Residues in Internal Repeats of the Rice Cation/H+ Exchanger Are Involved in the Transport and Selection of Cations

Takehiro Kamiya,Masayoshi Maeshima

doi:10.1074/jbc.m309726200

Abstract

In plants, the cation/H+ exchanger (CAX) translocates Ca2+ and other metal ions into vacuoles using the H+ gradient formed by H+-ATPase and H+-pyrophosphatase. Such exchangers carrying 11 transmembrane domains (TMs) have been isolated from plants, yeast, and bacteria. In this study, multiple sequence alignment of several CAXs revealed the presence of highly conserved 36-residue regions between TM3 and TM4 and between TM8 and TM9. These two repetitive motifs are designated repeats c-1 and c-2. Using site-directed mutagenesis, we generated 31 mutations in the repeats of the Oryza sativa CAX, which translocates Ca2+ and Mn2+. Mutant exchangers were expressed in a Saccharomyces cerevisiae strain that is sensitive to Ca2+ and Mn2+ because of the absence of vacuolar Ca2+-ATPase and the Ca2+/H+ exchanger. Mutant exchangers were classified into six classes according to their tolerance for Ca2+ and Mn2+. For example, the class III mutants had no tolerance for either ion, and the class IV mutants had tolerance only for Ca2+. The biochemical function of each residue was estimated. We investigated the membrane topology of the repeats using a method combining cysteine mutagenesis and sulfhydryl reagents. Our results suggest that repeat c-1 re-enters the membrane from the vacuolar luminal side and forms a solution-accessible region. Furthermore, several residues in repeats c-1 and c-2 were found to be conserved in animal Na+/Ca2+ exchangers. Finally, we suggest that these re-entrant repeats may form a vestibule or filter for cation selection.

Highlights

Regulation of the cytosolic concentrations of metal ions is essential for normal cell growth
Expression of the Rice cation/H؉ exchanger (CAX) in Yeast as a Functional Enzyme—To characterize a constitutively active form of OsCAX1a, we truncated a nucleotide sequence corresponding to the N-terminal autoinhibitory domain from the original cDNA
Immunoblotting of the crude membrane fraction prepared from the transformant with the anti-OsCAX1a antibody, which was prepared against the N-terminal region from positions 22 to 44, yielded a major band of 44 kDa and a minor band of 42 kDa (Fig. 1A)

Summary

Introduction

Regulation of the cytosolic concentrations of metal ions is essential for normal cell growth. The N-terminal regulatory region has been shown to suppress Ca2ϩ transport activity by interacting with its neighboring N-terminal sequence. This regulatory region was found in A. thaliana CAX1 and CAX3 and in mung bean (Vigna radiata) VCAX1 [10], but not in other exchangers. In CAX1, the 9-amino acid calcium domain exists in the hydrophilic loop between TM1 and TM2 This domain is thought to be involved in the selection of Ca2ϩ; the sequence has not been found in other CAXs. The C domain located in TM4 may be involved in the selection of Mn2ϩ by Arabidopsis CAX2, which is the only plant CAX known to be capable of Mn2ϩ transport [11]. The similarity of the conserved motifs of CAXs and the animal Naϩ/Ca2ϩ exchanger is discussed

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