A copper metallochaperone for photosynthesis and respiration reveals metal-specific targets, interaction with an importer, and alternative sites for copper acquisition.
A bacterial two-hybrid assay revealed interaction between a protein now designated bacterial Atx1 and amino-terminal domains of copper-transporting ATPases CtaA (cellular import) and PacS (thylakoid import) but not the related zinc (ZiaA) or cobalt (CoaT) transporters from the same organism (Synechocystis PCC 6803). The specificity of metallochaperone interactions coincides with metal specificity. After reconstitution in a N(2) atmosphere, bacterial Atx1 bound 1 mol of copper mol(-1), and apoPacS(N) acquired copper from copper-Atx1. Copper was displaced from Atx1 by p-(hydroxymercuri)phenylsulfonate, indicative of thiol ligands, and two cysteine residues were obligatory for two-hybrid interaction with PacS(N). This organism contains compartments (thylakoids) where the copper proteins plastocyanin and cytochrome oxidase reside. In copper super-supplemented mutants, photooxidation of cytochrome c(6) was greater in Deltaatx1DeltactaA than in DeltactaA, showing that Atx1 contributes to efficient switching from iron in cytochrome c(6) to copper in plastocyanin for photosynthetic electron transport. Cytochrome oxidase activity was also less in membranes purified from low [copper]-grown Deltaatx1 or DeltapacS, compared with wild-type, but the double mutant Deltaatx1DeltapacS was non-additive, consistent with Atx1 acting via PacS. Conversely, activity in Deltaatx1DeltactaA was less than in either respective single mutant, revealing that Atx1 can function without the major copper importer and consistent with a role in recycling endogenous copper.
Tottey
Stephen
S
Department of Biochemistry and Genetics, Medical School, University of Newcastle, Newcastle upon Tyne NE2 4HH, United Kingdom.
Rondet
Sabine A M
SA
Borrelly
Gilles P M
GP
Robinson
Pamela J
PJ
Rich
Peter R
PR
Robinson
Nigel J
NJ
D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D015139:Blotting, Southern; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003300:Copper / Q000737:chemistry*; D000458:Cyanobacteria / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics; D003580:Cytochromes / Q000378:metabolism; D045348:Cytochromes f; D004247:DNA / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D010641:Phenotype; D010788:Photosynthesis; D010970:Plastocyanin / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D020524:Thylakoids / Q000737:chemistry*; D013997:Time Factors; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; C093341:ATX1 protein, S cerevisiae; D001426:Bacterial Proteins; D002352:Carrier Proteins; D027682:Cation Transport Proteins; C090098:CtaA protein, bacteria; D003573:Cytochrome b Group; D003580:Cytochromes; D005656:Fungal Proteins; D008024:Ligands; D008565:Membrane Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D003300:Copper; D004247:DNA; D010970:Plastocyanin; D045348:Cytochromes f; D003576:Electron Transport Complex IV; D001616:beta-Galactosidase; D000251:Adenosine Triphosphatases; D003545:Cysteine; D010100:Oxygen