Phosphatidylinositol 4,5-Bisphosphate Mediates the Targeting of the Exocyst to the Plasma Membrane for Exocytosis in Mammalian Cells

Abstract

The exocyst is an evolutionarily conserved octameric protein complex that tethers post-Golgi secretory vesicles at the plasma membrane for exocytosis. To elucidate the mechanism of vesicle tethering, it is important to understand how the exocyst physically associates with the plasma membrane (PM). In this study, we report that the mammalian exocyst subunit Exo70 associates with the PM through its direct interaction with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ). Furthermore, we have identified key conserved residues at the C-terminus of Exo70 that are crucial for the interaction of Exo70 with PI(4,5)P 2 . Disrupting Exo70-PI(4,5)P 2 interaction abolished the membrane association of Exo70. We have also found that wild-type Exo70 but not the PI(4,5)P 2 -binding-deficient Exo70 mutant is capable of recruiting other exocyst components to the PM. Using the ts045 vesicular stomatitis virus glycoprotein trafficking assay, we demonstrate that Exo70-PI(4,5)P 2 interaction is critical for the docking and fusion of post-Golgi secretory vesicles, but not for their transport to the PM. INTRODUCTIONExocytosis is important for a variety of cellular functions, ranging from the release of hormones to the incorporation of membrane proteins for cell growth and morphogenesis. The late stage of exocytosis is a multistep process that includes directional transport, tethering, docking, and fusion of postGolgi secretory vesicles with the plasma membrane (PM). The tethering step, defined as the initial contact of secretory vesicles with the PM before SNARE-mediated docking and fusion (Pfeffer, 1999;Guo et al., 2000;Waters and Hughson, 2000;Whyte and Munro, 2002), is mediated by the exocyst, an evolutionarily conserved octameric complex composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 (for review, see Guo et al., 2000;Hsu et al., 2004;Munson and Novick, 2006;Wang and Hsu, 2006). In budding yeast, the exocyst components localize to the growing end of the daughter cell ("bud"), where active exocytosis and membrane addition take place (TerBush and Novick, 1995;Finger et al., 1998, Guo et al., 1999. This localization pattern contrasts that of the membrane fusion machine, the t-SNAREs, which are evenly distributed along both the mother and daughter cell membrane (Brennwald et al., 1994). In mammalian cells, the exocyst components were found in the cytosol, recycling endosomes and trans-Golgi network Fö lsch et al., 2003;Ang et al., 2004;Langevin et al., 2005). However, they are recruited to the PM during a number of cellular processes. For example, in epithelial cells, the exocyst is recruited to the adherens junction region upon cell-cell contact, where it mediates protein and membrane addition at the basolateral domain (Grindstaff et al., 1998;Yeaman et al., 2001); in developing neurons, the exocyst is localized to the growing neurites, where it mediates membrane expansion (Hazuka et al., 1999;Vega and Hsu, 2001); during cell migration, the exocyst is recruited to the leading edges of the PM (Rosse et al., 2006;...

Membrane Organization and Dynamics in Cell Polarity

“…It is likely that vesicular trafficking events such as transcytosis redirect membrane protein incorporation to the apical or basolateral membranes in response to a shift in the local ratio of phosphoinositides. As mentioned earlier, in both yeast and mammalian cells, exocyst components Sec3 and Exo70 interact directly with PI(4,5)P 2 , and this interaction is important for vesicle tethering at the plasma membrane during exocytosis (He et al 2007b;Liu et al 2007;Zhang et al 2008). It is therefore likely that secretion machinery such as the exocyst complex is recruited to specific domains of the plasma membrane for exocytosis.…”

“…Recently, it was found that the yeast Exo70 (He et al 2007b) and Sec3 ) bind directly to PI(4,5)P 2 on the plasma membrane. This proteinlipid interaction is conserved from yeast to mammals (Liu et al 2007). These interactions play a critical role in recruiting Exo70, Sec3, and ultimately the other exocyst components to the plasma membrane for vesicle tethering (Fig.…”

At the Intersection of the Pathways for Exocytosis and Autophagy

“…The GTPase proteins Rho3 and Cdc42 also interact with Exo70 and Sec3 to facilitate the assembly of the exocyst complex at the plasma membrane (He et al, 2007;Moskalenko et al, 2002). In mammalian cells, Exo70 facilitates exocyst association with the plasma membrane through its interaction with TC10 (the orthologue of Cdc42) (Inoue et al, 2003;Liu et al, 2007). In yeast, secretory vesicles acquire the Rab GTPase protein Sec4, which directly interacts with the exocyst, via the Sec15 subunit, thus allowing the secretory vesicle to be recognised by the exocyst plasma membrane complex.…”

Regulation of Gic2 Localization and Function by Phosphatidylinositol 4,5-Bisphosphate during the Establishment of Cell Polarity in Budding Yeast

“…Another example is the yeast protein Cla4, which interacts with both PI4P and Cdc42 and requires inputs from both signaling pathways (30). Recently, we found that PI(4,5)P 2 interacts with members of the exocyst complex and is important for polarized exocytosis (13,18,31). In particular, we have found that the exocyst component Sec3, through its N terminus, interacts with and is synergistically regulated by Cdc42 and PI(4,5)P 2 during polarized exocytosis (18).…”