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.
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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.2013.00187This article is viewable in ResearchGate's enriched formatRG Format enables you to read in context with side-by-side figures, citations, and feedback from experts in your field.
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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 · 3.64 Impact FactorThis article is viewable in ResearchGate's enriched formatRG Format enables you to read in context with side-by-side figures, citations, and feedback from experts in your field.
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ABSTRACT: Background Exocytosis is integral to root growth: trafficking components of systems that control growth (e.g., PIN auxin transport proteins) to the plasma membrane, and secreting materials that expand the cell wall to the apoplast. Spatiotemporal regulation of exocytosis in eukaryotes often involves the exocyst, an octameric complex that tethers selected secretory vesicles to specific sites on the plasma membrane and facilitates their exocytosis. We evaluated Arabidopsis lines with mutations in four exocyst components (SEC5, SEC8, EXO70A1 and EXO84B) to explore exocyst function in primary root growth.ResultsThe mutants have root growth rates that are 82% to 11% of wild-type. Even in lines with the most severe defects, the organization of the quiescent center and tissue layers at the root tips appears similar to wild-type, although meristematic, transition, and elongation zones are shorter. Reduced cell production rates in the mutants are due to the shorter meristems, but not to lengthened cell cycles. Additionally, mutants demonstrate reduced anisotropic cell expansion in the elongation zone, but not the meristematic zone, resulting in shorter mature cells that are similar in shape to wild-type. As expected, hypersensitivity to brefeldin A links the mutant root growth defect to altered vesicular trafficking. Several experimental approaches (e.g., dose¿response measurements, localization of signaling components) failed to identify aberrant auxin or brassinosteroid signaling as a primary driver for reduced root growth in exocyst mutants.Conclusions The exocyst participates in two spatially distinct developmental processes, apparently by mechanisms not directly linked to auxin or brassinosteroid signaling pathways, to help establish root meristem size, and to facilitate rapid cell expansion in the elongation zone.BMC Plant Biology 12/2014; 14(1):1594. DOI:10.1186/s12870-014-0386-0 · 3.94 Impact Factor