Molecular cloning and expression of three isoforms of the 100-kDa a subunit of the mouse vacuolar proton-translocating ATPase

Abstract

We have identified cDNAs encoding three isoforms (a1, a2, and a3) of the 100-kDa a subunit of the mouse vacuolar proton-translocating ATPase (V- ATPase). The predicted protein sequences of the three isoforms are 838, 856, and 834 amino acids, respectively, and they display approximately 50% identity between isoforms. Northern blot analysis demonstrated that all three isoforms are expressed in most tissues examined. However, the al isoform is expressed most heavily in brain and heart, a2 in liver and kidney, and a3 in liver, lung, heart, brain, spleen, and kidney. We also identified multiple alternatively spliced variants for each isoform. Reverse transcriptase- mediated polymerase chain reaction revealed that one splicing variant of the al isoform (al-I) was expressed only in brain, whereas two other variants (al-II and al-III) were expressed in tissues other than brain. These alternatively spliced forms differ in the presence or absence of 6-7 amino acid residues near the amino and carboxyl termini of the proteins encoded. The a3 isoform is also encoded by three alternatively spliced variants, two of which are predicted to encode a protein that is truncated near the border of the amino-and carboxyl-terminal domains of the a subunit and therefore lacks the integral transmembrane-spanning helices thought to participate in proton translocation. Expression of each isoform (with the exception of al-I) was detectable at all developmental stages investigated, with al-I absent only in day 7 embryos. The results obtained suggest that isoforms of the 100- kDa a subunit may contribute to tissue-specific functions of the V-ATPase.