THE VACUOLAR PROTON PUMP OF DICTYOSTELIUM-DISCOIDEUM - MOLECULAR-CLONING AND ANALYSIS OF THE 100-KDA SUBUNIT

The vacuolar proton pump is a highly-conserved multimeric enzyme that catalyzes the translocation of protons across the membranes of eukaryo tic cells, Its largest subunit (95-116 kDa) occurs in tissue and organ elle-specific isoforms and thus may be involved in targeting the enyzm e or modulating its function, In amoebae of Dictyostelium discoideum, proton pumps with a 100 kDa subunit are found in membranes of the cont ractile vacuole complex, an osmoregulatory organelle, We cloned the cD NA that encodes this 100 kDa protein and found that its sequence predi cts a protein 45% identical (68% similar) to the corresponding mammali an proton pump subunit, Like the mammalian protein, the predicted Dict yostelium sequence contains six possible transmembrane domains and a s ingle consensus sequence for N-linked glycosylation, Southern blot ana lysis detected only a single gene, which was designated vatM, Using ge nomic DNA and degenerate oligonucleotides based on conserved regions o f the protein as primers, we generated products by polymerase chain re action that included highly variable regions of this protein family, T he cloned products were identical in nucleotide sequence to vatM, argu ing that Dictyostelium cells contain only a single isoform of this pro ton pump subunit. Consistent with this interpretation, the amino acid sequences of peptides derived from a protein associated with endosomal membranes (Adessu et al, (1995) J, Cell Sci, 108, 3331-3337) match th e predicted sequence of the protein encoded by vatM. Thus, a single is oform of the 100 kDa proton pump subunit appears to serve in both the contractile vacuole system and the endosomal/lysosomal system of Dicty ostelium, arguing that this subunit is not responsible for regulating the differing abundance and function of proton pumps in these two comp artments, Gene targeting experiments suggest that this subunit plays i mportant (possibly essential) roles in Dictyostelium cells.