Molecular biological studies on active proton-translocators in celluar organelles

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1.V-ATPase in Acetabularia acetabulum(1)Molecular cloning of A and B subunits of V-ATpase : Two independent cDNA clones were obtained for V-A and V-B subunits, respectively, by direct ampilification of a cDNA library and RT-PCR technique : 2,169bp (D50528) and 2,084bp (D50529) as for V-A subunit, and 1,666bp (D50530) and 1,726bp (D50531) as for V-B subunit. The respective cDNA clones showed differences in 5'-, 3'-untranslated regions and their codon usages. Data suggested the presence of a small multigene family in V-ATPase of Acetabularia acetabulum as observed for higher plants.(2)Molecular cloning of proteolipid (C) subunit of V-ATPase : As the results of cDNA cloning, 6 independent clones (AACEVAPD1 to 6, AB003937 to AB003942) were isolated which putatively encoded the C subunit of V-ATPase.They were different in 5', 3'-untranslated regions and their codon usages. Deduced amino acid sequences were divied into 2 families, while their expression analyzed by Northern hybridization divided the cDNA clones into 4 categories.2.H^+-PPase in Acetabularia acetabulum(1)Molecular cloning of H^+-PPase : By 5'-and 3'-rapid amplification of cDNA ends, an almost complete gene that encodes H^+-PPase ( 3,104bp, 721 amino acid) was isolated from total RNA of the organism. The deduced amino acid sequence showed about 50% identity with higher plants H^+-PPase so far reported, while more similarity was observed between Aceabularia acetabulum H^+ PPase and R.rubrum H^+-PPase.