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Biochem. J. (2003) 369 (519–528) (Printed in Great Britain)

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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

Stephen W. INGRAM*¹ , Stephen T. SAFRANY† and Larry D. BARNES*²

*Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, U.S.A., and †School of Life Sciences, The University of Dundee, Dundee DD1 5EH, Scotland, U.K.

Schizosaccharomyces pombe Aps1 is an enzyme that degrades both diadenosine oligophosphates (Ap_nA, 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-InsP_x) in S. pombe using HPLC methods. Ap₅A was present at 0.06pmol/mg of protein (approx. 4nM). PP-InsP₅, [PP]_x-InsP_x 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-InsP_x. 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-InsP_x 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-InsP_x in vivo.

Key words: adenine nucleotide, diadenosine oligophosphate, fission yeast, inositol phosphate, nudix hydrolase.

Abbreviations used: Ap_nA, diadenosine 5´,5-P¹,Pⁿ-oligophosphate (n = 3–7); DAPI, 4´,6-diamidino-2-phenylindole; DIPP, diphosphoinositol polyphosphate phosphohydrolase; MM, minimal medium; nudix, nucleoside diphosphate X; p₄A, adenosine tetraphosphate; p₅A, adenosine pentaphosphate; PP-InsP₅, diphosphoinositol pentakisphosphate; [PP]₂-InsP₄, bisdiphosphoinositol tetrakisphosphate; [PP]_x-InsP_x, a novel diphosphoinositol polyphosphate that is probably an isomer of diphosphoinositol pentakisphosphate.

¹Present address: Tanox, Inc., Houston, TX 77025-5497, U.S.A.

²To whom correspondence should be addressed (e-mail barnesl@uthscsa.edu).

Received 8 May 2002/30 August 2002; accepted 18 October 2002

Published as BJ Immediate Publication 21 October 2002, DOI 10.1042/BJ20020733

The Biochemical Society, London © 2003