Cd2+ and the N-terminal metal-binding domain protect the putative membranous CPC motif of the Cd2+-ATPase of Listeria monocytogenes.
CadA, the Cd(2+)-ATPase of Listeria monocytogenes, contains four cysteine residues: two in the CTNC (Cys-Thr-Asn-Cys) sequence in the cytoplasmic metal-binding domain (MBD), and two in the CPC (Cys-Pro-Cys) sequence in the membrane domain. Taking advantage of DeltaMBD, a truncated version of CadA that lacks the MBD but which still acts as a functional Cd(2+)-ATPase [Bal, Mintz, Guillain and Catty (2001) FEBS Lett. 506, 249-252], we analysed the role of the membrane cysteine residues (studied using DeltaMBD) separately from that of the cysteine residues of the MBD, which were studied using full-length CadA. The role of the cysteines was assessed by reacting DeltaMBD and CadA with N -ethylmaleimide (NEM), an SH-specific reagent, in the presence or absence of Cd(2+). We show here that (i) in both DeltaMBD and CadA, the cysteine residues in the CPC motif are essential for phosphorylation; (ii) in both proteins, Cd(2+) protects against alkylation by NEM; and (iii) in the absence of Cd(2+), the MBD of CadA also protects against alkylation by NEM. Our results suggest that the CPC motif is present in the membrane Cd(2+) transport site(s) and that the MBD protects these site(s).
Bal
Nathalie
N
Commissariat à l'Energie Atomique, Département de Réponse et Dynamique Cellulaires, Laboratoire de Biophysique Moléculaire et Cellulaire, UMR CEA-CNRS-UJF 5090, 17 rue des Martyrs, 38054 Grenoble Cedex 09, France.
Wu
Chen Chou
CC
Catty
Patrice
P
Guillain
Florent
F
Mintz
Elisabeth
E
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D001693:Biological Transport, Active; D002104:Cadmium / Q000378:metabolism*; D002462:Cell Membrane; D003545:Cysteine / Q000737:chemistry; D005033:Ethylmaleimide / Q000737:chemistry; D008089:Listeria monocytogenes / Q000201:enzymology*; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D013439:Sulfhydryl Reagents / Q000737:chemistry
D013439:Sulfhydryl Reagents; D002104:Cadmium; D000251:Adenosine Triphosphatases; C059771:cadmium translocating ATPase; D003545:Cysteine; D005033:Ethylmaleimide