A Dissection of the Yeast ER Translocation Machine

蛋白质易位进入酵母内质网的分子机制将被详细阐述，从而对多肽如何穿越生物膜有更深入的了解。虽然通过遗传分析已经确定了蛋白质易位所需的几个因素，但这些成分如何协同作用以将前体蛋白穿过内质网膜仍然不清楚。每个易位复合物的成员将使用标准技术纯化，并使用新的生化互补测试进行分析。ATP驱动蛋白质易位的机制，可能是通过改变易位装置的亚复合物的构象和活性来实现的。易位复合体的活性成员的纯化将依赖于一种测定方法，在这种方法中，野生型蛋白质组分被测试其恢复蛋白质输入到重组蛋白脂质体的能力，这些重组蛋白脂质体是由相应基因中具有非功能性突变的细胞制备的。利用这种生化互补试验，成功地纯化了易位复合物的一个活性亚复合物。一旦复合体中的其他活性成分被分离，它们之间的相互作用，它们与前体蛋白和核糖体的关联，以及它们调节易位通道的能力将被研究。用复合体中必需的纯化因子重组的脂质体应该支持蛋白质的进口，并确定转运机器的哪些成员是刺激的或调节的。为了检查易位复合体的成分如何相互作用，准备了包含所需易位因子的亲和列。纯化的蛋白质或粗溶解膜提取物应用于基质，以识别相互作用的亚复合物或易位复合物的新成员。为了确定易位装置中的哪些因子与核糖体和前体蛋白接触，纯化的因子将被制成脂质体，并与放射性标记的核糖体或分泌蛋白混合，以评估它们的相互作用。这些接触的一个可能的候选者是易位通道Sec61p，目前其纯度为50%，并且在上文所述的重组试验中与sec61突变体进行生化互补。Sec61p与易位复合体中其他因子的相互作用，以及测量放射性标记肽进入含有Sec61p单独或与其他纯化蛋白结合的脂质体的摄取，为了解易位孔是如何调节的提供了见解。这些研究将剖析在内质网膜上执行分泌途径第一步的复杂的蛋白质机器。这项研究的结果将使我们能够理解蛋白质在膜上的运输是如何通过易位复合体促进的，这是一个能量不利的过程：迄今为止，这种复杂性的膜蛋白机器尚未得到充分的检查和重建。本文提出的工作将进一步理解能量需求运输系统、门控通道、热休克蛋白功能和分泌途径。最后，由于这些研究利用了遗传定义系统和生化方法，因此检测膜运输和其他细胞生物学现象的新技术将变得显而易见。细胞利用蛋白质复合物在空间上组织时间事件。这些蛋白质机器遍布整个细胞，帮助组织代谢序列、整体细胞结构和极性，以及将新合成的蛋白质运送到正确的位置。我们感兴趣的蛋白质机器就是后者的一个例子；它是一种识别新合成的蛋白质的细胞（分泌），并像杂货店店员一样将它们打包。有趣的是，它只吸收那些应该分泌的物质，所以它必须识别这些物质，只为它们打开袋子——而不是其他蛋白质。蛋白质进入内质网（ER）的运动（易位）是由这个消耗能量的蛋白质机器调节的。这项工作是解剖机器，纯化成分，并使用“合成”袋（脂质体）重建其活性。这种类型的重组以前没有做过。利用酵母遗传学的力量已经确定了机器的组成部分；如果其中一个部件因突变而失效，机器就会停止运行。机器对这些突变的敏感性可以在细胞（使用ER）和试管（使用脂质体）中进行研究。分析了这些组分的相互作用、它们的能量依赖性以及它们在试管中的自组装。研究结果可以应用于其他蛋白质机器如何组合在一起，以及细胞如何调节细胞内的进出。* * *

9506002 Brodsky The molecular mechanism of protein translocation into the yeast ER will be detailed, affording a deeper understanding of how polypeptides may cross biological membranes. Although several factors required for protein translocation have been identified through genetic analyses, how these components act coordinately to thread a precursor protein across the ER membrane remains obscure. Each member of the translocation complex will be purified using standard techniques and analyzed with a novel biochemical complementation test. The mechanism(s) whereby ATP drives protein translocation, perhaps by altering the conformation and activity of a subcomplex of the translocation apparatus will be investigated. The purification of active members of the translocation complex will rely on an assay in which wild type protein fractions are tested for their ability to restore protein import to reconstituted proteoliposomes prepared from cells with non-functional mutations in the corresponding genes. An active subcomplex of the translocation complex was successfully purified using this test of biochemical complementation. Once other active components in the complex are isolated, their interactions with one another, their associations with a precursor protein and ribosomes, and their ability to regulate the translocation channel will be investigated. A liposome reconstituted with essential purified factors in the complex should support protein import and determine which members of the translocation machine are stimulatory or regulatory. To examine how constituents of the translocation complex interact, affinity columns containing the required translocation factors are prepared. Purified proteins or crude solubilized membrane extracts are applied to the matrices to identify interacting subcomplexes or novel members of the translocation complex. To determine which factors in the translocation apparatus contact ribosomes and precursor proteins, purified factors will be r econstituted into liposomes and mixed with radiolabeled ribosomes or secreted proteins to assess their interaction. A likely candidate for these contacts is the translocation channel, Sec61p, which is presently 50% pure and biochemically complements the sec61 mutant in the reconstituted assay described above. The interaction of Sec61p with other factors in the translocation complex and measurements of the uptake of radiolabeled peptides into liposomes containing either Sec61p alone or combined with other purified proteins offer insights into how the translocation pore is regulated. These studies will dissect an intricate protein machine at the ER membrane that performs the first step in the secretory pathway. The results from this study will permit an understanding of how the transport of a protein across a membrane, an energetically unfavorable process, is facilitated by the translocation complex: a membrane protein machine of this complexity has not been fully examined and reconstituted to date. The work proposed here will further the comprehension of energy-requiring transport systems, of gated channels, of heat shock protein function, and of the secretory pathway. Finally, because these studies utilize a genetically defined system and biochemical methods, novel techniques for examining membrane transport and other cell biological phenomena will become evident. %%% The cell uses protein complexes to spacially organize temporal events. These protein machines are found throughout the cell and help to organize, for example, metabolic sequences, global cellular structure and polarity, and traffic of newly synthesized proteins to their correct locations. The protein machine of interest here is an example of the latter; it is one which recognizes newly synthesized proteins destined for delivery outside the cell (secretion) and, like a grocery clerk, bags them up. Interestingly, it only takes those items which should be secreted, so it must recognize those items and open up the bag for them only -- not other proteins. The movement (translocation) of proteins into the bag, or endoplasmic reticulum (ER), is regulated by this energy-consuming protein machine. This work is to dissect the machine, purify the components, and reconstitute its activity using a "synthetic" bag, a liposome. This type or reconstitution has not been done before. The components of the machine have been identified using the power of yeast genetics; if one of the components is made nonfunctional by mutation, the machine stops. The sensitivity of the machine to these mutations can be studied both in the cell (using ER) and in the test tube (using liposomes). The interaction of these components, their energy dependence, and their self-assembly in the test tube are analyzed. The results have application to how other protein machines fit together and how the cell regulates what gets out of and what stays inside the cell. ***