Genetic suppressor screen identifies Tgp1 (glycerophosphocholine transporter), Kcs1 (IP 6 kinase), and Plc1 (phospholipase C) as determinants of inositol pyrophosphate toxicosis in fission yeast

Abstract

ABSTRACT The inositol pyrophosphate signaling molecule 1,5-IP 8 is an agonist of RNA 3 ' -processing and transcription termination in fission yeast that regulates the expression of phosphate acquisition genes pho1 , pho84 , and tgp1 . IP 8 is synthesized from 5-IP 7 by the Asp1 N-terminal kinase domain and catabolized by the Asp1 C-terminal pyrophosphatase domain. asp1-STF mutations that delete or inactivate the Asp1 pyrophosphatase domain elicit growth defects in yeast extract with supplements (YES) medium ranging from severe sickness to lethality. We now find that the toxicity of asp1-STF mutants is caused by a titratable constituent of yeast extract. Via a genetic screen for spontaneous suppressors, we identified a null mutation of glycerophosphodiester transporter tgp1 that abolishes asp1-STF toxicity in YES medium. This result, and the fact that tgp1 mRNA expression is increased by >40-fold in asp1-STF cells, prompted discovery that: (i) glycerophosphocholine (GPC) recapitulates the toxicity of yeast extract to asp1-STF cells in a Tgp1-dependent manner, and (ii) induced overexpression of tgp1 in asp1 + cells also elicits toxicity dependent on GPC. asp1-STF suppressor screens yielded a suite of single missense mutations in the essential IP 6 kinase Kcs1 that generates 5-IP 7 , the immediate precursor to IP 8 . Transcription profiling of the kcs1 mutants in an asp1 + background revealed the downregulation of the same phosphate acquisition genes that were upregulated in asp1-STF cells. The suppressor screen also returned single missense mutations in Plc1, the fission yeast phospholipase C enzyme that generates IP 3 , an upstream precursor for the synthesis of inositol pyrophosphates. IMPORTANCE The inositol pyrophosphate metabolite 1,5-IP 8 governs repression of fission yeast phosphate homeostasis genes pho1 , pho84 , and tgp1 by lncRNA-mediated transcriptional interference. Asp1 pyrophosphatase mutations that increase IP 8 levels elicit precocious lncRNA termination, leading to derepression of the PHO genes. Deletions of the Asp1 pyrophosphatase domain result in growth impairment or lethality via IP 8 agonism of transcription termination. It was assumed that IP 8 toxicity ensues from dysregulation of essential genes. In this study, a suppressor screen revealed that IP 8 toxicosis of Asp1 pyrophosphatase mutants is caused by: (i) a >40-fold increase in the expression of the inessential tgp1 gene encoding a glycerophosphodiester transporter and (ii) the presence of glycerophosphocholine in the growth medium. The suppressor screen yielded missense mutations in two upstream enzymes of inositol polyphosphate metabolism: the phospholipase C enzyme Plc1 that generates IP 3 and the essential Kcs1 kinase that converts IP 6 to 5-IP 7 , the immediate precursor of IP 8 .