Physiology of mutants with reduced expression of plasma membrane H⁺‐ATPase

Abstract

Two mutations containing insertions and deletions in the promoter in the plasma membrane H+-ATPase gene (PMA1) of Saccharomyces cerevisiae have been introduced into the genome by homologous recombination, replacing the wild-type gene. The resulting strains have 15 and 23% of the wild-type ATPase content. Decreased levels of ATPase correlate with decreased rates of proton efflux and decreased uptake rates of amino acids, methylamine, hygromycin B and tetraphenylphosphonium. This supports a central role of the enzyme in yeast bioenergetics. However, the final accumulation gradient of tetraphenylphosphonium is not affected by the mutations and that of methylamine and 2-aminoisobutyric acid is only decreased in the most extreme mutant. Apparently, kinetic constraints seem to prevent the equilibration of yeast active transports with the electrochemical proton gradient. As expected from their transport defects, the ATPase-deficient mutants are more resistant to hygromycin B and more sensitive to acidification than wild-type yeast. Mutant cells are very elongated, suggesting a structural role of the ATPase in the yeast surface.

HOR7, a Multicopy Suppressor of the Ca2+-induced Growth Defect in Sphingolipid Mannosyltransferase-deficient Yeast

“…14 C]methylammonium uptake as an indicator of membrane potential (35,36). As expected, wild type cells preincubated with protonophore carbonyl cyanide p-chlorophenylhydrazone displayed a significant reduction in methylammonium uptake compared with untreated cells (Fig.…”

“…Overexpression of Hor7p, on the other hand, conferred an increased resistance to Na ϩ and sensitivity to low pH. Moreover, Hor7p-overproducing cells displayed a decreased uptake of methylammonium, an indicator of the plasma membrane potential (35,36). The latter finding suggests that Hor7p overexpression causes a depolarization of the plasma membrane.…”

“…Plasma membrane depolarization, low pH sensitivity, and an increased Na ϩ resistance have previously been reported for yeast strains bearing mutations in the plasma membrane H ϩ -ATPase, Pma1p (29,31). This activity is required for cytosolic pH homeostasis as well as for maintaining the electrochemical potential that drives transport of multiple nutrients and ions across the yeast plasma membrane (35,38,39). Pma1p function is dependent on Pmp1p and Pmp2p, two small (38-residue) and highly hydrophobic isoproteins whose removal causes a drastic reduction in the rate of plasma membrane-associated ATP hydrolysis (33).…”

Ion pumps as targets for therapeutic intervention: Old and new paradigms

“…As illustrated above, the P-type ATPases serve as effective therapeutic targets for clinically important problems. The fungal plasma membrane H + -ATPase is a proton pump that helps regulate intracellular pH (Sanders et al, 1981;Vallejo and Serrano, 1989) and maintains the transmembrane electrochemical proton gradient necessary for nutrient uptake (Prasad, 1991;Serrano, 1989). It is an essential enzyme (Serrano et al, Fink, 1986) consisting of a single large polypeptide subunit of Mr ~100 kDa that represents approximately 25% percent of the total plasma membrane protein.…”

Turnover of the K+ transport system in Saccharomyces cerevisiae

“…The results are means f SD of 4 experiments We observed that addition of cycloheximide to glucose growing yeast gave rise to an inactivation of the ATPase that occurred at a lower rate than the one observed in the case of the K' transport system. To investigate the effect that these changes in ATPase could produce in the K' transport activity we used two mutants with a reduced expression of plasma membrane ATPase [16] and found that a decrease in the activity of this enzyme of as much as 60% was accompanied by a decrease in the activity of the carrier of only 25% (Table I). Similar results have been reported by Ramos et al [12].…”