Molecular Recognition Involved in the Formation of Cell Organelles.

参与细胞器形成的分子识别。

基本信息

批准号：
01304058
负责人：
KATO Keitaro
金额：
$ 7.1万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Co-operative Research (A)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

1. A cytosolic factor which plays important roles in the import process of mitochondrial proteins, and the receptor of mitochondrial precursor proteins were purified with an affinity column chromatography using the presequence as a ligand. A precursor processing protease was purified to homogeneity from rat liver mitochondria and its properties were examined. 2. Full length cDNAs for three lysosomal membrane glycoproteins in rat liver were isolated and sequenced, and biosynthesis, processing and intracellular transport of these glycoproteins were studied using pulsechase experiments with primary cultured rat hepatocytes. 3. The structural requirements for the signal-anchor and stop-transfer sequences for the membrane of ER were examined using an in vitro protein translocation system. 4.70kDa peroxisomal membrane protein had the ATP-binding domain which was similar to those of a superfamily involved in membrane transport. On the treatment of isolated rat liver peroxidase with proteinase … More K, this domain (exposed to the cytosol) was cleaved off and acyl-CoA oxidase import was diminished. 5. Using two steps affinity chromatography, anti-DDDED-Sepharose and nucleoplasmin-Sepharose, a protein of 69kDa was purified from nuclear pore fraction and this protein reacted with nuclear transport signal sequences. 6. The primary structure of rat liver 5' -nucleotidase was deduced from the cDNA sequence. This enzyme was initially synthesized as a precursor, cotranslationally processed to an intermediate form containing a hydrophobic domain at the COOH terminus, and finally converted to a mature form by removal of the hydrophobic domain and by simultaneous replacement with glycosyl-phosphatidylinositol. 7. The subpopulation of endosomes was isolated by the high magnetic field separation and the density gradient centrifugation, and effect of microtubles on endocytosis of transferin was studied. 8. SARI, a yeast gene which was originally isolated as a multicopy suppressor of the sec12 temperature-sensitive mutation, encodes a 21kDa GTP-biding protein and is essential for ER-to-Golgi protein transport. Its product, Sarip, is a peripheral membrane protein mainly residing in the ER. Less

1.以前序列为配体，通过亲和柱层析纯化在线粒体蛋白输入过程中起重要作用的胞质因子和线粒体蛋白前体受体，将前体加工蛋白酶从大鼠肝线粒体中纯化至同质。 2.分离并测序了大鼠肝脏中三种溶酶体膜糖蛋白的全长cDNA，并对其进行了生物合成、加工和细胞内转运。使用原代培养的大鼠肝细胞进行脉冲追踪实验来研究糖蛋白。 3.使用具有 ATP 的体外蛋白质易位系统检查 ER 膜的信号锚定和停止转移序列的结构要求。与参与膜运输的超家族的结合域相似，在用蛋白酶 K 处理分离的大鼠肝过氧化物酶时，该域（暴露于细胞质）是5. 使用两步亲和层析，抗 DDDED-Sepharose 和核蓝蛋白-Sepharose，从核孔级分中纯化出 69kDa 的蛋白质，并且该蛋白质与核转运信号序列发生反应。大鼠肝脏 5'-核苷酸酶的一级结构是从 cDNA 序列中推导出来的，该酶最初是作为前体合成的。共翻译加工成在 COOH 末端含有疏水结构域的中间形式，最后通过去除疏水结构域并同时用糖基磷脂酰肌醇替换而转化为成熟形式。 7. 通过高磁场分离内体亚群。研究了分离和密度梯度离心，以及微管对转铁蛋白内吞作用的影响。 8. SARI，一种酵母基因，最初是作为转铁蛋白分离的。 sec12 温度敏感突变的多拷贝抑制因子，编码 21kDa GTP 结合蛋白，对于 ER 至高尔基体蛋白转运至关重要，其产物 Sarip 是一种主要存在于 ER Less 的外周膜蛋白。