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Related Experiment Videos

Structure-function analyses of the ATX1 metallochaperone.

M E Portnoy1, A C Rosenzweig, T Rae

  • 1Departments of Environmental Health Sciences and Biochemistry, Johns Hopkins University School of Public Health, Baltimore, Maryland 21205, USA.

The Journal of Biological Chemistry
|May 18, 1999
PubMed
Summary

Saccharomyces cerevisiae Atx1p protein delivers copper and also prevents oxidative damage. Researchers found these two functions of Atx1p are distinct, with separate mechanisms and structural attributes.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Saccharomyces cerevisiae Atx1p is a metallochaperone protein that transports copper within cells.
  • Atx1p delivers copper to the Ccc2p transporter and can also prevent oxidative damage when overproduced.
  • The precise relationship between Atx1p's copper trafficking and antioxidant roles was unclear.

Purpose of the Study:

  • To investigate the structural and mechanistic basis of Atx1p's dual functions: copper delivery and antioxidant activity.
  • To determine if the metallochaperone activity and antioxidant properties of Atx1p are mechanistically linked.
  • To elucidate the distinct attributes of Atx1p responsible for its different cellular roles.

Main Methods:

  • Site-directed mutagenesis was used to alter specific lysine residues in Atx1p, including Lys65 near the metal-binding site.

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  • Analysis of surface electrostatic potential distribution to identify conserved lysine residues.
  • In vitro assays to assess the reaction of purified copper-bound Atx1p (Cu-Atx1p) with superoxide anion.

    • Main Results:

    • Mutagenesis of lysine residues in the metal-binding region and on the lysine-rich face affected copper delivery to Ccc2p.
    • Copper trafficking to Ccc2p was dependent on the lysine-rich face of Atx1p.
    • Surprisingly, elimination of these lysine residues did not impair Atx1p's antioxidant activity, suggesting a non-metallochaperone mechanism.
    • Purified Cu-Atx1p directly reacted with superoxide anion in vitro in a noncatalytic manner.

    Conclusions:

    • The copper-trafficking and antioxidant functions of Saccharomyces cerevisiae Atx1p are mediated by distinct chemical and structural features.
    • Atx1p's antioxidant activity does not rely on its metallochaperone function; instead, it may directly consume superoxide.
    • These findings resolve the mechanistic overlap between Atx1p's copper escort and oxidative damage prevention roles.