Article

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: 3.01). 05/2005; 44(16):6302-11. DOI: 10.1021/bi048008z
Source: PubMed

ABSTRACT 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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    • "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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    • "Besides a role in tethering, exocyst subunits interact with SNARE and SM proteins to control SNARE complex assembly (57). The exocyst subunit Sec6, as a dimer, interacts with the Qbc-SNARE Sec9 and inhibits the formation of Qabc acceptor SNARE complexes (63). In addition, Sec6 interacts with the SM protein Sec1 when it is part of the exocyst complex mediated by N-terminal sites in Sec6, which functionally overlap with Sec9 binding sites (64). "
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    ABSTRACT: CAPS (Calcium-dependent Activator Protein for Secretion, aka CADPS) and Munc13 (Mammalian Unc-13) proteins function to prime vesicles for Ca(2+)-triggered exocytosis in neurons and neuroendocrine cells. CAPS and Munc13 proteins contain conserved C-terminal domains that promote the assembly of SNARE complexes for vesicle priming. Similarities of the C-terminal domains of CAPS/Munc13 proteins with Complex Associated with Tethering Containing Helical Rods domains in multi-subunit tethering complexes (MTCs) have been reported. MTCs coordinate multiple interactions for SNARE complex assembly at constitutive membrane fusion steps. We review aspects of these diverse tethering and priming factors to identify common operating principles.
    Frontiers in Endocrinology 12/2013; 4:187. DOI:10.3389/fendo.2013.00187
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    • "In vesicular fusion, an exocyst complex likely plays active roles in regulating SNARE assembly in addition to simply tethering two lipid bilayers [59,60]. An exocyst can bind to both SNAREs [61] and SM proteins [62], which are dedicated SNARE-binding proteins that control membrane fusion [63]. These kinds of binding can be used to couple tethering to SNARE-mediated membrane fusion for multi-subunit tethering complexes including exocyst [64]. "
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