Mechanism of N-terminal autoinhibition in the Arabidopsis Ca(2+)/H(+) antiporter CAX1.
Regulation of Ca(2+)/H(+) antiporters may be an important function in determining the duration and amplitude of cytosolic Ca(2+) oscillations. Previously the Arabidopsis Ca(2+)/H(+) transporter, CAX1 (cation exchanger 1), was identified by its ability to suppress yeast mutants defective in vacuolar Ca(2+) transport. Recently, a 36-amino acid N-terminal regulatory region on CAX1 has been identified that inhibits CAX1-mediated Ca(2+)/H(+) antiport. Here we show that a synthetic peptide designed against the CAX1 36 amino acids inhibited Ca(2+)/H(+) transport mediated by an N-terminal-truncated CAX1 but did not inhibit Ca(2+) transport by other Ca(2+)/H(+) antiporters. Ca(2+)/H(+) antiport activity measured from vacuolar-enriched membranes of Arabidopsis root was also inhibited by the CAX1 peptide. Through analyzing CAX chimeric constructs the region of interaction of the N-terminal regulatory region was mapped to include 7 amino acids (residues 56-62) within CAX1. The CAX1 N-terminal regulatory region was shown to physically interact with this 7-amino acid region by yeast two-hybrid analysis. Mutagenesis of amino acids within the N-terminal regulatory region implicated several residues as being essential for regulation. These findings describe a unique mode of antiporter autoinhibition and demonstrate the first detailed mechanisms for the regulation of a Ca(2+)/H(+) antiporter from any organism.
Pittman
Jon K
JK
Plant Physiology Group, United States Department of Agriculture/Agricultural Research Service Children's Nutrition Research Center, Baylor College of Medicine, 1100 Bates Street, Houston, Texas 77030, USA.
Shigaki
Toshiro
T
Cheng
Ning-Hui
NH
Hirschi
Kendal D
KD
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017920:Antiporters / Q000378:metabolism / Q000502:physiology*; D017360:Arabidopsis; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D027682:Cation Transport Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013329:Structure-Activity Relationship; D013912:Threonine / Q000378:metabolism
D017920:Antiporters; D002135:Calcium-Binding Proteins; D027682:Cation Transport Proteins; D011993:Recombinant Fusion Proteins; C027614:calcium-hydrogen antiporters; D013912:Threonine; D012694:Serine; D002118:Calcium