Manganese Specificity Determinants in the ArabidopsisMetal/H+ Antiporter CAX2

Abstract

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.

Golgi Manganese Transport Is Required for Rapamycin Signaling in Saccharomyces cerevisiae

“…This result suggests that lowering cytosolic Ca 21 or Mn 21 by transport to the vacuolar space does not restore rapamycin sensitivity of pmr1D. The Arabadopsis thaliana CAX1 and CAX2 genes encode Ca 21 /H 1 exchangers that localize to the yeast vacuole (Shigaki et al 2003). Cax2 transports Mn 21 better than Cax1, and expression of Cax1 and Cax2 decreases the Mn 21 toxicity phenotype of pmr1D (Shigaki et al 2003).…”

“…The Arabadopsis thaliana CAX1 and CAX2 genes encode Ca 21 /H 1 exchangers that localize to the yeast vacuole (Shigaki et al 2003). Cax2 transports Mn 21 better than Cax1, and expression of Cax1 and Cax2 decreases the Mn 21 toxicity phenotype of pmr1D (Shigaki et al 2003). Neither Cax1 nor Cax2 restored rapamycin sensitivity of pmr1 (supplemental data at http://www.genetics.org/supplemental/), confirming that lowering Ca 21 or Mn 21 in the cytoplasmic compartment by transport into the vacuole was not sufficient to restore rapamycin sensitivity.…”

Nutritional impact of elevated calcium transport activity in carrots

“…Future work needs to be done testing multiple lines and various growth regimes. Previously, using biochemical approaches, we have shown that sCAX1 can transport a wide range of substrates (9,30). Potentially, CAX-expressing crops could be used for enrichment of other nutrients (e.g., CAX-mediated Zn 2ϩ accumulation).…”

AtCCX3 Is an Arabidopsis Endomembrane H+-Dependent K+ Transporter

“…Yeast cells expressing all of the AtCCX3 and AtCCX4 variants were unable to suppress the Ca 2+ sensitivity of yeast strains deficient in vacuolar Ca 2+ transport (data not shown). Yeast cells expressing AtCAXs all show increased Ca 2+ transport when truncated in their N-terminal domain (Hirschi, 1999;Shigaki et al, 2001Shigaki et al, , 2003Pittman et al, 2004b). A lack of N-terminal regulation, the ability to suppress Na + -sensitive phenotypes, and the inability to suppress Ca 2+ -sensitive phenotypes in yeast suggests functions for AtCCX3 and AtCCX4 that differ from those of CAXs.…”

“…The AtsCAX2 plasmid was cloned previously (Shigaki et al, 2003). The Arabidopsis Na + /H + exchanger AtNHX1 was PCR cloned from plasmid DNA with the primers listed in Supplemental Table S1 (primers 6 and 7) and cloned into yeast expression vectors.…”