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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.

以前我们分离到一个温度敏感的分泌突变体uso1，它的蛋白质从内质网到高尔基体的运输在37ﾟC被阻断。推导的Uso1蛋白氨基酸序列表明，该蛋白含有1790个氨基酸残基和一个由1100个氨基酸组成的C-末端卷曲部分。在本研究中，我们从酿酒酵母的裂解物中纯化了Uso1蛋白，并观察了一种纤维蛋白的理化性质。最近，我们成功地在电子显微镜下直接看到了蛋白质的形状(与东京大学的T.Wakabayashi教授合作)。Uso1蛋白看起来像肌球蛋白重链，由两个头部和150 nm长的杆状区域组成，恰好与预测的卷曲部分重合。至于Uso1蛋白的功能，它将不是一个马达蛋白，因为我们在预测的头部部分找不到ATP结合序列。在抑制基因突变的研究中，Uso1蛋白被认为参与了分泌囊泡到高尔基体膜上的膜融合步骤。在我们之前的工作中，我们揭示了布雷菲尔丁A(BFA)特异性地阻断了白念珠菌内质网和高尔基体之间的蛋白质运输。随后我们发现裂殖酵母在0.004%的SD存在下对黄曲霉属敏感。在裂殖酵母的宿主-载体系统中，我们克隆了几个使宿主对黄曲霉毒素处于多拷贝状态的DNA片段。我们对一个DNA片段进行了测序，发现bfr1^+(编码1530个氨基酸)与BFA抗性有关。该基因与哺乳动物多药耐药基因mdr有一定的相似性。我们还筛选了BHK细胞-新城疫病毒系统细胞内蛋白转运的抑制剂。结果表明，Folimycin和SS33410为有效的抑制剂，其靶标分子为V型H^+-ATPase。