A Copper Metallochaperone for Photosynthesis and Respiration Reveals Metal-specific Targets, Interaction with an Importer, and Alternative Sites for Copper Acquisition

Stephen Tottey,Sabine A.M Rondet,Gilles P.M Borrelly,Pamela J Robinson,Peter R Rich,Nigel J Robinson

doi:10.1074/jbc.m105857200

Stephen Tottey, Sabine A.M Rondet + Show 4 more

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https://doi.org/10.1074/jbc.m105857200

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Abstract

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.

Highlights

A bacterial two-hybrid assay revealed interaction between a protein designated bacterial Atx1 and amino-terminal domains of copper-transporting ATPases CtaA and PacS but not the related zinc (ZiaA) or cobalt (CoaT) transporters from the same organism (Synechocystis PCC 6803)
In copper super-supplemented mutants, photooxidation of cytochrome c6 was greater in ⌬atx1⌬ctaA than in ⌬ctaA, showing that Atx1 contributes to efficient switching from iron in cytochrome c6 to copper in plastocyanin for photosynthetic electron transport
We recently described two such copper transporters in Synechocystis PCC 6803, CtaA and PacS, both of which are required for efficient switching to the use of copper in plastocyanin rather than heme iron in cytochrome c6 for photosynthetic electron transport [15]

Summary

Introduction

A bacterial two-hybrid assay revealed interaction between a protein designated bacterial Atx 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). Copper was displaced from Atx by p-(hydroxymercuri)phenylsulfonate, indicative of thiol ligands, and two cysteine residues were obligatory for two-hybrid interaction with PacSN This organism contains compartments (thylakoids) where the copper proteins plastocyanin and cytochrome oxidase reside. Copper-transporting CPx-type ATPases have been described in bacteria, including cyanobacteria [21, 22], S. cerevisiae, higher plants, and man; representatives of this protein family (reviewed in Ref. 23) are known that transport cadmium [24], zinc and lead [25,26,27], silver [28], and cobalt [29]. We recently described two such copper transporters in Synechocystis PCC 6803, CtaA and PacS, both of which are required for efficient switching to the use of copper in plastocyanin rather than heme iron in cytochrome c6 for photosynthetic electron transport [15].

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