Functional Analysis of the Plasma Membrane H

Plasma membrane H+-ATPases of fungi, yeasts, and plants act as proton pumps to generate an electrochemical gradient, which is essential for secondary transport and intracellular pH maintenance. Saccharomyces cerevisiae has two genes (PMA1 and PMA2) encoding H+-ATPases. In contrast, plants have a larger number of genes for H+-ATPases. In Ustilago maydis, a biotrophic basidiomycete that infects corn and teosinte, the presence of two H+-ATPase-encoding genes has been described, one with high identity to the fungal enzymes (pma1, UMAG_02851), and the other similar to the plant H+-ATPases (pma2, UMAG_01205). Unlike S. cerevisiae, these two genes are expressed jointly in U. maydis sporidia. In the present work, mutants lacking one of these genes (Δpma1 and Δpma2) were used to characterize the role of each one of these enzymes in U. maydis physiology and to obtain some of their kinetic parameters. To approach this goal, classical biochemical assays were performed. The absence of any of these H+-ATPases did not affect the growth or fungal basal metabolism. Membrane potential tests showed that the activity of a single H+-ATPase was enough to maintain the proton-motive force. Our results indicated that in U. maydis, both H+-ATPases work jointly in the generation of the electrochemical proton gradient, which is important for secondary transport of metabolites and regulation of intracellular pH.