title: Mammalian exocyst complex is required for the docking step of insulin vesicle exocytosis.
abstract: Glucose stimulates insulin secretion from pancreatic beta cells by inducing the recruitment and fusion of insulin vesicles to the plasma membrane. However, little is currently known about the mechanism of the initial docking or tethering of insulin vesicles prior to fusion. Here, we examined the role of the SEC6-SEC8 (exocyst) complex, implicated in trafficking of secretory vesicles to fusion sites in the plasma membrane in yeast and in regulating glucose-stimulated insulin secretion from pancreatic MIN6 beta cells. We show first that SEC6 is concentrated on insulin-positive vesicles, whereas SEC5 and SEC8 are largely confined to the cytoplasm and the plasma membrane, respectively. Overexpression of truncated, dominant-negative SEC8 or SEC10 mutants decreased the number of vesicles at the plasma membrane, whereas expression of truncated SEC6 or SEC8 inhibited overall insulin secretion. When single exocytotic events were imaged by total internal reflection fluorescence microscopy, the fluorescence of the insulin surrogate, neuropeptide Y-monomeric red fluorescent protein brightened, diffused, and then vanished with kinetics that were unaffected by overexpression of truncated SEC8 or SEC10. Together, these data suggest that the exocyst complex serves to selectively regulate the docking of insulin-containing vesicles at sites of release close to the plasma membrane.
authors.affiliation: Henry Wellcome Laboratories for Integrated Cell Signalling and Department of Biochemistry, School of Medical Sciences, University Walk, University of Bristol, Bristol BS8 1TD, United Kingdom.
authors.forename: Takashi
authors.initials: T
authors.lastname: Tsuboi
authors.forename: Magalie A
authors.initials: MA
authors.lastname: Ravier
authors.forename: Hao
authors.initials: H
authors.lastname: Xie
authors.forename: Marie-Ann
authors.initials: MA
authors.lastname: Ewart
authors.forename: Gwyn W
authors.initials: GW
authors.lastname: Gould
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Baldwin
authors.forename: Guy A
authors.initials: GA
authors.lastname: Rutter
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology*; D015870:Gene Expression; D007328:Insulin / Q000378:metabolism*; D007340:Insulinoma; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D008322:Mammals; D008565:Membrane Proteins; D051379:Mice; D016296:Mutagenesis; D022142:Secretory Vesicles / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D007328:Insulin; D008565:Membrane Proteins; C490683:Sec6l1 protein, mouse; C489608:Sec8l1 protein, mouse