The F658G substitution in Saccharomyces cerevisiae cohesin Irr1/Scc3 is semi-dominant in the diploid and disturbs mitosis, meiosis and the cell cycle

The sister chromatid cohesion complex of Saccharomyces cerevisiae includes chromosomal ATPases Smc1p and Smc3p, the kleisin Mcd1p/Scc1p, and Irr1p/Scc3p, the least studied component. We have created an irr1-1 mutation (F658G substitution) which is lethal in the haploid and semi-dominant in the heterozygous diploid irr1-1/IRR1. The mutated Irr1-1 protein is present in the nucleus, its level is similar to that of wild-type Irr1p/Scc3p and it is able to interact with chromosomes. The irr1-1/IRR1 diploid exhibits mitotic and meiotic chromosome segregation defects, irregularities in mitotic divisions and is severely affected in meiosis. These defects are gene-dosage dependent, and experiments with synchronous cultures suggest that they may result from the malfunctioning of the spindle assembly checkpoint. The partial structure of Irr1p/Scc3p was predicted and the F658G substitution was found to induce marked changes in the general shape of the predicted protein. Nevertheless, the mutant protein retains its ability to interact with Scc1p, another component of the cohesin complex, as shown by coimmunoprecipitation.     

Additional copies of the NOG2 and IST2 genes suppress the deficiency of cohesin Irr1p/Scc3p in Saccharomyces cerevisiae

The protein encoded by the IRR1/SCC3 gene is an element of the cohesin complex of Saccharomyces cerevisiae, responsible for establishing and maintaining sister chromatid cohesion during mitotic cell division. We noticed previously that lowering the level of expression of IRR1/SCC3 affects colony formation on solid support. Here we describe two dosage suppressors (IST2, NOG2) overcoming the inability to form colonies of an Irr1p-deficient strain. Ist2 is probably involved in osmotolerance, Nog2p is a putative GTPase required for 60S ribosomal subunit maturation, but may also participate in mRNA splicing.     

The G1 cyclin Cln3p controls vacuolar biogenesis in Saccharomyces cerevisiae

How organelle biogenesis and inheritance is linked to cell division is poorly understood. In the budding yeast Saccharomyces cerevisiae the G(1) cyclins Cln1,2,3p control initiation of cell division. Here we show that Cln3p controls vacuolar (lysosomal) biogenesis and segregation. First, loss of Cln3p, but not Cln1p or Cln2p, resulted in vacuolar fragmentation. Although the vacuoles of cln3delta cells were fragmented, together they occupied a large space, which accounted for a significant fraction of the overall cell size increase in cln3delta cells. Second, cytosol prepared from cells lacking Cln3p had reduced vacuolar homotypic fusion activity in cell-free assays. Third, vacuolar segregation was perturbed in cln3delta cells. Our findings reveal a novel role for a eukaryotic G(1) cyclin in cytoplasmic organelle biogenesis and segregation.     

Incorporation of Sed1p into the cell wall of Saccharomyces cerevisiae involves KRE6

KRE6 (YPR159W) encodes a Golgi membrane protein required for normal beta-1,6-glucan levels in the cell wall. A functional Kre6p is necessary for cell wall protein accumulation in response to changing metabolic conditions. The product of the SED1 (YDR077W) gene is a stress-induced GPI-cell wall protein. Successful incorporation of HA-tagged Sed1p into the cell wall involves KRE6. The double-mutant sed1 kre6 has a reduced growth rate, increased flocculation and increased sensitivity to Zymolyase. A similar phenotype is found in mutants defective in glycosyl-phosphatidyl-insositol (GPI) anchor assembly. These findings support the theory that Kre6p could function as a transglucosylase that allows the incorporation of proteins with a GPI anchor into the cell wall.     

Amyloid-like properties of Saccharomyces cerevisiae cell wall glucantransferase Bgl2p

Glucantransferase Bgl2p is a major conserved cell wall constituent described for a wide range of yeast species. In the baker’s yeast Saccharomyces cerevisiae it is the only non-covalently bound cell wall protein that cannot be released from cell walls by sequential SDS and trypsin treatment. It contains 7 amyloidogenic determinants. Circular dichroism analysis and fluorescence spectroscopy with thioflavin T indicate the presence of β-sheet structures in Bgl2p isolates. Bgl2p forms fibrils, a process that is enforced in the presence of other cell wall components. Thus the data obtained is the first evidence for amyloid-like properties of yeast cell wall protein – glucantransferase Bgl2p.     

