Suppression of inositol pyrophosphate toxicosis and hyper-repression of the fission yeast PHO regulon by loss-of-function mutations in chromatin remodelers Snf22 and Sol1.

Inositol pyrophosphates are signaling molecules that regulate cellular phosphate homeostasis in eukaryal taxa. In fission yeast, where the phosphate regulon (comprising phosphate acquisition genes pho1, pho84, and tgp1) is repressed under phosphate-replete conditions by lncRNA-mediated transcriptional interference, mutations of inositol pyrophosphatases that increase IP8 levels derepress the PHO regulon by eliciting precocious termination of lncRNA transcription. Asp1 pyrophosphatase mutations resulting in too much IP8 are cytotoxic in YES medium owing to overexpression of glycerophosphodiester transporter Tgp1. IP8 toxicosis is ameliorated by mutations in cleavage/polyadenylation and termination factors, perturbations of the Pol2 CTD code, and mutations in SPX domain proteins that act as inositol pyrophosphate sensors. Here, we show that IP8 toxicity is alleviated by deletion of snf22+, the gene encoding the ATPase subunit of the SWI/SNF chromatin remodeling complex, by an ATPase-inactivating snf22-(D996A-E997A) allele, and by deletion of the gene encoding SWI/SNF subunit Sol1. Deletion of snf22+ hyper-repressed pho1 expression in phosphate-replete cells; suppressed the pho1 derepression elicited by mutations in Pol2 CTD, termination factor Seb1, Asp1 pyrophosphatase, and 14-3-3 protein Rad24 (that favor precocious prt lncRNA termination); and delayed pho1 induction during phosphate starvation. RNA analysis and lack of mutational synergies suggest that Snf22 is not impacting 3'-processing/termination. Using reporter assays, we find that Snf22 is important for the activity of the tgp1 and pho1 promoters, but not for the promoters that drive the synthesis of the PHO-repressive lncRNAs. Transcription profiling of snf22∆ and snf22-(D996A-E997A) cells identified an additional set of 66 protein-coding genes that were downregulated in both mutants.IMPORTANCERepression of the fission yeast PHO genes tgp1, pho1, and pho84 by lncRNA-mediated interference is sensitive to inositol pyrophosphate dynamics. Cytotoxic asp1-STF alleles derepress the PHO genes via the action of IP8 as an agonist of precocious lncRNA 3'-processing/termination. IP8 toxicosis is alleviated by mutations of the Pol2 CTD and the 3'-processing/termination machinery that dampen the impact of toxic IP8 levels on termination. In this study, a forward genetic screen revealed that IP8 toxicity is suppressed by mutations of the Snf22 and Sol1 subunits of the SWI/SNF chromatin remodeling complex. Genetic and biochemical evidence indicates that the SWI/SNF is not affecting 3'-processing/termination or lncRNA promoter activity. Rather, SWI/SNF is critical for firing the PHO mRNA promoters. Our results implicate the ATP-dependent nucleosome remodeling activity of SWI/SNF as necessary to ensure full access of PHO-activating transcription factor Pho7 to its binding sites in the PHO mRNA promoters.

Comprehensive analysis of non-selective and selective autophagy in yeast atg mutants and characterization of autophagic activity in the absence of the Atg8 conjugation system.

Most autophagy-related genes, or ATG genes, have been identified through studies using budding yeast. Although the functions of the ATG genes are well understood, the contributions of individual genes to non-selective and various types of selective autophagy remain to be fully elucidated. In this study, we quantified the activity of non-selective autophagy, the cytoplasm-to-vacuole targeting (Cvt) pathway, mitophagy, endoplasmic reticulum (ER)-phagy and pexophagy in all Saccharomyces cerevisiae atg mutants. Among the mutants of the core autophagy genes considered essential for autophagy, the atg13 mutant and mutants of the genes involved in the two ubiquitin-like conjugation systems retained residual autophagic functionality. In particular, mutants of the Atg8 ubiquitin-like conjugation system (the Atg8 system) exhibited substantial levels of non-selective autophagy, the Cvt pathway and pexophagy, although mitophagy and ER-phagy were undetectable. Atg8-system mutants also displayed intravacuolar vesicles resembling autophagic bodies, albeit at significantly reduced size and frequency. Thus, our data suggest that membranous sequestration and vacuolar delivery of autophagic cargo can occur in the absence of the Atg8 system. Alongside these findings, the comprehensive analysis conducted here provides valuable datasets for future autophagy research.

