MOLECULAR ANALYSIS OF SECRETORY PROTEIN TRANSLOCATION

分泌蛋白易位的分子分析

• 批准号: 3295023
• 负责人: DAVID I MEYER
• 金额: $ 29.11万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-09-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3295023
• 关键词: affinity chromatography; binding proteins; cell free system; cell fusion; density gradient ultracentrifugation; endoplasmic reticulum; enzyme inhibitors; enzyme mechanism; fungal genetics; gel filtration chromatography; gene expression; genetic manipulation; genetic transcription; genetic translation; immunologic techniques; ion exchange chromatography; laboratory mouse; laboratory rabbit; membrane permeability; membrane proteins; molecular cloning; molecular genetics; nucleic acid hybridization; protein signal sequence; protein transport; secretion; secretory protein; yeasts

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.

蛋白质穿过或进入内质网的靶向 (ER)是分泌的第一步，也是许多 膜。 除了SRP外，对接蛋白（SRP 受体）和信号肽酶，关于 所涉及的组件或该过程的要求。 这是 在很大程度上，由于进一步解剖的困难， 重建如此复杂的过程。 增强生化反应 用基因的方法将允许进一步的， 在这一领域取得更快的进展。 酵母细胞，其 分泌途径非常类似于高等真核生物的分泌途径， 有一个明显的优势， 遗传的 最近已经完成了对急诊室的定位 在源自酵母的无细胞系统中。 的目的 本提案中所述的研究是酵母的开发 遗传学，结合我们现有的生物化学专业知识， 以识别和表征介导 靶向和跨膜转运 儿 我们建议改进同源酵母无细胞系统， 能够分离涉及的胞质和膜蛋白 在易位。 重要的胞质组分将是 在裂解物依赖性翻译后测定中鉴定。 将采用生物化学和免疫学方法， 分析粗糙ER特异性蛋白在易位中的作用。 验证这些蛋白质参与分泌 该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，缺陷的靶向和易位， 一种重要的细胞质酶的表达作为信号 序列嵌合体 以这种方式产生的突变体 然后在体外系统中进行生物化学表征。