Membrane topology of the p1258 CadA Cd(II)/Pb(II)/Zn(II)-translocating P-type ATPase.
Plasmid p1258 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 p1258 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.
Tsai
Kan-Jen
KJ
School of Medical Technology, Chung Shan Medical University, Taichung, Taiwan, Republic of China. kjt@csmu.edu.tw
Lin
Yung-Feng
YF
Wong
Marco D
MD
Yang
Henry Hung-Chi
HH
Fu
Hsueh-Liang
HL
Rosen
Barry P
BP
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D003600:Cytosol; D017930:Genes, Reporter; D008565:Membrane Proteins / Q000737:chemistry*; D008566:Membranes; D008958:Models, Molecular; D010957:Plasmids / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013211:Staphylococcus aureus / Q000201:enzymology
D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D000251:Adenosine Triphosphatases; C059771:cadmium translocating ATPase