tRNA production links nutrient conditions to the onset of sexual differentiation through the TORC1 pathway

Target of rapamycin (TOR) kinase controls cell growth and metabolism in response to nutrient availability. In the fission yeast Schizosaccharomyces pombe, TOR complex 1 (TORC1) promotes vegetative growth and inhibits sexual differentiation in the presence of ample nutrients. Here, we report the isolation and characterization of mutants with similar phenotypes as TORC1 mutants, in that they initiate sexual differentiation even in nutrient‐rich conditions. In most mutants identified, TORC1 activity is downregulated and the mutated genes are involved in tRNA expression or modification. Expression of tRNA precursors decreases when cells undergo sexual differentiation. Furthermore, overexpression of tRNA precursors prevents TORC1 downregulation upon nitrogen starvation and represses the initiation of sexual differentiation. Based on these observations, we propose that tRNA precursors operate in the S. pombe TORC1 pathway to switch growth mode from vegetative to reproductive.     

Functional characterization of the fission yeast phosphatidylserine synthase gene, pps1, reveals novel cellular functions for phosphatidylserine

To investigate the contributions of phosphatidylserine to the growth and morphogenesis of the rod-shaped fission yeast Schizosaccharomyces pombe, we have characterized the single gene in this organism, pps1, encoding a predicted phosphatidylserine synthase. S. pombe pps1Delta mutants grow slowly in rich medium and are inviable in synthetic minimal medium. They do not produce detectable phosphatidylserine in vivo and possess negligible in vitro phosphatidylserine synthase activity, indicating that pps1 encodes the major phosphatidylserine synthase activity in S. pombe. Supplementation of growth medium with ethanolamine partially suppresses the growth-defective phenotype of pps1Delta cells, reflecting the likely importance of phosphatidylserine as a precursor for phosphatidylethanolamine in S. pombe. In medium lacking ethanolamine, pps1Delta mutants exhibit striking cell morphology, cytokinesis, actin cytoskeleton, and cell wall remodeling and integrity defects. Overexpression of pps1 likewise leads to defects in cell morphology and cytokinesis, thus implicating phosphatidylserine as a dosage-dependent regulator of these processes. During log-phase growth, green fluorescent protein-Pps1p fusion proteins are concentrated at the cell and nuclear peripheries as well as presumptive endoplasmic reticulum membranes, while in stationary-phase cells, they are redistributed to unusual cytoplasmic structures of unknown origin. Moreover, stationary-phase pps1Delta cultures retain very poor viability relative to wild-type S. pombe cells, even in medium containing ethanolamine, demonstrating a role for phosphatidylserine in the physiological adaptations required for stationary-phase survival. Our findings reveal novel cellular functions for phosphatidylserine and emphasize the usefulness of S. pombe as a model organism for elucidating potentially conserved biological and molecular functions of this phospholipid.     

Pattern of end growth of the fission yeast Schizosaccharomyces pombe

The patterns of end growth of individual cells of Schizosaccharomyces pombe, wild-type cells (strain 972 h-), cells exposed to 8 mM hydroxyurea, and cdc mutants (cdc11-123 and cdc2-33), were investigated by time-lapse photomicrography. It was reconfirmed that there are three patterns of end growth: cells growing at the old end, at the new end, and at both ends from the beginning of the cell cycle. Cells that initiated growth at the old (new) end increased their growth rate at the new (old) end and became constant in their growth rate at the old (new) end when cells had their growth rate higher than a critical value: 0.08, 0.09, 0.08, and 0.11 microns/min in wild-type cells, cells exposed to hydroxyurea, cdc11-123 cells, and cdc2-33 cells, respectively. The critical value is proportional to the doubling time in length.     