Mitophagy in Yeast: Molecular Mechanism and Regulation.

Mitophagy is a type of autophagy that selectively degrades mitochondria. Mitochondria, known as the "powerhouse of the cell", supply the majority of the energy required by cells. During energy production, mitochondria produce reactive oxygen species (ROS) as byproducts. The ROS damage mitochondria, and the damaged mitochondria further produce mitochondrial ROS. The increased mitochondrial ROS damage cellular components, including mitochondria themselves, and leads to diverse pathologies. Accordingly, it is crucial to eliminate excessive or damaged mitochondria to maintain mitochondrial homeostasis, in which mitophagy is believed to play a major role. Recently, the molecular mechanism and physiological role of mitophagy have been vigorously studied in yeast and mammalian cells. In yeast, Atg32 and Atg43, mitochondrial outer membrane proteins, were identified as mitophagy receptors in budding yeast and fission yeast, respectively. Here we summarize the molecular mechanisms of mitophagy in yeast, as revealed by the analysis of Atg32 and Atg43, and review recent progress in our understanding of mitophagy induction and regulation in yeast.

Mitophagy regulation mediated by the Far complex in yeast.

Mitochondrial autophagy (mitophagy) selectively degrades mitochondria and plays an important role in mitochondrial homeostasis. In the yeast Saccharomyces cerevisiae, the phosphorylation of the mitophagy receptor Atg32 by casein kinase 2 is essential for mitophagy, whereas this phosphorylation is counteracted by the protein phosphatase Ppg1. Although Ppg1 functions cooperatively with the Far complex (Far3, Far7, Far8, Vps64/Far9, Far10 and Far11), their relationship and the underlying phosphoregulatory mechanism of Atg32 remain unclear. Our recent study revealed: (i) the Far complex plays its localization-dependent roles, regulation of mitophagy and target of rapamycin complex 2 (TORC2) signaling, via the mitochondria- and endoplasmic reticulum (ER)-localized Far complexes, respectively; (ii) Ppg1 and Far11 form a subcomplex, and Ppg1 activity is required to assemble the sub- and core-Far complexes; (iii) association and dissociation between the Far complex and Atg32 are crucial determinants for mitophagy regulation. Here, we summarize our findings and discuss unsolved issues.

Association and dissociation between the mitochondrial Far complex and Atg32 regulate mitophagy.

Mitophagy plays an important role in mitochondrial homeostasis. In yeast, the phosphorylation of the mitophagy receptor Atg32 by casein kinase 2 is essential for mitophagy. This phosphorylation is counteracted by the yeast equivalent of the STRIPAK complex consisting of the PP2A-like protein phosphatase Ppg1 and Far3-7-8-9-10-11 (Far complex), but the underlying mechanism remains elusive. Here we show that two subpopulations of the Far complex reside in the mitochondria and endoplasmic reticulum, respectively, and play distinct roles; the former inhibits mitophagy via Atg32 dephosphorylation, and the latter regulates TORC2 signaling. Ppg1 and Far11 form a subcomplex, and Ppg1 activity is required for the assembling integrity of Ppg1-Far11-Far8. The Far complex preferentially interacts with phosphorylated Atg32, and this interaction is weakened by mitophagy induction. Furthermore, the artificial tethering of Far8 to Atg32 prevents mitophagy. Taken together, the Ppg1-mediated Far complex formation and its dissociation from Atg32 are crucial for mitophagy regulation.

Regulatory Mechanisms of Mitochondrial Autophagy: Lessons From Yeast.

Mitochondria produce the majority of ATP required by cells via oxidative phosphorylation. Therefore, regulation of mitochondrial quality and quantity is important for maintaining cellular activities. Mitophagy, the selective degradation of mitochondria, is thought to contribute to control of mitochondrial quality and quantity. In recent years, the molecular mechanism of mitophagy has been extensively studied in yeast and mammalian cells. In particular, identification of the mitophagy receptor Atg32 has contributed to substantial progress in understanding of mitophagy in yeast. This review summarizes the molecular mechanism of mitophagy in yeast and compares it to the mechanism of mitophagy in mammals. We also discuss the current understanding of mitophagy in plants.