Interaction of alpha-agglutinin with Saccharomyces cerevisiae a cells.

Binding of Saccharomyces cerevisiae alpha-agglutinin to target a cells was assayed by agglutination inhibition and 125I-alpha-agglutinin binding. The assays showed characteristics of equilibrium binding, namely saturability, competability, and the establishment of a kinetic endpoint in the presence of free alpha-agglutinin and free receptor. The binding was heterogeneous, displaying strong binding (10(9) liters/mol) and a weaker interaction. There were about 2 X 10(4) strong binding sites per a cell. Denaturing gels displayed identical labeled species binding to the a cells in the weak and strong interactions. Furthermore, weakly bound material could subsequently bind tightly to fresh a cells, implying that the same species of alpha-agglutinin was bound in the two states.     

Mating type-specific cell-cell recognition of Saccharomyces cerevisiae: cell wall attachment and active sites of a- and alpha-agglutinin.

Mating type-specific agglutination of Saccharomyces cerevisiae a and alpha cells depends on the heterophilic interaction of two cell surface glycoproteins, the gene products of AG alpha 1 and AGA2. Evidence is presented with immunogold labelling that the alpha-agglutinin is part of the outer fimbrial cell wall coat. The a-agglutinin is bound via two S-S bridges (Cys7 and Cys50) to a cell wall component, most probably the gene product of AGA1. His273 of alpha-agglutinin has previously been shown to be essential for a- and alpha-agglutinin interaction and a model based on two opposing ion-pairs had been proposed. By site-directed mutagenesis this possibility has now been excluded. With the help of various peptides, either chemically synthesized, obtained by proteolysis of intact glycosylated a-agglutinin or prepared from a fusion protein expressed in Escherichia coli, the biologically active region of a-agglutinin was located at the C-terminus of the molecule. A peptide consisting of the C-terminal 10 amino acids (GSPIN-TQYVF) was active in nanomolar concentrations. Saccharide moieties, therefore, are not essential for the mating type-specific cell-cell interaction; glycosylated peptides are, however, four to five times more active than non-glycosylated ones. Comparisons of the recognition sequences of the S. cerevisiae agglutinins with that of the Dictyostelium contact site A glycoprotein (gp80), as well as with those of the various families of cell adhesion molecules of higher eucaryotes, have been made and are discussed.     

Cell surface anchorage and ligand-binding domains of the Saccharomyces cerevisiae cell adhesion protein alpha-agglutinin, a member of the immunoglobulin superfamily.

alpha-Agglutinin is a cell adhesion glycoprotein expressed on the cell wall of Saccharomyces cerevisiae alpha cells. Binding of alpha-agglutinin to its ligand a-agglutinin, expressed by a cells, mediates cell-cell contact during mating. Analysis of truncations of the 650-amino-acid alpha-agglutinin structural gene AG alpha 1 delineated functional domains of alpha-agglutinin. Removal of the C-terminal hydrophobic sequence allowed efficient secretion of the protein and loss of cell surface attachment. This cell surface anchorage domain was necessary for linkage to a glycosyl phosphatidylinositol anchor. A construct expressing the N-terminal 350 amino acid residues retained full a-agglutinin-binding activity, localizing the binding domain to the N-terminal portion of alpha-agglutinin. A 278-residue N-terminal peptide was inactive; therefore, the binding domain includes residues between 278 and 350. The segment of alpha-agglutinin between amino acid residues 217 and 308 showed significant structural and sequence similarity to a consensus sequence for immunoglobulin superfamily variable-type domains. The similarity of the alpha-agglutinin-binding domain to mammalian cell adhesion proteins suggests that this structure is a highly conserved feature of adhesion proteins in diverse eukaryotes.     

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.     

Identification of glycoprotein components of alpha-agglutinin, a cell adhesion protein from Saccharomyces cerevisiae.

Several glycoproteins which inhibit the agglutinability of Saccharomyces cerevisiae mating type a cells were partially purified from extracts of mating type alpha cells. These proteins, called alpha-agglutinin, were labeled with 125I-Bolton-Hunter reagent. The labeled alpha-agglutinin showed specific binding to a cells. Such specific binding approached saturation with respect to agglutinin or cells and was inhibited in the presence of excess unlabeled alpha-agglutinin. Nonspecific binding was similar in a and alpha cells, was neither saturable nor competable, and was three- to fourfold less than the specific binding to a cells at maximum tested agglutinin concentrations. The major a-specific binding species had a low electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels and had an apparent molecular weight of 155,000 by rate zonal centrifugation. Endo-N-acetylglucosaminidase H digestion of the purified glycoprotein complex converted the low-mobility material to four major and several minor bands which were resolved by polyacrylamide gel electrophoresis. All but two minor peptides bound specifically to a cells. Analyses of agglutinin from mnn mutants confirmed the deglycosylation results in suggesting that the N-linked carbohydrate portion of alpha-agglutinin was not necessary for activity.     

