Membrane Topology of the pl258 CadA Cd(II)/Pb(II)/Zn(II)-Translocating P-Type ATPase

Journal of bioenergetics - Tập 34 - Trang 147-156 - 2002
Kan-Jen Tsai1, Yung-Feng Lin2, Marco D. Wong3, Henry Hung-Chi Yang2, Hsueh-Liang Fu2, Barry P. Rosen3
1School of Medical Technology, Chung Shan Medical University, Taichung, Taiwan, Republic of China
2Graduate Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, Republic of China
3Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit

Tóm tắt

Plasmid pl258 carries the cadA gene that confers resistance to cadmium, lead, and zinc. CadA catalyzes ATP-dependent cadmium efflux from cells of Staphylococcus aureus. It is a member of the superfamily of P-type ATPases and belongs to the subfamily of soft metal ion pumps. In this study the membrane topology of this P-type ATPase was determined by constructing fusions with the topological reporter genes phoA or lacZ. A series of 44 C-terminal truncated CadAs were fused with one or the other reporter gene, and the activity of each chimeric protein was determined. In addition, the location of the first transmembrane segment was determined by immunoblot analysis. The results are consistent with the pl258 CadA ATPase having eight transmembrane segments. The first 109 residues is a cytosolic domain that includes the Cys(X)2Cys motif that distinguishes soft metal ion-translocating P-type ATPases from their hard metal ion-translocating homologues. Another feature of soft metal ion P-type ATPases is the CysProCys motif, which is found in the sixth transmembrane segment of CadA. The phosphorylation site and ATP binding domain conserved in all P-type ATPases are situated within the large cytoplasmic loop between the sixth and seventh transmembrane segments.

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