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

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