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...
