The patB gene of Dictyostelium discoideum encodes a P-type H(+)-ATPase isoform essential for growth and development under acidic conditions.
During growth and early development of Dictyostelium discoideum, the amoebae exhibit transient pH changes in their cytosol (pHi) and external medium which correlate with the extrusion of H+ from the cell by a plasma membrane pump. Moreover, the changes in pHi have been postulated to influence early prestalk/prespore differentiation during development. To learn more about the role of H+ fluxes in Dictyostelium, we cloned and analysed cDNAs of the gene patB, which appears to encode a P-type H(+)-ATPase. The patB ORF encodes a protein (termed PAT2) of 1058 amino acids with a calculated molecular mass of 117,460 Da. When aligned with other P-type ion-transport ATPases, PAT2 showed the greatest amino acid sequence identity with plasma membrane H(+)-ATPases of plants and fungi and considerably lower identity with other monovalent cation pumps and with Ca2+ pumps. Northern and Western analyses revealed that patB is expressed at very low levels in cells growing at neutral pH, but it is up-regulated rapidly and dramatically when the cells are shifted to an acidic medium. Immunofluorescence analysis indicated that PAT2 resides on the plasma membrane. When patB was disrupted by homologous recombination, the cells grew and developed normally at neutral and slightly alkaline pHs but they were unable to grow or develop at pH 5.0, and they slowly died. In growth medium at pH 6.8, patB+ and patB cells exhibited similar levels of vanadate-sensitive ATPase activity. However, when the cells were shifted to pH 5.0, this activity rapidly increased about twofold in the control cells but not in the mutant cells. Despite the lower ATPase activity in patB cells, they showed relatively normal H+ fluxes and only a slight decrease in pHi when incubated in acidic medium. Together, these results suggest that patB encodes an acid-inducible P-type H(+)-ATPase which is indispensable for the survival of Dictyostelium cells in moderately acidic external environments.
Coukell
M Barrie
MB
Department of Biology, York University,North York, Ontario,Canada M3J 1P3.
Moniakis
John
J
Department of Biology, York University,North York, Ontario,Canada M3J 1P3.
Cameron
Anne M
AM
Department of Biology, York University,North York, Ontario,Canada M3J 1P3.
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D005656:Fungal Proteins / Q000096:biosynthesis*; D005800:Genes, Fungal; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D006180:Proton-Translocating ATPases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011995:Recombination, Genetic; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013172:Spores, Fungal; D014638:Vanadates / Q000494:pharmacology
D005656:Fungal Proteins; D007527:Isoenzymes; D014638:Vanadates; D006180:Proton-Translocating ATPases