Summary auto-generated
This study identifies and characterizes the patB gene in Dictyostelium discoideum, which encodes PAT2, a P-type proton-ATPase essential for growth under acidic conditions. The researchers cloned patB and determined it produces a 1058-amino-acid protein of 117.5 kDa that shares greatest sequence identity with plant and fungal plasma membrane H+-ATPases. Northern and Western blot analyses revealed patB expression is dramatically upregulated when cells shift from neutral to acidic pH (5.0), with protein levels increasing within 1 hour and peaking at 4 hours. Immunofluorescence confirmed PAT2 localizes to the plasma membrane. Gene disruption experiments showed patB-null cells grew normally at neutral pH but exhibited slow growth at pH 6.0 and complete growth failure at pH 5.0, eventually dying. Despite reduced vanadate-sensitive ATPase activity in mutant cells at acidic pH, proton fluxes remained relatively normal, suggesting PAT2 has specialized functions beyond simple proton transport. The findings indicate patB encodes an acid-inducible H+-ATPase critical for Dictyostelium survival in moderately acidic environments.
Key findings
- patB encodes PAT2, a 117.5 kDa P-type H+-ATPase with greatest sequence similarity to fungal and plant plasma membrane H+-ATPases
- patB expression is rapidly upregulated (fourfold increase) when cells shift from neutral to acidic pH, with protein levels peaking within 4 hours
- patB-null mutants grow normally at neutral pH but cannot survive at pH 5.0, demonstrating the gene is essential for acidic stress tolerance
- PAT2 localizes to the plasma membrane, and vanadate-sensitive ATPase activity increases twofold in wild-type cells but not mutants when exposed to acidic pH
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Abstract
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 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 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.