PROTEIN TRANSLOCATION ACROSS THE ENDOPLASMIC RETICULUM

跨内质网的蛋白质易位

• 批准号: 3288722
• 负责人: JAMES REID GILMORE
• 金额: $ 18万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3288722
• 关键词: binding proteins; crosslink; dogs; endoplasmic reticulum; guanine nucleotide binding protein; laboratory rabbit; lipid bilayer membrane; liposomes; membrane permeability; membrane proteins; membrane transport proteins; monoclonal antibody; mutant; polymerase chain reaction; protein reconstitution; protein signal sequence; protein structure; ribonucleoproteins; ribosomes; site directed mutagenesis; tissue /cell culture

The research described in this proposal is directed towards elucidating the mechanism by which proteins are translocated across the rough endoplasmic reticulum. Particular emphasis will be placed on (a) the analysis of GTP-dependent events that occur during early phases of the protein translocation reaction and on (b) the biochemical, molecular and functional characterization of a 34 kD integral membrane protein that can be crosslinked to in vitro assembled translocation intermediates. GTP binding proteins perform a pivotal role at early stages of the protein translocation reaction. Three subunits of the signal recognition particle (SRP) and the SRP receptor are predicted to be GTP-binding proteins. Insight into the precise role of these proteins will be obtained by defining all three GTP hydrolysis cycles with respect to (a) regulatory components that initiate guanine nucleotide exchange, (b) downstream effector proteins or targets and (c) proteins that activate GTP hydrolysis., The GTP hydrolysis cycle of the SRP receptor alpha subunit will be investigated by combining site directed mutagenesis of the protein with in vitro analysis of SRP receptor function. Assays will be developed to determine the significance of the GTP binding sites in the 54 kD subunit of SRP and the beta subunit of the SRP receptor. Reaction intermediates will be trapped by deleting GTP or substituting nonhydrolyzable GTP analogues. GTP binding and hydrolysis assays will be developed to identify the proteins that regulate the GTP hydrolysis cycle of SRP54. The long term goal of this portion of the project is to learn how sequential or interlocking GTP hydrolysis cycles control the selective delivery of ribosomes to me surface of the rough endoplasmic reticulum. Proteins that are proposed to mediate nascent chain transport have been identified in mammalian and yeast systems using distinct experimental approaches. To date, proteins identified in the mammalian transport reaction do not have obvious homologues in the yeast transport reaction. Chemical crosslinking has been used to detect an integral membrane protein (imp34) that is adjacent to polypeptides undergoing transport across mammalian microsomal membranes. Imp-34 will be purified from canine microsomal membranes and a cDNA clone encoding the protein will be sequenced to allow comparison with the yeast Sec6l, Sec62 and Sec63 proteins. More importantly, liposome reconstitution assays will be used to evaluate the role of imp-34 in the protein transport reaction. The long term goal of this project is to understand how proteins are selectively transported across membrane bilayers. Once all of the necessary components have been identified, these protein translocation components can be reconstituted into phospholipid vesicles and the transport process can be analyzed in detail.

本提案中所述的研究旨在阐明 蛋白质在粗糙表面上转移的机制 内质网 将特别强调（a） 分析在早期阶段发生的GTP依赖性事件， 蛋白质易位反应和（B）生物化学、分子和 34 kD整合膜蛋白的功能表征， 与体外组装的易位中间体交联。 GTP 结合蛋白在蛋白质的早期阶段起关键作用， 易位反应 信号识别的三个亚基 颗粒（SRP）和SRP受体被预测为GTP结合 proteins. 深入了解这些蛋白质的确切作用将是 通过定义关于（a）的所有三个GTP水解循环获得， 启动鸟嘌呤核苷酸交换的调节组分，（B） 下游效应蛋白或靶和（c）激活 GTP水解，SRP受体α的GTP水解循环 亚基将通过结合定点诱变进行研究， 该蛋白具有体外分析SRP受体功能。 测定将 以确定GTP结合位点在 SRP的54 kD亚基和SRP受体的β亚基。 反应中间体将通过删除GTP或取代 不可水解的GTP类似物。 GTP结合和水解试验将在 用于鉴定调节GTP水解循环的蛋白质 SRP54 本项目这一部分的长期目标是学习 如何顺序或联锁GTP水解周期控制 选择性地将核糖体传递到粗面内质网表面 网状细胞。 介导新生链转运的蛋白质类 已经在哺乳动物和酵母系统中使用不同的 实验方法。 到目前为止，在哺乳动物中鉴定的蛋白质 运输反应在酵母运输中没有明显的同源物 反应 化学交联已用于检测积分 膜蛋白（imp34），其邻近经历 通过哺乳动物微粒体膜的转运。 杂质-34将被纯化 和编码该蛋白的cDNA克隆 将被测序以允许与酵母Sec 61、Sec 62和 Sec 63蛋白。 更重要的是，脂质体重建测定将是 用于评价IMP-34在蛋白质转运反应中的作用。 这个项目的长期目标是了解蛋白质是如何 选择性跨膜转运。 一旦所有的 必要的成分已经确定，这些蛋白质易位 组分可重构为磷脂囊泡， 运输过程可以详细分析。