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Mitochondria perform critical functions, including respiration, ATP production, small molecule metabolism, and anti-oxidation, and they are involved in a number of human diseases. While the mitochondrial genome contains a small number of protein-coding genes, the vast majority of mitochondrial proteins are encoded by nuclear genes. In fission yeast Schizosaccharomyces pombe, we screened 457 deletion (del) mutants deficient in nuclear-encoded mitochondrial proteins, searching for those that fail to form colonies in culture medium containing low glucose (0.03–0.1%; low-glucose sensitive, lgs), but that proliferate in regular 2–3% glucose medium. Sixty-five (14%) of the 457 deletion mutants displayed the lgs phenotype. Thirty-three of them are defective either in dehydrogenases, subunits of respiratory complexes, the citric acid cycle, or in one of the nine steps of the CoQ10 biosynthetic pathway. The remaining 32 lgs mutants do not seem to be directly related to respiration. Fifteen are implicated in translation, and six encode transporters. The remaining 11 function in anti-oxidation, amino acid synthesis, repair of DNA damage, microtubule cytoskeleton, intracellular mitochondrial distribution or unknown functions. These 32 diverse lgs genes collectively maintain mitochondrial functions under low (1/20–1/60× normal) glucose concentrations. Interestingly, 30 of them have homologues associated with human diseases.

Isolation and introduction of genetic mutations is the primary approach to characterize gene functions in model yeasts. Although this approach has proven very powerful, it is not applicable to all genes in these organisms. For example, introducing defective mutations into essential genes causes lethality upon loss of function. To circumvent this difficulty, conditional and partial repression of target transcription is possible. While transcriptional regulation techniques, such as promoter replacement and 3′ untranslated region (3′UTR) disruption, are available for yeast systems, CRISPR–Cas-based technologies have provided additional options. This review summarizes these gene perturbation technologies, including recent advances in methods based on CRISPR–Cas systems for Schizosaccharomyces pombe. We discuss how biological resources afforded by CRISPRi can promote fission yeast genetics.

Mitochondria are essential for regulation of cellular respiration, energy production, small molecule metabolism, anti-oxidation and cell ageing, among other things. While the mitochondrial genome contains a small number of protein-coding genes, the great majority of mitochondrial proteins are encoded by chromosomal genes. In the fission yeast Schizosaccharomyces pombe, 770 proteins encoded by chromosomal genes are located in mitochondria. Of these, 195 proteins, many of which are implicated in translation and transport, are absolutely essential for viability. We isolated and characterized eight temperature-sensitive (ts) strains with mutations in essential mitochondrial proteins. Interestingly, they are also sensitive to limited nutrition (glucose and/or nitrogen), producing low-glucose-sensitive and ‘super-housekeeping' phenotypes. They fail to produce colonies under low-glucose conditions at the permissive temperature or lose cell viability under nitrogen starvation at the restrictive temperature. The majority of these ts mitochondrial mutations may cause defects of gene expression in the mitochondrial genome. mrp4 and mrp17 are defective in mitochondrial ribosomal proteins. ppr3 is defective in rRNA expression, and trz2 and vrs2 are defective in tRNA maturation. This study promises potentially large dividends because mitochondrial quiescent functions are vital for human brain and muscle, and also for longevity.

Catalytically inactive mutant of Cas9 (dCas9) can repress gene transcription. This gene knockdown method, called CRISPRi, is useful because it can repress an arbitrary target gene only by changing the sgRNA sequence that determines target gene specificity. Here, dCas9-mediated CRISPRi is implemented for the deeply studied model organism fission yeast, and a method to design sgRNAs for efficient CRISPRi is offered. This will facilitate revealing gene functions and developing tools based on dCas9 technology in Schizosaccharomyces pombe

Target of rapamycin (TOR) kinases form two distinct complexes, TORC1 and TORC2, which are evolutionarily conserved among eukaryotes. These complexes control intracellular biochemical processes in response to changes in extracellular nutrient conditions. Previous studies using the fission yeast, Schizosaccharomyces pombe, showed that the TORC2 signaling pathway, which is essential for cell proliferation under glucose-limited conditions, ensures cell-surface localization of a high-affinity hexose transporter, Ght5, by downregulating its endocytosis. The TORC2 signaling pathway retains Ght5 on the cell surface, depending on the presence of nitrogen sources in medium. Ght5 is transported to vacuoles upon nitrogen starvation. In this review, we discuss the molecular mechanisms underlying this regulation to cope with nutritional stress, a response which may be conserved from yeasts to mammals.

