The Mammalian Exocyst, a Complex Required for Exocytosis, Inhibits Tubulin Polymerization

Abstract

The exocyst is a 734-kDa complex essential for development. Perturbation of its function results in early embryonic lethality. Extensive investigation has revealed that this complex participates in multiple biological processes, including protein synthesis and vesicle/ protein targeting to the plasma membrane. In this article we report that the exocyst may also play a role in modulating microtubule dynamics. Using monoclonal antibodies, we observed that endogenous exocyst subunits co-localized with microtubules and mitotic spindles in normal rat kidney cells. To test for a functional relationship between the exocyst complex and microtubules, we established an in vitro exocyst reconstitution assay and studied exocyst effect on microtubule dynamics. We found that the exocyst complex reconstituted from eight recombinant exocyst subunits inhibited tubulin polymerization in vitro. Deletion of exocyst subunit sec5, sec6, sec15, or exo70 diminished its tubulin polymerization inhibition activity. Surprisingly, exocyst subunit exo70 itself was also capable of inhibiting tubulin polymerization, although exocyst complex with exo70 deletion did not lose its activity completely. Overexpression of exo70 in NRK cells resulted in microtubule network disruption and the formation of filopodia-like plasma membrane protrusions. The formation of these membrane protrusions was greatly hampered by stabilizing microtubules with taxol. Overexpression of exo84, an exocyst subunit that did not show tubulin polymerization inhibition activity, did not cause this phenotype. Results shown in this article, along with a previous report that localized microtubule instability induces plasma membrane addition, implicates a novel role for the exocyst in modulating microtubule dynamics underlying exocytosis.

Interaction of BIG2, a brefeldin A-inhibited guanine nucleotide-exchange protein, with exocyst protein Exo70

“…There is no information regarding an involvement of BIG2 in these processes. Exo70 was found to induce depolymerization of microtubules in vitro (25), but similar action in mitotic cells has apparently not been reported.…”

“…They reported that only Exo70-GFP appeared confined to the lateral plasma membrane, although it was not localized like the endogenous protein, and it failed to generate tight intercellular contacts. Wang et al (25) found that overexpression of GFP-Exo70 (but not GFP-Exo84) resulted in the disruption of microtubule architecture in the area of cytoplasm under regions of plasma membrane protrusions, and microtubule polymerization in vitro was inhibited by recombinant Exo70. The interaction of BIG2 with Exo70 in these processes suggests a mechanism for the congenital defects described in two families with mutations in the BIG2 gene (7).…”

Molecular Mechanisms, Biological Actions, and Neuropharmacology of the Growth-Associated Protein GAP-43

“…Inhibition of phospholipase D2 impaired L1-stimulated neurite outgrowth in cerebellar granule neurons [95]. The exocyst refers to a 734 kDa complex, consisting of at least eight subunits, that is required for exocytosis [94]. RalA is also associated with refilling the readily releasable pool (RRP) of synaptic vesicles [77], and has been shown to bind to the actin-binding protein filamin and thereby induce formation of filopodia [69].…”

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

“…As shown in Figure 1A, GFP-Exo70 was clearly detected at the PM as revealed by serial optical sectioning from different axis ( Figure 1A). In addition, cells expressing GFP-Exo70 displayed filopodia-like structures as previously reported (Wang et al, 2004;Xu et al, 2005;Zuo et al, 2006). We next mapped the region of Exo70 that is required for its association with the PM by examining the localization of a series of GFP-tagged Exo70 truncates in HeLa cells.…”