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Cellular factors that assist in membrane protein anchoring

协助膜蛋白锚定的细胞因子

基本信息

批准号：
07044197
负责人：
ITO Koreaki
金额：
$ 1.54万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07044197/
关键词：
membrane protein FtsH Sec Y protein translocation secretion Escherichia coli fluorescent label proteolysis 分子シャペロン

项目摘要

Recent studies suggest that a translocating secretory protein moves through an aqueous pore. However, little is known about protein integration into phospholipid bilayrs. This project was aimed at identifying the cellular factors that are involved in the processes of membrane protein integration. Our genetic studies suggested that the function of FtsH,a membrane-bound ATPase, might be involved in the stop transfer process of a membrane protein segment. We characterized physiological roles of FtsH,and founed that it has multiple functions. It possesses a protease activity against unassembled subunits of membrane proteins complexes, such as the SecY protein and subunit a of the F_0 sector of proton ATPase. It also possesses a chaperone-like activities, since its mutational phenotypes was suppressible by overproduction of certain chaperones and it had an ability to bind to some denatured proteins without degrading them. Furthermore, we found that FtsH forms a complex with a membrane prote … More in complex, HflKC,which seems to serve as a modulator of FtsH functions.To study the stop transfer integration reaction in vitro, we constructed a model protein in which a transmembrane segment derived from the lactose permease was attached to the C-terminal region of proOmpA protein. This protein underwent partial translocation into the inverted E.coli membrane vesicles as well as into proteoliposomes containing partially purified SecY-SecE-SecG complex (in conjunction with the SecA ATPase). It was thus suggested that the SecYEG translocator complex itself has an ability to mediate the stop transfer reaction. It remains to be established whether integration into the lipid phase of the membrane occurred in this system. This hybrid protein between the differently localizing protein segments may titrate out some cellular components related to membrane protein targeting and/or integration and is toxic in vivo.Direct measurement of hydropathic nature of the translocation environment will be essential as an assay of membrane protein integration. The site-directed introduction of a fluorescent-modified lysine into the nascent precursor polypeptide has been successful for characterization of the ER translocation system of mammalian cells. To adopt this fluorescence technique to the post-translational E.coli system, we have been using the proOmpA protein to create a translocation intermediate, in which translocation has been aborted at or near an artificially created disulfide loop. We succeeded to utilize the established source of the fluorescence-modified amino acid, the yeast Lys-tRNA,for translation of this protein. Less

最近的研究表明，一种移位的分泌蛋白通过水孔移动。然而，对蛋白质与磷脂双层的整合知之甚少。该项目的目的是确定参与膜蛋白整合过程的细胞因子。我们的遗传学研究表明，膜结合ATPase FtsH的功能可能参与了膜蛋白片段的停止转移过程。我们对FtsH的生理作用进行了表征，发现它具有多种功能。它对膜蛋白复合体的未组装亚基，如SecY蛋白和质子ATPase F0区的a亚基具有较强的蛋白酶活性。它还具有伴侣样活性，因为它的突变表型可以被某些伴侣的过度生产所抑制，并且它能够结合一些变性蛋白而不降解它们。此外，我们还发现FtsH与膜蛋白…形成了一个络合物。更复杂的是，HflKC，它似乎是FtsH功能的调节器。为了研究体外停止转移整合反应，我们构建了一个模型蛋白，其中来自乳糖渗透酶的跨膜片段连接到proOmpA蛋白的C-末端区域。该蛋白被部分转位到倒置的大肠杆菌膜小泡以及含有部分纯化的SecY-SecE-SecG复合体(与SecA ATPase结合)的蛋白脂质体中。因此，我们认为SecYEG转运子复合体本身具有介导停止转移反应的能力。在这个系统中，是否整合到膜的脂相中仍有待确定。这种位于不同定位蛋白片段之间的杂交蛋白可能滴定出与膜蛋白靶向和/或整合相关的一些细胞成分，并且在活体中是有毒的。直接测量转位环境的水路性质对于膜蛋白整合的分析是必不可少的。将荧光修饰的赖氨酸定向引入到新生前体多肽中，成功地表征了哺乳动物细胞的内质网易位系统。为了将这种荧光技术应用于翻译后大肠杆菌系统，我们一直在使用proOmpA蛋白来创建易位中间体，在该中间体中，易位已经在人工创建的二硫键环处或附近中止。我们成功地利用已建立的荧光修饰氨基酸来源酵母Lys-tRNA来翻译该蛋白质。较少