Structural Determinants of Ca2+ Transport in the Arabidopsis H+/Ca2+Antiporter CAX1

Abstract

Ca2؉ 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. 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.

The Protein Kinase SOS2 Activates the Arabidopsis H+/Ca2+ Antiporter CAX1 to Integrate Calcium Transport and Salt Tolerance

“…Full-length CAX1 and CAX3 and N-terminal truncated sCAX1 and sCAX3, both lacking the first 36 amino acids, were used from previous studies (13,17,25). Full-length CAX1 point mutants (CAX1-S10A, CAX1-S25A, CAX1-S25T, CAX1-S25D, CAX1-E7A/E13A, and CAX1-R26A/E27A) and sCAX1/sCAX3 chimeras (sCAX1-␣ and sCAX3-␣1) were constructed in previous studies (14,16,17). Yeast strain K667 expressing wild-type CAX1, CAX1 mutants, or sCAX1/sCAX3 chimeras, which are hypersensitive to high concentrations of Ca 2ϩ , were transformed with SOS2 (7) or SOS2 variants (18), which were cloned into the yeast expression vector p2UGpd (19).…”

“…This strain has the following additional mutations: ade2-1, can1-100, his3-11, 15 leu2-3, 112 trp1-1, and ura 3-1 (10). Full-length CAX1 and CAX3 and N-terminal truncated sCAX1 and sCAX3, both lacking the first 36 amino acids, were used from previous studies (13,17,25). Full-length CAX1 point mutants (CAX1-S10A, CAX1-S25A, CAX1-S25T, CAX1-S25D, CAX1-E7A/E13A, and CAX1-R26A/E27A) and sCAX1/sCAX3 chimeras (sCAX1-␣ and sCAX3-␣1) were constructed in previous studies (14,16,17).…”

“…Ca 2ϩ Tolerance Assay-Yeast cells expressing a full-length CAX gene (CAX1, CAX2, CAX3, CAX4, chimeric sCAX, or CAX1 mutant constructs) with the SOS2 gene or SOS2 variants were assayed on YPD media supplemented with 200 or 250 mM CaCl 2 and grown at 30°C for 3 days (14,17).…”

“…A CAX1 homolog called CAX3 is unable to suppress the K667 Ca 2ϩ sensitivity phenotype, even if its N terminus is truncated to give sCAX3 (17,25). Neither sCAX3 alone nor sCAX3 co-expressed with SOS2 was able to suppress the yeast Ca 2ϩ sensitivity (Fig.…”

“…3A). We have previously generated various chimeric constructs between sCAX1 and sCAX3, some of which retain the sCAX1-like property of being able to transport Ca 2ϩ whereas others, such as sCAX3, are inactive despite all chimeras having equivalent protein stability (17). SOS2 co-expressed with an inactive sCAX3/sCAX1 chimeric construct that contained the central region and C terminus of CAX1 (construct sCAX1-␣) was also unable to suppress the Ca 2ϩ -sensitive phenotype (Fig.…”