Dimerization of the Exocyst Protein Sec6p and Its Interaction with the t-SNARE Sec9p †

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Biochemistry (Impact Factor: 3.02). 05/2005; 44(16):6302-11. DOI: 10.1021/bi048008z
Source: PubMed


Vesicles in eukaryotic cells transport cargo between functionally distinct membrane-bound organelles and the plasma membrane for growth and secretion. Trafficking and fusion of vesicles to specific target sites are highly regulated processes that are not well understood at the molecular level. At the plasma membrane, tethering and fusion of secretory vesicles require the exocyst complex. As a step toward elucidation of the molecular architecture and biochemical function(s) of the exocyst complex, we expressed and purified the exocyst subunit Sec6p and demonstrated that it is a predominantly helical protein. Biophysical characterization of purified Sec6p by gel filtration and analytical ultracentrifugation experiments revealed that Sec6p is a dimer. Limited proteolysis defined an independently folded C-terminal domain (residues 300-805) that equilibrated between a dimer and monomer in solution. Removal of residues 300-410 from this construct yielded a well-folded, monomeric domain. These results demonstrate that residues 300-410 are necessary for dimerization, and the presence of the N-terminal region (1-299) increases dimer stability. Moreover, we found that the dimer of Sec6p binds to the plasma membrane t-SNARE Sec9p and inhibits the interaction between Sec9p and its partner t-SNARE Sso1p. This direct interaction between the exocyst complex and the t-SNARE implicates the exocyst in SNARE complex regulation.

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    • "*These authors contributed equally to this work recycling vesicles (Takahashi et al., 2012), we asked which SNAREs are responsible for recycling of Tfn–TfnR downstream of the exocyst. Previous studies in yeasts showed that an exocyst subunit, Sec6p, interacts with Sec9p in vitro (Morgera et al., 2012; Sivaram et al., 2005); Sec9p is homologous to mammalian PM-localizing Q bc -SNAREs, SNAP23 and SNAP25. Therefore, we first addressed whether the exocyst-SNARE interaction is conserved in mammalian cells. "
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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
    • "Therefore, it was postulated that assembly of both subcomplexes to the holocomplex leads to tethering of secretory vesicles and control of SNARE assembly [112] [113] [114]. The exocyst Sec6 interacts with Sec9 Q bc -SNARE [113]. The tethering mechanism of exocyst thereby seems to be similar to the HOPS-recruitment of the Vam7-(Q c )SNARE, even though Vam7 is not part of the HOPS complex . "
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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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    • "Sec6p also contributes to anchor the exocyst complex at sites of secretion – possibly via interaction with PM-associated proteins (Songer and Munson, 2009). Besides facilitating exocytosis by interactions with Sec9p, a Qbc exocytic t-SNARE protein (Sivaram et al., 2005), and with Sec1, a protein from the Sec1/Munc18 family regulating SNARE functions (Morgera et al., 2012), the exocyst also interacts with the vesicles transporting myosin Myo2p (also a known Sec4p interactor) via the Sec15p subunit that directly binds the motor and allows for its release after vesicle tethering (Jin et al., 2011; Donovan and Bretscher, 2012). "
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    ABSTRACT: Delivery and final fusion of the secretory vesicles with the relevant target membrane are hierarchically organized and reciprocally interconnected multi-step processes involving not only specific protein-protein interactions, but also specific protein-phospholipid interactions. The exocyst was discovered as a tethering complex mediating initial encounter of arriving exocytic vesicles with the plasma membrane. The exocyst complex is regulated by Rab and Rho small GTPases, resulting in docking of exocytic vesicles to the plasma membrane (PM) and finally their fusion mediated by specific SNARE complexes. In model Opisthokont cells, the exocyst was shown to directly interact with both microtubule and microfilament cytoskeleton and related motor proteins as well as with the PM via phosphatidylinositol 4, 5-bisphosphate specific binding, which directly affects cortical cytoskeleton and PM dynamics. Here we summarize the current knowledge on exocyst-cytoskeleton-PM interactions in order to open a perspective for future research in this area in plant cells.
    Frontiers in Plant Science 01/2014; 4:543. DOI:10.3389/fpls.2013.00543 · 3.95 Impact Factor
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