PROTEIN TRANSLOATION ACROSS THE ENDOPLASMIC RETICULUM

跨内质网的蛋白质翻译

• 批准号: 2178007
• 负责人: JAMES REID GILMORE
• 金额: $ 19.88万
• 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/2178007
• 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.

该提案中描述的研究旨在阐明 蛋白质在粗糙的机制中易位的机制 内质网。 特别强调（a） 分析GTP依赖性事件，这些事件发生在早期的早期阶段 蛋白质易位反应以及（b）生化，分子和 34 kD积分膜蛋白的功能表征可以 交联至体外组装的易位中间体。 GTP 结合蛋白在蛋白质的早期阶段发挥关键作用 易位反应。 信号识别的三个亚基 粒子（SRP）和SRP受体被预测为GTP结合 蛋白质。 深入了解这些蛋白质的确切作用将是 通过定义所有三个GTP水解周期相对于（a）而获得 启动鸟嘌呤核苷酸交换的调节成分，（b） 下游效应蛋白或靶标和（C）激活的蛋白 GTP水解，SRP受体α的GTP水解循环 将通过将位点定向诱变进行研究 具有SRP受体功能的体外分析的蛋白质。 测定将 开发以确定GTP结合位点的重要性 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或替代而被困 不可水解的GTP类似物。 GTP结合和水解测定将是 开发以识别调节GTP水解循环的蛋白质 SRP54。 该项目的这一部分的长期目标是学习 顺序或互锁的GTP水解循环如何控制 核糖体的选择性输送给我的粗糙内质表面 网状。 建议介导新生链转运的蛋白质 已经在哺乳动物和酵母系统中使用不同的 实验方法。 迄今为止，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），毗邻多肽 跨哺乳动物微粒体膜的运输。 IMP-34将被净化 从犬微粒体膜和编码蛋白质的cDNA克隆 将测序以允许与酵母Sec6L，Sec62和 SEC63蛋白。 更重要的是，脂质体重构测定将是 用于评估IMP-34在蛋白质转运反应中的作用。 该项目的长期目标是了解蛋白质的蛋白质 有选择地跨膜双层运输。 一旦全部 已经确定了必要的成分，这些蛋白质易位 可以将成分重构为磷脂囊泡，并将其重组 可以详细分析运输过程。