Manganese specificity determinants in the Arabidopsis metal/H+ antiporter CAX2.
In plants and fungi, vacuolar transporters help remove potentially toxic cations from the cytosol. Metal/H(+) antiporters are involved in metal sequestration into the vacuole. However, the specific transport properties and the ability to manipulate these transporters to alter substrate specificity are poorly understood. The Arabidopsis thaliana cation exchangers, CAX1 and CAX2, can both transport Ca(2+) into the vacuole. There are 11 CAX-like transporters in Arabidopsis; however, CAX2 was the only characterized CAX transporter capable of vacuolar Mn(2+) transport when expressed in yeast. To determine the domains within CAX2 that mediate Mn(2+) specificity, six CAX2 mutants were constructed that contained different regions of the CAX1 transporter. One class displayed no alterations in Mn(2+) or Ca(2+) transport, the second class showed a reduction in Ca(2+) transport and no measurable Mn(2+) transport, and the third mutant, which contained a 10-amino acid domain from CAX1 (CAX2-C), showed no reduction in Ca(2+) transport and a complete loss of Mn(2+) transport. The subdomain analysis of CAX2-C identified a 3-amino acid region that is responsible for Mn(2+) specificity of CAX2. This study provides evidence for the feasibility of altering substrate specificity in a metal/H(+) antiporter, an important family of transporters found in a variety of organisms.
Shigaki
Toshiro
T
United States Department of Agriculture/Agricultural Research Service Children's Nutrition Research Center, Baylor College of Medicine, Houston, Texas 77030, USA.
Pittman
Jon K
JK
Hirschi
Kendal D
KD
D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D027682:Cation Transport Proteins; D006863:Hydrogen-Ion Concentration; D008345:Manganese / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017920:Antiporters; D029681:Arabidopsis Proteins; D002135:Calcium-Binding Proteins; D027682:Cation Transport Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C027614:calcium-hydrogen antiporters; D008345:Manganese; D002118:Calcium