MOLECULAR ANALYSIS OF SECRETORY PROTEIN TRANSLOCATION

蛋白质跨过或进入内质网的靶向 (Er)是分泌的第一步，也是许多 膜。除SRP外，对接蛋白(SRP 受体)和信号肽酶，人们对此知之甚少。 所涉及的组件或此过程的要求。这是 这在很大程度上是由于进一步解剖和分析 重新构建这样一个复杂的过程。增强了生物化学 用基因的方法将允许进一步的，并有可能 在这一领域将取得更快、更快的进展。酵母细胞，其 分泌途径与高等真核生物的分泌途径非常相似， 有一个明显的优势，那就是它们很容易被操纵 从基因上来说。最近，针对ER的目标已经完成 在一个从酵母中提取的无细胞系统中。该计划的目标是 这项提案中描述的研究是酵母的开发 遗传学，结合我们现有的生化专业知识， 识别和表征调解的组件 靶向和跨膜转位 呃。 我们建议改进同源酵母无细胞系统，以 能够分离所涉及的细胞质和膜蛋白 在移位中。重要的胞浆成分将是 在依赖裂解物的翻译后化验中被鉴定。 将采用生化和免疫学方法来 分析粗面内质网特异蛋白在易位中的作用。 对这些蛋白质参与分泌的验证 这一过程将通过基因破坏技术在体内完成 在酵母中是可行的。同时，我们将选择新的 分泌突变体，靶向和易位有缺陷，由 一种关键的细胞质酶作为信号的表达 带有序列的嵌合体。以这种方式产生的突变体将 然后在体外系统中进行生化表征。

The targeting of proteins across or into the endoplasmic reticulum (ER) is the first step in secretion and in the assembly of many membranes. With the exception of SRP, docking protein (SRP receptor) and the signal peptidase, little is known about the components involved or the requirements of this process. This is due, in large part, to the difficulty of further dissecting and reconstituting such a complex process. Augmenting a biochemical approach with a genetic one would allow further, and potentially more rapid, progress to be made in this area. Yeast cells, whose secretory pathway closely resembles that of higher eukaryotes, have the obvious advantage that they can be easily manipulated genetically. Recently targeting to the ER has been accomplished in a cell-free system derived from yeast. The objective of the research described in this proposal is the exploitation of yeast genetics, in combination with our existing biochemical expertise, to identify and characterize the components that mediate targeting to, and the translocation across, the membrane of the ER. We propose to refine the homologous yeast cell-free system to enable the isolation of cytosolic and membrane proteins involved in translocation. Important cytosolic components will be identified in a lysate-dependent post-translational assay. Biochemical and immunological methods will be employed to analyze the role of rough ER-specific proteins in translocation. Verification of the participation of these proteins in the secretory process will be accomplished in vivo by gene disruption techniques that are feasible in yeast. In parallel, we will select for new secretory mutants, defective in targeting and translocation, by the expression of a crucial cytoplasmic enzyme as a signal sequence-bearing chimera. Mutants produced in this way will then be characterized biochemically in the in vitro system.