Growth control strength and active site of yeast plasma membrane ATPase studied by site‐directed mutagenesis

Abstract

Several amino acids which are conserved in cation-pumping ATPases with phosphorylated intermediate have been mutagenized in the yeast plasma membrane H +-ATPase. The mutant genes have been selectively expressed in a yeast strain where the wild-type ATPase is only expressed in galactose nicdium. A scrics of mutants with decreasing levels of activity demonstrates that the ATPase is rate-limiting for growth and that decreased ATPase activity correlates with decreased intracellular pH. Enzymatic and transport studies of mutant ATPases indicate that (a) Lys474 is the target for the inhibitor fluorescein 5'-isothiocyanate and this residue can be replaced by either arginine or histidine with partial retention of activity; (b) the sensitivity to inhibition by vanadate is affected by the mutations Thr231 --f Gly, Cys376 --f Leu, Lys379 -+ Gin and Asp634 + Asn; (c) the mutation Ser234 + Ala causes uncoupling between ATP hydrolysis and proton transport and reduces the ATP content of the cells; (d) the mutation Asp730 --f Asn, which affects a polar residuc conserved in hydrophobic stretches of H ' -ATPases, abolishes ATPase activity and proton transport but not the formation of a phosphorylated intermediate.The yeast plasma membrane H+-ATPase belongs to a family of cation pumps characterized by the formation of a phosphorylated intermediate. This family, referred to here as (E-P)ATPases, includes bacterial K +

An Arabidopsis thaliana Plasma Membrane Proton Pump Is Essential for Pollen Development

“…The role of this domain was conis the first among the AHA family members to have a clear and essential phenotype, and similar observations firmed by several studies, which showed that truncation or displacement of the C terminus of plant H ϩ -ATPases with other single AHA knockouts have not been seen (Young et al 2001). yielded a constitutively activated enzyme when expressed in yeast (Portillo and Serrano 1989;Palmgren et al On the basis of previous work describing AHA3 localization in companion cells, we predicted a role for this 1990; Portillo et al 1991;Palmgren and Christensen 1993;Regenberg et al 1995). Activation in vivo is initigene in phloem loading, and consequently that an AHA3 knockout plant would be deficient in this process.…”

“…The most likely role of AHA3 in growing microspores is the mutants mad2 and mad3 are defective during pollen mitosis I, which occurs after reabsorption of the vacuoles control of nutrient uptake. As their dependence on the surrounding sporophytic tissues (tapetum) for nourishin vacuolated microspores (Grini et al 1999); mad1 is defective during pollen mitosis II (Grini et al 1999), ment decreases, early vauolated microspores rely increasingly on the activity of the plasma membrane H ϩ -ATPase as is ttd38 (Procissi et al 2001); mad4 is defective in pollen tube elongation (Grini et al 1999); and raringfor the transport of nutrients. If this protein is nonfunctional, the microspores starve and die.…”

Molecular and biochemical characterization of the Dio‐9‐resistant pma1‐1 mutation of the H+‐ATPase from Saccharomyces cerevisiae

“…The black boxes are regions which show more than 15% mean identity in pairwise comparisons of sequence segments of skeletal Ca2+-ATPase, sheep Na'+/K+-ATPase, yeast H+-ATPase and E. coli K+-ATPase. The circled residues are those from S. cerevisiae PMAI, which after mutation lead to vanadate resistance of ATPase activity [7,22,23,301 bition patterns exhibited by vanadate, even though the molecular site of action of this inhibitor in the phosphorylation site is likely to be identical. In this study we confirm that in yeast H +-ATPase, vanadate is a typical non-competitive inhibitor with a Ki of 4.3 pM.…”

“…The plasma-membrane H +-ATPase is essential [4] and rate limiting for growth, about 30% of the wildtype activity being the threshold required for normal growth [23,311. Since the K, values for the mutant enzyme are strongly modified within the physiological range of cellular pH and ATP, VjK, seems to be a more appropriate index to measure ATPase efficiency than the specific activity.…”

Energy, control and DNA structure in the living cell

“…In yeast the plasma membrane H+-ATPase exerts a strong control on growth rate [58]. The metabolic role of this Hf-ATPase, however, is quite different, not lying on the main route of free-energy transduction but serving to control the intracellular pH.…”