PEROXISOME BIOGENESIS IN YEAST

酵母中的过氧化物酶体生物合成

• 批准号: 2770393
• 负责人: James Michael Cregg
• 金额: $ 21.45万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2770393
• 关键词: affinity chromatography; cytogenetics; developmental genetics; electron microscopy; fungal genetics; genetic regulation; human genetic material tag; immunoprecipitation; molecular cloning; northern blottings; nucleic acid sequence; peroxisome; phenotype; protein biosynthesis; protein sequence; protein structure function; temperature sensitive mutant; western blottings; yeast two hybrid system; yeasts

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.

过氧化物酶体是一种普遍存在的真核细胞器，参与多种 重要的代谢途径。 在人类中，过氧化物酶体是必不可少的 对于某些脂质的代谢，以及它们的生物合成中的缺陷， 导致了一系列致命的遗传疾病，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 过氧化物酶体生物发生 一种结合了分子遗传学和生物化学 攻击已经开始使用年毕赤酵母作为模型 系统 近年来，十几种不同的PEX基因和 已经描述了它们的产品。 然而，令人信服的证据表明， 其中只有少数几种在生物发生中具有特殊作用。 该提案的一个主要目标是确定两个机构的具体职能， P. Pex2p和Pex8p在过氧化物酶体生物合成中的作用。 Pex8p是位于过氧化物酶体内膜上的蛋白质， 可能起到从它们的细胞释放新输入的基质蛋白的作用。 受体。 该模型的证据将通过识别和 Pex8p上负责导入特定子集的解剖结构域 过氧化物酶体蛋白和与特定输入的相互作用 受体。 Pex2p属于一类相关的过氧化物酶体积分 P.pastoris是Zellweger基因的直系同源物。 这个家族的成员被假设为形成了 过氧化物酶体蛋白质易位装置 脉冲追踪研究将 对温度敏感的pex2突变体进行，以获得证据， Pex2p功能是蛋白质导入所必需的。 的组织 推定的进口设备将通过识别 Pex2p相互作用蛋白。 一种涉及到 双杂交系统，抑制突变体的分析和纯化 将使用含Pex2p的络合物。 最后，一个高效的 选择方案将用于鉴定巴斯德毕赤酵母缺陷突变体 新的PEX基因。 PEX基因将被克隆， 他们的产品序列将用于搜索数据库， 他们的人类PEX cDNA直向同源物。 人PEX cDNA也是 Zellweger基因将通过其恢复正常的能力来鉴定 过氧化物酶体与特定Zellweger细胞系的关系。 因此，这种酵母系统 为研究过氧化物酶体生物发生提供了一个极好的模型， 了解这种疾病的分子病因。