Interaction of alpha-agglutinin and a-agglutinin, Saccharomyces cerevisiae sexual cell adhesion molecules

alpha-Agglutinin and a-agglutinin are complementary cell adhesion glycoproteins active during mating in the yeast Saccharomyces cerevisiae. They bind with high affinity and high specificity: cells of opposite mating types are irreversibly bound by a few pairs of agglutinins. Equilibrium and surface plasmon resonance kinetic analyses showed that the purified binding region of alpha-agglutinin interacted similarly with purified a-agglutinin and with a-agglutinin expressed on cell surfaces. At 20 degrees C, the K(D) for the interaction was 2 x 10(-9) to 5 x 10(-9) M. This high affinity was a result of a very low dissociation rate ( approximately 2.6 x 10(-4) s(-1)) coupled with a low association rate (= 5 x 10(4) M(-1) s(-1)). Circular-dichroism spectroscopy showed that binding of the proteins was accompanied by measurable changes in secondary structure. Furthermore, when binding was assessed at 10 degrees C, the association kinetics were sigmoidal, with a very low initial rate. An induced-fit model of binding with substantial apposition of hydrophobic surfaces on the two ligands can explain the observed affinity, kinetics, and specificity and the conformational effects of the binding reaction.     

Saccharomyces cerevisiae structural cell wall mannoprotein

A novel mannoprotein fraction with an average molecular weight of 180 000 has been isolated from Saccharomyces cerevisiae mnn9 mutant cell wall that was solubilized by beta-glucanase digestion. The same material could be extracted from purified wall fragments with 1% sodium dodecyl sulfate. The protein component, 12% by weight, is rich in proline, whereas the carbohydrate, mainly mannose, is about evenly distributed between asparagine and hydroxyamino acids. Endoglucosaminidase H digestion of the isolated mannoprotein reduced its average molecular weight to 150 000, but the mannoprotein, while still embedded in the cell wall, was inaccessible to the enzyme. Biosynthesis and translocation of the mannoprotein were investigated by following incorporation of [3H]proline into this fraction. In the presence of tunicamycin, both mnn9 and wild-type X2180 cells made a mannoprotein fraction with an average molecular weight of 140 000, whereas in the absence of the glycosylation inhibitor, the mnn9 mutant made material with a molecular weight of 180 000 and the mannoprotein made by wild-type cells was too large to penetrate the polyacrylamide gel. Although the cell wall mannoprotein was resistant to heat and proteolytic enzymes, attempts to isolate the carbohydrate-free component failed to yield any characteristic peptide material.     

Saccharomyces cerevisiae a- and alpha-agglutinin: characterization of their molecular interaction.

An O-glycosylated protein of approximately 18 kDa responsible for mating type specific agglutination has been isolated from Saccharomyces cerevisiae a cells, purified to homogeneity and via peptide sequences the gene was cloned by PCR. An open reading frame codes for a protein of 69 amino acids. A minimum of five serine and five threonine residues of the mature protein are glycosylated. alpha-Agglutinin is a highly N-glycosylated protein of approximately 250 kDa. Both purified agglutinins form a specific 1:1 complex in vitro. Pretreatment of alpha-agglutinin, but not of alpha-agglutinin, with diethylpyrocarbonate (DEPC) prevents formation of the complex; treatment of alpha-agglutinin in the presence of alpha-agglutinin protects the former from DEPC inactivation. By carboxy terminal shortening of the alpha-agglutinin gene and by replacing three of its eight histidyl residues by arginine, the active region of alpha-agglutinin for interaction with alpha-agglutinin has been defined. Neither the N- nor the O-linked saccharides of the two agglutinins seem to be essential for their interaction.     

Morphogenetic pathway of spore wall assembly in Saccharomyces cerevisiae

The Saccharomyces cerevisiae spore is protected from environmental damage by a multilaminar extracellular matrix, the spore wall, which is assembled de novo during spore formation. A set of mutants defective in spore wall assembly were identified in a screen for mutations causing sensitivity of spores to ether vapor. The spore wall defects in 10 of these mutants have been characterized in a variety of cytological and biochemical assays. Many of the individual mutants are defective in the assembly of specific layers within the spore wall, leading to arrests at discrete stages of assembly. The localization of several of these gene products has been determined and distinguishes between proteins that likely are involved directly in spore wall assembly and probable regulatory proteins. The results demonstrate that spore wall construction involves a series of dependent steps and provide the outline of a morphogenetic pathway for assembly of a complex extracellular structure.     

