Meiosis-specific localization of the exocytic Rab Ypt2 in fission yeast.
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ABSTRACT:
SUBMITTER: Imada K
PROVIDER: S-EPMC7204522 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:A genetic screen for mutations synthetically lethal with fission yeast calcineurin deletion led to the identification of Ypt3, a homolog of mammalian Rab11 GTP-binding protein. A mutant with the temperature-sensitive ypt3-i5 allele showed pleiotropic phenotypes such as defects in cytokinesis, cell wall integrity, and vacuole fusion, and these were exacerbated by FK506-treatment, a specific inhibitor of calcineurin. Green fluorescent protein (GFP)-tagged Ypt3 showed cytoplasmic staining that was concentrated at growth sites, and this polarized localization required the actin cytoskeleton. It was also detected as a punctate staining in an actin-independent manner. Electron microscopy revealed that ypt3-i5 mutants accumulated aberrant Golgi-like structures and putative post-Golgi vesicles, which increased remarkably at the restrictive temperature. Consistently, the secretion of GFP fused with the pho1(+) leader peptide (SPL-GFP) was abolished at the restrictive temperature in ypt3-i5 mutants. FK506-treatment accentuated the accumulation of aberrant Golgi-like structures and caused a significant decrease of SPL-GFP secretion at a permissive temperature. These results suggest that Ypt3 is required at multiple steps of the exocytic pathway and its mutation affects diverse cellular processes and that calcineurin is functionally connected to these cellular processes.
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Project description:Meiosis-specific mRNAs are transcribed in vegetative fission yeast, and these meiotic mRNAs are selectively removed from mitotic cells to suppress meiosis. This RNA elimination system requires degradation signal sequences called determinant of selective removal (DSR), an RNA-binding protein Mmi1, polyadenylation factors, and the nuclear exosome. However, the detailed mechanism by which meiotic mRNAs are selectively degraded in mitosis but not meiosis is not understood fully. Here we report that Red1, a novel protein, is essential for elimination of meiotic mRNAs from mitotic cells. A red1 deletion results in the accumulation of a large number of meiotic mRNAs in mitotic cells. Red1 interacts with Mmi1, Pla1, the canonical poly(A) polymerase, and Rrp6, a subunit of the nuclear exosome, and promotes the destabilization of DSR-containing mRNAs. Moreover, Red1 forms nuclear bodies in mitotic cells, and these foci are disassembled during meiosis. These results demonstrate that Red1 is involved in DSR-directed RNA decay to prevent ectopic expression of meiotic mRNAs in vegetative cells.
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Project description:Cytokinesis in all organisms involves the creation of membranous barriers that demarcate individual daughter cells. In fission yeast, a signaling module termed the septation initiation network (SIN) plays an essential role in the assembly of new membranes and cell wall during cytokinesis. In this study, we have characterized Slk1p, a protein-kinase related to the SIN component Sid2p. Slk1p is expressed specifically during meiosis and localizes to the spindle pole bodies (SPBs) during meiosis I and II in a SIN-dependent manner. Slk1p also localizes to the forespore membrane during sporulation. Cells lacking Slk1p display defects associated with sporulation, leading frequently to the formation of asci with smaller and/or fewer spores. The ability of slk1 Delta cells to sporulate, albeit inefficiently, is fully abolished upon compromise of function of Sid2p, suggesting that Slk1p and Sid2p play overlapping roles in sporulation. Interestingly, increased expression of the syntaxin Psy1p rescues the sporulation defect of sid2-250 slk1 Delta. Thus, it is likely that Slk1p and Sid2p play a role in forespore membrane assembly by facilitating recruitment of components of the secretory apparatus, such as Psy1p, to allow membrane expansion. These studies thereby provide a novel link between the SIN and vesicle trafficking during cytokinesis.
