Downstream are overlapping binding sites for Sec15p and Ypt32p-GTP, which compete against each other to hole to Sec2p. of Sec2p, adjacent to the PI(4)P binding site, and that addition of PI(4)P inhibits Sec2p phosphorylation by Yck2p. Loss of Yck1p and Yck2p function leads to accumulation of an intracellular pool of the secreted glucanase Bgl2p, as well as to accumulation of Golgi-related structures in the cytoplasm. We propose that Sec2p is phosphorylated after it has been recruited to secretory vesicles and the level of PI(4)P continues to be reduced. p32 Inhibitor M36 This promotes Sec2p function by stimulating its interaction with Sec15p. Finally, Sec2p is dephosphorylated very late in the exocytic reaction to facilitate recycling. == INTRO == Rab GTPases are nucleotide-dependent switches that serve as master regulators of membrane traffic (Hutagalung and Novick, 2011). Each Rab recruits a specific set of effectors that, in turn, directs different actions of a vesicular transport reaction. Because effectors, by definition, bind preferentially to the GTP-bound form of a Rab, guanine nucleotide exchange factors (GEFs) that catalyze the displacement of GDP from the p32 Inhibitor M36 Rab and thereby promote binding of GTP are essential intended for Rab function. Controlling when and where a GEF is active will therefore define the temporal and spatial regulation of its substrate Rab and downstream effectors. We focus here on the final stage from the secretory pathway in the budding yeastSaccharomyces cerevisiae. Secretory vesicles derived from the late Golgi are delivered to sites of polarized cell surface growth, such as the tip p32 Inhibitor M36 of small buds or the neck of large-budded cells, by the type V myosin Myo2p. They are then tethered to the cell cortex at these sites by the octomeric exocyst complex and finally fuse with the plasma membrane through the formation of atranssolubleN-ethylmaleimidesensitive element attachment protein receptor (SNARE) complex. The Rab GTPase Sec4p controls these three steps through direct interactions of Sec4p-GTP with Myo2p, the exocyst subunit, Sec15p, and the SNARE regulator Sro7p (Guoet al., 1999; Grosshanset al., 2006; Jinet al., 2011). Activation of Sec4p requires the function of Sec2p, a highly effective and specific Sec4p GEF (Walch-Solimenaet al., 1997). The N-terminus of Sec2p (amino acids [aa] 1160) forms a homodimeric coiled-coil structure that catalyzes the nucleotide exchange reaction Rabbit polyclonal to Sca1 on Sec4p, whereas the remainder of Sec2p (aa 160759) interacts with several different ligands that together determine its subcellular localization. Both Sec4p and Sec2p are highly concentrated on the surface of secretory vesicles yet exhibit no detectable association with the Golgi membranes from which these vesicles originate. The recruitment of Sec2p to secretory vesicles requires two different signals working in concert. One signal is the upstream, late Golgiassociated Rab Ypt32p (Ortizet al., 2002; Medkovaet al., 2006). The region of Sec2p covering aa 160 to 258 binds to the GTP-bound form of Ypt32p, and this interaction is essential intended for Sec2p localization. We termed this recruitment mechanism a Rab GEF cascade, and there are now several analogous good examples in which a GEF for one Rab is recruited by the GTP-bound form of the preceding Rab on a membrane traffic pathway (reviewed inMizuno-Yamasakiet al., 2012). Sec2p localization also requires the Golgi-associated pool of phosphatidylinositol-4-phosphate (PI(4)P) (Mizuno-Yamasakiet al., 2010). This phosphoinositide binds to several polybasic patches within a region of Sec2p downstream of the Ypt32p binding site. Mutation of those polybasic sites or loss of function from the Golgi-associated phosphatidylinositol 4-kinase Pik1p blocks Sec2p recruitment to secretory vesicles. In addition to binding to Ypt32p-GTP and PI(4)P, Sec2p also interacts with the Sec4p effector Sec15p (Medkovaet al., 2006). The interaction of a GEF with an effector of its substrate Rab.
Downstream are overlapping binding sites for Sec15p and Ypt32p-GTP, which compete against each other to hole to Sec2p
