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HMA2. A Transmembrane Zn<sup>2+</sup> Transporting ATPase from Arabidopsis thaliana

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P<SUB>1B</SUB>-type ATPases transport a number of monovalent and divalent heavy metals (Cu<SUP>+</SUP>, Cu<SUP>2+</SUP>, Ag<SUP>+</SUP>, Zn<SUP>2+</SUP>, Cd<SUP>2+</SUP>, Pb<SUP>2+</SUP> and Co<SUP>+2</SUP>) across biological membranes. These ATPases are found in archea, bacteria and eukaryotes and are one of the key elements required for maintaining metal homeostasis. Plants have an unusually high number of P<SUB>1B</SUB>-type ATPases with distinct metal selectivity compared to other eukaryotes that usually have one or two Cu<SUP>+</SUP>-ATPases. Higher plants are the only eukaryotes where Zn<SUP>2+</SUP>-ATPases have been identified. Towards understanding the physiological roles of plant Zn<SUP>2+</SUP>-ATPases, we characterized <I>Arabidopsis thaliana</I> HMA2. We expressed HMA2 in yeast and measured the metal dependent ATPase activity in membranes. We showed that HMA2 is a Zn<SUP>2+</SUP>-ATPase that is also activated by Cd<SUP>2+</SUP>. Zn<SUP>2+</SUP> transport determinations showed that this enzyme drives the efflux of metal from the cytoplasm. Analysis of HMA2 mRNA levels showed that the enzyme is present in all plant organs. We analyzed the effect of removal of HMA2 full-length transcript in whole plants by gene knock out. Although <I>hma2</I> mutants did not show a different visible phenotype from the wild type plants, we observed increased levels of Zn<SUP>2+</SUP> or Cd<SUP>2+</SUP>. The observed phenotype of <I>hma2</I> mutants and plasma membrane location of HMA2, mainly in vasculature (Hussain et al., 2004), indicates that this ATPase might have a central role in Zn<SUP>2+</SUP> uploading into the phloem. P<SUB>1B</SUB>-type ATPases have cytoplasmic regulatory metal binding domains (MBDs) in addition to transmembrane metal binding sites (TMBDs). Plant Zn<SUP>2+</SUP>-ATPases have distinct sequences in both their N- and C-termini that might contribute to novel metal binding sites. These ATPases contain long C-terminal sequences rich in CC dipeptides and His repeats. Removal of the C-terminus (C-MBD) of HMA2 leads to a 50% reduction in the enzyme turnover suggesting a regulatory role for this domain. Atomic Absorption Spectroscopy (AAS) analysis showed that Zn<SUP>2+</SUP> binds to C-MBD with a stoichiometry of three (3 Zn/C-MBD). Chemical modification studies and Zn K-edge X-ray Absorption Spectroscopy (XAS) of Zn-C-MBD showed that Zn<SUP>2+</SUP> is likely coordinated by His in two sites and the third site slightly differs from the others involving a Cys together with three His. All plant Zn<SUP>2+</SUP>-ATPases lack the typical CXXC signature sequences observed in Cu<SUP>+</SUP>-ATPases and some bacterial Zn<SUP>2+</SUP>-ATPases N-terminus metal binding domains (N-MBDs). Instead, these have conserved CCXXE sequences. Truncation of HMA2 N-MBD results in a 50% decrease in enzyme Vmax suggesting that N-MBD is also a regulatory domain. The results indicate that the N-MBD binds Zn<SUP>2+</SUP> with a stoichiometry of one (1 Zn/N-MBD). Metal binding analysis of individual N-MBD mutants Cys17Ala, Cys18Ala and Glu21Ala/Cys prevented Zn<SUP>+2</SUP> binding to HMA2 N-MBD suggesting the involvement of all these residues in metal coordination. ATPase activity measurements with HMA2 carrying the mutations Cys17Ala, Cys18Ala and Glu21Ala/Cys showed a reduction in the enzyme activity similar to that observed the truncated protein indicating that the enzyme activity reduction observed in the N-terminus truncated forms of the enzyme is related to the removal of the metal binding capability. Summaryzing, these studies show the central role of HMA2 in plant Zn<SUP>2+</SUP> homeostasis. They also describe the mechanism and direction of Zn<SUP>2+</SUP> transport. Finally, they establish the presence of novel metal binding domains in the cytoplasmic portion of the enzyme. Metal binding to these domains is required for full enzymatic activity.

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贡献者
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  • English
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  • etd-010507-150007
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  • 2007
Date created
  • 2007-01-05
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  • 2021-01-27

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