Article

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

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.

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    • "Two of the eight exocyst subunits are responsible for the initial attachment to the PM at the future site of exocytosis: Sec3 and Exo70. Both interact with PI(4,5)P 2 in the inner lipid layer of the PM via polybasic sequences, located either at the N terminus (for Sec3) or at the C terminus (for Exo70) (He et al. 2007;Liu et al. 2007;Zhang et al. 2008). This appears to be also true for mammals (He and Guo 2009) and plants (L. "
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    ABSTRACT: Unconventional protein secretion (UPS) is a collective term for mechanisms by which cytosolic proteins that lack a signal peptide ("leaderless secretory proteins" (LSPs)) can gain access to the cell exterior. Numerous examples of UPS have been well documented in animal and yeast cells. In contrast, our understanding of the mechanism(s) and function of UPS in plants is very limited. This review evaluates the available literature on this subject. The apparent large numbers of LSPs in the plant secretome suggest that UPS also occurs in plants but is not a proof. Although the direct transport of LSPs across the plant plasma membrane (PM) has not yet been described, it is possible that as in other eukaryotes, exosomes may be released from plant cells through fusion of multivesicular bodies (MVBs) with the PM. In this way, LSPs, but also small RNAs (sRNAs), that are passively taken up from the cytosol into the intraluminal vesicles of MVBs, could reach the apoplast. Another possible mechanism is the recently discovered exocyst-positive organelle (EXPO), a double-membrane-bound compartment, distinct from autophagosomes, which appears to sequester LSPs.
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    • "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.
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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.
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