Diversity of mouse proton-translocating ATPase: presence of multiple isoforms of the C, d and G subunits.

Vacuolar-type proton-translocating ATPases (V-ATPases), multimeric proton pumps, are involved in a wide variety of physiological processes. For their diverse functions, V-ATPases utilize a specific subunit isoform(s). Here, we reported the molecular cloning and characterization of three novel subunit isoforms, C2, d2 and G3, of mouse V-ATPase. These isoforms were expressed in a tissue-specific manner, in contrast to the ubiquitously expressed C1, d1 and G1 isoforms. C2 was expressed predominantly in lung and kidney, and d2 and G3 specifically in kidney. We introduced these isoforms into yeasts lacking the corresponding genes. Although the G3 and d2 did not rescue the vmaDelta phenotype, d1 and the two C isoforms functionally complemented the Deltavma6 and Deltavma5, respectively, indicating that they are bona fide subunits of V-ATPase.

A human gene, ATP6E1, encoding a testis-specific isoform of H(+)-ATPase subunit E.

We have identified a novel human gene, ATP6E, encoding an E subunit isoform of vacuolar-type proton-translocating ATPase (V-ATPase). ATP6E1 was mapped to approximately 2p16-p12 on chromosome 2, and has a simple genomic organization: a noncoding exon and a coding one for an E1 isoform separated by a 6.1 kb intron, with boundaries following the GT-AG rule. Transcription initiation sites were found at -375 and -158 bases upstream of the translation initiation codon. Northern blotting analysis demonstrated that ATP6E1 is specifically transcribed in testis as 1.1 kb and 2.2 kb mRNAs, whereas the previously reported ATP6E2 (E2) is expressed in all tissues tested. E1 exhibited 76.9% identity with ubiquitously expressed E2, and both isoforms functionally complemented null mutations of the yeast counterpart VMA4, indicating that they are bona fide subunits of the V-ATPase complex.

A proton pump ATPase with testis-specific E1-subunit isoform required for acrosome acidification.

The vacuolar-type H(+)-ATPases (V-ATPases) are a family of multimeric proton pumps involved in a wide variety of physiological processes. We have identified two novel mouse genes, Atp6e1 and Atp6e2, encoding testis-specific (E1) and ubiquitous (E2) V-ATPase subunit E isoforms, respectively. The E1 transcript appears about 3 weeks after birth, corresponding to the start of meiosis, and is expressed specifically in round spermatids in seminiferous tubules. Immunohistochemistry with isoform-specific antibodies revealed that the V-ATPase with E1 and a2 isoforms is located specifically in developing acrosomes of spermatids and acrosomes in mature sperm. In contrast, the E2 isoform was expressed in all tissues examined and present in the perinuclear compartments of spermatocytes. The E1 isoform exhibits 70% identity with the E2, and both isoforms functionally complemented a null mutation of the yeast counterpart VMA4, indicating that they are bona fide V-ATPase subunits. The chimeric enzymes showed slightly lower K(m)(ATP) than yeast V-ATPase. Consistent with the temperature-sensitive growth of Deltavma4-expressing E1 isoform, vacuolar membrane vesicles exhibited temperature-sensitive coupling between ATP hydrolysis and proton transport. These results suggest that E1 isoform is essential for energy coupling involved in acidification of acrosome.