Ectopic expression of syncollin in INS-1 beta-cells sorts it into granules and impairs regulated secretion.
Ectopic expression of syncollin in INS-1 beta-cells sorts it into granules and impairs regulated secretion.
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INS-1 β 细胞中 Syncollin 的异位表达将其分类为颗粒并损害调节的分泌。
DOI：
10.1021/bi048894d
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Li,GuoDong
中科院分区：
文献类型：
--
作者：
Li,Jingsong;Luo,Ruihua;Hooi,ShingChuan;Ruga,Pilar;Zhang,Jiping;Meda,Paolo;Li,GuoDong
Syncollin最初被证明是一种能够在无细胞系统中影响颗粒融合的蛋白质，但后来的研究表明其在酶原颗粒中的腔定位。为了确定其在完整细胞胞吐中的可能作用，将syncollin和截短形式的蛋白质（缺乏N-末端疏水结构域）稳定转染到胰岛素分泌型INS-1细胞中，因为这些充分研究的胞吐细胞似乎不表达蛋白质本身。通过亚细胞分级分离分析，双重免疫荧光染色和电子显微镜检查的研究表明，syncollin的转染在胰岛素分泌颗粒中产生强烈的信号，而从转染截短的syncollin的产物主要与高尔基体和较小程度的内质网。表达产物与细胞膜相关，而不是细胞质或细胞器内腔中的可溶性组分。重要的是，在表达syncollin的细胞中，由各种促分泌素刺激的胰岛素释放严重受损，但不受表达截短syncollin的影响。syncollin的转染似乎并不妨碍胰岛素的生物合成和加工，因为胰岛素原和胰岛素的细胞内容物和分泌颗粒的数量没有改变。此外，调节胰岛素分泌的早期信号（膜去极化和Ca 2+反应）不受影响。这些发现表明syncollin可能是针对胰岛素分泌颗粒特异性和损害调节分泌的远端阶段。
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 Ca2+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.