De-repression of Pho1 in erh1Δ cells requires the synthesis of inositol 1-pyrophosphates by Asp1 kinase. (A) Growth of S. pombe strains with the indicated erh1, asp1, and aps1 alleles. Cells were inoculated in YES broth and grown at 30°C. Exponentially growing cultures were adjusted to A600 of 0.1, and aliquots (3 µL) of serial fivefold dilutions were spotted on YES agar and then incubated at the temperatures specified. (B,C) S. pombe strains with the indicated erh1, asp1, and aps1 alleles were grown to A600 of 0.5 to 0.8 in liquid culture in YES medium at 30°C. Cells were then harvested, washed with water, and assayed for Pho1 acid phosphatase activity by conversion of p-nitrophenylphosphate to p-nitrophenol. Activity is expressed as the ratio of A410 (p-nitrophenol production) to A600 (input cells). Each datum in the bar graph is the average of assays using cells from at least three independent cultures ± SEM. (D) Structures of the 5-IP7, 1-IP7, and 1,5-IP8 are shown. Asp1 kinase converts 5-IP7 to IP8 (and also IP6 to 1-IP7) and the Asp1 pyrophosphatase reverses this process.