Repair of UV damage in the fission yeast Schizosaccharomyces pombe

This review is concerned with repair and tolerance of UV damage in the fission yeast, Schizosaccharomyces pombe and with the differences between Sch. pombe and budding yeast, Saccharomyces cerevisiae in their response to UV irradiation. Sch. pombe is not as sensitive to ultra-violet radiation as Sac. cerevisiae nor are any of its mutants as sensitive as the most sensitive Sac. cerevisiae mutants. This can be explained in part by the fact that Sch. pombe, unlike budding yeast or mammalian cells, has an extra pathway (UVER) for excision of UV photoproducts in addition to nucleotide excision repair (NER). However, even in mutants lacking this additional pathway, there are significant differences between the two yeasts. Sch. pombe mutants that lack the alternative pathway are still more UV-resistant than wild-type Sac. cerevisiae; recombination mutants are significantly UV sensitive (unlike their Sac. cerevisiae equivalents); mutants lacking the second pathway are sensitized to UV by caffeine; and checkpoint mutants are relatively more sensitive than the budding yeast equivalents. In addition, Sch. pombe has no photolyase. Thus, the response to UV in the two yeasts has a number of significant differences, which are not accounted for entirely by the existence of two alternative excision repair pathways. The long G2 in Sch. pombe, its well-developed recombination pathways and efficient cell cycle checkpoints are all significant components in survival of UV damage.     

Radicicol binding to Swo1/Hsp90 and inhibition of growth of specific temperature-sensitive cell cycle mutants of fission yeast.

A panel screening using cdc mutants of Schizosaccharomyces pombe identified radicicol as a potent growth inhibitor of certain mutants at the permissive temperature. The strains sensitive to radicicol were cdc7, cdc11, and cdc14, all of which are defective in early septum formation. Cytokinesis but not nuclear division of these mutants was inhibited by radicicol, but that of cells with the wild-type background was not. A biologically active derivative of radicicol with a biotin moiety at the C-11 position bound Swo1, an Hsp90 homologue in S. pombe. Increased Swo1 expression partially suppressed radicicol sensitivity of cdc14 and almost completely rescued morphological abnormalities in cdc14 and cdc7 cells induced by radicicol at the permissive temperature. On the other hand, the increased Swo1 expression did not restore septum formation at the nonpermissive temperature. These results suggest that Swo1, as a molecular chaperone, plays a role in stabilizing these temperature-sensitive proteins at the permissive temperature or in activating the cytokinesis signaling cascade.     

Radicicol Binding to Swo1/Hsp90 and Inhibition of Growth of Specific Temperature-sensitive Cell Cycle Mutants of Fission Yeast

A panel screening using cdc mutants of Schizosaccharomyces pombe identified radicicol as a potent growth inhibitor of certain mutants at the permissive temperature. The strains sensitive to radicicol were cdc7, cdc11, and cdc14, all of which are defective in early septum formation. Cytokinesis but not nuclear division of these mutants was inhibited by radicicol, but that of cells with the wild-type background was not. A biologically active derivative of radicicol with a biotin moiety at the C-11 position bound Swo1, an Hsp90 homologue in S. pombe. Increased Swo1 expresion partially suppressed radicicol sensitivity of cdc14 and almost completely rescued morphological abnormalities in cdc14 and cdc7 cells induced by radicicol at the permissive temperature. On the other hand, the increased Swo1 expression did not restore septum formation at the nonpermissive temperature. These results suggest that Swo1, as a molecular chaperone, plays a role in stabilizing these temperature-sensitive proteins at the permissive temperature or in activating the cytokinesis signaling cascade.     

BED1, a gene encoding a galactosyltransferase homologue, is required for polarized growth and efficient bud emergence in Saccharomyces cerevisiae

The ellipsoidal shape of the yeast Saccharomyces cerevisiae is the result of successive isotropic/apical growth switches that are regulated in a cell cycle-dependent manner. It is thought that growth polarity is governed by the remodeling of the actin cytoskeleton that is itself under the control of the cell cycle machinery. The cell cycle and the morphogenesis cycle are tightly coupled and it has been recently suggested that a morphogenesis/polarity checkpoint control monitors bud emergence in order to maintain the coupling of these two events (Lew, D. J., and S. I. Reed. 1995. J. Cell Biol. 129:739-749). During a screen based on the inability of cells impaired in the budding process to survive when the morphogenesis checkpoint control is abolished, we identified and characterized BED1, a new gene that is required for efficient budding. Cells carrying a disrupted allele of BED1 no longer have the wild-type ellipsoidal shape characteristic of S. cerevisiae, are larger than wild-type cells, are deficient in bud emergence, and depend upon an intact morphogenesis checkpoint control to survive. These cells show defects in polarized growth despite the fact that the actin cytoskeleton appears normal. Our results suggest that Bed1 is a type II membrane protein localized in the endoplasmic reticulum. BED1 is significantly homologous to gma12+, a S. pombe gene coding for an alpha-1,2,-galactosyltransferase, suggesting that glycosylation of specific proteins or lipids could be important for signaling in the switch to polarized growth and in bud emergence.     

