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

Publisher Website
Google Scholar
Abstract
Schizosaccharomyces pombe Aps1 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 Aps1 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 0.06pmol/mg of protein (approx. 4nM). PP-InsP5, [PP]x-InsPx and [PP]2-InsP4 were present at 15pmol/mg (approx. 1.1μM), 15pmol/mg (approx. 1.1μM) and 30pmol/mg (approx. 2.2μM) respectively, while the intracellular concentration of InsP6 was 0.5nmol/mg of protein (approx. 36μM). Disruption of aps1 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 aps1 resulted in no detectable change in morphology or growth rate in minimal or rich media at 30°C. Overexpression of aps1 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 aps1 also resulted in a reduced growth rate and in morphological changes, including swollen, rounded and multiseptate cells. No phenotypic changes or changes in intracellular Ap5A occurred upon overexpression of aps1E93Q, which encodes a mutated Aps1 lacking significant enzymic activity. We conclude that Aps1 degrades PP-InsP5 and [PP]x-InsPxin vivo.