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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: 4.55). 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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    • "Next, we examined the migration properties of MDA-MB-231 cells by wound healing assays (Figures 3C and 3D). We have previously generated siRNA oligos that selectively knock down Exo70 (Zuo et al., 2006; Liu et al., 2007, 2009). Here, we found that cells with their endogenous Exo70 knocked down by siRNA took more time for wound closure than control cells. "
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    ABSTRACT: Epithelial-mesenchymal transition (EMT) is an important developmental process hijacked by cancer cells for their dissemination. Here, we show that Exo70, a component of the exocyst complex, undergoes isoform switching mediated by ESRP1, a pre-mRNA splicing factor that regulates EMT. Expression of the epithelial isoform of Exo70 affects the levels of key EMT transcriptional regulators such as Snail and ZEB2 and is sufficient to drive the transition to epithelial phenotypes. Differential Exo70 isoform expression in human tumors correlates with cancer progression, and increased expression of the epithelial isoform of Exo70 inhibits tumor metastasis in mice. At the molecular level, the mesenchymal-but not the epithelial-isoform of Exo70 interacts with the Arp2/3 complex and stimulates actin polymerization for tumor invasion. Our findings provide a mechanism by which the exocyst function and actin dynamics are modulated for EMT and tumor invasion.
    Developmental Cell 12/2013; 27(5):560-573. DOI:10.1016/j.devcel.2013.10.020 · 10.37 Impact Factor
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    • "A role for PIP 2 in trafficking to the plasma membrane or between intracellular compartments is also emerging, as PIP 2 is synthesized on intracellular membrane compartments (Vicinanza et al., 2008) and PIP 2 generation modulates E-cadherin sorting to the basolateral membrane from the recycling endosome (Ling et al., 2007). Exocyst complex components also bind PIP 2 and may regulate trafficking (Liu et al., 2007). This suggests that the exocyst complex coordinates with PIP 2 synthesizing enzymes to modulate integrin trafficking during cell migration. "
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    ABSTRACT: Polarized delivery of signaling and adhesion molecules to the leading edge is required for directional migration of cells. Here, we describe a role for the PIP(2)-synthesizing enzyme, PIPKIγi2, in regulation of exocyst complex control of cell polarity and polarized integrin trafficking during migration. Loss of PIPKIγi2 impaired directional migration, formation of cell polarity, and integrin trafficking to the leading edge. Upon initiation of directional migration, PIPKIγi2 via PIP(2) generation controls the integration of the exocyst complex into an integrin-containing trafficking compartment that requires the talin-binding ability of PIPKIγi2, and talin for integrin recruitment to the leading edge. A PIP(2) requirement is further emphasized by inhibition of PIPKIγi2-regulated directional migration by an Exo70 mutant deficient in PIP(2) binding. These results reveal how phosphoinositide generation orchestrates polarized trafficking of integrin in coordination with talin that links integrins to the actin cytoskeleton, processes that are required for directional migration.
    Developmental Cell 01/2012; 22(1):116-30. DOI:10.1016/j.devcel.2011.10.030 · 10.37 Impact Factor
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    • "The PM targeting of Exo70 has been shown to be critical for exocyst-mediated polarized exocytosis (He et al., 2007a, 2007b; Liu et al., 2007). Several lines of evidence support that the membrane targeting of Exo70 depends on the binding of PI4,5P 2 in various organisms, including yeast (He et al., 2007b; He and Guo, 2009), fruit flies (Fabian et al., 2010), and mammalian cells (Liu et al., 2007). "
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    ABSTRACT: E-Cadherin-mediated formation of adherens junctions (AJs) is essential for the morphogenesis of epithelial cells. However, the mechanisms underlying E-cadherin clustering and AJ maturation are not fully understood. Here we report that type Iγ phosphatidylinositol-4-phosphate 5-kinase (PIPKIγ) associates with the exocyst via a direct interaction with Exo70, the exocyst subunit that guides the polarized targeting of exocyst to the plasma membrane. By means of this interaction, PIPKIγ mediates the association between E-cadherin and Exo70 and determines the targeting of Exo70 to AJs. Further investigation revealed that Exo70 is necessary for clustering of E-cadherin on the plasma membrane and extension of nascent E-cadherin adhesions, which are critical for the maturation of cohesive AJs. In addition, we observed phosphatidylinositol-4,5-bisphosphate (PI4,5P(2)) accumulation at E-cadherin clusters during the assembly of E-cadherin adhesions. PIPKIγ-generated PI4,5P(2) is required for recruiting Exo70 to newly formed E-cadherin junctions and facilitates the assembly and maturation of AJs. These results support a model in which PIPKIγ and PIPKIγ-generated PI4,5P(2) pools at nascent E-cadherin contacts cue Exo70 targeting and orient the tethering of exocyst-associated E-cadherin. This could be an important mechanism that regulates E-cadherin clustering and AJ maturation, which is essential for the establishment of solid, polarized epithelial structures.
    Molecular biology of the cell 11/2011; 23(1):87-98. DOI:10.1091/mbc.E11-05-0449 · 5.98 Impact Factor
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