Mkp1 and Mkp2, two MAPKAP-kinase homologues in<Emphasis Type="Italic"> Schizosaccharomyces pombe</Emphasis>, interact with the MAP kinase Sty1

Mkp1 ( MAPKAP kinase Schizosaccharomyces pombe 1) and Mkp2 are two members from fission yeast of the sub-class of putative MAPK-activated protein kinases in yeasts, the other known members being Rck1 and Rck2 from Saccharomyces cerevisiae. The Mkp1 protein is readily co-immunoprecipitated with Sty1 from S. pombe extracts; Mkp2 shows a weaker interaction with Sty1. In mkp1 mutants, conjugation and meiosis proceed more readily and rapidly than in wild-type cells, in analogy to what was previously found for S. cerevisiae rck1 mutants. Conversely, overexpression of mkp1(+) delays meiosis. Mkp1 is phosphorylated in vivo in a sty1(+)-dependent manner; this modification is removed when cells are starved for nitrogen, a condition that is conducive to entry into stationary phase and meiosis. Overexpression of mkp1(+), like a sty1 mutation, also causes vegetative cells to elongate. The level of Mkp1 phosphorylation drops as cells enter mitosis. We have localised Mkp1 to the cytoplasm, excluded from the nucleus, in vegetative cells. The Mkp1 protein accumulates in zygotic asci and is concentrated within spores. The mkp2(+) gene has no noticeable impact on meiosis. Mkp2 is excluded from the nucleus in vegetative cells, and is concentrated at the septa of dividing cells. Mkp2 does not accumulate in meiotic cells.     

Human p53 inhibits growth in Schizosaccharomyces pombe.

Overexpression of wild-type p53 in mammalian cells blocks growth. We show here that the overexpression of wild-type human p53 in the fission yeast Schizosaccharomyces pombe also blocks growth, whereas the overexpression of mutant forms of p53 does not. The p53 polypeptide is located in the nucleus and is phosphorylated at both the cdc2 site and the casein kinase II site in S. pombe. A new dominant mutation of p53, resulting in the change of a cysteine to an arginine at amino acid residue 141, was identified. The results presented here demonstrate that S. pombe could provide a simple system for studying the mechanism of action of human p53.     

Study of cyclin proteolysis in anaphase-promoting complex (APC) mutant cells reveals the requirement for APC function in the final steps of the fission yeast septation initiation network

Cytokinesis in eukaryotic cells requires the inactivation of mitotic cyclin-dependent kinase complexes. An apparent exception to this relationship is found in Schizosaccharomyces pombe mutants with mutations of the anaphase-promoting complex (APC). These conditional lethal mutants arrest with unsegregated chromosomes because they cannot degrade the securin, Cut2p. Although failing at nuclear division, these mutants septate and divide. Since septation requires Cdc2p inactivation in wild-type S. pombe, it has been suggested that Cdc2p inactivation occurs in these mutants by a mechanism independent of cyclin degradation. In contrast to this prediction, we show that Cdc2p kinase activity fluctuates in APC cut mutants due to Cdc13/cyclin B destruction. In APC-null mutants, however, septation and cutting do not occur and Cdc13p is stable. We conclude that APC cut mutants are hypomorphic with respect to Cdc13p degradation. Indeed, overproduction of nondestructible Cdc13p prevents septation in APC cut mutants and the normal reorganization of septation initiation network components during anaphase.     

