Abstract
The CrCAX1 gene encoding a Ca2+/H+ and Na+/H+ exchanger was cloned and characterized from the unicellular green alga Chlamydomonas reinhardtii to begin to understand the mechanisms of cation homeostasis in this model organism. CrCAX1 was more closely related to fungal cation exchanger (CAX) genes than those from higher plants but has structural characteristics similar to plant Ca2+/H+ exchangers including a long N-terminal tail. When CrCAX1-GFP was expressed in Saccharomyces cerevisiae, it localized at the vacuole. CrCAX1 could suppress the Ca2+-hypersensitive phenotype of a yeast mutant and mediated proton gradient-dependent Ca2+/H+ exchange activity in vacuolar membrane vesicles. Ca2+ transport activity was increased following N-terminal truncation of CrCAX1, suggesting the existence of an N-terminal auto-regulatory mechanism. CrCAX1 could also provide tolerance to Na+ stress when expressed in yeast or Arabidopsis thaliana because of Na+/H+ exchange activity. This Na+/H+ exchange activity was not regulated by the N terminus of the CrCAX1 protein. A subtle tolerance by CrCAX1 in yeast to Co2+ stress was also observed. CrCAX1 was transcriptionally regulated in Chlamydomonas cells grown in elevated Ca2+ or Na+. This study has thus uncovered a novel eukaryotic proton-coupled transporter, CrCAX1, that can transport both monovalent and divalent cations and that appears to play a role in cellular cation homeostasis by the transport of Ca2+ and Na+ into the vacuole.
Highlights
Example, tightly controlled levels of Ca2ϩ play a critical role in cell signaling, but high concentrations of Ca2ϩ are very toxic to the cell [1]
Identification of a cation/Hϩ exchanger (CAX) Gene from C. reinhardtii—Analysis of sequence from Chlamydomonas expressed sequence tag cDNA clones and the completed genome sequence has identified the presence of open reading frames homologous to Arabidopsis and S. cerevisiae CAX genes
PCR primers were designed to amplify the predicted CrCAX1 cDNA from C. reinhardtii 137Cϩ RNA isolated from cells grown in liquid TAP medium
Summary
DNA Manipulations—CrCAX1 and N-terminally truncated CrCAX1 (sCrCAX1) cDNA was amplified by PCR from cDNA template derived from isolated Chlamydomonas RNA using primers CrCAX1F, CrCAX1R, and sCrCAX1F (see supplemental Table S1 for primer sequences). For comparison with AtCAX1, an AtCAX1 cDNA that encodes the first 67 amino acids was amplified using primers CAX1TnTF and CAX1aa67R This PCR product was fused to a previously amplified rsGFP sequence containing a 5Ј BglII site and 3Ј SacI site [23]. To generate C-terminal green fluorescent protein (GFP) fusions to CrCAX1 and sCrCAX1, a HindIII site was introduced into the 3Ј end of the cDNA using the primer CrCAX1HR in combination with CrCAX1F or sCrCAX1F. Transformed yeast strains were grown in synthetic defined medium minus appropriate amino acids for selective growth for the expression plasmid and the mutations. To determine CrCAX1 expression in transgenic Arabidopsis lines or yeast cells, RT-PCR was performed using sCrCAX1F and CrCAX1TnTR primers and actin or tubulin primers as a constitutive control. The fluorescent yeast vacuole marker stain carboxy-5- and 6-carboxy-2Ј,7Ј-dichlorofluorescein diacetate [30] was used to visualize yeast vacuoles and was detected using a L4 fluorescein isothiocyanate filter cube (Leica Microsystems)
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