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 Km 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.
Ghislain
M
M
Unité de Biochimie Physiologique, Université Catholique de Louvain, Belgium.
De Sadeleer
M
M
Goffeau
A
A
D002462:Cell Membrane / Q000201:enzymology*; D004352:Drug Resistance, Microbial / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D005998:Glycine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry; D009154:Mutation; D009930:Organic Chemicals; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D014475:Uncoupling Agents / Q000494:pharmacology*; D014638:Vanadates / Q000494:pharmacology
D009930:Organic Chemicals; D014475:Uncoupling Agents; C100056:DIO 9; D014638:Vanadates; D006180:Proton-Translocating ATPases; D005947:Glucose; D008239:Lysine; D005998:Glycine