Junpei Ishiguro

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)β-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)β-D-glucan synthesis at cytokinesis. We have studied the in vivo physiological localization of Bgs1p in a bgs1Δ 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)β-D-glucan of the primary septum, as well as a similar lineal (1,3)β-D-glucan when other processes of cell wall growth or repair are needed.

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

Schizosaccharomyces pombe contains four putative (1,3)β-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 β-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 β-glucan alterations induce compensatory mechanisms from other Bgs subunits and (1,3)α-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)β-D-glucan necessary to preserve cell integrity when cell wall synthesis or repair are needed.

An actin point‐mutation neighboring the ‘hydrophobic plug’ causes defects in the maintenance of cell polaroty and septum organization in the fission yeast Schizosaccharomyces pombe

The fission yeast cps8 mutation gives rise to abnormally enlarged and dispolarized cells, each of which contains several nuclei with aberrant multisepta. Molecular cloning and sequence analysis of the cps8 gene indicated that it encodes an actin with an amino acid substitution of aspartic acid for glycine at residue 273 in the hydrophobic loop that is located between actin subdomains 3 and 4. Fluorescence microscopy using phalloidin and anti‐actin antibody revealed changes in the F‐actin structure and distribution in the mutant cells. These results indicate that the hydrophobic loop plays an essential role for creating normal F‐actin structure, only by which cell polarity and the late mitotic events can be maintained properly.

Fission yeast Mor2/Cps12, a protein similar to Drosophila Furry, is essential for cell morphogenesis and its mutation induces Wee1-dependent G2 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 G2 delay at 36°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 G2 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.

Altered ribosomal protein S11 from the SUP46 suppressor of yeast

Abstract The dominant suppressor SUP46 of the yeast Saccharomyces cerevisiae was shown to act on a wide range of mutations (preceding paper by Ono et al., 1981). Masurekar et al. (1981) demonstrated that ribosomes from the SUP46 strain make an abnormally high rate of errors in a cell-free translation system. These findings indicated that SUP46 suppression was the result of abnormal ribosomes misreading mutant codons. We have used two-dimensional polyacrylamide gel electrophoresis to show that the S11 protein from the 40 S ribosomal subunit has an altered electrophoretic mobility. Thus the gene product of the SUP46 locus is either the S11 ribosomal protein or an enzyme that modifies the S11 protein. These results demonstrate that the altered S11 protein is responsible for the suppression by misreading.

Schizosaccharomyces pombe Pmr1p Is Essential for Cell Wall Integrity and Is Required for Polarized Cell Growth and Cytokinesis

ABSTRACT The cps5-138 fission yeast mutant shows an abnormal lemon-like morphology at 28°C in minimal medium and a lethal thermosensitive phenotype at 37°C. Cell growth is completely inhibited at 28°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°C). The green fluorescent protein-tagged Cps5 proteins, which are expressed under physiological conditions (an integrated single copy with its own promoter in the cps5Δ strain), display a localization pattern typical of endoplasmic reticulum proteins. Biochemical analyses show that 1,3-β-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.

Study on proteins from yeast cytoplasmic ribosomes by two-dimensional gel electrophoresis

SummaryProteins of yeast cytoplasmic ribosomes were analyzed by two different methods of two-dimensional gel electrophoresis: run at pH 8.6 in 1-D1 and at pH 4.6 in 2-D (Method A); run at pH 5.0 in 1-D and in the presence of sodium dodecyl sulfate in 2-D (Method B). The numbers of proteins estimated were 28 (Method A) and 29 or 30 (Method B) in the 40S small subunit, and 40 (Method A) and 41 (Method B) in the 60S large subunit, respectively. Molecular weights of proteins in the small and the large subunits were found to be less than 40,000 and 60,000 respectively.

An abnormal cell division cycle in an AIR carboxylase-deficient mutant of the fission yeast Schizosaccharomyces pombe.

SummaryAdenine-requiring mutant strains of S. pombe enter the stationary phase after depleting a culture medium of adenine or its analogues. Stationary phase cells of six mutants defective at different stages of the purine nucleotide synthetic pathway were examined for cell volume and DNA content, and then compared in these respects with those of a prototrophic wild-type strain. The cell cycle of the wild-type strain was arrested in the G2 phase (2C state) in the nitrogen rich medium, as is evident from DNA content per cell (0.0425 pg) and cell volume (47.7 μm3). An AIR carboxylase-deficient (ade6) mutant strain was found to have an unusual cell volume (307.4 μm3) and DNA content (0.1187 pg). By DAPI fluorescence microscopy, each mutant cell was seen to contain only one enlarged nucleus, which indicates the absence of cell populations containing cells in the 4C state of the S phase following nuclear division. It then follows that in ade6 mutant cells, DNA synthesis occurs in the absence of a completed nuclear division. Thus in S. pombe cells, the completion of nuclear division is not necessarily required for the next cycle initiation of DNA synthesis under certain physiological conditions.

Genetic and biochemical characterization of antisuppressor mutants in the yeast Saccharomyces cerevisiae.

SummaryFive mutants carrying antisuppressors which reduce the efficiency of SUP46 suppressor of the yeast, Saccharomyces cerevisiae, were isolated. One of them was found to have an altered protein, S27, of 40S ribosomal subunit by using two-dimensional gel electrophoresis. A single recessive gene, designated asu11, was responsible for the mutation, and the heterodiploid (asu11/+) strain was shown to contain both altered and normal S27 proteins, suggesting that the asu11 locus codes for the S27 protein. The mutant was resistant to an aminoglycoside antibiotic, paromomycin which has been known to induce translational errors. These results together with the previous finding that SUP46 suppressor mutant contains an altered S11 ribosomal protein (Ishiguro et al. 1981) suggest that the secondary mutation in the ribosomal protein, S27, renders the restrictive effect to increased translational ambiguity caused by the alteration of S11 protein.

Characterization of blasticidin S — resistant mutants of Saccharomyces cerevisiae

SummaryBlasticidin S-resistant mutants of S. cerevisiae were isolated and characterized. Resistant mutations were found to fall into two complementation groups. A single recessive nuclear gene was responsible for each group, donated as bls1 and bls2, respectively. A gene bls1 was linked to an ilv3 gene located on the right arm of chromosome X. The resistant phenotypes from both genes were not associated with ribosomes known to be target sites of Blasticidin S, when analyzed by poly(U)-directed polyphenylalanine synthesis. The resistant mechanisms of the mutations are discussed in this paper.