Structural determinants of Ca2+ transport in the Arabidopsis H+/Ca2+ antiporter CAX1.
Ca(2+) levels in plants, fungi, and bacteria are controlled in part by H(+)/Ca(2+) exchangers; however, the relationship between primary sequence and biological activity of these transporters has not been reported. The Arabidopsis H(+)/cation exchangers, CAX1 and CAX2, were identified by their ability to suppress yeast mutants defective in vacuolar Ca(2+) transport. CAX1 has a much higher capacity for Ca(2+) transport than CAX2. An Arabidopsis thaliana homolog of CAX1, CAX3, is 77% identical (93% similar) and, when expressed in yeast, localized to the vacuole but did not suppress yeast mutants defective in vacuolar Ca(2+) transport. Chimeric constructs and site-directed mutagenesis showed that CAX3 could suppress yeast vacuolar Ca(2+) transport mutants if a nine-amino acid region of CAX1 was inserted into CAX3 (CAX3-9). Biochemical analysis in yeast showed CAX3-9 had 36% of the H(+)/Ca(2+) exchange activity as compared with CAX1; however, CAX3-9 and CAX1 appear to differ in their transport of other ions. Exchanging the nine-amino acid region of CAX1 into CAX2 doubled yeast vacuolar Ca(2+) transport but did not appear to alter the transport of other ions. This nine-amino acid region is highly variable among the plant CAX-like transporters. These findings suggest that this region is involved in CAX-mediated Ca(2+) specificity.
Shigaki
T
T
Baylor College of Medicine, Plant Physiology Group, United States Department of Agriculture/ARS Children's Nutrition Research Center, Houston, Texas 77030, USA.
Cheng
N H
NH
Pittman
J K
JK
Hirschi
K
K
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D017920:Antiporters / Q000737:chemistry* / Q000378:metabolism*; D017360:Arabidopsis / Q000737:chemistry*; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D027682:Cation Transport Proteins; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013997:Time Factors; D014617:Vacuoles / Q000378:metabolism
D000596:Amino Acids; D017920:Antiporters; D002135:Calcium-Binding Proteins; D027682:Cation Transport Proteins; D002412:Cations; D011993:Recombinant Fusion Proteins; C027614:calcium-hydrogen antiporters; D006859:Hydrogen; D002118:Calcium