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. 2019 Jul 5;47(16):8452–8469. doi: 10.1093/nar/gkz567

Figure 1.

Figure 1.

Opposite effects of IPP kinase and IPP pyrophosphatase mutations on pho1 expression. (A) Models for the pho1 hyper-repressed (top panel) and de-repressed (bottom panel) states of the prt–pho1 locus under phosphate-replete conditions. (B) Growth of S. pombe strains with the indicated 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 5-fold dilutions were spotted on YES agar and then incubated at the temperatures specified. (C) The indicated fission yeast strains 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) Total RNA from fission yeast cells with the indicated asp1 genotypes was analyzed by reverse transcription primer extension using a mixture of radiolabeled primers complementary to the pho84, act1, and pho1 mRNAs (Top panel) or the tgp1 mRNA (Bottom panel). The reaction products were resolved by denaturing PAGE and visualized by autoradiography. The positions and sizes (nt) of DNA markers are indicated on the right. (E) The indicated single and double mutant haploid progeny of genetic crosses were spot-tested for growth on YES agar at 30°C. The IPP pyrophosphatase-dead aps1Δ and asp1-H397A alleles were synthetically lethal. (F) The indicated strains were assayed for Pho1 acid phosphatase activity. The results show that the de-repression of Pho1 expression in aps1Δ cells (lacking Aps1 IPP pyrophosphatase) is erased by asp1Δ and asp1-D333A mutations that ablate Asp1 IPP kinase activity. (G) Structures of the predominant forms of IP7 and IP8 are shown. Asp1 kinase converts IP7 to IP8 and the Asp1 pyrophosphatase reverses this process (reviewed in (35)).