SIW14 IS A NOVEL 5-DIPHOSPHOINOSITOL PENTAKISPHOSPHATE (5PP-IP5) PHOSPHATASE THAT NEGATIVELY REGULATES THE STRESS RESPONSE

Abstract

Inositol pyrophosphates are high energy signaling molecules involved in cellular processes such as energetic metabolism, telomere maintenance, vesicle trafficking, and the stress response. While the inositol kinases underlying inositol pyrophosphate biosynthesis are well characterized, the phosphatases that selectively regulate their cellular pools are not fully described. The DIPP (diphosphoinositol phosphate phosphohydrolase) enzymes of the Nudix protein family have been demonstrated to dephosphorylate inositol pyrophosphates; however, the Saccharomyces cerevisiae homolog Ddp1 prefers inorganic polyphosphate over inositol pyrophosphates. Additionally, a proper functioning cellular stress response is important for cell survival. In yeast, the presence of inositol pyrophosphates is a requirement for the induction of a stress response. I identified a novel phosphatase of the recently discovered Atypical Dual-Specificity Phosphatase family as a physiologic inositol pyrophosphate phosphatase. Purified recombinant Siw14 hydrolyzes the β-phosphate from 5-diphosphoinositol pentakisphosphate (5PP-IP5) in vitro. In vivo, siw14Δ yeast mutants possess increased IP7 levels while heterologous SIW14 overexpression eliminates IP7 from cells. IP7 levels increased proportionately when siw14Δ was combined with ddp1Δ or vip1Δ, indicating independent activity by the enzymes encoded by these genes. Interestingly, we also found that the stress response is partially on in the unstressed siw14∆ mutant strain and it can still mount a stress response. I show this by using microarray analysis, reverse transcriptase-quantitative PCR (qRT-PCR), and cell growth assays under heat, osmotic, and oxidative treatments. Consistent with these data and role of Siw14 as an IP7 phosphatase, the levels of inositol pyrophosphates change when wild-type cells are under stress. I conclude that Siw14 is a physiologic phosphatase that modulates inositol pyrophosphates metabolism by dephosphorylating the IP7 isoform 5PP-IP5 to IP6 which is the reason why the deletion mutant is resistant to stresses.