ORGANELLAR H-ATPASE - SITE-DIRECTED MUTAGENESIS AND SUPPRESSOR MUTANTS()

The oligomeric state of the proteolipid subunit of V-ATPase from Sacch aromyces cerevisiae was studied using hemagglutinine (HA) epitope-tag. Like with several other highly hydrophobic proteins, the proteolipid tends to aggregate in the presence of sodium dodecyl sulfate (SDS). We observed that the oligomeric state of the proteolipid predetermined i ts tendency for aggregation. Recently we discovered a novel V-ATPase s ubunit, denoted as M16 for the mammalian enzyme and Vma10p for the yea st enzyme, that is homologous to the b subunit of the membrane sector of F-ATPases. It is assumed that the structure of Vma10p resembles tha t of subunit b which is basically two anti parallel helices. We mutate d the VMA10 gene to change charges on the protein in helices and to in troduce helix braking instead of helix forming amino acids. The functi onality of the mutated VMA10 was analyzed by growing the transformed y east cells on a YPD medium buffered at PH 7.5. Two inactive site-direc ted mutants we used for obtaining second-site suppressors. Mutagenesis with EMS was utilized to get an equal chance of obtaining intra and e xtragene second-site suppressors. To our surprise the number of coloni es that grew at pH 7.5 was too large to account for mutations in V-ATP ase subunits. Apparently, mutations that are situated in genes that do not encode V-ATPase subunits could reverse the phenotype of V-ATPase null mutations resulting in growth at pH 7.5. The large number of colo nies that grew at pH 7.5 after EMS treatment suggest a big complex wit h multiple subunits as a target for mutagenesis. The observed phenomen on is very intriguing. If the responsible protein complex is identifie d, it may shed light on an important and novel cell biology subject.