Disruption and overexpression of the Schizosaccharomyces pombe aps1 gene, and effects on growth rate, morphology and intracellular diadenosine 5′,5‴-P1,P5-pentaphosphate and diphosphoinositol polyphosphate concentrations

Abstract

Schizosaccharomyces pombe Apsl is an enzyme that degrades both diadenosine oligophosphates (ApnA, n = 5 or 6) and diphosphoinositol polyphosphates {diphosphoinositol pentakisphosphate (PP-InsP5) and bisdiphosphoinositol tetrakisphosphate ([PP]2-InsP4)} in vitro. The in vivo substrates of Apsl are unknown. We report here the identification of Ap5A, PP-InsP5, [PP]2-InsP4 and a novel diphosphoinositol polyphosphate ([PP]x-InsPx) in S. pombe using HPLC methods. Ap5A was present at 0.06 pmol/mg of protein (approx. 4 nM). PP-InsP5, [PP]x-InsPx and [PP]2-InsP4 were present at 15 pmol/mg (approx. 1.1 μM), 15 pmol/mg (approx. 1.1 μM) and 30 pmol/mg (approx. 2.2 μM) respectively, while the intracellular concentration of InsP6 was 0.5 nmol/mg of protein (approx. 36 μM). Disruption of apsl resulted in a 52% decrease in Ap6A hydrolase activity in vitro, no detectable change in the intracellular Ap5A concentration, and 3-fold increased intracellular concentrations of PP-InsP5 and [PP]x-InsPx. Disruption of apsl resulted in no detectable change in morphology or growth rate in minimal or rich media at 30°C. Overexpression of apsl via two different plasmids that resulted in 60% and 6-fold increases above wild-type enzymic activity in vitro caused no detectable changes in the intracellular concentrations of [PP]2-InsP4, [PP]x-InsPx or PP-InsP5, but paradoxical increases of approx. 2.5- and 55-fold respectively in the intracellular Ap5A concentration. Overexpression of apsl 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 Ap5A occurred upon overexpression of apsl E93Q, which encodes a mutated Apsl lacking significant enzymic activity. We conclude that Apsl degrades PP-InsP5 and [PP]x-InsPx in vivo.