Bph1p, the Saccharomyces cerevisiae homologue of CHS1/beige, functions in cell wall formation and protein sorting

Mutations in the Chediak-Higashi syndrome gene (CHS1) and its murine homologue Beige result in the formation of enlarged lysosomes. BPH1 (Beige Protein Homologue 1) encodes the Saccharomyces cerevisiae homologue of CHS1/Beige. BPH1 is not essential and the encoded protein was found to be both cytosolic and peripherally bound to a membrane. Neither disruption nor overexpression of BPH1 affected vacuole morphology as assessed by fluorescence microscopy. The deltabph1 strain showed an impaired growth on defined synthetic media containing potassium acetate buffered below pH 4.25, increased sensitivity to calcofluor white, and increased agglutination in response to low pH. A library screen identified VPS9, FLO1, FLO9, BTS1 and OKP1 as high copy suppressors of the growth defect of deltabph1 on both low pH potassium acetate and calcofluor white. The deltabph1 strain demonstrated a mild defect in sorting vacuolar components, including increased secretion of carboxypeptidase Y and missorting of alkaline phosphatase. Overexpression of VPS9, BTS1 and OKP1 suppressed the carboxypeptidase Y secretion defect of deltabph1. Overexpression of BPH1 was found to suppress the calcofluor white sensitivity of a class E VPS deletion strain, deltavta1. Together, these data suggest that Bph1p associates with a membrane and is involved in protein sorting and cell wall formation.     

Dynamics of cell wall structure in Saccharomyces cerevisiae

The cell wall of Saccharomyces cerevisiae is an elastic structure that provides osmotic and physical protection and determines the shape of the cell. The inner layer of the wall is largely responsible for the mechanical strength of the wall and also provides the attachment sites for the proteins that form the outer layer of the wall. Here we find among others the sexual agglutinins and the flocculins. The outer protein layer also limits the permeability of the cell wall, thus shielding the plasma membrane from attack by foreign enzymes and membrane-perturbing compounds. The main features of the molecular organization of the yeast cell wall are now known. Importantly, the molecular composition and organization of the cell wall may vary considerably. For example, the incorporation of many cell wall proteins is temporally and spatially controlled and depends strongly on environmental conditions. Similarly, the formation of specific cell wall protein-polysaccharide complexes is strongly affected by external conditions. This points to a tight regulation of cell wall construction. Indeed, all five mitogen-activated protein kinase pathways in bakers' yeast affect the cell wall, and additional cell wall-related signaling routes have been identified. Finally, some potential targets for new antifungal compounds related to cell wall construction are discussed.     

Structure of the glucan-binding sugar chain of Tip1p, a cell wall protein of Saccharomyces cerevisiae

Tip1p is one of the major cell wall mannoproteins of Saccharomyces cerevisiae and is presumed to be synthesized as a glycosylphosphatidylinositol (GPI)-anchored form. We purified Tip1p from a glucanase extract of yeast cell walls and analyzed the sugar chain involved in the cell wall linkage. One mol of glucanase-extracted Tip1p contained 7.5 mol of glucose derived from glucan and 1 mol of ethanolamine, a component of the GPI anchor. One mol of the C-terminal peptide of Tip1p digested with Achromobacter protease I also contained 7.9 mol of glucose and 1 mol of ethanolamine. On the other hand, Tip1p contained no glucosamine, which is a component of the GPI anchor. The glucan-binding sugar chain of Tip1p was released by hydrazinolysis and isolated. This sugar chain contained ethanolamine with a free amino group and a glucose reducing end, but no mannose reducing end. Phosphodiesterase treatment eliminated the free amino group from this sugar chain, suggesting that a phosphodiester bond exists between the ethanolamine and the glucan remnant. These results indicate (1) the glucan-binding sugar chain of Tip1p is a GPI derivative, and (2) the GPI anchor is cleaved at the glycosyl moiety, and the resultant mannose reducing end is probably used to link Tip1p to cell wall glucan.     

Saccharomyces cerevisiae Pra1p/Yip3p interacts with Yip1p and Rab proteins.

The regulation of membrane traffic involves the Rab family of Ras-related GTPases, of which there are a total of 11 members in the yeast Saccharomyces cerevisiae. Previous work has identified PRA1 as a dual prenylated Rab GTPase and VAMP2 interacting protein [Martinic et al. (1999) J. Biol. Chem. 272, 26991-26998]. In this study we demonstrate that the yeast counterpart of PRA1 interacts with Rab proteins and with Yip1p, a membrane protein of unknown function that has been reported to interact specifically with the Rab proteins Ypt1p and Ypt31p. Yeast Pra1p/Yip3p is a factor capable of biochemical interaction with a panel of different Rab proteins and does not show in vitro specificity for any particular Rab. The interactions between Pra1p/Yip3p and Rab proteins are dependent on the presence of the Rab protein C-terminal cysteines and require C-terminal prenylation.     

G1/S cyclin-dependent kinase regulates small GTPase Rho1p through phosphorylation of RhoGEF Tus1p in Saccharomyces cerevisiae

Rho1p is an essential small GTPase that plays a key role in the morphogenesis of Saccharomyces cerevisiae. We show here that the activation of Rho1p is regulated by a cyclin-dependent kinase (CDK). Rho1p is activated at the G1/S transition at the incipient-bud sites by the Cln2p (G1 cyclin) and Cdc28p (CDK) complex, in a process mediated by Tus1p, a guanine nucleotide exchange factor for Rho1p. Tus1p interacts physically with Cln2p/Cdc28p and is phosphorylated in a Cln2p/Cdc28p-dependent manner. CDK phosphorylation consensus sites in Tus1p are required for both Cln2p-dependent activation of Rho1p and polarized organization of the actin cytoskeleton. We propose that Cln2p/Cdc28p-dependent phosphorylation of Tus1p is required for appropriate temporal and spatial activation of Rho1p at the G1/S transition.     

