title: Two serine residues control sequential steps during catalysis of the yeast copper ATPase through different mechanisms that involve kinase-mediated phosphorylations.
abstract: Ccc2, the yeast copper-transporting ATPase, pumps copper from the cytosol to the Golgi lumen. During its catalytic cycle, Ccc2 undergoes auto-phosphorylation on Asp(627) and uses the energy gained to transport copper across the cell membrane. We previously demonstrated that cAMP-dependent protein kinase (PKA) controls the energy interconversion (Cu)E∼P → E-P + Cu when Ser(258) is phosphorylated. We now demonstrate that Ser(258) is essential in vivo for copper homeostasis in extremely low copper and iron concentrations. The S258A mutation abrogates all PKA-mediated phosphorylations of Ccc2, whereas the S971A mutation leads to a 100% increase in its global regulatory phosphorylation. With S258A, the first-order rate constant of catalytic phosphorylation by ATP decreases from 0.057 to 0.030 s(-1), with an 8-fold decrease in the burst of initial phosphorylation. With the S971A mutant, the rate constant decreases to 0.007 s(-1). PKAi(5-24) decreases the amount of the aspartylphosphate intermediate (EP) in Ccc2 wt by 50% within 1 min, but not in S258A, S971A, or S258A/S971A. The increase of the initial burst and the extremely slow phosphorylation when the "phosphomimetic" mutant S258D was assayed (k = 0.0036 s(-1)), indicate that electrostatic and conformational (non-electrostatic) mechanisms are involved in the regulatory role of Ser(258). Accumulation of an ADP-insensitive form in S971A demonstrates that Ser(971) is required to accelerate the hydrolysis of the E-P form during turnover. We propose that Ser(258) and Ser(971) are under long-range intramolecular, reciprocal and concerted control, in a sequential process that is crucial for catalysis and copper transport in the yeast copper ATPase.
authors.affiliation: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21949-900 Rio de Janeiro, Brazil.
authors.forename: Rafael H F
authors.initials: RH
authors.lastname: Valverde
authors.forename: Thiago
authors.initials: T
authors.lastname: Britto-Borges
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lowe
authors.forename: Marcelo
authors.initials: M
authors.lastname: Einicker-Lamas
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Mintz
authors.forename: Martine
authors.initials: M
authors.lastname: Cuillel
authors.forename: Adalberto
authors.initials: A
authors.lastname: Vieyra
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000378:metabolism; D002384:Catalysis; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003300:Copper / Q000378:metabolism*; D000079922:Copper Transport Proteins; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D006706:Homeostasis / Q000502:physiology; D007313:Insecta; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000502:physiology; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
chemical_list: C092974:CCC2 protein, S cerevisiae; D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; C032197:beta-aspartyl phosphate; D001224:Aspartic Acid; D012694:Serine; D003300:Copper; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases