Disruption and overexpression of the Schizosaccharomyces pombe aps1 gene, and effects on growth rate, morphology and intracellular diadenosine 5 ',5 '''-P-1,P-5-pentaphosphate and diphosphoinositol polyphosphate concentrations

摘要

Schizosaccharomyces pombe Aps1 is an enzyme that degrades both diadenosine oligophosphates (Ap(n)A, n = 5 or 6) and diphosphoinositol polyphosphates {diphosphoinositol pentakisphosphate (PP-InsP(5)) and bisdiphosphoinositol tetrakisphosphate ([PP](2)-InsP(4))} in vitro. The in vivo substrates of Aps1 are unknown. We report here the identification of Ap(5)A, PP-InsP(5), [PP](2)-InsP(4) and a novel diphosphoinositol polyphosphate ([PP],-InsP.) in S. pombe using HPLC methods. Ap(5)A was present at 0.06 pmol/mg of protein (approx. 4 nM). PP-InsP(5), [PP](x)-InsP(x) and [PP](2)-InsP(4) were present at 15 pmol/mg (approx. 1.1 muM), 15 pmol/mg (approx. 1.1 muM) and 30 pmol/ mg (approx. 2.2 muM) respectively, while the intracellular concentration of InsP(6) was 0.5 nmol/mg of protein (approx. 36 muM). Disruption of aps1 resulted in a 52 % decrease in Ap(6)A hydrolase activity in vitro, no detectable change in the intracellular Ap(5)A concentration, and 3-fold increased intracellular concentrations of PP-InsP. and [PP],-InsP., Disruption of aps1 resulted in no detectable change in morphology or growth rate in minimal or rich media at 30 degreesC. Overexpression of aps1 via two different plasmids that resulted in 60 % and 6-fold increases above wildtype enzymic activity in vitro caused no detectable changes in the intracellular concentrations of [PP](2)-InsP(4), [PP](x)-InsP(x) or PP-InsP(5), but paradoxical increases of approx. 2.5- and 55-fold respectively in the intracellular Ap(5)A concentration. Overexpression of aps1 also resulted in a reduced growth rate and in morphological changes, including swollen, rounded and multi-septate cells. No phenotypic changes or changes in intracellular Ap(5)A occurred upon overexpression of aps1E93Q, which encodes a mutated Aps1 lacking significant enzymic activity. We conclude that Aps1 degrades PP-InsP(5) and [PP](x)-InsP(x) in vivo.