Yeast Rim11 kinase responds to glutathione-induced stress by regulating the transcription of phospholipid biosynthetic genes.

Glutathione (GSH), a tripeptide composed of glycine, cysteine, and glutamic acid, is an abundant thiol found in a wide variety of cells, ranging from bacterial to mammalian cells. Adequate levels of GSH are essential for maintaining iron homeostasis. The ratio of oxidized/reduced GSH is strictly regulated in each organelle to maintain the cellular redox potential. Cellular redox imbalances cause defects in physiological activities, which can lead to various diseases. Although there are many reports regarding the cellular response to GSH depletion, studies on stress response to high levels of GSH are limited. Here, we performed genome-scale screening in the yeast Saccharomyces cerevisiae and identified RIM11, BMH1, and WHI2 as multicopy suppressors of the growth defect caused by GSH stress. The deletion strains of each gene were sensitive to GSH. We found that Rim11, a kinase important in the regulation of meiosis, was activated via autophosphorylation upon GSH stress in a glucose-rich medium. Furthermore, RNA-seq revealed that transcription of phospholipid biosynthetic genes was downregulated under GSH stress, and introduction of multiple copies of RIM11 counteracted this effect. These results demonstrate that S. cerevisiae copes with GSH stress via multiple stress-responsive pathways, including a part of the adaptive pathway to glucose limitation.

TAQing2.0 for genome reorganization of asexual industrial yeasts by direct protein transfection.

Genomic rearrangements often generate phenotypic diversification. We previously reported the TAQing system where genomic rearrangements are induced via conditional activation of a restriction endonuclease in yeast and plant cells to produce mutants with marked phenotypic changes. Here we developed the TAQing2.0 system based on the direct delivery of endonucleases into the cell nucleus by cell-penetrating peptides. Using the optimized procedure, we introduce a heat-reactivatable endonuclease TaqI into an asexual industrial yeast (torula yeast), followed by a transient heat activation of TaqI. TAQing2.0 leads to generation of mutants with altered flocculation and morphological phenotypes, which exhibit changes in chromosomal size. Genome resequencing suggested that torula yeast is triploid with six chromosomes and the mutants have multiple rearrangements including translocations having the TaqI recognition sequence at the break points. Thus, TAQing2.0 is expected as a useful method to obtain various mutants with altered phenotypes without introducing foreign DNA into asexual industrial microorganisms.

The fission yeast synaptobrevin ortholog Syb1 plays an important role in forespore membrane formation and spore maturation.

Synaptobrevin, also called vesicle-associated membrane protein (VAMP), is a component of the plasma membrane N-methylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which plays a key role in intracellular membrane fusion. Previous studies have revealed that, similar to synaptobrevin in other organisms, the fission yeast synaptobrevin ortholog Syb1 associates with post-Golgi secretory vesicles and is essential for cytokinesis and cell elongation. Here, we report that Syb1 has a role in sporulation. After nitrogen starvation, green fluorescent protein (GFP)-Syb1 is found in intracellular dots. As meiosis proceeds, GFP-Syb1 accumulates around the nucleus and then localizes at the forespore membrane (FSM). We isolated a syb-S1 mutant, which exhibits a defect in sporulation. In syb1-S1 mutants, the FSM begins to form but fails to develop a normal morphology. Electron microscopy shows that an abnormal spore wall is often formed in syb1-S1 mutant spores. Although most syb1-S1 mutant spores are germinated, they are less tolerant to ethanol than wild-type spores. The syb1-S1 allele carries a missense mutation, resulting in replacement of a conserved cysteine residue adjacent to the transmembrane domain, which reduces the stability and abundance of the Syb1 protein. Taken together, these results indicate that Syb1 plays an important role in both FSM assembly and spore wall formation.

Endocytosis is essential for dynamic translocation of a syntaxin 1 orthologue during fission yeast meiosis.

Syntaxin is a component of the target soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex, which is responsible for fusion of membrane vesicles at the target membrane. The fission yeast syntaxin 1 orthologue Psy1 is essential for both vegetative growth and spore formation. During meiosis, Psy1 disappears from the plasma membrane (PM) and dramatically relocalizes on the nascent forespore membrane, which becomes the PM of the spore. Here we report the molecular details and biological significance of Psy1 relocalization. We find that, immediately after meiosis I, Psy1 is selectively internalized by endocytosis. In addition, a meiosis-specific signal induced by the transcription factor Mei4 seems to trigger this internalization. The internalization of many PM proteins is facilitated coincident with the initiation of meiosis, whereas Pma1, a P-type ATPase, persists on the PM even during the progression of meiosis II. Ergosterol on the PM is also important for the internalization of PM proteins in general during meiosis. We consider that during meiosis in Schizosaccharomyces pombe cells, the characteristics of endocytosis change, thereby facilitating internalization of Psy1 and accomplishing sporulation.