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Project description:Sexual development in Schizosaccharomyces pombe culminates in meiosis and sporulation. We used ribosome profiling to investigate the translational landscape of this process. We show that the translation efficiency of hundreds of genes is regulated in complex patterns, often correlating with changes in RNA levels. Ribosome-protected fragments show a three-nucleotide periodicity that identifies translated sequences and their reading frame. Using this property, we identified 46 new translated genes and found that 24% of noncoding RNAs are actively translated. We also detected 19 nested antisense genes, in which both DNA strands encode translated mRNAs. Finally, we identified 1,735 translated upstream open reading frames (ORFs) in leader sequences. In S. pombe, in contrast with Saccharomyces cerevisiae, sexual development is not accompanied by large increases in upstream ORF use, thus suggesting that this is an organism-specific adaptation, not a general feature of developmental processes.
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Project description:In eukaryotes, the cyclin-dependent kinase Cdk1p (Cdc2p) plays a central role in entry into and progression through nuclear division during mitosis and meiosis. Cdk1p is activated during meiotic nuclear divisions by dephosphorylation of its tyrosine-15 residue. The phosphorylation status of this residue is largely determined by the Wee1p kinase and the Cdc25p phosphatase. In fission yeast, the forkhead-type transcription factor Mei4p is essential for entry into the first meiotic nuclear division. We recently identified cdc25(+) as an essential target of Mei4p in the control of entry into meiosis I. Here, we show that wee1(+) is another important target of Mei4p in the control of entry into meiosis I. Mei4p bound to the upstream region of wee1(+) in vivo and in vitro and inhibited expression of wee1(+), whereas Mei4p positively regulated expression of the adjacent pseudogene. Overexpression of Mei4p inhibited expression of wee1(+) and induced that of the pseudogene. Conversely, deletion of Mei4p did not decrease expression of wee1(+) but inhibited that of the pseudogene. In addition, deletion of Mei4p-binding regions delayed repression of wee1(+) expression as well as induction of expression of the pseudogene. These results suggest that repression of wee1(+) expression is primarily owing to Mei4p-mediated transcriptional interference.
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Project description:From yeast to human, TOR (target of rapamycin) kinase plays pivotal roles in coupling extracellular stimuli to cell growth and metabolism. TOR kinase functions in two distinct protein complexes, TOR complex 1 (TORC1) and 2 (TORC2), which phosphorylate and activate different AGC-family protein kinases. TORC1 is controlled by the small GTPase Rheb, but little is known about TORC2 regulators.We have identified the Ryh1 GTPase, a human Rab6 ortholog, as an activator of TORC2 signaling in the fission yeast Schizosaccharomyces pombe. Mutational inactivation of Ryh1 or its guanine nucleotide exchange factor compromises the TORC2-dependent phosphorylation of the AGC-family Gad8 kinase. In addition, the effector domain of Ryh1 is important for its physical interaction with TORC2 and for stimulation of TORC2 signaling. Thus, GTP-bound Ryh1 is likely to be the active form stimulatory to TORC2-Gad8 signaling. Consistently, expression of the GTP-locked mutant Ryh1 is sufficient to promote interaction between TORC2 and Gad8 and to induce Gad8 hyperphosphorylation. The loss of functional Ryh1, TORC2, or Gad8 brings about similar vacuolar fragmentation and stress sensitivity, further corroborating their involvement in a common cellular process. Human Rab6 can substitute Ryh1 in S. pombe, and therefore Rab6 may be a potential activator of TORC2 in mammals.In its GTP-bound form, Ryh1, an evolutionarily conserved Rab GTPase, activates TORC2 signaling to the AGC kinase Gad8. The Ryh1 GTPase and the TORC2-Gad8 pathway are required for vacuolar integrity and cellular stress resistance in S. pombe.
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Project description:Meiotic homologous pairing is crucial to proper homologous recombination, which secures subsequent reductional chromosome segregation. We have identified a novel meiosis-specific protein of fission yeast Schizosaccharomyces pombe, Meu13p, to be a molecule that is required for proper homologous pairing and recombination. Rec12p (homologue of Saccharomyces cerevisiae Spo11p), which is essential for the initiation of meiotic recombination, is also shown for the first time to participate in the pairing process of S.pombe. Meu13p, however, contributes to pairing through a recombination-independent mechanism, as disruption of the meu13(+) gene reduces pairing whether the rec12(+) gene is deleted or not. We also demonstrate a dynamic nature of homologous pairing in living meiotic cells, which is markedly affected by meu13 deletion. Meu13p is not required for telomere clustering and the nuclear movement process, which are well known requirements for efficient pairing in S.pombe. Based on these results, together with the localization of Meu13p on meiotic chromatin, we propose that Meu13p directly promotes proper homologous pairing and recombination.
