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 a moebae exhibit transient pH changes in their cytosol (pH(i)) and exter nal medium which correlate with the extrusion of H+ from the cell by a plasma membrane pump. Moreover, the changes in pH(i) have been postul ated to influence early prestalk/prespore differentiation during devel opment. 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 105 8 amino acids with a calculated molecular mass of 117460 Da. When alig ned 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 catio n pumps and with Ca2+ pumps. Northern and Western analyses revealed th at pats is expressed at very low levels in cells growing at neutral pH , but it is up-regulated rapidly and dramatically when the cells are s hifted to an acidic medium. Immunofluorescence analysis indicated that PAT2 resides on the plasma membrane. When patB was disrupted by homol ogous recombination, the cells grew and developed normally at neutral and slightly alkaline pHs but they were unable to grow or develop at p H 5.0, and they slowly died. in growth medium at pH 6.8, patB(+) and p atB cells exhibited similar levels of vanadate-sensitive ATPase activi ty. However, when the cells were shifted to pH 5.0, this activity rapi dly increased about twofold in the control cells but not in the mutant cells. Despite the lower ATPase activity in patB cells, they showed r elatively normal H+ fluxes and only a slight decrease in pH, when incu bated in acidic medium. Together, these results suggest that patB enco des an acid-inducible P-type H+-ATPase which is indispensable for the survival of Dictyostelium cells in moderately acidic external environm ents.