Cellular factors that assist in membrane protein anchoring

最近的研究表明，易位分泌蛋白通过水孔移动。然而，很少有人知道蛋白质整合到磷脂双层。该项目旨在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，FtsH是一种膜结合ATP酶，其功能可能参与了膜蛋白片段的停止转运过程。我们对FtsH的生理作用进行了表征，发现它具有多种功能。它对膜蛋白复合物的未组装亚基如SecY蛋白和质子ATP酶F_0区的a亚基具有蛋白酶活性。它还具有分子伴侣样活性，因为它的突变表型可通过过量产生某些分子伴侣来抑制，并且它具有与一些变性蛋白结合而不降解它们的能力。此外，我们发现FtsH与膜蛋白形成复合物， ...更多信息 为了在体外研究终止转移整合反应，我们构建了一个模型蛋白，其中来自乳糖通透酶的跨膜片段连接到proOmpA蛋白的C-末端区域。该蛋白质部分转运到倒置的大肠杆菌膜囊泡中以及含有部分纯化的SecY-SecE-SecG复合物（与SecA ATP酶结合）的蛋白脂质体中。因此表明SecYEG转运蛋白复合物本身具有介导停止转移反应的能力。它仍然有待确定是否整合到脂质相的膜发生在这个系统中。这种不同定位蛋白片段之间的杂合蛋白可以滴定出一些与膜蛋白靶向和/或整合相关的细胞组分，并且在体内是有毒的。将荧光修饰的赖氨酸定点引入新生前体多肽中已成功用于表征哺乳动物细胞的ER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蛋白来产生易位中间体，其中易位在人工产生的二硫环处或附近被中止。我们成功地利用已建立的荧光修饰氨基酸来源，酵母Lys-tRNA，翻译这种蛋白质。少

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