Functional expression of mung bean Ca2+/H+ antiporter in yeast and its intracellular localization in the hypocotyl and tobacco cells

The Ca2+-transport activity and intracellular localization of the translati on product of cDNA for mung bean Ca2+/H+ antiporter (VCAX1) were examined. When the cDNA was expressed in Saccharomyces cerevisiae that lacked its own genes for vacuolar Ca2+-ATPase and the antiporter, VCAX1 complemented the active Ca2+ transporters, and the microsomal membranes from the transforman t showed high activity of the Ca2+/H+ antiporter. Treatment of the vacuolar membranes with a cross-linking reagent resulted in a clear band of the dim er detected with antibody specific for VCAX1p. The antibody was also used f or immunolocalization of the antiporter in fractions obtained by sucrose-de nsity-gradient centrifugation of the microsomal fraction from mung bean. Th e immunostained band was detected in the vacuolar membrane fraction and the slightly heavy fractions that exhibited activity of the Golgi marker enzym e. A fusion protein of VCAX1p and green fluorescent protein was expressed i n tobacco cells. The green fluorescence was clearly observed on the vacuola r membrane and, in some cases, in the small vesicles. The subcellular fract ionation of transformed tobacco cells confirmed the vacuolar membrane local ization of the fusion protein. These results confirm that VCAX1p functions in the vacuolar membrane as a Ca2+/H+ antiporter and also suggest that VCAX 1p may exist in the Golgi apparatus.