Poster: Membrane Transport

Abs # 1206: Mutational analysis of the highly conserved regions of rice cation/H⁺ exchanger

Presenter:	Kamiya, Takehiro , i012016m@mbox.nagoya-u.ac.jp
Authors	Kamiya, Takehiro  (A)   Maeshima, Masayoshi  (A)
Affiliations:	(A): Graduate School of Bioagricultural Sciences, Nagoya university

The cation/H⁺ exchanger (CAX) transports Ca²⁺ and heavy metals such as Cd²⁺ and Mn²⁺ into vacuoles by using the H⁺ gradient formed by H⁺-PPase and H⁺-ATPase. It has 11 transmembrane domains, and has been isolated from plants, yeast and bacteria. Amino acid sequence data of the CAXs have suggested the presence of highly conserved regions between domains 3 and 4 and between domains 8 and 9. We generated 31 mutations in the CAX from Oryza sativa (OsCAX1a) by site-directed mutagenesis and analyzed the conserved amino acids and hydrophilic residues in these regions. We introduced the mutant proteins into Saccharomyces cerevisiae (yeast) strain K665, which is sensitive to Ca²⁺ and Mn²⁺ because it lacks vacuolar Ca²⁺-ATPase and Ca²⁺/H⁺ exchanger. The 31 OsCAX1a mutant proteins could be classified into 6 types from the tolerance of the yeast expressing each mutant protein: that is, the transgenic yeast had either the same tolerance to both Mn²⁺ and Ca²⁺ as the wild type (Class 1), reduced tolerance to both ions (Class 2), no tolerance to either ion (Class 3), the same tolerance to Ca²⁺ as the wild type but no tolerance to Mn²⁺ (Class 4), more tolerance to Mn²⁺ than the wild type, but the same tolerance to Ca²⁺ as the wild type (Class 5) or the same tolerance to Mn²⁺ as the wild type, but reduced tolerance to Ca²⁺ (Class 6). Twenty-three of the 31 transgenic yeasts had altered ion transport and selectivity. These results suggested that the regions in which these mutations were generated are the regions responsible for ion translocation.