Molecular cloning and chromosomal assignment of the porcine 54 and 56 kDa vacuolar H(+)-ATPase subunit gene (V-ATPase)

Vacuolar proton-translocating ATPases (V-ATPase) are multisubunit enzyme co mplexes located in the membranes of eukaryotic cells regulating cytoplasmic pH. So far, nothing is known about the genomic organization and chromosoma l location of the various subunit genes in higher eukaryotes. Here we descr ibe the isolation and analysis of a cDNA coding for the 54- and 56-kDa porc ine V-ATPase subunit alpha and beta isoforms. We have determined the genomi c structure of the V-ATPase subunit gene spanning at least 62 kb on Chromos ome (Chr) 4q14-q16. It consists of 14 exons with sizes ranging from 54 bp t o 346 bp, with a non-coding first exon and an alternatively spliced seventh exon leading to two isoforms. The 5' end of the V-ATPase cDNA was isolated by RACE-PCR. The V-ATPase alpha isoform mRNA, lacking the seventh exon, ha s an open reading frame of 1395 nucleotides encoding a hydrophilic protein of 465 amino acids with a calculated molecular mass of 54.2 kDa and a pi of 7.8, whereas the beta isoform has a length of 1449 nucleotides encoding a protein of 483 amino acids with a calculated molecular mass of 55.8 kDa. Am ino acid and DNA sequence comparison revealed that the porcine V-ATPase sub unit exhibits a significant homology to the VMA13 subunit of Saccharomyces cerevisiae V-ATPase complex and V-ATPase subunit of Caenorhabditis elegans.