MECHANISMS OF POLYTOPIC PROTEIN BIOGENESIS IN THE ER

真核多跨整体膜的生物发生与组装 蛋白质被认为是通过共同翻译的作用发生的 指导顺序易位的独立拓扑发生序列 内质网的启动、终止和膜整合事件 薄膜。然而，最近的研究表明，在 这种生物合成途径。对于天然多跨蛋白质的子集， 合作交互而不是顺序的独立活动可以 暂时延迟链整合到膜中，直到 多个跨膜片段的合成已经完成。 值得注意的是，还发现了新的拓扑致病决定因素 单一多肽的多种拓扑异构体的直接生物发生 链条。这些发现对结构和 不同的膜蛋白的功能，需要详细的重新检查 它们组装过程中所涉及的分子机制。 更好地理解多面体利用的不同生物发生途径 蛋白质，拟议的研究将：i)确定信息的性质 编码在新生链中的序列决定因素中，该序列决定了 不同的转运和膜整合事件，II)决定如何 该信息由移位机制和/或 内质网膜上的伴侣作用于不同的组装机制， 三)研究不同生物发生途径对最终结果的影响 蛋白质结构和功能。三种不同的蛋白质，人类MDR1， CHIP28和MIWC，每一个都展示了不同的生物发生途径 将使用无细胞翻译系统、非洲爪哇卵母细胞和 培养的哺乳动物细胞。拓扑性序列决定因素将是 以确定的蛋白质嵌合体为特征，并在自然环境中 确定负责易位特异性的关键信息并 膜集成。这些决定因素之间的相互作用和 运输机械的部件将通过以下方式确定 内质网蛋白在特定阶段与新生链的交联 生物发生学。最后，不同的拓扑异构体在分子生物学中的作用 将研究蛋白质的成熟度和功能。 这些研究将实现三个重要目标。首先，他们 系统地定义引导不同事件的决定因素 拓扑成熟。其次，他们确定了易位机制和 这些决定因素通过细胞伴侣发挥作用。第三，他们 不同的生物发生机制与蛋白质需求的关系 功能。这项工作将提供对细胞的详细理解 调节多聚体蛋白组装并允许进一步 对这些通路如何在后天或后天受到干扰的调查 遗传性人类疾病。

Biogenesis and assembly of eukaryotic multispanning integral membrane proteins is proposed to occur cotranslationally through the action of independent topogenic sequences which direct sequential translocation initiation, termination and membrane integration events at the ER membrane. Recent studies, however, have demonstrated new complexities in this biosynthetic pathway. For a subset of native multispanning proteins, cooperative interactions rather than sequential independent activities may transiently delay integration of the chain into the membrane until synthesis of multiple transmembrane segments has been completed. Remarkably, novel topogenic determinants have also been identified which direct biogenesis of multiple topologic isoforms from a single polypeptide chain. These findings have important implications for the structure and function of diverse membrane proteins and require a detailed reexamination of the molecular mechanisms involved in their assembly. To better understand different biogenesis pathways utilized by polytopic proteins, the proposed studies will: i) identify the nature of information encoded within sequence determinants in the nascent chain which direct different translocation and membrane integration events, ii) determine how this information is interpreted by translocation machinery and/or chaperones at the ER membrane to effect different assembly mechanisms and, iii) investigate the effects of different biogenesis pathways on final protein structure and function. Three different proteins, human MDR1, CHIP28, AND MIWC, each of which demonstrate different biogenesis pathways will be studied using cell-free translation systems, Xenopus oocytes and cultured mammalian cells. Topogenic sequence determinants will be characterized in defined protein chimeras and in native contexts to identify key information responsible for translocation specificity and membrane integration. Interactions between these determinants and components of the translocation machinery will be identified by crosslinking ER proteins to nascent chains at specific stages of biogenesis. Finally, the role of different topological isoforms in protein maturation and function will be investigated. These studies will accomplish three important goals. First, they systematically define determinants which direct distinct events of topological maturation. Second, they identify translocation machinery and cellular chaperones through which these determinants act. Third, they correlate different biogenesis mechanisms with requirements for protein function. This work will provide a detailed understanding of cellular mechanisms which regulate polytopic protein assembly and allow further investigation into how these pathways might be disrupted in acquired or inherited human diseases.