komen, Jeffrey S. Van; Irshad, Hassan; Liu, Song; Wilson, Kirilee A.; McNew, James A.

doi:10.1083/jcb.200405018

Cited by 107 publications

(195 citation statements)

References 48 publications

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“…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 antibod- ies 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.…”

Section: Mso1-gfp Localizes To Sites Of Exocytosis In Mitosis and To supporting

confidence: 74%

“…The expression of soluble His 6 -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.…”

Section: Pull-down Experiments and Immunoprecipitationsmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

“…The oligonucleotides used for the generation of all constructs are available upon request. Plasmids pJM146 (from James McNew, Rice University, Houston, TX) and pTGroE (from Shunsuke Ishii, RIKEN Tsukuba Institute, Ibaraki, Japan) for bacterial expression of His 6 -Sec1p and GroE, respectively, have been described previously (Yasukawa et al, 1995;Scott et al, 2004). …”

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confidence: 99%

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Molecular Interactions Position Mso1p, a Novel PTB Domain Homologue, in the Interface of the Exocyst Complex and the Exocytic SNARE Machinery in Yeast

Knop

Miller

Mazza

et al. 2005

MBoC

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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. INTRODUCTIONEvolutionarily 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 . 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;Kaup...

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Section: Mso1-gfp Localizes To Sites Of Exocytosis In Mitosis and To supporting

confidence: 74%

Section: Pull-down Experiments and Immunoprecipitationsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Molecular Interactions Position Mso1p, a Novel PTB Domain Homologue, in the Interface of the Exocyst Complex and the Exocytic SNARE Machinery in Yeast

Knop

Miller

Mazza

et al. 2005

MBoC

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“…These liposomes can selectively interact with other liposomes bearing a reconstituted "vesicle," or v-, SNARE to allow selective lipid mixing (5), vesicle content mixing (6), or lysis (7,8). This reconstituted reaction shows impressive specificity for cognate SNARE pairs (9) and can be directly promoted by other fusion-regulatory factors such as synaptotagmin and calcium (10) or Sec1p (11) under conditions that minimize lysis (12).…”

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confidence: 99%

Stringent 3Q·1R Composition of the SNARE 0-Layer Can Be Bypassed for Fusion by Compensatory SNARE Mutation or by Lipid Bilayer Modification

Fratti¹,

Collins²,

Hickey

et al. 2007

Journal of Biological Chemistry

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SNARE proteins form bundles of four ␣-helical SNARE domains with conserved polar amino acids, 3Q and 1R, at the "0-layer" of the bundle. Previous studies have confirmed the importance of 3Q⅐1R for fusion but have not shown whether it regulates SNARE complex assembly or the downstream functions of assembled SNAREs. Yeast vacuole fusion requires regulatory lipids (ergosterol, phosphoinositides, and diacylglycerol), the Rab Ypt7p, the Rab-effector complex HOPS, and 4 SNAREs: the Q-SNAREs Vti1p, Vam3p, and Vam7p and the R-SNARE Nyv1p. We now report that alterations in the 0-layer Gln or Arg residues of Vam7p or Nyv1p, respectively, strongly inhibit fusion. Vacuoles with wild-type Nyv1p show exquisite discrimination for the wild-type Vam7p over Vam7 Q283R , yet Vam7 Q283R is preferred by vacuoles with Nyv1 R191Q . Rotation of the position of the arginine in the 0-layer increases the K m for Vam7p but does not affect the maximal rate of fusion. Vam7 Q283R forms stable 2Q⅐2R complexes that do not promote fusion. However, fusion is restored by the lipophilic amphiphile chlorpromazine or by the phospholipase C inhibitor U73122, perturbants of the lipid phase of the membrane. Thus, SNARE function as regulated by the 0-layer is intimately coupled to the lipids, which must rearrange for fusion. SNARE 4 proteins (1) are vital for membrane fusion. Initially discovered in neuronal tissues, SNAREs are found on all organelles of the exocytic and endocytic pathways, from yeast to humans. Their characteristic feature is a heptad-repeat "SNARE domain," flanked by varied N-domains and by C-terminal membrane anchors, either a single membrane-spanning apolar polypeptide or an acyl anchor. SNAREs form 4-helical complexes through their SNARE domains (2). Although the residues in each SNARE that face the others in a 4-helical complex are generally apolar, 3 glutamine (Q) and 1 arginine (R) near the center of the 4-complexed SNARE domains form a conserved and polar "0-layer" (3). Recombinant neuronal SNAREs spontaneously assemble into stable bundles, yet SNARE complex assembly in vivo requires SNARE-binding proteins of the Sec1-Munc18 "SM" family. SNARE complexes are disassembled by two chaperones: Sec17p/␣-SNAP, which binds directly to SNARE complexes, and Sec18p/NSF, which binds to Sec17p/␣-SNAP and couples the energy of ATP binding and hydrolysis to SNARE complex disassembly (4). SNARE complexes form in cis, with each SNARE anchored to the same membrane, or in trans, with SNAREs anchored to apposed, "tethered" membranes.SNARE function has been studied in vivo, on isolated organelles, and through the reconstitution of recombinant SNAREs into proteoliposomes. When several SNAREs, which are found in vivo on a "target" membrane, are co-reconstituted into one population of liposomes, they form a t-SNARE complex. These liposomes can selectively interact with other liposomes bearing a reconstituted "vesicle," or v-, SNARE to allow selective lipid mixing (5), vesicle content mixing (6), or lysis (7,8). This reconstituted reaction shows impress...

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Molecular engineering of exocytic vesicle traffic enhances the productivity of Chinese hamster ovary cells

Peng

Fussenegger

2008

Biotech & Bioengineering

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A complex vesicle trafficking system manages the precise and regulated distribution of proteins, membranes and other molecular cargo between cellular compartments as well as the secretion of (heterologous) proteins in mammalian cells. Sec1/Munc18 (SM) proteins are key components of the system by regulating membrane fusion. However, it is not clear how SM proteins contribute to the overall exocytosis. Here, functional analysis of the SM protein Sly1 and Munc18c suggested a united, positive impact upon SNARE-based fusion of ER-to-Golgi- and Golgi-to-plasma membrane-addressed exocytic vesicles and increased the secretory capacity of different therapeutic proteins in Chinese hamster ovary cells up to 40 pg/cell/day. Sly1- and Munc18c-based vesicle traffic engineering cooperated with Xbp-1-mediated ER/Golgi organelle engineering. Our study supports a model for united function of SM proteins in stimulating vesicle trafficking machinery and provides a generic secretion engineering strategy to improve biopharmaceutical manufacturing of important protein therapeutics.

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Sec1p directly stimulates SNARE-mediated membrane fusion in vitro

Cited by 107 publications

References 48 publications

Molecular Interactions Position Mso1p, a Novel PTB Domain Homologue, in the Interface of the Exocyst Complex and the Exocytic SNARE Machinery in Yeast

Molecular Interactions Position Mso1p, a Novel PTB Domain Homologue, in the Interface of the Exocyst Complex and the Exocytic SNARE Machinery in Yeast

Stringent 3Q·1R Composition of the SNARE 0-Layer Can Be Bypassed for Fusion by Compensatory SNARE Mutation or by Lipid Bilayer Modification

Molecular engineering of exocytic vesicle traffic enhances the productivity of Chinese hamster ovary cells

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