Functional Analysis of the Plasma Membrane H+-ATPases of Ustilago maydis

Abstract

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.

Ustilago maydis Yeast Mutant Produces Cytosolic Melanin by Tyrosine-Tyrosinase Activity with Stain Biosorption Capability

“…These results suggest that the accumulation of melanin did not affect the cell division processes and vegetative growth. Furthermore, deletion of the pma1 gene did not affect the glucose or oxygen consumption [31]. On the other hand, we noticed that the ∆PMA1 mutant was stable after long periods of storage at −80 • C.…”

“…In contrast, in the yeast Saccharomyces cerevisiae, only one gene (PMA1) is expressed [45]. In a recent publication about the U. maydis H+ ATPase, mutation of any of these genes did not affect the proton pumping or internal pH changes [31].…”

“…In the process of generating null mutants of the two plasma membrane proton-ATPases (H+-ATPase, PMA1 or PMA2), the cells exhibited a pigmented phenotype initially. However, a new construction of these mutants resulted in null mutants without the pigmented phenotype [31]. Initially, the pigmented cells did not display any noticeable differences from the wild cells.…”

The Plasma Membrane H+ ATPase CsPMA2 Regulates Lipid Droplet Formation, Appressorial Development and Virulence in Colletotrichum siamense

“…There are two plasma membrane H + -ATPases, PMA1 and PMA2, in fungi [10][11][12]. Many studies have shown that PMA1 is localized in lipid microdomains of the plasma membrane and plays an important role in cell growth and virulence in pathogenic fungi [16,17]. However, PMA2 is generally believed to have little effect on the growth and pathogenicity of fungi due to its extremely low expression in cells [22].…”

“…The structure and function of plasma membrane H + -ATPases, especially PMA1, have been extensively studied in some fungi, such as Fusarium graminearum, Valsa mali, and Ustilago maydis [14,15]. Most studies have shown that PMA1 is localized to lipid microdomains of the plasma membrane and plays a central role in the regulation of the pH homeostasis system, and it is responsible for cell growth and virulence in pathogenic fungi [16,17], such as PMA1 in Neurospora crassa [18], PMA1 in F. graminearum [19], PMA2 in Magnaporthe grisea [20], PMA1 in Leptosphaeria maculans [21], and PMA1 in U. maydis [17]. Unlike PMA1, it is generally believed that PMA2 has little effect on the growth and pathogenicity of fungi due to its extremely low expression in cells [22].…”