title: Functional and Biochemical Characterization of Cucumber Genes Encoding Two Copper ATPases CsHMA5.1 and CsHMA5.2.
abstract: Plant copper P1B-type ATPases appear to be crucial for maintaining copper homeostasis within plant cells, but until now they have been studied mostly in model plant systems. Here, we present the molecular and biochemical characterization of two cucumber copper ATPases, CsHMA5.1 and CsHMA5.2, indicating a different function for HMA5-like proteins in different plants. When expressed in yeast, CsHMA5.1 and CsHMA5.2 localize to the vacuolar membrane and are activated by monovalent copper or silver ions and cysteine, showing different affinities to Cu(+) (Km ∼1 or 0.5 μM, respectively) and similar affinity to Ag(+) (Km ∼2.5 μM). Both proteins restore the growth of yeast mutants sensitive to copper excess and silver through intracellular copper sequestration, indicating that they contribute to copper and silver detoxification. Immunoblotting with specific antibodies revealed the presence of CsHMA5.1 and CsHMA5.2 in the tonoplast of cucumber cells. Interestingly, the root-specific CsHMA5.1 was not affected by copper stress, whereas the widely expressed CsHMA5.2 was up-regulated or down-regulated in roots upon copper excess or deficiency, respectively. The copper-induced increase in tonoplast CsHMA5.2 is consistent with the increased activity of ATP-dependent copper transport into tonoplast vesicles isolated from roots of plants grown under copper excess. These data identify CsHMA5.1 and CsHMA5.2 as high affinity Cu(+) transporters and suggest that CsHMA5.2 is responsible for the increased sequestration of copper in vacuoles of cucumber root cells under copper excess.
authors.affiliation: Institute of Experimental Biology, Department of Plant Molecular Physiology, Kanonia 6/8, 50-328 Wroclaw, Poland. Electronic address: mmigocka@biol.uni.wroc.pl.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Migocka
authors.affiliation: Institute of Experimental Biology, Department of Plant Molecular Physiology, Kanonia 6/8, 50-328 Wroclaw, Poland.
authors.forename: Ewelina
authors.initials: E
authors.lastname: Posyniak
authors.affiliation: Institute of Experimental Biology, Department of Genetics and Cell Physiology, Kanonia 6/8, 50-328 Wroclaw, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Maciaszczyk-Dziubinska
authors.affiliation: Institute of Experimental Biology, Department of Plant Molecular Physiology, Kanonia 6/8, 50-328 Wroclaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Papierniak
authors.affiliation: University of Wroclaw, Kanonia 6/8, 50-328 Wroclaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Kosieradzaka
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003300:Copper / Q000378:metabolism; D018553:Cucumis sativus / Q000201:enzymology / Q000235:genetics; D007425:Intracellular Membranes / Q000201:enzymology; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000201:enzymology / Q000235:genetics
chemical_list: D026901:Membrane Transport Proteins; D010940:Plant Proteins; D003300:Copper; D000251:Adenosine Triphosphatases
keywords: P1B-ATPases; copper transport; gene expression; membrane transport; metal homeostasis; vacuolar ATPase