PROTEIN TRANSLOCATION ACROSS THE ENDOPLASMIC RETICULUM

这项建议中描述的研究旨在澄清 蛋白质在荒野中转移的机制 内质网。我们会特别强调(A) 对早期阶段发生的GTP依赖事件的分析 蛋白质易位反应和(B)生化、分子和 一种34kD的完整膜蛋白的功能鉴定 与体外组装的易位中间体发生交联。GTP 结合蛋白在蛋白质的早期阶段起着关键作用。 易位反应。信号识别的三个子单元 颗粒(SRP)和SRP受体被预测为GTP结合 蛋白质。对这些蛋白质的确切作用的洞察将是 通过定义关于(A)的所有三个GTP水解循环而获得 启动鸟嘌呤核苷酸交换的调节成分，(B) 下游效应蛋白或靶蛋白以及(C)激活的蛋白 GTP水解度，即SRP受体α的GTP水解期 亚基将通过结合定点突变进行研究。 对该蛋白进行体外SRP受体功能分析。化验结果会 被开发来确定GTP结合位点在 SRP的54kD亚基和SRP受体的β亚基。 反应中间体将通过删除GTP或替换 非水解性GTP类似物。GTP结合和水解分析将是 用于鉴定调节GTP水解酶循环的蛋白质 SRP54。该项目这一部分的长期目标是学习 顺序或联锁的GTP水解循环如何控制 核糖体选择性输送至粗面内质膜表面 网状结构。建议用来调节新生链式转运的蛋白质 在哺乳动物和酵母系统中使用截然不同的 实验方法。到目前为止，在哺乳动物中发现的蛋白质 酵母转运中的转运反应没有明显的同源性 反应。化学交联法被用来检测一种积分 与多肽相邻的膜蛋白(Imp34) 通过哺乳动物微粒体膜的运输。IMP-34将被提纯 从犬微粒体膜和编码该蛋白的cDNA克隆 将进行测序，以便与酵母Sec61、Sec62和 Sec63蛋白。更重要的是，脂质体重建分析将是 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 选择性地跨膜双层转运。一旦所有的 已经确定了必要的成分，这些蛋白质易位 成分可以重组成磷脂微囊，而 可以对输送过程进行详细分析。

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.