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Project description:During meiosis, the centrosome/spindle pole body (SPB) must be regulated in a manner distinct from that of mitosis to achieve a specialized cell division that will produce gametes. In this paper, we demonstrate that several SPB components are localized to SPBs in a meiosis-specific manner in the fission yeast Schizosaccharomyces pombe. SPB components, such as Cut12, Pcp1, and Spo15, which stay on the SPB during the mitotic cell cycle, disassociate from the SPB during meiotic prophase and then return to the SPB immediately before the onset of meiosis I. Interestingly, the polo kinase Plo1, which normally localizes to the SPB during mitosis, is excluded from them in meiotic prophase, when meiosis-specific, horse-tail nuclear movement occurs. We found that exclusion of Plo1 during this period was essential to properly remodel SPBs, because artificial targeting of Plo1 to SPBs resulted in an overduplication of SPBs. We also found that the centrin Cdc31 was required for meiotic SPB remodeling. Thus Plo1 and a centrin play central roles in the meiotic SPB remodeling, which is essential for generating the proper number of meiotic SPBs and, thereby provide unique characteristics to meiotic divisions.
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Project description:Alternative splicing increases the diversity of transcriptomes and proteomes in metazoans. The extent to which alternative splicing is active and functional in unicellular organisms is less understood. Here, we exploit a single-molecule long-read sequencing technique and develop an open-source software program called SpliceHunter to characterize the transcriptome in the meiosis of fission yeast. We reveal 14,353 alternative splicing events in 17,669 novel isoforms at different stages of meiosis, including antisense and read-through transcripts. Intron retention is the major type of alternative splicing, followed by alternate "intron in exon." Seven hundred seventy novel transcription units are detected; 53 of the predicted proteins show homology in other species and form theoretical stable structures. We report the complexity of alternative splicing along isoforms, including 683 intra-molecularly co-associated intron pairs. We compare the dynamics of novel isoforms based on the number of supporting full-length reads with those of annotated isoforms and explore the translational capacity and quality of novel isoforms. The evaluation of these factors indicates that the majority of novel isoforms are unlikely to be both condition-specific and translatable but consistent with the possibility of biologically functional novel isoforms. Moreover, the co-option of these unusual transcripts into newly born genes seems likely. Together, the results of this study highlight the diversity and dynamics at the isoform level in the sexual development of fission yeast.
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Project description:Polarized morphogenesis is achieved by targeting or inhibiting growth in distinct regions. Rod-shaped fission yeast cells grow exclusively at their ends by restricting exocytosis and secretion to these sites. This growth pattern implies the existence of mechanisms that prevent exocytosis and growth along nongrowing cell sides. We previously identified a set of 50-100 megadalton-sized node structures along the sides of fission yeast cells that contained the interacting proteins Skb1 and Slf1. Here, we show that Skb1-Slf1 nodes contain the syntaxin-like soluble N-ethylmaleimide-sensitive factor attachment protein receptor Psy1, which mediates exocytosis in fission yeast. Psy1 localizes in a diffuse pattern at cell tips, where it likely promotes exocytosis and growth, but is sequestered in Skb1-Slf1 nodes at cell sides where growth does not occur. Mutations that prevent node assembly or inhibit Psy1 localization to nodes lead to aberrant exocytosis at cell sides and increased cell width. Genetic results indicate that this Psy1 node mechanism acts in parallel to actin cables and Cdc42 regulation. Our work suggests that sequestration of syntaxin-like Psy1 at nongrowing regions of the cell cortex reinforces cell morphology by restricting exocytosis to proper sites of polarized growth.
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