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CERLP AND PROTEIN FOLDING AND TRANSLOCATION

CERLP 和蛋白质折叠和易位

基本信息

批准号：
2677495
负责人：
PETER H VON HIPPEL
金额：
$ 14.93万
依托单位：
UNIVERSITY OF OREGON
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-08-01 至 2000-07-31
项目状态：
已结题

项目摘要

Protein translocation and folding are facilitated in the cell by helper proteins known as molecular chaperones. The heat shock protein 70 family (hsp70) of molecular chaperones is central to these processes. The goal of this research proposal is to elucidate the functions of a newly discovered chaperone called Cer1p, found in the endoplasmic reticulum (ER). Cer1p has limited homology with known hsp70s and may have some overlapping functions with the ER hsp70, Kar2p. Cer1p, like Kar2p, is important for translocation, though only for those proteins being translocated posttranslationally. A combination of genetic, molecular biological, and biochemical approaches will be employed to investigate the role of Cer1p in translocation and its restriction to a subset of the import routes. By generating rapid onset mutations long term adaptation to loss of Cer1p can be eliminated, and the direct functions of Cer1p can more conclusively be revealed. Multiple strategies are proposed to generate rapid onset mutations of cer1. Several approaches will be used to investigate the molecular basis for Cer1p's restriction to the posttranslational import pathway. Biochemical fractionation of detergent extracts will be performed to identify potential interacting partners with Cer1p and to determine if these interactions are pathway specific. Whether Cer1p directly interacts with translocating chains will be determined by crosslinking assays using microsomes. Initial pulse chase experiments suggest that Cer1p plays a role in protein folding. Funds are requested to complete a series of in vivo folding experiments to assess the relative contributions of Cer1p, Kar2p, and the hsp40 homolog Scj1p in protein folding in the ER. In addition, biochemical experiments with purified components are proposed to directly compare the properities and regulation of Cer1p and the true ER hsp70, Kar2p. Together, these studies are designed to investigate how the activities of these related chaperones are controlled to function in the translation and folding pathway in the ER.

蛋白质的移位和折叠在细胞中是由辅助蛋白促进的。分子伴侣蛋白。热休克蛋白70 分子伴侣家族（hsp70）在这些过程中起中心作用。这项研究的目的是阐明一个功能，新发现的伴侣蛋白Cer1p，发现于内质网网织膜（ER）。 Cer1p与已知的hsp70同源性有限，与ER hsp70、Kar2p有部分功能重叠。 Cer1p，就像 Kar2p对易位很重要，尽管仅对那些蛋白质被转移到了后期。一种基因，将采用分子生物学和生物化学方法，研究Cer1p在易位中的作用及其对进口路线的子集。通过产生快速突变，可以消除Cer1p缺失的术语适应， Cer1p的功能可以更明确地揭示。多提出了产生CER 1的快速起始突变的策略。几种方法将被用来调查的分子基础， Cer1p对翻译后输入途径的限制。将对洗涤剂浸提液进行生化分馏，确定Cer1p的潜在相互作用伙伴，并确定这些相互作用是途径特异性的。Cer1p是否直接与易位链的相互作用将由交联决定使用微粒体的测定。最初的脉冲追踪实验表明，Cer1p在蛋白质折叠要求提供资金，以完成一系列体内试验，折叠实验以评估Cer1p的相对贡献， Kar2p和热休克蛋白40同源物Scj1p在内质网中的蛋白质折叠。在此外，还提出了纯化组分的生化实验直接比较Cer1p和真核生物的性质和调控， ER hsp70，Kar2p。这些研究旨在调查如何控制这些相关伴侣的活动，在ER的翻译和折叠途径中发挥作用。