Functional analyzes of molecular chaperons concerning on protein translocation across the ER membrane

分子伴侣对蛋白质跨内质网易位的功能分析

基本信息

批准号：
07458254
负责人：
KOHNO Kenji
金额：
$ 1.34万
依托单位：
Nara Institute of Science and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07458254/
关键词：
HSP70 Stress Response BiP yeast HSE Microtubule Tubulin hsp70 Ssalp Green Fluorescent Protein 核の配分

项目摘要

Bip is the member of HSP70 family that is localized in the lumen of the endoplasmic reticulum (ER) and plays an important role in translocation of nascent proteins across the ER membrane and their subsequent folding and assembly in the ER lumen. BiP is induced at the transcriptional level by various environmental stresses such as high temperatue, block of the protein glycosylation, intracellular Ca^<2+> disturbance, the addition of the recuded agents, those are leading to the accumulation of unfolded proteins in the ER.We found that depletion of intracellular Ssa1p induced KAR2 gene expression at the transcriptional level. By analyzing internal deletion mutants of the KAR2 promoter, heat shock element (HSE) is necessary for KAR2 gene induction in response to the depletion of Ssa1p. a temperature-sensitive ssal mutant transformed with HSE-CYCl-lacZ fusion vector exhibited the highly induced beta-galactosidase activity compared with that of wild-type control cells at restrictive temperature. These results show that the consentration of functional Ssalp in the cytosol participates in the regulation of KAR2 gene expression through HSE-mediated pathway and also support the idea that SSAl gene expression is auto-regulated. Another findings is that, after shift to the restrictive temperature (37ﾟC), ssal temperature-sensitive mutant cells showed abnormal distribution of nucleus and accumulated as large-budded cells with 2N DNA contents accompanied by aberrant microtubule structure. This result suggests llthat Ssalp is involved in function of microtubules.

BIP是HSP70家族的成员，该家族位于内质网（ER）的腔内，在跨ER膜的新生蛋白易位及其随后在ER管腔中的折叠和组装中起着重要作用。 BIP是通过各种环境应力在转录水平上诱导的，例如高温，蛋白质糖基化的块，细胞内Ca^<2+>灾难，添加了被审查药物的灾难，它们正在导致ER中展开的蛋白质积累。我们发现，固定的SSA1P诱导的KAR2 Gene cene cene cene cene cenee cenee cenee cenee cenee cenee cenee cenee cenee cenee cenee cenee cenee cenee cenee cenee cent cenee centercy contriptiation。通过分析KAR2启动子的内部缺失突变体，热休克元件（HSE）对于响应SSA1P的部署而言是KAR2基因诱导所必需的。与在限制性温度下的野生型对照细胞相比，用HSE-CYCL-LACZ融合载体转化的温度敏感的SSAL突变体表现出高度诱导的β-半乳糖苷酶活性。这些结果表明，在细胞质中对功能性SSALP的考虑参与了通过HSE介导的途径调节Kar2基因表达，还支持SSAL基因表达自动调节的想法。另一个发现是，在转移到限制性温度（37 c）之后，SSAL温度敏感的突变细胞表现出异常的核分布，并积聚为具有2N DNA含量的大型细胞，并伴有异常微管结构。该结果表明，ssalp参与了微管的功能。