Enolase is present in the cell wall of Saccharomyces cerevisiae

We have reported that the macrophage-like cell line J774.1, when infected with the periodontopathic bacterium Actinobacillus actinomycetemcomitans, undergoes apoptosis. In this study, we examined whether stimulation of J774.1 cells with lipopolysaccharide (LPS) before the infection affects the subsequent apoptosis. Cytotoxicity on the LPS-stimulated cells was about half of the unstimulated control cells. DNA fragmentation in the LPS-stimulated cells was also significantly lower than in the control cells, whereas it was increased to a level similar to that of the control cells by addition of a nitric oxide (NO) inhibitor. In addition, significantly smaller numbers of live A. actinomycetemcomitans were recovered from the LPS-stimulated macrophages at 8 h after the infection as compared with the control cells. These findings suggest that the inhibitory effect of LPS on apoptosis results from an enhanced NO-mediated bactericidal activity.     

Protein N-glycosylation determines functionality of the Saccharomyces cerevisiae cell wall integrity sensor Mid2p

The fungal cell wall is a highly dynamic structure that is essential to maintain cell shape and stability. Hence in yeasts and fungi cell wall integrity is tightly controlled. The Saccharomyces cerevisiae plasma membrane protein Mid2p is a putative mechanosensor that responds to cell wall stresses and morphological changes during pheromone induction. The extracellular domain of Mid2p, which is crucial to sensing, is highly O- and N-glycosylated. We showed that O-mannosylation is determining stability of Mid2p. If and how N-glycosylation is linked to Mid2p function was unknown. Here we demonstrate that Mid2p contains a single high mannose N-linked glycan at position Asn-35. The N -glycan is located close to the N-terminus and is exposed from the plasma membrane towards the cell wall through a highly O-mannosylated domain that is predicted to adopt a rod-like conformation. In contrast to O-mannosylation, lack of the N-linked glycan affects neither, stability of Mid2p nor distribution at the plasma membrane during vegetative and sexual growth. However, non-N-glycosylated Mid2p fails to perceive cell wall challenges. Our data further demonstrate that both the extent of the N-linked glycan and its distance from the plasma membrane affect Mid2p function, suggesting the N -glycan to be directly involved in Mid2p sensing.     

Regulation of cell wall biogenesis in Saccharomyces cerevisiae: the cell wall integrity signaling pathway

The yeast cell wall is a strong, but elastic, structure that is essential not only for the maintenance of cell shape and integrity, but also for progression through the cell cycle. During growth and morphogenesis, and in response to environmental challenges, the cell wall is remodeled in a highly regulated and polarized manner, a process that is principally under the control of the cell wall integrity (CWI) signaling pathway. This pathway transmits wall stress signals from the cell surface to the Rho1 GTPase, which mobilizes a physiologic response through a variety of effectors. Activation of CWI signaling regulates the production of various carbohydrate polymers of the cell wall, as well as their polarized delivery to the site of cell wall remodeling. This review article centers on CWI signaling in Saccharomyces cerevisiae through the cell cycle and in response to cell wall stress. The interface of this signaling pathway with other pathways that contribute to the maintenance of cell wall integrity is also discussed.     

The protein complex Ppz1p/Hal3p and nonsense suppression efficiency in the yeast Saccharomyces cerevisiae

It is known that nonsense suppression efficiency in yeast is controlled both genetically and epigenetically. As many components of translation machinery are represented by phosphoproteins, it depends, in particular, on the activity of kinases and phosphatases. The Ppz1p/Hal3p complex is among them. In this complex, the Ppz1p phosphatase is a catalytic subunit and Hal3p negatively regulates its function. The aim of this work was to study mechanisms which relate the activity of Ppz1p/Hal3p complex to nonsense suppression efficiency. In this study we used a genetic approach consisting of the analysis of nonsense suppression phenotype of strains over-expressing HAL3 or PPZ1 genes and also bearing deletions or mutant alleles of genes which presumably could participate in the manifestation of these over-expressions. We have shown that Hal3p inhibits not only Ppz1p, but also the homologous phosphatase Ppz2p. Our data indicate that Ppz2p is also involved in the control of nonsense suppression efficiency. In the course of search for Ppz1p target protein, it was shown that Ppz1p dephosphorylates at least two proteins participating in translation. Moreover, Ppz1p affects nonsense suppression efficiency not only due to its phosphatase activity but also due to another mechanism triggered by its interaction with Hsp70 chaperones.