PEROXISOME BIOGENESIS IN YEAST

过氧化物酶体是真核生物中普遍存在的一种细胞器，参与多种 重要的新陈代谢途径。在人类中，过氧化物体是必不可少的。 对于某些脂肪的新陈代谢，其生物发生的缺陷是 对一个家族的致命性遗传疾病负责，统称为 齐薇格综合征。该计划的主要长期目标是 在分子水平上理解控制机制 过氧化物酶的生物发生。一种分子遗传学和生化相结合的方法 以巴斯德毕赤酵母为模型的攻击已经开始 系统。近年来，十几种不同的PEX基因和 对他们的产品进行了描述。然而，令人信服的证据表明 其中只有几个在生物发生中起到了特定的作用。 这项提案的一个主要目标是确立两个 巴斯德毕赤酵母PEX蛋白Pex2p和Pex8p在过氧化体生物发生中的作用 Pex8p是一种位于过氧化物酶体内膜上的蛋白质，它可以 可能起到释放新进口的基质蛋白的作用 感受器。这一模式的证据将通过确定和 在Pex8p上解剖负责导入特定子集的域 过氧化体蛋白及其与特定进口产品的相互作用 感受器。Pex2p属于一族相关的过氧化体积分族 是Zellweger基因的巴斯德毕赤酵母的同源基因。 这个家庭的成员被假设为形成 过氧化体蛋白转运仪。脉冲追逐研究将是 对温度敏感的pex2突变体进行研究，以获得证据 Pex2p功能是蛋白质导入所必需的。的组织结构 假定的进口设备将通过鉴定 Pex2p相互作用蛋白。一种涉及到 双杂交系统、抑制子突变体的分析与纯化 将使用含Pex2p的络合物。最后，一个高效的 将利用选择方案来鉴定有缺陷的巴斯德酵母突变株 在新的PEX基因中。PEX基因将被克隆，初级 他们的产品序列将被用来搜索数据库 他们的人PEX基因的同源基因。人类PEX cDNA也是 齐薇格基因将通过其恢复正常的能力来识别 对特定的Zellweger细胞系具有过氧化物体。因此，这个酵母系统 为研究过氧化物酶体的生物发生和 了解这种疾病的分子病因学。

Peroxisomes are ubiquitous eukaryotic organelles involved in a variety of important metabolic pathways. In humans, peroxisomes are essential for the metabolism of certain lipids, and defects in their biogenesis are responsible for a family of lethal genetic disorders, collectively termed Zellweger syndrome. The primary long-term goal of this program is to understand, at the molecular level, the mechanisms controlling peroxisomal biogenesis. A combined molecular genetic and biochemical attack has been initiated using the years Pichia pastoris as the model system. In recent years, more than a dozen different PEX genes and their products have been described. However, convincing evidence for a specific role in biogenesis has been provided for only a few of these. A major goal of this proposal is to establish the specific function of two P. pastoris PEX proteins, Pex2p and Pex8p, in peroxisome biogenesis. Pex8p is a protein located on the inner membrane of the peroxisome that may function to release newly imported matrix proteins from their receptors. Evidence for this model will be obtained by identifying and dissecting domains on Pex8p responsible for import of specific subsets of peroxisomal proteins and for interaction with specific import receptors. Pex2p belongs to a family of related peroxisomal integral membrane proteins and is the P. pastoris ortholog of a Zellweger gene. Members of this family are hypothesized to form the core of the peroxisomal protein translocation apparatus. Pulse-chase studies will be conducted on temperature-sensitive pex2 mutants to obtain evidence that Pex2p function is required for protein import. The organization of the putative import apparatus will be elucidated through the identification of Pex2p interacting proteins. A combination of approaches involving the two-hybrid system, suppressor mutant analysis and purification of Pex2p-containing complexes will be employed. Finally, a highly efficient selection scheme will be utilized to identify P. pastoris mutants defective in novel PEX genes. The PEX genes will be cloned, and the primary sequences of their products will be used to search the databases for their human PEX cDNA orthologs. Human PEX cDNAs that are also Zellweger genes will be identified by their ability to restore normal peroxisomes to specific Zellweger cell lines. Thus, this yeast system provides an excellent model for investigating peroxisome biogenesis and understanding the molecular etiology of this disease.