EXPRESSION OF 2 RELATED VACUOLAR H-ATPASE 16-KILODALTON PROTEOLIPID GENES IS DIFFERENTIALLY REGULATED IN A TISSUE-SPECIFIC MANNER()

The 16-kD proteolipid subunit is the principal integral membrane prote in of the vacuolar H+-ATPase (V-ATPase) complex that forms the proton channel responsible for translocating protons across lipid bilayers. T wo degenerate synthetic oligonucleotides, COT11 and COT12, correspondi ng to highly conserved transmembrane domains in all 16-kD subunits seq uenced so far, were used to amplify a partial cDNA of the V-ATPase pro teolipid subunit from cotton (Gossypium hirsutum L.) by polymerase cha in reaction (PCR). These PCR products were used to isolate two full-le ngth cDNAs from a -3 d postanthesis cotton ovule library. Both clones, CVA16.2 and CVA16.4, consisting of 816 and 895 bp, respectively, enco de the 16-kD proteolipid subunit of the V-ATPase. At the nucleotide le vel, the complete sequences of the two clones show 73.5% identity, but share about 95% identity within the coding region, although the two p olypeptides differ by only one amino acid. Comparison of deduced amino acid sequences of the proteolipid subunits revealed that the four tra nsmembrane domains and the two cytosolic extramembrane domains are hig hly conserved in all eukaryotes. Southern blot analysis of cotton geno mic DNA showed that these clones belong to small gene families in rela ted diploid and allotetraploid species. Northern blot analysis suggest ed that the three major V-ATPase subunits (69, 60, and 16 kD) are coor dinately regulated, in part, at the transcriptional level. RNA analysi s and reverse-transcription PCR established that 16-kD proteolipid tra nscripts differentially accumulate in different tissues and increase d ramatically in tissues undergoing rapid expansion, particularly in ant hers, ovules, and petals. The CVA16.4 proteolipid transcript is the mo st prevalent of the two proteolipid messages in expanding ovules harve sted 10 d postanthesis. In contrast, the two proteolipid mRNAs accumul ate to similar levels in developing petals.