Altered plasma membrane H⁺‐ATPase from the Dio‐9‐resistant pmal‐2 mutant of Schizosaccharomyces pombe

Abstract

The pmul-2 mutation affecting the plasma membrane H + -ATPase of Schizosacchuromycespombe has been selected for resistance to the antibiotic Dio-9. In membrane fractions purified from glucosestarved cells, the mutant ATPase activity is reduced by 96%, is insensitive to inhibition by vanadate and has a pH profile displaced in the acidic pH range when compared to the wild type. The maximum velocity of the H + -ATPase activity of plasma membranes from glucose-activated pmul-2 cells is activated 20-fold. This is in striking contrast with the wild-type ATPase activity, the maximal velocity of which is not affected by glucose. However, similar to the wild-type enzyme, glucose activation of thepmal-2 mutant Hf-ATPase reduces the K , for MgATP 9 -2 mM and shifts the optimal pH from The pmal-2 mutation modifies Lys250 to a threonine, which is highly conserved in fungal and plant H+-ATPases. These results, compared to those reported for mutations of neighbour residues in yeast or mammalian P-type ATPases, suggest that Lys250 could play a significant role, not only in phosphate binding and/or in the EIP-E2P conformational isomerisation, but also in glucose activation of the HC-ATPase. [7] and Candida albicans [S]. The closely related polypeptides are folded similarly in the plasma membrane, with 8 -12 putative transmembrane segments delimiting two major cytoplasmic regions. The largest hydrophilic region contains the nucleotide-binding and phosphorylation domains [9]. A smaller hydrophilic domain, rcferred to as an energy-transduction or P-stranded [lo] domain, is believed to be essential for protein phosphatase activity [9] and/or for conformational changes at the level of the phosphoenzyme [I 1 I. The pmal-1 mutation, affecting the fission yeast H +-ATPase, was originally selected on the basis of Dio-9 resistance [12] and was later shown to result from rcplacemcnt of Gly268 by an aspartate [7]. The ATPase activity is decreased and highly resistant to vanadate [13]. The kinetic properties of the mutant enzyme are opposite to changes resulting from activation of the S. cerevisiae H ' -ATPase by glucose [14]. It has therefore been suggested that the pmal-1 mutation modifies the physiological regulation of ATPase activity by glucose [15].In this paper, we report on the properties of the novel Dio-9-rcsistant mutation pmal-2 which affects the residue Lys250Correspondence to A. Goffeau, Unite de Biochimie Physiologiquc, Place Croix du Sud 2/20, B-I 348 Louvain-la-Neuvc, BelgiumEnzyme. H+-ATPase, ATP phosphohydrolase (EC 3.6.1.35).located close to Gly268 within the small hydrophilic domain. We also compare the glucose activation of the pmal-1 and pmaf-2 mutant H+-ATPases. MATERIALS AND METHODS Yeast strains and mediaThepmal-1 strain (JV66) andpmal-2 strains (JV27, JV28, JV29 and JV37) are derivatives of the wild-type strain 97211-ade7-413. The mutants were isolated after mutagenesis with Nmethyl-N'-nitro-N-nitrosoguanidine and selection for growth resistance to 131. Yeast strains were grown in 5.8% (massivol.) glucose, 2% (ma...

Carbohydrate and energy-yielding metabolism in non-conventional yeasts: Figure 1

“…When the coding region of pma2 + is expressed from a strong promoter, it is able to complement the lethal phenotype of an S. pombe pma1 + mutant [266]. In S. pombe , as it happens in S. cerevisiae , the activity of the plasma membrane H + ‐ATPase is increased by glucose [267]. This activation is likely due to a phosphorylation and could implicate the MAP kinase encoded by the spm1 + / pmk1 + and the protein phosphatase encoded by the pzh1 + gene.…”

Pma1, a P-type Proton ATPase, Is a Determinant of Chronological Life Span in Fission Yeast

“…Analysis of the ATPase Activity of the L18 Mutant-Pma1 is an essential P-type ATPase in the plasma membrane and functions as a hydrogen ion pump (17,18). To characterize the nature of the Pma1-L18 protein, the H ϩ -ATPase activity of the L18 mutant was analyzed.…”

The Pzh1 protein phosphatase and the Spm1 protein kinase are involved in the regulation of the plasma membrane H+‐ATPase in fission yeast

“…As shown in Fig. 4 A, the ATPase in spm1 pzh1 cells is slightly less active in the absence of glucose and, in addition, is virtually unable to show the characteristic activation in response to glucose [24]. As expected for yeast cells with low levels of ATPase activity, spm1 pzh1 double mutants displayed a growth defect on acidic medium (below pH 4).…”