|Budget Amount *help
¥6,300,000 (Direct Cost : ¥6,300,000)
Fiscal Year 1994 : ¥2,300,000 (Direct Cost : ¥2,300,000)
Fiscal Year 1993 : ¥4,000,000 (Direct Cost : ¥4,000,000)
Since 1990, there have been a lot of drug efflux proteins found in bacteria. So, the molecular biological studies on tetracycline efflux protein (Tet) , which is now a unique protein of which molecular mechanisms are studied, becomes more important as a paradigm of such drug efflux proteins. In this study, we revealed the molecular mechanism of Tet mainly by gene engineering methods.
There is a well-known sequence motif widely conserved in secondary membrane transporters such as mammalian glucose transporters, bacterial sugar/H^+ symporters and bacterial drug exporters, which is depicted as GXXSDRXGRR.However, the precise role of this motif in the transporter function is still unknown. We first studied the role of this motif in this study by using site-directed mutagenesis technique. As a result, the 5th Asp66 and 9th Arg70 are essential for the function. As to Arg70, Lys70 mutant retained about 30% the wild type transport activity, whereas the mutants to a neutral or acidic residues lost the transport activity except for Cys70 mutant. Cys70 mutant retained the activity comparable to Lys70 mutant. The unexpectedly high activity of Cys70 mutant is due to the mercaptide formation with a divalent cation which acts as a cationic side chain.
Among 8 Cys mutants of this motif, only Cys65 and Cys70 mutants were inactivated by NEM.The binding of NEM to these mutants was stimulated by addition of tetracycline, indicating that the motif, which is located on the cytoplasmic surface, is exposed to the medium by the substrate. In contrast, NEM binding to Cys97, which is located on the periplasmic surface, was inhibited by tetracycline, indicating that the periplasmic residue is hidden by a substrate-induced conformational change. These observations confirm our model for tetracycline/H^+ antiport, in which tetracycline makes Tet protein an inside-open/outside-closed conformation.