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.

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.

Calcofluor is a fluorochrome that exhibits antifungal activity and a high affinity for yeast cell wall chitin. We isolated Saccharomyces cerevisiae mutants resistant to Calcofluor. The resistance segregated in a Mendelian fashion and behaved as a recessive character in all the mutants analyzed. Five loci were defined by complementation analysis. The abnormally thick septa between mother and daughter cells caused by Calcofluor in wild-type cells were absent in the mutants. The Calcofluor-binding capacity, observed by fluorescence microscopy, in a S. cerevisiae wild-type cells during alpha-factor treatment was also absent in some mutants and reduced in others. Staining of cell walls with wheat germ agglutinin-fluorescein complex indicated that the chitin uniformly distributed over the whole cell wall in vegetative or in alpha-factor-treated cells was almost absent in three of the mutants and reduced in the two others. Cell wall analysis evidenced a five- to ninefold reduction in the amount of chitin in mutants compared with that in the wild-type strain. The total amounts of cell wall mannan and beta-glucan in wild-type and mutant strains were similar; however, the percentage of beta-glucan that remained insoluble after alkali extraction was considerably reduced in mutant cells. The susceptibilities of the mutants and the wild-type strains to a cell wall enzymic lytic complex were rather similar. The in vitro levels of chitin synthase 2 detected in all mutants were similar to that in the wild type. The significance of these results is discussed in connection with the mechanism of chitin synthesis and cell wall morphogenesis in S. cerevisiae.

The major coat protein of the L-A double-stranded RNA virus of Saccharomyces cerevisiae covalently binds m7 GMP from 5' capped mRNAs in vitro. We show that this cap binding also occurs in vivo and that, while this activity is required for expression of viral information (killer toxin mRNA level and toxin production) in a wild-type strain, this requirement is suppressed by deletion of SKI1/XRN1/SEP1. We propose that the virus creates decapped cellular mRNAs to decoy the 5'-->3' exoribonuclease specific for cap- RNA encoded by XRN1. The SKI2 antiviral gene represses the copy numbers of the L-A and L-BC viruses and the 20S RNA replicon, apparently by specifically blocking translation of viral RNA. We show that SKI2, SKI3, and SKI8 inhibit translation of electroporated luciferase and beta-glucuronidase mRNAs in vivo, but only if they lack the 3' poly(A) structure. Thus, L-A decoys the SKI1/XRN1/SEP1 exonuclease directed at 5' uncapped ends, but translation of the L-A poly(A)- mRNA is repressed by Ski2,3,8p. The SKI2-SKI3-SKI8 system is more effective against cap+ poly(A)- mRNA, suggesting a (nonessential) role in blocking translation of fragmented cellular mRNAs.