A constitutive role for GPI anchors in Saccharomyces cerevisiae: cell wall targeting

GPI anchors are widely represented among organisms and have several cellular functions. It has been proposed that in yeast there are two groups of GPI proteins: plasma membrane-resident proteins, such as Gas1p or Yap3p, and cell wall-targeted proteins, such as Tir1p or alpha-agglutinin. A model has been proposed for the plasma membrane retention of proteins from the first group because of a dibasic motif located just upstream of the GPI-anchoring signal. The results we report here are not in agreement with such a model as we show that constructs containing the C-terminal parts of Gas1p and Yap3p are also targeted to the cell wall. We also detect the genuine Gas1p after cell wall treatment with Quantazyme or Glucanex glycanases. In addition, we show that the GPI-anchoring signal from the human placental alkaline phosphatase (PLAP) is not compatible with the yeast machinery unless the human transamidase hGpi8p is co-expressed. In this condition, this human signal is able to target a protein to the cell wall. Moreover, TIR1 proved to be a multicopy suppressor of Deltagas1 mutation. The present findings suggest a constitutive role for GPI anchors in yeast: the cell wall targeting of proteins.     

Identification of two Saccharomyces cerevisiae cell wall mannan chemotypes

We have obtained evidence for two structurally and antigenically different Saccharomyces cerevisiae cell wall mannans. One, which occurs widely and is found in S. cerevisiae strain 238C, is already known to be a neutral mannan which yields mannose, mannobiose, mannotriose, and mannotetraose on acetolysis of the (1 --> 6)-linked backbone. The other, which was found in S. cerevisiae brewer's strains, is a phosphomannan with a structure very similar to that of Kloeckera brevis mannan. S. cerevisiae (brewer's yeast strain) was agglutinated by antiserum prepared against Kloeckera brevis cells. The mannan, isolated from a proteolytic digest of the cell wall of the former, did not react with S. cerevisiae 238C antiserum, whereas it cross-reacted strongly with K. brevis antiserum. Controlled acetolysis cleaved the (1 --> 6)-linkages in the polysaccharide backbone and released mannose, mannobiose, mannotriose, and mannotriose phosphate. Mild acid treatment of the phosphomannan hydrolyzed the phosphodiester linkage, yielding phosphomonoester mannan and mannose. The resulting phosphomonoester mannan reacted with antiserum prepared against K. brevis possessing monoester phosphate groups on the cell surface. alpha-d-Mannose-1-phosphate completely inhibited the precipitin reaction between brewer's yeast mannan and the homologous antiserum. Flocculent and nonflocculent strains of this yeast were shown to have similar structural and immunological properties.     

Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity.

A beta-glucanase (Z-glucanase) from Zymolyase was freed from a protease (Z-protease) by affinity chromatography on alpha 2-macroglobulin-Sepharose columns and used to solubilize proteins from isolated cell walls of Saccharomyces cerevisiae. The cell wall proteins were labeled with 125I and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The bulk of the labeled material had very low mobility. Its mannoprotein nature was demonstrated by precipitation with specific antibodies and by conversion to a band with an average molecular weight of 94,000 after incubation with endo-beta-N-acetylglucosaminidase. The intact mannoproteins were hydrolyzed by Z-protease, but were resistant to the enzyme when the carbohydrate was first removed by endo-beta-N-acetylglucosaminidase. In intact cells, lysis of cell walls by Z-glucanase required a previous incubation with z-protease, which led to solubilization of most of the 125I-labeled proteins. Other proteases that did not attack the cell wall mannoproteins were unable to substitute for Z-protease. The specific effect of Z-protease is consistent with the notion that mannoproteins form a surface layer of the cell wall that penetrates the wall to some depth and shields glucans from attack by Z-glucanase. Mannoproteins, however, do not appear to cover the inner face of the cell wall, because isolated cell walls, in contrast to intact cells, were completely solubilized by Z-glucanase in the absence of protease. The function of mannoproteins in determining cell wall porosity was highlighted by the finding that horseradish peroxidase (Mr, 40,000) causes lysis of cells that had been treated with Z-protease. Depletion of mannoproteins by Z-protease also resulted in the disappearance of a darkly stained surface layer of the cell wall, as observed by electron microscopy. Other agents that facilitate cell lysis by Z-glucanase, such as 2-mercaptoethanol, digitonin, and high concentrations of salts, caused little or no solubilization of mannoprotein. We assume that they perturb and loosen the structure of the mannoprotein network, thereby increasing its porosity. The implications of our results for the construction of the yeast cell wall and the anchoring of mannoprotein to the cell are discussed.     