The cell must utilise nutrients to generate energy as a means of sustaining its life. As the environment is not necessarily abundant in nutrients and oxygen, the cell must be able to regulate energy metabolism to adapt to changes in extracellular and intracellular conditions. Recently, several key regulators of energy metabolism have been reported. This review describes the recent advances in molecular regulation of energy metabolism, focusing mainly on glycolysis and its shunt pathways. Human diseases, such as cancer and neurodegenerative disorders, are also discussed in relation to failure of energy metabolism regulation. [ABSTRACT FROM AUTHOR]

The fission yeast, Schizosaccharomyces pombe, is an excellent eukaryote model organism for studying essential biological processes. Its genome contains ~1,200 genes essential for cell viability, most of which are evolutionarily conserved. To study these essential genes, resources enabling conditional perturbation of target genes are required. Here, we constructed comprehensive arrayed libraries of plasmids and strains to knock down essential genes in S. pombe using dCas9-mediated CRISPRi. These libraries cover ~98% of all essential genes in fission yeast. We estimate that in ~60% of these strains, transcription of a target gene was repressed so efficiently that cell proliferation was significantly inhibited. To demonstrate the usefulness of these libraries, we performed metabolic analyses with knockdown strains and revealed flexible interaction among metabolic pathways. Libraries established in this study enable comprehensive functional analyses of essential genes in S. pombe and will facilitate the understanding of essential biological processes in eukaryotes. [ABSTRACT FROM AUTHOR]

In the fission yeast, Schizosaccharomyces pombe, the high-affinity hexose transporter, Ght5, must be transcriptionally upregulated and localized to the cell surface for cell division under limited glucose. Although cell-surface localization of Ght5 depends on Target of rapamycin complex 2 (TORC2), the molecular mechanisms by which TORC2 ensures proper localization of Ght5 remain unknown. We performed genetic screening for gene mutations that restore Ght5 localization on the cell surface in TORC2-deficient mutant cells, and identified a gene encoding an uncharacterized α-arrestin-like protein, Aly3/SPCC584.15c. α-arrestins are thought to recruit a ubiquitin ligase to membrane-associated proteins. Consistently, Ght5 is ubiquitylated in TORC2-deficient cells, and this ubiquitylation is dependent on Aly3. TORC2 supposedly enables cell-surface localization of Ght5 by preventing Aly3-dependent ubiquitylation and subsequent ubiquitylation-dependent translocation of Ght5 to vacuoles. Surprisingly, nitrogen starvation, but not glucose depletion, triggers Aly3-dependent transport of Ght5 to vacuoles in S. pombe, unlike budding yeast hexose transporters, vacuolar transport of which is initiated upon changes in hexose concentration. This study provides new insights into the molecular mechanisms controlling the subcellular localization of hexose transporters in response to extracellular stimuli. [ABSTRACT FROM AUTHOR]

Fission yeast, Schizosaccharomyces pombe, possesses eight hexose transporters, Ght1~8. In order to clarify the role of each hexose transporter on glucose uptake, a glucose uptake assay system was established and the actual glucose uptake activity of each hexose transporter-deletion mutant was measured. Under normal growth condition containing 2% glucose, ∆ght5 and ∆ght2 mutants showed large and small decrease in glucose uptake activity, respectively. On the other hand, the other deletion mutants did not show any decrease in glucose uptake activity indicating that, in the presence of Ght5 and Ght2, the other hexose transporters do not play a significant role in glucose uptake. To understand the relevance between glucose uptake and lifespan regulation, we measured the chronological lifespan of each hexose transporter deletion mutant, and found that only ∆ght5 mutant showed a significant lifespan extension. Based on these results we showed that Ght5 is mainly involved in the glucose uptake in Schizosaccharomyces pombe, and suggested that the ∆ght5 mutant has prolonged lifespan due to physiological changes similar to calorie restriction.