Dimerization of the exocyst protein Sec6p and its interaction with the t-SNARE Sec9p
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.
- SourceAvailable from: Lukas Synek
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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). "
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). "
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  and SM proteins , which are dedicated SNARE-binding proteins that control membrane fusion . These kinds of binding can be used to couple tethering to SNARE-mediated membrane fusion for multi-subunit tethering complexes including exocyst . "
ABSTRACT: The use of exocytosis for membrane expansion at nerve growth cones is critical for neurite outgrowth. TC10 is a Rho family GTPase that is essential for specific types of vesicular trafficking to the plasma membrane. Recent studies have shown that TC10 and its effector Exo70, a component of the exocyst tethering complex, contribute to neurite outgrowth. However, the molecular mechanisms of the neuritogenesis-promoting functions of TC10 remain to be established. Here, we propose that GTP hydrolysis of vesicular TC10 near the plasma membrane promotes neurite outgrowth by accelerating vesicle fusion by releasing Exo70. Using Förster resonance energy transfer (FRET)-based biosensors, we show that TC10 activity at the plasma membrane decreased at extending growth cones in hippocampal neurons and nerve growth factor (NGF)-treated PC12 cells. In neuronal cells, TC10 activity at vesicles was higher than its activity at the plasma membrane, and TC10-positive vesicles were found to fuse to the plasma membrane in NGF-treated PC12 cells. Therefore, activity of TC10 at vesicles is presumed to be inactivated near the plasma membrane during neuronal exocytosis. Our model is supported by functional evidence that constitutively active TC10 could not rescue decrease in NGF-induced neurite outgrowth induced by TC10 depletion. Furthermore, TC10 knockdown experiments and colocalization analyses confirmed the involvement of Exo70 in TC10-mediated trafficking in neuronal cells. TC10 frequently resided on vesicles containing Rab11, which is a key regulator of recycling pathways and implicated in neurite outgrowth. In growth cones, most of the vesicles containing the cell adhesion molecule L1 had TC10. Exocytosis of Rab11- and L1-positive vesicles may play a central role in TC10-mediated neurite outgrowth. The combination of this study and our previous work on the role of TC10 in EGF-induced exocytosis in HeLa cells suggests that the signaling machinery containing TC10 proposed here may be broadly used for exocytosis.PLoS ONE 11/2013; 8(11):e79689. DOI:10.1371/journal.pone.0079689 · 3.23 Impact Factor