ALTERED PLASMA-MEMBRANE H+-ATPASE FROM THE DIO-9-RESISTANT PMA1-2 MUTANT OF SCHIZOSACCHAROMYCES-POMBE

The pma1-2 mutation affecting the plasma membrane H+-ATPase of Schizosaccharomyces pombe has been selected for resistance to the antibiotic Dio-9. In membrane fractions purified from glucose-starved 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 pma1-2 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 the pma1-2 mutant H+-ATPase reduces the K(m) for MgATP 9 - 2 mM and shifts the optimal pH from 4.8 to 6.0 - 6.5. The pma1-2 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 E1P-E2P conformational isomerisation, but also in glucose activation of the H+-ATPase.