Mechanism for insertion of tail-anchored proteins into the endoplasmic reticulum

将尾部锚定蛋白插入内质网的机制

基本信息

批准号：
09680704
负责人：
MASAKI Ryuichi
金额：
$ 1.79万
依托单位：
Kansai Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680704/
关键词：
HPC-1 syntaxin 1A Tail-anchored protein ER integration Intracellular transport msALDH 小胞体膜挿入 GFP COS細胞細胞膜

项目摘要

HPC-1/syntaxin lA (HPC-l), which plays an important role in vesicular transport to the plasma membrane, possesses a hydrophobic sequence at its C terminus, In the present study, we investigated the role of the C-terminal portion of HPC-1 in intracellular localization. When expressed from cDNA in COS cells, wild-type HPC-1 was localized in the Golgi complex and the plasma membrane. HPC-l localized in the Golgi complex was chased away to the plasma membrane in the presence of cycloheximide. Truncation of the hydrophobic domain resulted in the cytoplasmic localization of the mutant, thus indicating that the domain indeed functions as a membrane anchor. The fusion protein with the N-terminal region of bovine opsin (the 0P3 extension), which contains the N-glycosylation sites, was glycosylated in transfected COS cells. In addition, we found that a chimeric protein consisting of Escherichia coil maltose binding protein with the last 24 amino acids of HPC-1 and the 0P3 extension is also glycosylated and localized along the exocytic pathway of transfected cells similar to HPC- 1. Taken together, these indicate that HPC-1 has a transmembrane structure, and suggest that the protein is first inserted into the endoplasmic reticulum and then transported to the plasma membrane via the exocytic pathway. In addition, our results indicate that the C-terminal portion of HPC-1is important for its intracellular localization.

HPC-1/Syntaxin LA（HPC-L）在囊泡转运到质膜中起重要作用，在其C末端具有疏水序列，在本研究中，我们研究了我们研究的C-末端部分的作用细胞内定位中的HPC-1。当在COS细胞中用cDNA表达时，野生型HPC-1位于高尔基体配合物和质膜中。在存在环己酰亚胺的情况下，将位于高尔基体配合物中的HPC-L被追赶到质膜。疏水结构域的截断导致突变体的细胞质定位，因此表明该结构域确实是膜锚。在转染的cos细胞中糖基化了含有牛蛋白的N末端区域的融合蛋白（0P3延伸）。此外，我们发现，由HPC-1的最后24个氨基酸和03扩展的嵌合蛋白也是糖基化并沿着类似于HPC-1的转染细胞的胞外途径进行糖基化并局部定位的嵌合麦芽糖结合蛋白。，这些表明HPC-1具有跨膜结构，并表明该蛋白质首先插入内质网中，然后通过外生途径转运到质膜。此外，我们的结果表明，HPC-11的C末端部分对于其细胞内定位很重要。