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...
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