啤酒酵母自溶与细胞壁关系

啤酒酿造过程中正常情况下的酵母不会发生自溶,但如果细胞衰老死亡或操作不当则会出现酵母自溶现象.虽然自溶的细胞在生产中占的比例较小,但自溶后的酵母将胞内物质释放到酒液中,产生酵母味,严重影响了啤酒的口感和外观质量.早期研究认为酵母细胞壁在酵母自溶过程中不发生水解,即自溶后的细胞仅剩下空的细胞外壳.现在研究发现酵母在自溶时其细胞壁也会发生水解作用,葡聚糖酶将构成细胞壁成分的葡聚糖进行分解,分解产物最终释放到酒液里和细胞质内物质共同影响啤酒的质量.     

Identification of three mannoproteins in the cell wall of Saccharomyces cerevisiae.

Three glucanase-extractable cell wall proteins from Saccharomyces cerevisiae were purified, and their N-terminal amino acid sequences were determined. With this information, we were able to assign gene products to three known open reading frames (ORFs). The N-terminal sequence of a 55-kDa mannoprotein corresponded with the product of ORF YKL096w, which we named CWP1 (cell wall protein 1). A 80-kDa mannoprotein was identified as the product of the TIP1 gene, and a 180-kDa mannoprotein corresponded to the product of the ORF YKL444, which we named CWP2. CWP1, TIP1, and CWP2 encode proteins of 239, 210, and 92 amino acids, respectively. The C-terminal regions of these proteins all consist for more than 40% of serine/threonine and contain putative glycosylphosphatidylinositol attachment signals. Furthermore, Cwp1p and Tip1p were shown to carry a beta 1,6-glucose-containing side chain. The cwp2 deletion mutant displayed an increased sensitivity to Congo red, calcofluor white, and Zymolyase. Electron microscopic analysis of the cwp2 deletion mutant showed a strongly reduced electron-dense layer on the outside of the cell wall. These results indicate that Cwp2p is a major constituent of the cell wall and plays an important role in stabilizing the cell wall. Depletion of Cwp1p or Tip1p also caused increased sensitivities to Congo red and calcofluor white, but the effects were less pronounced than for cwp2 delta. All three cell wall proteins show a substantial homology with Srp1p, which also appears to be localized in the cell wall. We conclude that these four proteins are small structurally related cell wall proteins.     

A glutamine synthetase mutant of Saccharomyces cerevisiae shows defect in cell wall

To study the organization and biosynthesis of the yeast cell wall, hypo-osmolarity-sensitive mutants of Saccharomyces cerevisiae were analyzed. Cells of JS4 were irregular in shape and fragile. Calcofluor staining and quantitative analysis indicated that the chitin content was reduced. By DNA cloning and genetic analysis, the mutation hpo1-1 was found to be allelic to GLN1 which encodes glutamine synthetase. The glutamine content was significantly low in JS4, and the mutant was recovered from the cell wall defect by supplying glutamine in the medium. Partial inhibition of glutamine synthetase by phosphinothricin also induced defects in the cell wall. These results indicate that the shortage of glutamine affects cell wall integrity prior to other cellular functions.     

Cell wall assembly in Saccharomyces cerevisiae.

An extracellular matrix composed of a layered meshwork of beta-glucans, chitin, and mannoproteins encapsulates cells of the yeast Saccharomyces cerevisiae. This organelle determines cellular morphology and plays a critical role in maintaining cell integrity during cell growth and division, under stress conditions, upon cell fusion in mating, and in the durable ascospore cell wall. Here we assess recent progress in understanding the molecular biology and biochemistry of cell wall synthesis and its remodeling in S. cerevisiae. We then review the regulatory dynamics of cell wall assembly, an area where functional genomics offers new insights into the integration of cell wall growth and morphogenesis with a polarized secretory system that is under cell cycle and cell type program controls.     

Alteration of cell wall structure in Saccharomyces cerevisiae and Saccharomyces bayanus during autolysis

Summary After culture in a synthetic and in a wine medium, the autolysis of Saccharomyces cerevisiae and Saccharomyces bayanus produced typical cell wall alterations depending on the yeast growth conditions. After growth in a wine medium, cell wall thickness did not change in either of the two yeasts even when there is an important loss of amino acids and glucans. This loss of wall material and especially of glucan involved a slackening of wall structures. The thickness of cell wall of yeast grown in a synthetic medium decreased by 50% after autolysis. This change was the consequence of a loss of amino acids and sugars which more specifically were constituents of the peripheral layer of the wall.     

Chromosomal mutants of Saccharomyces cerevisiae affecting the cell wall binding site for killer factor.

Fifty-two killer, factor-resistant, nuclear mutants were isolated from sensitive strains of yeast and assorted into three functional groups. All but one mutant owed their resistance to an alteration in the cell wall binding site for killer. In several mutant strains, an alteration at the site of killer binding was associated with a change in the susceptibility of the cell wall to degradation by glusulase. The killer-binding site could be inactivated by periodate but not by pronase treatment. The nature of the site is discussed.