MOLECULAR ANALYSIS OF SECRETORY PROTEIN TRANSLOCATION

分泌蛋白易位的分子分析

• 批准号: 3295017
• 负责人: DAVID I MEYER
• 金额: $ 24.77万
• 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 1992-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3295017
• 关键词: affinity chromatography; cell free system; cell fusion; density gradient ultracentrifugation; endoplasmic reticulum; fungal genetics; gel filtration chromatography; gene expression; genetic manipulation; genetic transcription; genetic translation; immunologic techniques; ion exchange chromatography; membrane permeability; molecular cloning; molecular genetics; secretion; 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.

将蛋白质靶向跨或进入内质网 （er）是分泌的第一步，也是许多人组装 膜。 除了SRP，停靠蛋白（SRP）以外（SRP） 受体）和信号肽酶，对 涉及的组件或此过程的要求。 这是 在很大程度上是由于难以进一步解剖和 重建这样一个复杂的过程。 增强生化 与遗传的接近将允许进一​​步 更快，在这一领域取得进展。 酵母细胞，其 分泌途径与较高的真核生物相似， 具有明显的优势，可以轻松地操纵它们 遗传。 最近针对急诊室已经完成了 在衍生自酵母的无细胞系统中。 目的 该提案中描述的研究是对酵母的开发 遗传学，结合我们现有的生化专业知识， 识别和表征介导的组件 定位到跨易位的膜 嗯。 我们建议将无酵母细胞系统完善 使涉及的胞质和膜蛋白的分离 在易位。 重要的胞质成分将是 在裂解物依赖性的翻译后测定中鉴定。 将采用生化和免疫学方法 分析粗糙的ER特异性蛋白在易位中的作用。 验证这些蛋白在分泌中的参与 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， 至关重要的细胞质酶作为信号的表达 带序的嵌合体。 以这种方式产生的突变体将 然后在体外系统中以生化为特征。