Fission yeast TOR signaling is essential for the down-regulation of a hyperactivated stress-response MAP kinase under salt stress.

TOR (target of rapamycin) signaling regulates cell growth and division in response to environmental stimuli such as the availability of nutrients and various forms of stress. The vegetative growth of fission yeast cells, unlike other eukaryotic cells, is not inhibited by treatment with rapamycin. We found that certain mutations including pmc1Δ (Ca(2+)-ATPase), cps9-193 (small GTPase, Ryh1) and cps1-12 (1,3-ß-D-glucan synthase, Bgs1) confer a rapamycin-sensitive phenotype to cells under salt stress with potassium chloride (>0.5 M). Cytometric analysis revealed that the mutant cells were unable to enter the mitotic cell cycle when treated with the drug under salt stress. Gene cloning and overexpression experiments revealed that the sensitivity to rapamycin was suppressed by the ectopic expression of tyrosine phosphatases, Pyp1 and Pyp2, which are negative regulators of Spc1/Sty1 mitogen-activated protein kinase (MAPK). The level of tyrosine phosphorylation on Spc1 was higher and sustained substantially longer in these mutants than in the wild type under salt stress. The hyperphosphorylation was significantly suppressed by overexpression of pyp1 (+) with concomitant resumption of the mutant cells' growth. In fission yeast, TOR signaling has been thought to stimulate the stress-response pathway, because mutations of TORC2 components such as Tor1, Sin1 and Ste20 result in similar sensitive phenotypes to environmental stress. The present study, however, strongly suggests that TOR signaling is required for the down-regulation of a hyperactivated Spc1 for reentry into the mitotic cell cycle. This finding may shed light on our understanding of a new stress-responsive mechanism in TOR signaling in higher organisms.

Characterization of a fission yeast P(5)-type ATPase homologue that is essential for Ca(2+)/Mn(2+ )homeostasis in the absence of P(2)-type ATPases.

In the fission yeast Schizosaccharomyces pombe, three P-type ATPases, namely Cta4p, Pmr1p, and Pmc1p, have been shown to be essential for Ca(2+) homeostasis and are required for specific cellular functions as well. Here, we show that the simultaneous deletion of pmc1(+) and SPAC29A4.19c, which encodes a putative P(5)-type ATPase, causes a hypersensitive growth to either high concentrations of Ca(2+) in a medium, or the antiarrhythmic drug amiodarone, which has been known to cause a disruption of Ca(2+) homeostasis. On the other hand, simultaneous deletion of pmr1(+) and SPAC29A4.19c causes a hypersensitive growth to Mn(2+) depletion in a medium. The green fluorescent protein-tagged SPAC29A4.19c protein reveals a typical localization pattern of the Golgi proteins, but the SPAC29A4.19c protein is not exchangeable in function with Pmr1p, which is required for Ca(2+)/Mn(2+) homeostasis in secretory pathways. These results suggest that the putative P(5)-type ATPase encoded by SPAC29A4.19c is essential for Ca(2+) and Mn(2+ )homeostasis in the absence of P(2)-type ATPases, Pmc1p or Pmr1p, respectively. According to the precedent nomenclature of calcium/cation transporting ATPase in fission yeast, SPAC29A4.19 was named cta5(+) in this study.

Inhibitory activity of 1-farnesylpyridinium on the spatial control over the assembly of cell wall polysaccharides in Schizosaccharomyces pombe.

The modes of actions of 1-farnesylpyridinium (FPy) on yeast cell growth were investigated on the basis of its effects on cell cycle progression, morphogenesis and the related events for construction of cell wall architecture in Schizosacchromyces pombe. FPy predominantly inhibited the growth of the yeast cells after various cycles of cell division so that cells were arrested at the phase of separation into daughter cells accompanying morphological changes to swollen spherical cells at 24 h of incubation. FPy-treated cells were osmotically stable but were susceptible to the lytic action of (1, 3) beta-D-glucanases, and characterized by serious damages to the cell wall architecture as represented by a rough and irregular surface outlook. The isolated cell wall fraction gave a similar hexose composition with or without FPy treatment, suggesting that FPy did not inhibit the synthesis of each cell wall polysaccharide. FPy was permissive for the extracellular accumulation of amorphous cell wall materials and septum development in protoplasts, but absolutely interfered with the following morphogenetic process for construction of the rod-shaped cell wall architecture. Our results suggest the inhibitory activity of FPy on the spatial control over the assembly of cell wall polysaccharides.

