During growth and early development of the amoebae exhibit transient pH changes in their cytosol (pH) and external medium which correlate with the extrusion of H from the cell by a plasma membrane pump. Moreover, the changes in pH have been postulated to influence early prestalk/prespore differentiation during development. To learn more about the role of H fluxes in we cloned and analysed cDNAs of the gene which appears to encode a P-type H-ATPase. The ORF encodes a protein (termed PAT2) of 1058 amino acids with a calculated molecular mass of 117460 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 Ca 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 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, and 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 cells, they showed relatively normal H fluxes and only a slight decrease in pH when incubated in acidic medium. Together, these results suggest that encodes an acid-inducible P-type H-ATPase which is indispensable for the survival of cells in moderately acidic external environments.


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