SEVERAL DISTINCT GENES ENCODE NEARLY IDENTICAL 16 KDA PROTEOLIPIDS OFTHE VACUOLAR H-ATPASE FROM ARABIDOPSIS-THALIANA()

To understand the subcellular roles and the regulation of vacuolar H+- ATPases, we have begun to identify the genes encoding the major subuni ts and to determine their patterns of expression in Arabidopsis thalia na. Two distinct cDNAs (AVA-P1 and AVA-P2) and one genomic sequence (A VA-P3) encoding the 16 kDa subunit have been isolated. The 16 kDa prot eolipid is a major component of the membrane integral sector that form s the proton conductance pathway and is required for assembly of the V -ATPase complex. Interestingly, the open reading frame of one full-len gth cDNA (AVA-P1) and a genomic sequence (AVA-P3) encoded an identical polypeptide of 164 amino acids with a molecular mass of 16 570. The d educed amino acid sequences of the two cDNAs were nearly identical (99 %) and hydropathy plots suggested a molecule with four membrane-spanni ng domains characteristic of V-ATPase proteolipids. The three genes di ffered mainly in their codon usage and in their 3'-untranslated region s. The coding region of the genomic sequence, AVA-P3, was interrupted by two introns located at the codons for Cys-26 and Arg-121. The prese nce of additional 16 kDa proteolipid genes was suggested from several polymerase chain reaction (PCR)-amplified fragments that differed from one another in the size of the second intron. PCR 1 had an intron of ca. 800 bp and its identity as AVA-P4, a fourth member of the gene fam ily, was confirmed from sequence analyses of an EST cDNA. The mRNAs of three genes (AVA-P1, AVA-P2 and AVA-P3) were detected in Arabidopsis leaf, root, flower and silique; yet expression of AVA-P1 and AVA-P2 wa s lower in roots. All three genes were expressed in light- or dark-gro wn seedlings; however mRNA levels of AVA-P2 were enhanced in etiolated plants. Arabidopsis thaliana, therefore, has at least four distinct g enes encoding nearly identical 16 kDa proteolipids, and the enhanced e xpression of AVA-P2 transcript in etiolated seedlings suggests that an increase in V-ATPase could accompany cell expansion.