The novel fission yeast (1,3)beta-D-glucan synthase catalytic subunit Bgs4p is essential during both cytokinesis and polarized growth.

Schizosaccharomyces pombe contains four putative (1,3)beta-D-glucan synthase (GS) catalytic subunits, Bgs1p-4p. In this work, we cloned bgs4+ and show that Bgs4p is the only subunit found to be a part of the GS enzyme and essential for maintaining cell integrity during cytokinesis and polarized growth. Here we show that bgs4+, cwg1+ (cwg1-1 shows reduced cell-wall beta-glucan and GS catalytic activity) and orb11+ (orb11-59 is defective in cell morphogenesis) are the same gene. bgs4+ is essential for spore germination and bgs4+ shut-off produces cell lysis at growing poles and mainly at the septum prior to cytokinesis, suggesting that Bgs4p is essential for cell wall growth and to compensate for an excess of cell wall degradation during cytokinesis. Shut-off and overexpression analysis suggest that Bgs4p forms part of a GS catalytic multiprotein complex and that Bgs4p-promoted cell-wall beta-glucan alterations induce compensatory mechanisms from other Bgs subunits and (1,3)alpha-D-glucan synthase. Physiological localization studies showed that Bgs4p localizes to the growing ends, the medial ring and septum, and at each stage of wall synthesis or remodeling that occurs during sexual differentiation: mating, zygote and spore formation, and spore germination. Bgs4p timing and requirements for proper positioning during cytokinesis and its localization pattern during spore maturation differ from those of Bgs1p. Bgs4p localizes overlapping the contractile ring once Bgs1p is present and a Calcofluor white-stained septum material is detected, suggesting that Bgs4p is involved in a late process of secondary or general septum synthesis. Unlike Bgs1p, Bgs4p needs the medial ring but not the septation initiation network proteins to localize with the other septation components. Furthermore, Bgs4p localization depends on the polarity establishment proteins. Finally, F-actin is necessary for Bgs4p delocalization from and relocalization to the growing regions, but it is not needed for the stable maintenance of Bgs4p at the growing sites, poles and septum. All these data show for the first time an essential role for a Bgs subunit in the synthesis of a (1,3)beta-D-glucan necessary to preserve cell integrity when cell wall synthesis or repair are needed.

Schizosaccharomyces pombe Pmr1p is essential for cell wall integrity and is required for polarized cell growth and cytokinesis.

The cps5-138 fission yeast mutant shows an abnormal lemon-like morphology at 28 degrees C in minimal medium and a lethal thermosensitive phenotype at 37 degrees C. Cell growth is completely inhibited at 28 degrees C in a Ca2+-free medium, in which the wild type is capable of growing normally. Under these conditions, actin patches become randomly distributed throughout the cell, and defects in septum formation and subsequent cytokinesis appear. The mutant cell is hypersensitive to the cell wall-digesting enzymatic complex Novozym234 even under permissive conditions. The gene SPBC31E1.02c, which complements all the mutant phenotypes described above, was cloned and codes for the Ca2+-ATPase homologue Pmr1p. The gene is not essential under optimal growth conditions but is required under conditions of low Ca2+ (<0.1 mM) or high temperature (>35 degrees C). The green fluorescent protein-tagged Cps5 proteins, which are expressed under physiological conditions (an integrated single copy with its own promoter in the cps5Delta strain), display a localization pattern typical of endoplasmic reticulum proteins. Biochemical analyses show that 1,3-beta-D-glucan synthase activity in the mutant is decreased to nearly half that of the wild type and that the mutant cell wall contains no detectable galactomannan when the cells are exposed to a Ca2+-free medium. The mutant acid phosphatase has an increased electrophoretic mobility, suggesting that incomplete protein glycosylation takes place in the mutant cells. These results indicate that S. pombe Pmr1p is essential for the maintenance of cell wall integrity and cytokinesis, possibly by allowing protein glycosylation and the polarized actin distribution to take place normally. Disruption and complementation analyses suggest that Pmr1p shares its function with a vacuolar Ca2+-ATPase homologue, Pmc1p (SPAPB2B4.04c), to prevent lethal activation of calcineurin for cell growth.

