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Chimeras of P1-type ATPases and their transcriptional regulators: Contributions of a cytosolic amino-terminal domain to metal specificity

Lookup NU author(s): Dr Gilles Borrelly, Dr Stephen Tottey, Professor Nigel Robinson

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Abstract

Zn2+-responsive repressor ZiaR and Co2+-responsive activator CoaR modulate production of P1-type Zn2+-(ZiaA) and Co2+- (CoaT) ATPases respectively. What dictates metal selectivity? We show that ΔziaΔcoa double mutants had similar Zn2+ resistance to Δzia single mutants and similar Co 2+ resistance to Δcoa single mutants. Controlling either ziaA or coaT with opposing regulators restored no resistance to metals sensed by the regulators, but coincident replacement of the deduced cytosolic amino-terminal domain CoaTN with ZiaAN (in ziaR-pziaA- ziaANcoaT) conferred Zn2+ resistance to ΔziaΔcoa, Zn2+ content was lowered and residual Co 2+ resistance lost. Metal-dependent molar absorptivity under anaerobic conditions revealed that purified ZiaAN binds Co 2+ in a pseudotetrahedral two-thiol site, and Co2+ was displaced by Zn2+. Thus, the amino-terminal domain of ZiaA inverts the metals exported by zinc-regulated CoaT from Co2+ to Zn 2+, and this correlates simplistically with metal-binding preferences; KZiaAN Zn2+ tighter than Co2+. However, Zn2+ did not bleach Cu+-ZiaAN, and only Cu+ co-migrated with ZiaAN after competitive binding versus Zn2+. Bacterial two-hybrid assays that detected interaction between the Cu+-metallochaperone Atx1 and the amino-terminal domain of Cu+-transporter PacSN detected no interaction with the analogous, deduced, ferredoxin-fold subdomain of ZiaAN. Provided that there is no freely exchangeable cytosolic Cu+, restricted contact with the Cu+-metallochaperone can impose a barrier impairing the formation of otherwise favoured Cu+-ZiaAN complexes.


Publication metadata

Author(s): Borrelly GPM, Rondet SAM, Tottey S, Robinson NJ

Publication type: Article

Publication status: Published

Journal: Molecular Microbiology

Year: 2004

Volume: 53

Issue: 1

Pages: 217-227

ISSN (print): 0950-382X

ISSN (electronic): 1365-2958

Publisher: Wiley-Blackwell

URL: http://dx.doi.org/10.1111/j.1365-2958.2004.04106.x

DOI: 10.1111/j.1365-2958.2004.04106.x

PubMed id: 15225316


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