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 eukaryotic cells. Its largest subunit (95-116 kDa) occurs in tissue and organelle-specific isoforms and thus may be involved in targeting the enzyme or modulating its function. In amoebae of Dictyostelium discoideum, proton pumps with a 100 kDa subunit are found in membranes of the contractile vacuole complex, an osmoregulatory organelle. We cloned the cDNA that encodes this 100 kDa protein and found that its sequence predicts a protein 45% identical (68% similar) to the corresponding mammalian proton pump subunit. Like the mammalian protein, the predicted Dictyostelium sequence contains six possible transmembrane domains and a single consensus sequence for N-linked glycosylation. Southern blot analysis detected only a single gene, which was designated vatM. Using genomic DNA and degenerate oligonucleotides based on conserved regions of the protein as primers, we generated products by polymerase chain reaction that included highly variable regions of this protein family. The cloned products were identical in nucleotide sequence to vatM, arguing that Dictyostelium cells contain only a single isoform of this proton 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 the predicted sequence of the protein encoded by vatM. Thus, a single isoform of the 100 kDa proton pump subunit appears to serve in both the contractile vacuole system and the endosomal/lysosomal system of Dictyostelium, arguing that this subunit is not responsible for regulating the differing abundance and function of proton pumps in these two compartments. Gene targeting experiments suggest that this subunit plays important (possibly essential) roles in Dictyostelium cells.
Liu
T
T
Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City 73104, USA.
Clarke
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M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004023:Dictyostelium / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry*; D005800:Genes, Fungal; D005815:Genetic Code; D016681:Genome, Fungal; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification*; D017494:Proton Pumps / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis; D012689:Sequence Homology, Nucleic Acid; D014617:Vacuoles / Q000378:metabolism*
D005656:Fungal Proteins; D010446:Peptide Fragments; D017494:Proton Pumps; D012333:RNA, Messenger