Bob1, a Gim5/MM-1/Pfd5 homolog, interacts with the MAP kinase kinase Byr1 to regulate sexual differentiation in the fission yeast, Schizosaccharomyces pombe

The MAPKK Byr1 is an essential component of a Ras-dependent MAPK module required for sexual differentiation in the fission yeast, Schizosaccharomyces pombe. Here we describe the genetic and molecular characterization of a highly conserved protein, Bob1, which was identified from a two-hybrid screen for Byr1-interacting proteins. Byrl and Bobl proteins coprecipitate from S. pombe cell lysates, and both proteins localize to the tips and septa of S. pombe cells. S. pombe bob1 null (bob1delta) mutants lack obvious growth defects but exhibit a significant mating deficiency, which can be suppressed by overexpression of Byrl. Overexpression of Bob1 also leads to inhibition of mating in S. pombe, and this defect is likewise suppressed by Byrl overexpression. Bob1 is highly homologous in structure to the mammalian MM-1/Pfd5 and budding yeast Gim5/Pfd5-Sc proteins, which have been implicated as regulators of actin and tubulins. Similar to budding yeast gim5/pfd5-Sc mutants, S. pombe bob1delta cells have cytoskeletal defects, as judged by hypersensitivity to cytoskeletal disrupting drugs. byr1delta mutants do not share this characteristic with bob1delta mutants, and byr1delta bob1delta mutants are not significantly more sensitive to cytoskeletal disrupting drugs than cells carrying only the bob1delta mutation. Taken together, our results suggest that Bob1 has Byr1-related function(s) required for proper mating response of S. pombe cells and Byrl-independent function(s) required for normal cytoskeletal control. We show that the human MM-1/Pfd5 protein can substitute for its counterpart in fission yeast, providing evidence that the functions of Bob1-related proteins have been highly conserved through evolution. Our results lead us to propose that Bob1-related proteins may play diverse roles in eukaryotic organisms.     

Mid2p stabilizes septin rings during cytokinesis in fission yeast

Septins are filament-forming proteins with a conserved role in cytokinesis. In the fission yeast Schizosaccharomyces pombe, septin rings appear to be involved primarily in cell-cell separation, a late stage in cytokinesis. Here, we identified a protein Mid2p on the basis of its sequence similarity to S. pombe Mid1p, Saccharomyces cerevisiae Bud4p, and Candida albicans Int1p. Like septin mutants, mid2delta mutants had delays in cell-cell separation. mid2delta mutants were defective in septin organization but not contractile ring closure or septum formation. In wild-type cells, septins assembled first during mitosis in a single ring and during septation developed into double rings that did not contract. In mid2delta cells, septins initially assembled in a single ring but during septation appeared in the cleavage furrow, forming a washer or disc structure. FRAP studies showed that septins are stable in wild-type cells but exchange 30-fold more rapidly in mid2delta cells. Mid2p colocalized with septins and required septins for its localization. A COOH-terminal pleckstrin homology domain of Mid2p was required for its localization and function. No genetic interactions were found between mid2 and the related gene mid1. Thus, these studies identify a new factor responsible for the proper stability and function of septins during cytokinesis.     

Role of bud6p and tea1p in the interaction between actin and microtubules for the establishment of cell polarity in fission yeast

BACKGROUND: In many cell types, microtubules are thought to direct the spatial distribution of F-actin in cell polarity. Schizosaccharomyces pombe cells exhibit a regulated program of polarized cell growth: after cell division, they grow first in a monopolar manner at the old end, and in G2 phase, initiate growth at the previous cell division site (the new end). The role of microtubule ends in cell polarity is highlighted by the finding that the cell polarity factor, tea1p, is present on microtubule plus ends and cell tips [1]. RESULTS: Here, we characterize S. pombe bud6p/fat1p, a homolog of S. cerevisiae Bud6/Aip3. bud6Delta mutant cells have a specific defect in the efficient initiation of growth at the new end and like tea1Delta cells, form T-shaped cells in a cdc11 background. Bud6-GFP localizes to both cell tips and the cytokinesis ring. Maintenance of cell tip localization is dependent upon actin but not microtubules. Bud6-GFP localization is tea1p dependent, and tea1p localization is not bud6p dependent. tea1Delta and bud6Delta cells generally grow in a monopolar manner but exhibit different growth patterns. tea1(Delta)bud6Delta mutants resemble tea1Delta mutants. Tea1p and bud6p coimmunoprecipitate and comigrate in large complexes. CONCLUSIONS: Our studies show that tea1p (a microtubule end-associated factor) and bud6p (an actin-associated factor) function in a common pathway, with bud6p downstream of tea1p. To our knowledge, bud6p is the first protein shown to interact physically with tea1p. These studies delineate a pathway for how microtubule plus ends function to polarize the actin cytoskeleton through actin-associated polarity factors.     

