title: A mutational study in the transmembrane domain of Ccc2p, the yeast Cu(I)-ATPase, shows different roles for each Cys-Pro-Cys cysteine.
abstract: Ccc2p is homologous to the human Menkes and Wilson copper ATPases and is herein studied as a model for human copper transport. Most studies to date have sought to understand how mutations in the human Menkes or Wilson genes impair copper homeostasis and induce disease. Here we analyze whether eight conserved amino acids of the transmembrane domain are important for copper transport. Wild-type Ccc2p and variants were expressed in a ccc2-Delta yeast strain to check whether they were able to restore copper transport by complementation. Wild-type Ccc2p and variants were also expressed in Sf9 cells using baculovirus to study their enzymatic properties on membrane preparations. The latter system allowed us to measure a copper-activated ATPase activity of about 20 nmol/mg/min for the wild-type Ccc2p at 37 degrees C. None of the variants was as efficient as the wild type in restoring copper homeostasis. The mutation of each cysteine of the (583)CPC(585) motif into a serine resulted in nonfunctional proteins that could not restore copper homeostasis in yeast and had no ATPase activity. Phosphorylation by ATP was still possible with the C583S variant, although it was not possible with the C585S variant, suggesting that the cysteines of the CPC motif have a different role in copper transport. Cys(583) would be necessary for copper dissociation and/or enzyme dephosphorylation and Cys(585) would be necessary for ATP phosphorylation, suggesting a role in copper binding.
authors.affiliation: Laboratorio de Fisico-Quimica Biologica Aida Hasson-Voloch, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, Brasil.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lowe
authors.forename: Adalberto
authors.initials: A
authors.lastname: Vieyra
authors.forename: Patrice
authors.initials: P
authors.lastname: Catty
authors.forename: Florent
authors.initials: F
authors.lastname: Guillain
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Mintz
authors.forename: Martine
authors.initials: M
authors.lastname: Cuillel
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000502:physiology; D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001692:Biological Transport; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D003300:Copper / Q000737:chemistry; D000079922:Copper Transport Proteins; D000073840:Copper-Transporting ATPases; D003545:Cysteine / Q000737:chemistry*; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005816:Genetic Complementation Test; D007313:Insecta; D008024:Ligands; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D010618:Phenanthrolines / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011804:Quinolines / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000502:physiology*; D013696:Temperature; D013997:Time Factors
chemical_list: C092974:CCC2 protein, S cerevisiae; D027682:Cation Transport Proteins; D002614:Chelating Agents; D000079922:Copper Transport Proteins; D008024:Ligands; D010618:Phenanthrolines; D011804:Quinolines; D029701:Saccharomyces cerevisiae Proteins; C028559:bathocuproine sulfonate; D003300:Copper; D000255:Adenosine Triphosphate; C047117:bicinchoninic acid; D000251:Adenosine Triphosphatases; D000073840:Copper-Transporting ATPases; D003545:Cysteine