Localization of the (1,3)beta-D-glucan synthase catalytic subunit homologue Bgs1p/Cps1p from fission yeast suggests that it is involved in septation, polarized growth, mating, spore wall formation and spore germination.

Schizosaccharomyces pombe Bgs1p/Cps1p has been identified as a putative (1,3)beta-D-glucan synthase (GS) catalytic subunit with a possible function during cytokinesis and polarized growth. To study this possibility, double mutants of cps1-12 and cdc septation mutants were made. The double mutants displayed several hypersensitive phenotypes and altered actin distribution. Epistasis analysis showed mutations prior to septum synthesis were dominant over cps1-12, while cps1-12 was dominant over the end of septation mutant cdc16-116, suggesting Bgs1p is involved in septum cell-wall (1,3)beta-D-glucan synthesis at cytokinesis. We have studied the in vivo physiological localization of Bgs1p in a bgs1delta strain containing a functional GFP-bgs1(+) gene (integrated single copy and expressed under its own promoter). During vegetative growth, Bgs1p always localizes to the growing zones: one or both ends during cell growth and contractile ring and septum during cytokinesis. Bgs1p localization in cdc septation mutants indicates that Bgs1p needs the medial ring and septation initiation network (SIN) proteins to localize properly with the rest of septation components. Bgs1p localization in the actin mutant cps8-188 shows it depends on actin localization. In addition, Bgs1p remains polarized in the mislocalized growing poles and septa of tea1-1 and tea2-1 mutants. During the meiotic process of the life cycle, Bgs1p localizes to the mating projection, to the cell-to-cell contact zone during cell fusion and to the neck area during zygote formation. Also, Bgs1p localization suggests that it collaborates in forespore and spore wall synthesis. During spore germination, Bgs1p localizes first around the spore during isotropic growth, then to the zone of polarized growth and finally, to the medial ring and septum. At the end of spore-cell division, the Bgs1p displacement to the old end occurs only in the new cell. All these data show that Bgs1p is localized to the areas of polarized cell wall growth and so we propose that it might be involved in synthesizing the lineal (1,3)beta-D-glucan of the primary septum, as well as a similar lineal (1,3)beta-D-glucan when other processes of cell wall growth or repair are needed.

Fission yeast Mor2/Cps12, a protein similar to Drosophila Furry, is essential for cell morphogenesis and its mutation induces Wee1-dependent G(2) delay.

Fission yeast cells identify growing regions at the opposite ends of the cell, producing the rod-like shape. The positioning of the growth zone(s) and the polarized growth require CLIP170-like protein Tip1 and the Ndr kinase Orb6, respectively. Here, we show that the mor2/cps12 mutation disrupts the localization of F-actin at the cell ends, producing spherical cells and concomitantly inducing a G(2) delay at 36 degrees C. Mor2 is important for the localization of F-actin at the cell end(s) but not at the medial region, and is essential for the restriction of the growth zone(s) where Tip1 targets. Mor2 is homologous to the Drosophila Furry protein, which is required to maintain the integrity of cellular extensions, and is localized at both cell ends and the medial region of the cell in an actin-dependent fashion. Cellular localization of Mor2 and Orb6 was interdependent. The tyrosine kinase Wee1 is necessary for the G(2) delay and maintenance of viability of the mor2 mutant. These results indicate that Mor2 plays an essential role in cell morphogenesis in concert with Orb6, and the mutation activates the mechanism coordinating morphogenesis with cell cycle progression.