Canavanine resistance and the mechanism of arginine uptake in the fission yeast Schizosaccharomyces pombe

The mechanism of resistance to the arginine analogue L-canavanine, and of arginine uptake, were examined in the fission yeast Schizosaccharomyces pombe. Two mutants with increased resistance to canavanine were analysed genetically: both were double mutants, and in each case one mutation conferred resistance to canavanine, while the other enhanced this resistance. Evidence is presented that can 1.1 strains are defective in one system for arginine uptake, which presumably prevents entry of canavanine into the cell. This system operates in the wild-type whether the nitrogen source supplied is ammonium or glutamate. Double mutants carrying can 1.1 and an arginine requirement are unable to grow on ammonium medium even when supplied with exogenous argine, while growth can occur on glutamate plus arginine. This suggested the existence of a second uptake system for arginine which is absent during growth on ammonium, and direct measurements of the rates of arginine uptake under various conditions confirmed this. Our observations closely parallel those made on the budding yeast Saccharomyces cerevisiae. The ability to select for or against function of the can 1 gene should facilitate certain types of genetical analysis in S.pombe.     

Postreplication repair and PCNA modification in Schizosaccharomyces pombe

Ubiquitination of proliferating cell nuclear antigen (PCNA) plays a crucial role in regulating replication past DNA damage in eukaryotes, but the detailed mechanisms appear to vary in different organisms. We have examined the modification of PCNA in Schizosaccharomyces pombe. We find that, in response to UV irradiation, PCNA is mono- and poly-ubiquitinated in a manner similar to that in Saccharomyces cerevisiae. However in undamaged Schizosaccharomyces pombe cells, PCNA is ubiquitinated in S phase, whereas in S. cerevisiae it is sumoylated. Furthermore we find that, unlike in S. cerevisiae, mutants defective in ubiquitination of PCNA are also sensitive to ionizing radiation, and PCNA is ubiquitinated after exposure of cells to ionizing radiation, in a manner similar to the response to UV-irradiation. We show that PCNA modification and cell cycle checkpoints represent two independent signals in response to DNA damage. Finally, we unexpectedly find that PCNA is ubiquitinated in response to DNA damage when cells are arrested in G2.     

Pseudo-exponential growth in length of the fission yeast, Schizosaccharomyces pombe

The growth patterns of individual cells of the fission yeast (Schizosaccharomyces pombe wild-type cells, strain 972 h-; cells exposed to hydroxyurea; and cdc mutants, 11-123, 2-33) were investigated by time-lapse photomicrography. Wild-type cells showed one, two, or three linear-growth segments followed by a constant-length stage. Cells with two segments were most frequent. Hydroxyurea cells that divided as oversized cells (about three times the birth length) had three linear-growth segments in a cycle. Mutant cdc11-123 cells did not divide but had a constant-length stage separating the cycles; both the first and second cycles consisted of two linear-growth segments, and cells were oversized at the second constant-length stage (about 3.5 times the birth length). Elongating cdc2-33 cells that did not divide and were oversized (about five times the birth length) while under observation, showed four linear-growth segments. Cells of all strains showed 30 to 40% increase in growth rate at the rate-change point and maintained approximate exponential (pseudo-exponential) growth. We conclude that the normal growth pattern of individual fission-yeast cells is the pseudo-exponential pattern.     

Colcemid sensitivity of fission yeast: permeability and detoxification properties of resistant mutants.

Mutants of Schizosaccharomyces pombe, which were resistant to concentrations of colcemid inhibitory to the wild-type parent, were not diminished in their permeability to colcemid or colchicine. They also did not modify colcemid to a detectable derivative or to a product that was incapable of binding to brain tubulin. The resistance of these mutants was therefore via mechanisms different from those of permeability and detoxification known for animal cells.