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

Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA.
Molecular Biology of the Cell (Impact Factor: 4.47). 12/2007; 18(11):4483-92. DOI: 10.1091/mbc.E07-05-0461
Source: PubMed


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.

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    • "Taken together with our study, it is likely that fusion with the PM of recycling vesicles and that of constitutive secretory vesicles are mediated by distinct sets of SNARE proteins. On the other hand, cell surface expression of vesicular stomatitis virus G (VSVG) protein, a well-studied marker for the constitutive secretory pathway, was reported to be significantly retarded by knockdown of Exo70 (Liu et al., 2007), suggesting that the exocyst is required for constitutive exocytosis of VSVG at the PM. It is therefore an issue to be addressed in future studies how different kinds of vesicles have proper command of distinct tethering and/or fusion machineries. "
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    ABSTRACT: We recently showed that Rab11 is involved not only in formation of recycling vesicles containing the transferrin (Tfn)-transferrin receptor (TfnR) complex at perinuclear recycling endosomes but also in tethering of recycling vesicles to the plasma membrane (PM) in concert with the exocyst tethering complex. We here aimed at identifying SNARE proteins responsible for fusion of Tfn-TfnR-containing recycling vesicles with the PM, downstream of the exocyst. We showed that exocyst subunits, Sec6 and Sec8, can interact with SNAP23 and SNAP25, both of which are PM-localizing Qbc-SNAREs, and that depletion of SNAP23 and/or SNAP25 in HeLa cells suppresses fusion of Tfn-TfnR-containing vesicles with the PM, leading to accumulation of the vesicles at the cell periphery. We also found that VAMP2, an R-SNARE, is colocalized with endocytosed Tfn on punctate endosomal structures, and that its depletion in HeLa cells suppresses recycling vesicle exocytosis. These observations indicate that fusion of recycling vesicles with the PM downstream of the exocyst is mediated by SNAP23/25 and VAMP2, and provide novel insight into non-neuronal roles of VAMP2 and SNAP25. © 2015. Published by The Company of Biologists Ltd.
    Biology Open 06/2015; 4(7). DOI:10.1242/bio.012146 · 2.42 Impact Factor
    • "Interestingly, it can form two stable subcomplexes. The subunits Exo70 and Sec3 are anchored to the plasma membrane via Rab and Rho GTPases and the phosphoinositide PI(4,5)P 2 , whereas the hexameric subcomplex of the other six subunits is thought to bind via Sec4 to the vesicle [109] [110] [111]. "
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    ABSTRACT: The HOPS multisubunit tethering factor (MTC) is a macromolecular protein complex composed of six different subunits. It is one of the key components in the perception and subsequent fusion of multivesicular bodies and vacuoles. Electron microscopy studies indicate structural flexibility of the purified HOPS complex. Inducing higher rigidity into HOPS by biochemically modifying the complex declines the potential to mediate SNARE-driven membrane fusion. Thus, we propose that integral flexibility seems to be not only a feature, but of essential need for the function of HOPS. This review focuses on the general features of membrane tethering and fusion. For this purpose, we compare the structure and mode of action of different tethering factors to highlight their common central features and mechanisms. Copyright © 2015. Published by Elsevier B.V.
    FEBS letters 06/2015; 589(19). DOI:10.1016/j.febslet.2015.06.001 · 3.17 Impact Factor
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    • "Recruitment of Exo70 and Sec8 was markedly stronger around internalized parasite regions (Fig. 1A,B). The exocyst complex regulates the exocytosis of post-Golgi vesicles (Guo et al., 1999; Guo et al., 2000; He et al., 2007; Hsu et al., 2004; Liu et al., 2007), so we investigated whether post- Golgi vesicles contribute to the formation of the T. cruzi parasitophorous vacuole. HeLa cells were transduced with a construct encoding temperature-sensitive VSVG-ts045–YFP before infection. "
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    ABSTRACT: Host cell invasion by Trypanosoma cruzi shares mechanistic elements with plasma membrane injury and repair. Both processes require Ca2+-triggered exocytosis of lysosomes, exocytosis of acid sphingomyelinase, and formation of ceramide-enriched endocytic compartments. T. cruzi invades at peripheral sites, suggesting a need for spatial regulation of membrane traffic. Here we show that Exo70 and Sec8, components of the exocyst complex, accumulate in nascent T. cruzi vacuoles and at sites of mechanical wounding. Exo70 or Sec8 depletion inhibits T. cruzi invasion and Ca2+-dependent resealing of mechanical wounds, but does not affect repair of smaller lesions caused by pore-forming toxins. Thus, T. cruzi invasion and mechanical lesion repair share a unique requirement for the exocyst, consistent with a dependence on targeted membrane delivery.
    Journal of Cell Science 11/2014; 128(1). DOI:10.1242/jcs.150573 · 5.43 Impact Factor
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