title: Ectopic expression of syncollin in INS-1 beta-cells sorts it into granules and impairs regulated secretion.
abstract: Syncollin was first demonstrated to be a protein capable of affecting granule fusion in a cell-free system, but later studies revealed its luminal localization in zymogen granules. To determine its possible role in exocytosis in the intact cell, syncollin and a truncated form of the protein (lacking the N-terminal hydrophobic domain) were stably transfected in insulin-secreting INS-1 cells since these well-studied exocytotic cells appear not to express the protein per se. Studies by subcellular fractionation analysis, double immunofluorescence staining, and electron microscopy examination revealed that transfection of syncollin produced strong signals in the insulin secretory granules, whereas the product from transfecting the truncated syncollin was predominantly associated with the Golgi apparatus and to a lesser degree with the endoplasmic reticulum. The expressed products were associated with membranes and not the soluble fractions in either cytoplasm or the lumens of organelles. Importantly, insulin release stimulated by various secretagogues was severely impaired in cells expressing syncollin, but not affected by expressing truncated syncollin. Transfection of syncollin appeared not to impede insulin biosynthesis and processing, since cellular contents of proinsulin and insulin and the number of secretory granules were not altered. In addition, the early signals (membrane depolarization and Ca(2+) responses) for regulated insulin secretion were unaffected. These findings indicate that syncollin may be targeted to insulin secretory granules specifically and impair regulated secretion at a distal stage.
authors.affiliation: Cardiovascular Research Institute, National University Medical Institutes, Singapore.
authors.forename: Jingsong
authors.initials: J
authors.lastname: Li
authors.forename: Ruihua
authors.initials: R
authors.lastname: Luo
authors.forename: Shing Chuan
authors.initials: SC
authors.lastname: Hooi
authors.forename: Pilar
authors.initials: P
authors.lastname: Ruga
authors.forename: Jiping
authors.initials: J
authors.lastname: Zhang
authors.forename: Paolo
authors.initials: P
authors.lastname: Meda
authors.forename: GuoDong
authors.initials: G
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D045744:Cell Line, Tumor; D005576:Colforsin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000235:genetics; D005822:Genetic Vectors; D005947:Glucose / Q000037:antagonists & inhibitors / Q000494:pharmacology; D007328:Insulin / Q000096:biosynthesis / Q000378:metabolism; D007329:Insulin Antagonists / Q000494:pharmacology; D000078790:Insulin Secretion; D008564:Membrane Potentials / Q000235:genetics; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011188:Potassium / Q000037:antagonists & inhibitors / Q000494:pharmacology; D011384:Proinsulin / Q000096:biosynthesis / Q000378:metabolism; D051381:Rats; D022142:Secretory Vesicles / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D017384:Sequence Deletion; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D007328:Insulin; D007329:Insulin Antagonists; D008565:Membrane Proteins; C107454:Sycn protein, rat; D005576:Colforsin; D011384:Proinsulin; D005947:Glucose; D011188:Potassium; D002118:Calcium