Abstract
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.
Highlights
CAX1 and CAX3 Colocalize—Previously we have shown that CAX3 does not suppress yeast mutants defective in vacuolar
The colocalization of CAX1 and CAX3 is supported by the comigration of the two proteins in sucrose density gradients of membrane preparations obtained from yeast cells expressing the tagged proteins (Fig. 1)
Computer modeling studies, which attempt to ascribe transport function based on primary sequence information, are hindered by the lack of any biological studies focusing on the functional domains required for Hϩ/Ca2ϩ exchange
Summary
Vol 276, No 46, Issue of November 16, pp. 43152–43159, 2001 Printed in U.S.A. Structural Determinants of Ca2؉ Transport in the Arabidopsis H؉/Ca2؉ Antiporter CAX1*. The Arabidopsis H؉/cation exchangers, CAX1 and CAX2, were identified by their ability to suppress yeast mutants defective in vacuolar Ca2؉ transport. 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 Ca2؉ transport. Exchanging the nine-amino acid region of CAX1 into CAX2 doubled yeast vacuolar Ca2؉ transport but did not appear to alter the transport of other ions. In the yeast vacuolar Hϩ/Ca2ϩ antiporter, VCX1, amino acid residues within membrane-spanning domains appear to help mediate ion transport. Two Arabidopsis thaliana Hϩ/Ca2ϩ transporters, CAX1 (cation exchanger 1), and CAX2, were identified [9, 12] by their ability to sequester Ca2ϩ into yeast vacuoles in Saccharomyces cerevisiae mutants deleted for the vacuolar Ca2ϩ-ATPase (PMC1) and Hϩ/Ca2ϩ antiporter.
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