CERLP AND PROTEIN FOLDING AND TRANSLOCATION

蛋白质在细胞内的转运和折叠是由辅助分子促进的 被称为分子伴侣的蛋白质。热休克蛋白70 分子伴侣家族(HSP70)是这些过程的核心。 这项研究提案的目的是阐明一种 新发现的伴侣蛋白Cer1p，在内质中发现 网状结构(ER)。Cer1p与已知的Hsp70同源性有限，可能 与内质网HSP70、Kar2p有一些重叠的功能。Cer1p，就像 Kar2p对易位很重要，尽管只对这些蛋白质 被翻译后移位的。基因的组合， 将采用分子生物学和生化方法来 研究Cer1p在易位中的作用及其对 导入路线的子集。通过产生快速发作的突变Long 可以消除对Cer1p丢失的术语适应，并直接 可以更确切地揭示Cer1p的功能。多重 提出了产生快速发作的cer1突变的策略。 几种方法将被用来研究分子基础 Cer1p对翻译后输入途径的限制。 将对洗涤剂提取物进行生化分级，以 确定与Cer1p互动的潜在合作伙伴，并确定 这些相互作用是特定于路径的。Cer1p是否直接 与转移链的相互作用将由交联性决定 使用微粒体法进行分析。 最初的脉冲追逐实验表明Cer1p在 蛋白质折叠。需要资金才能完成一系列体内试验 折叠实验评估Cer1p的相对贡献， Kar2p和Hsp40的同源物Scj1p在内质网的蛋白质折叠中。在……里面 此外，还提出了对纯化成分进行生化实验的建议。 直接比较Cer1p和True的性质和调节 呃，HSP70，Kar2p。总而言之，这些研究旨在调查 这些相关的伴侣的活动是如何控制的 在内质网的翻译和折叠途径中发挥作用。

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.