Open AccessFunctional expression of mung bean Ca 2+ /H + antiporter in yeast and its intracellular localization in the hypocotyl and tobacco cells
Author(s) -
UeokaNakanishi Hanayo,
Tsuchiya Tomohiro,
Sasaki Maki,
Nakanishi Yoichi,
Cunningham Kyle W.,
Maeshima Masayoshi
Publication year - 2000
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2000.01343.x
Subject(s) - antiporter , biochemistry , golgi apparatus , cell fractionation , biology , differential centrifugation , endoplasmic reticulum , complementary dna , microbiology and biotechnology , membrane , gene
The Ca 2+ ‐transport activity and intracellular localization of the translation product of cDNA for mung bean Ca 2+ /H + antiporter ( VCAX1 ) were examined. When the cDNA was expressed in Saccharomyces cerevisiae that lacked its own genes for vacuolar Ca 2+ ‐ATPase and the antiporter, VCAX1 complemented the active Ca 2+ transporters, and the microsomal membranes from the transformant showed high activity of the Ca 2+ /H + antiporter. Treatment of the vacuolar membranes with a cross‐linking reagent resulted in a clear band of the dimer detected with antibody specific for VCAX1p. The antibody was also used for immunolocalization of the antiporter in fractions obtained by sucrose‐density‐gradient centrifugation of the microsomal fraction from mung bean. The immunostained band was detected in the vacuolar membrane fraction and the slightly heavy fractions that exhibited activity of the Golgi marker enzyme. A fusion protein of VCAX1p and green fluorescent protein was expressed in tobacco cells. The green fluorescence was clearly observed on the vacuolar membrane and, in some cases, in the small vesicles. The subcellular fractionation of transformed tobacco cells confirmed the vacuolar membrane localization of the fusion protein. These results confirm that VCAX1p functions in the vacuolar membrane as a Ca 2+ /H + antiporter and also suggest that VCAX1p may exist in the Golgi apparatus.