Molecular and biochemical properties of two P1B2-ATPases, CsHMA3 and CsHMA4, from cucumber

Abstract

P1B-ATPases (heavy metal ATPases, HMAs) constitute a multigenic subfamily of P-ATPases involved in the transport of monovalent and divalent heavy metals in plant cells. Here, we present the organization of genes encoding the HMA family in the cucumber genome and report the function and biochemical properties of two cucumber proteins homologous to the HMA2-4-like plant HMAs. Eight genes encoding putative P1B-ATPases were identified in the cucumber genome. Among them, CsHMA3 was predominantly expressed in roots and up-regulated by Pb, Zn and Cd excess, whereas the CsHMA4 transcript was most abundant in roots and flowers of cucumber plants, and elevated under Pb and Zn excess. Expression of CsHMA3 in Saccharomyces cerevisiae enhanced yeast tolerance to Cd and Pb, whereas CsHMA4 conferred increased resistance of yeast cells to Cd and Zn. Immunostaining with specific antibodies raised against cucumber proteins revealed tonoplast localization of CsHMA3 and plasma membrane localization of CsHMA4 in cucumber root cells. Kinetic studies of CsHMA3 and CsHMA4 in yeast membranes indicated differing heavy metal cation affinities of these two proteins. Altogether, the results suggest an important role of CsHMA3 and CsHMA4 in Cd and Pb detoxification and Zn homeostasis in cucumber cells. 'In this work we identified genes encoding the family of P1B-ATPases in cucumber genome and performed a comparative functional analysis of two members of this family, designated as CsHMA3 and CsHMA4. Immunostaining with specific antibodies raised against cucumber proteins revealed tonoplast localization of CsHMA3 and plasma membrane localization of CsHMA4 in cucumber root cells. Kinetic studies of CsHMA3 and CsHMA4 in yeast membranes indicated differing heavy metal cation affinities of these two proteins. Altogether the results suggest an important role of CsHMA3 and CsHMA4 in Cd and Pb detoxification and Zn homeostasis in cucumber cells.'