GOLGI TARGETING AND RETENTION OF YEAST KEX2 PROTEASE

酵母 KEX2 蛋白酶的高尔基体靶向和保留

• 批准号: 6180456
• 负责人: ROBERTA S. FULLER
• 金额: $ 25.6万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180456
• 关键词: Golgi apparatus; biological signal transduction; endopeptidases; fungal genetics; gene mutation; membrane proteins; molecular cloning; nucleic acid sequence; protein localization; protein sequence; protein structure function; tissue /cell culture; yeasts

The nature and mechanism of action of signals that determine protein localization in cells are a primary focus of research in eukaryotic cell biology. The overall goal of this project is to elucidate in molecular detail the signal-dependent mechanisms by which transmembrane proteins are localized to the trans Golgi network (TGN) in eukaryotic cells, using localization of Kex2 protease in yeast as a model. Kex2p is the prototype of an important family of proprotein-processing proteases that function in late compartments of the secretory pathway in all eukaryotic cells. Thus this research is of significance not only for the knowledge it may provide about general principles of signal-dependent localization in the secretory pathway, but also because of the importance of understanding the cellular organization of proprotein processing, which is involved in a wide array of both normal and pathological phenomena in biology. During the current funding period, we have made significant progress in several areas. We have identified three genes, SOI1, SOI2 and SOI3, involved in localization of Kex2p and other transmembrane proteins to the yeast TGN. We have conducted detailed studies of the function of the SOI1 gene, which we found to encode a novel, conserved protein of 3144 residues that appears to function at both the TGN and the prevacuolar compartment (PVC, equivalent to the late endosome in animal cells) to promote the cycling of Kex2p and other TGN membrane proteins between the two compartments. We have identified two TGN localization signals (TLSs) in the Kex2p cytosolic tail. Soilp was shown to function through TLS1 to promote retrieval of Kex2p from the PVC and to function at the TGN to inhibit TLS2-dependent retention of Kex2p in the TGN. The work proposed here will expand on these results both to identify additional molecules involved in signal-dependent localization of TGN membrane proteins and to test aspects of the TGN-PVC cycling model. The specific aims of the project are: 1. to pursue a molecular, genetic and biochemical analysis of yeast Soilp and higher cell Soilp homologues; 2. to identify proteins that interact with Soilp using both genetic and biochemical approaches; 3. to clone and analyze the SOI2 and SOI3 genes; and 4. to identify and study additional genes involved in TGN localization through the isolation of multicopy suppressors of the Tyr713Ala mutation in the Kex2p TLS1 and identification of genes required for TLS2 function.

决定蛋白质的信号的性质和作用机制 细胞定位是真核细胞研究的主要焦点 生物学。 该项目的总体目标是从分子角度阐明 详细说明跨膜蛋白的信号依赖性机制 定位于真核细胞中的反高尔基体网络（TGN）， 使用 Kex2 蛋白酶在酵母中的定位作为模型。 Kex2p 是 一个重要的前蛋白加工蛋白酶家族的原型 在所有真核生物分泌途径的晚期区室中的功能 细胞。 因此，这项研究不仅具有知识意义 它可以提供信号相关定位的一般原理 在分泌途径中，还因为 了解前蛋白加工的细胞组织， 涉及广泛的正常和病理现象 在生物学中。 在本轮融资期间，我们取得了重大进展 多个领域取得进展。 我们已经鉴定出三个基因，SOI1、SOI2 和 SOI3，参与 Kex2p 和其他跨膜的本地化 酵母 TGN 的蛋白质。 我们对此进行了详细的研究 SOI1 基因的功能，我们发现该基因编码一种新的、保守的 3144 个残基的蛋白质似乎在 TGN 和 前液泡室（PVC，相当于晚期内体） 动物细胞）促进Kex2p和其他TGN膜的循环 两个区室之间的蛋白质。 我们已经确定了两个 TGN Kex2p 胞质尾部的定位信号 (TLS)。 土壤是 显示通过 TLS1 发挥作用，以促进从 PVC 并在 TGN 上发挥作用，抑制 TLS2 依赖性保留 TGN 中的 Kex2p。 这里提出的工作将扩展这些结果 两者都可以识别参与信号依赖性的其他分子 TGN 膜蛋白的定位并测试 TGN-PVC 的各个方面 自行车模型。 该项目的具体目标是： 1. 对酵母进行分子、遗传和生化分析 Soilp 和高等细胞 Soilp 同系物； 2. 利用基因和技术鉴定与 Soilp 相互作用的蛋白质 生化方法； 3. SOI2和SOI3基因的克隆和分析；和 4. 识别和研究参与 TGN 定位的其他基因 通过分离 Tyr713Ala 突变的多拷贝抑制子 在 Kex2p TLS1 中并鉴定 TLS2 所需的基因 功能。