Studies on the intracellular protein transport in eukaryotic cells.

真核细胞内蛋白质转运的研究。

基本信息

批准号：
04403023
负责人：
YAMASAKI Makari
金额：
$ 12.99万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-04403023/
关键词：
yeast intracellular protein transport coiled-coil protein Uso1 protein brefeldin A inhibitors for intracellular protein transport folimycin membrane fusion パン酵母分裂酵母ユンカナマイシンA SS33410 コンカナマイシンA 蛋白質膜通過ピュロマイシンブレフェルデインA ブレフェ

项目摘要

Formerly we isolated a temperature-sensitive secretion mutant, uso1, in which protein transport from the ER to the Golgi apparatus is blocked at 37ﾟC.The uso1 gene was cloned and sequenced. The deduced amino acid sequence of Uso1 protein suggests that the protein contains 1790 amino acid residues and a C-terminal coiled-coil portion composed of 1100 amino acids. In this study we purified Uso1 protein from the lysate of Saccharomyces cerevisiae and observed the physico-chemical properties characteristic of a fibrous protein. Recently we succeeded to see directly the shape of the protein under the electron microscope (cooperative work with Prof.T.Wakabayashi of Univ.Tokyo). The Uso1 protein looks like myosin heavy chain composed of two heads and a long rod region of 150 nm just coincided with the predicted coiled-coil portion. As to the function of Uso1 protein it will not be a motor protein because we cannot find ATP binding sequence in the predicted head portion. Uso1 protein is suggested to participate in the membrane fusion step of secretory vesicles to the Golgi membrane from the study of suppressor mutation.In our previous work, we revealed that brefeldin A (BFA) specifically blocks intracellular protein transport between the ER and the Golgi apparatus in the yeast Candida albicans. Later we found that Schizosaccharomyces pombe became BFA-sensitive in the presence of 0.004% SDS.In the host-vector system of S.pombe, we cloned several DNA fragments which endowed the host BFA-resistance at the multi-copy state. We sequenced one DNA fragment and bfr1^+ (encoding 1530 amino acids) was found to be responsible for BFA-resistance. The gene has some similarity to the mammalian multi-drug resistance gene mdr. We also screened inhibitors of intracellular protein transport by a BHK cell-NDV virus system. Folimycin and SS33410 were selected as effective inhibitors and their targert molecule was found to be V-type H^+-ATPase.

以前，我们分离了一个对温度敏感的分泌突变体USO1，其中从ER到高尔基体的蛋白质转运在37°C处被阻断。CUSO1基因被克隆并测序。推导的USO1蛋白的氨基酸序列表明，该蛋白包含1790个氨基酸和由1100个氨基酸组成的C末端盘绕螺旋部分。在这项研究中，我们从酿酒酵母的裂解物中纯化了USO1蛋白，并观察到了纤维蛋白的物理化学特性。最近，我们成功地直接看到了电子显微镜下蛋白质的形状（与Univ.tokyo的Wakabayashi教授合作）。 USO1蛋白看起来像是由两个头部组成的肌球蛋白重链，一个150 nm的长杆区域与预测的盘绕膜部分相吻合。至于USO1蛋白的功能，它不会是运动蛋白，因为我们在预测的头部部分中找不到ATP结合序列。建议从抑制突变的研究中，USO1蛋白参与秘密蔬菜向高尔基膜的膜融合步骤。在我们先前的工作中，我们透露，Brefeldin A（BFA）特异性地阻止了ER和Golgi在Yeast Yeast sandida sandida sandida sandida bistida bindida bindida bistida albicans中的细胞内蛋白质转运。后来，我们发现在存在0.004％SDS的情况下，Schizosaccharomyces Pombe变得对BFA敏感。在S.Pombe的宿主向量系统中，我们克隆了几个DNA片段，这些DNA片段在多层拷贝状态下赋予宿主BFA抗性。我们对一个DNA片段进行了测序，并发现BFR1^+（编码1530个氨基酸）是负责BFA抗性的。该基因与哺乳动物多药抗性基因MDR具有一定的相似性。我们还通过BHK细胞-NDV病毒系统筛选了细胞内蛋白转运的抑制剂。选择叶霉素和SS33410作为有效抑制剂，发现其Targert分子为V型H^+ATPase。