| Research Abstract |
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
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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
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