Multiple Genes for Vacuolar-type ATPase Proteolipids inCaenorhabditis elegans: A NEW GENE, vha-3, HAS A DISTINCT CELL-SPECIFIC DISTRIBUTION

Toshihiko Oka,Ryuji Yamamoto,Masamitsu Futai

doi:10.1074/jbc.273.35.22570

Abstract

In the vacuolar-type H+-ATPase (V-ATPase), highly hydrophobic subunits known as the proteolipids are components of the integral membrane V0 sector. Previously, we described the identification of three different proteolipid genes in Caenorhabditis elegans (Oka, T., Yamamoto, R., and Futai, M. (1997) J. Biol. Chem. 272, 24387-24392): vha-1 and vha-2 encoded 16-kDa subunits, and vha-4, a 23-kDa isoform. We report here that a third 16-kDa gene, vha-3, has been identified on chromosome IV. This is the first example in which four proteolipid genes have been found in a single organism. vha-2 and vha-3 exhibited 85% nucleotide identity within the open reading frames which encoded the identical amino acid sequence. Northern blot analysis indicated that all four genes were expressed in a similar pattern during the worm life cycle; however, studies with transgenic worms indicated that the vha-3 gene was expressed differently from other proteolipid genes in a cell-specific manner. These results implied that the isoforms of the proteolipids may be related to functional differences of V-ATPases in various cell types. Another new gene, vha-11, contained seven exons and was found to be located immediately downstream of vha-3. The two genes constitute a single transcriptional unit. The VHA-11 protein had 384 amino acids and shared strong sequence similarities with the C subunit, a component of the peripheral V1 sector of the V-ATPase, from yeast, bovine, and human. Expression of the vha-11 cDNA complemented a null mutation of VMA5, the yeast C subunit gene, thus demonstrating that vha-11 was the functional C subunit of C. elegans.

Highlights

In the vacuolar-type H؉-ATPase (V-ATPase), highly hydrophobic subunits known as the proteolipids are components of the integral membrane V0 sector
One PCR product was highly similar to the vha-2 gene, and analysis of the C. elegans genome data base indicated that the PCR product matched the Y38F2 YAC genomic clone from chromosome IV
Similar results were obtained in transgenic worms carrying the reporter gene fused to the 5Ј upstream region of vha-11, supporting that vha-3 and vha-11 are transcribed as a polycistronic unit

Summary

The abbreviations used are

V-ATPase, vacuolar-type Hϩ-ATPase; bp, base pair(s); EST, expressed sequence tag; GFP, green fluorescent protein; kb, kilobase(s); PCR, polymerase chain reaction; RT-PCR, reverse transcription PCR; SL, spliced leader. V0 is the integral membrane sector making up the proton pathway and consists of the proteolipid (16 and 23 kDa) and 100- and 36-kDa subunits [13]. We reported that Caenorhabditis elegans has three genes (vha-1, vha-2, and vha-4; Ref. 18) At this time, the number of proteolipid genes in mammalian cells is unclear. We demonstrate that a third 16-kDa proteolipid gene (vha-3) is present in the C. elegans genome. The vha-3 and vha-2 genes exhibit strong similarity and encode identical polypeptides but are expressed differently in a cellspecific manner Another newly described gene, vha-11, is located just downstream of vha-3 and codes for a hydrophilic polypeptide very similar to the V-ATPase C subunits of yeast [20, 21], bovine [22], and human [23]. The results demonstrate that the vha-11 gene product is the C subunit

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION