Functional analysis of the heavy metal binding domains of the Zn/Cd-transporting ATPase, HMA2, in Arabidopsis thaliana.
The Zn/Cd-transporting ATPase, HMA2, has N- and C-terminal domains that can bind Zn ions with high affinity. Mutant derivatives were generated to determine the significance of these domains to HMA2 function in planta. Mutant derivatives, with and without a C-terminal GFP tag, were expressed from the HMA2 promoter in transgenic hma2,hma4, Zn-deficient, plants to test for functionality. A deletion mutant lacking the C-terminal 244 amino acids rescued most of the hma2,hma4 Zn-deficiency phenotypes with the exception of embryo or seed development. Root-to-shoot Cd translocation was fully rescued. The GFP-tagged derivative was partially mis-localized in the root pericycle cells in which it was expressed. Deletion derivatives lacking the C-terminal 121 and 21 amino acids rescued all phenotypes and localized normally. N-terminal domain mutants localized normally but failed to complement the hma2,hma4 phenotypes. These observations suggest that the N-terminal domain of HMA2 is essential for function in planta while the C-terminal domain, although not essential for function, may contain a signal important for the subcellular localization of the protein.
Wong
Chong Kum Edwin
CKE
Department of Genetics, The University of Melbourne, Parkville, Australia, 3010.
Jarvis
Renée S
RS
Department of Genetics, The University of Melbourne, Parkville, Australia, 3010.
Sherson
Sarah M
SM
Department of Genetics, The University of Melbourne, Parkville, Australia, 3010.
Cobbett
Christopher S
CS
Department of Genetics, The University of Melbourne, Parkville, Australia, 3010.
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001693:Biological Transport, Active; D002104:Cadmium / Q000493:pharmacokinetics; D009154:Mutation; D018517:Plant Roots / Q000378:metabolism; D018520:Plant Shoots / Q000737:chemistry; D030821:Plants, Genetically Modified; D017434:Protein Structure, Tertiary; D011868:Radioisotopes; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015032:Zinc / Q000032:analysis
D029681:Arabidopsis Proteins; D011868:Radioisotopes; D002104:Cadmium; D000251:Adenosine Triphosphatases; C494967:HMA2 protein, Arabidopsis; D015032:Zinc