Sexual agglutination in budding yeasts: structure, function, and regulation of adhesion glycoproteins.

The sexual agglutinins of the budding yeasts are cell adhesion proteins that promote aggregation of cells during mating. In each yeast species, complementary agglutinins are expressed by cells of opposite mating type that interact to mediate aggregation. Saccharomyces cerevisiae alpha-agglutinin and its analogs from other yeasts are single-subunit glycoproteins that contain N-linked and O-linked oligosaccharides. The N-glycosidase-sensitive carbohydrate is not necessary for activity. The proposed binding domain of alpha-agglutinin has features characteristic of the immunoglobulin fold structures of cell adhesion proteins of higher eukaryotes. The C-terminal region of alpha-agglutinin plays a role in anchoring the glycoprotein to the cell surface. The S. cerevisiae alpha-agglutinin and its analogs from other species contain multiple subunits; one or more binding subunits, which interact with the opposite agglutinin, are disulfide bonded to a core subunit, which mediates cell wall anchorage. The core subunits are composed of 80 to 95% O-linked carbohydrate. The binding subunits have less carbohydrate, and both carbohydrate and peptide play roles in binding. The alpha-agglutinin and alpha-agglutinin genes from S. cerevisiae have been cloned and shown to be regulated by the mating-type locus, MAT, and by pheromone induction. The agglutinins are necessary for mating under conditions that do not promote cell-cell contact. The role of the agglutinins therefore is to promote close interactions between cells of opposite mating type and possibly to facilitate the response to phermone, thus increasing the efficiency of mating. We speculate that they mediate enhanced response to sex pheromones by providing a synapse at the point of cell-cell contact, at which both pheromone secretion and cell fusion occur.     

Cell-cell recognition in yeast. Molecular nature of the sexual agglutinin from Saccharomyces kluyveri 17-cells

A previous study of Saccharomyces kluyveri 17-cell sexual agglutinin (alpha-agglutinin), solubilized by zymolyase (beta-glucanase) digestion of 17-cells and purified by affinity adsorption to immobilized 16-cell agglutinin (alpha-agglutinin), suggested that the major active component was a glycoprotein of 60,000 daltons and that a minor active component of 40,000 daltons was also present, possibly the result of proteolysis (Pierce, M., and Ballou, C. E. (1983) J. Biol. Chem. 258, 3576-3582). We now show that both of these active components are proteolytic fragments of a larger form with a molecular weight of 134,000, and that the latter is produced by proteolysis of a still larger species with a molecular weight of more than 200,000. Washed 17-cell wall fragments were labeled with 125I and digested with purified protease-free beta-1,3-glucanase, and the solubilized alpha-agglutinin was precipitated with antiserum raised against purified agglutinin containing a mixture of the 60,000- and 134,000-dalton forms. Gel electrophoresis in sodium dodecyl sulfate revealed a radioactive material with Mr greater than 200,000 that, on digestion with zymolyase containing an active protease, was converted sequentially to radioactive components with Mr = 134,000, 60,000, and 40,000.     

Alpha-agglutinin expression in Saccharomyces cerevisiae

A polyclonal antiserum raised against purified alpha-agglutinin was made specific for alpha-agglutinin after adsorption with a cells. The adsorbed antiserum identified alpha-agglutinin peptides on Western blots and bound to cell surface alpha-agglutinin, inhibiting the binding of alpha cells to a cells. Using the antibody, we have determined that 1) the surface distribution of alpha-agglutinin on alpha cells is polar, 2) about 5 x 10(4) molecules/cell are constitutively expressed on strain X2180-1B (alpha) cells, and 3) treatment of alpha cells with the sex pheromone a-factor causes an increase in cell surface alpha-agglutinin, consistent with the a-factor induced increase in cell agglutinability.     

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.     

Delineation of functional regions within the subunits of the Saccharomyces cerevisiae cell adhesion molecule a-agglutinin

a-Agglutinin from Saccharomyces cerevisiae is a cell adhesion glycoprotein expressed on the surface of cells of a mating type and consists of an anchorage subunit Aga1p and a receptor binding subunit Aga2p. Cell wall attachment of Aga2p is mediated through two disulfide bonds to Aga1p (Cappellaro, C., Baldermann, C., Rachel, R., and Tanner, W. (1994) EMBO J. 13, 4737-4744). We report here that purified Aga2p was unstable and had low molar specific activity relative to its receptor alpha-agglutinin. Aga2p co-expressed with a 149-residue fragment of Aga1p formed a disulfide-linked complex with specific activity 43-fold higher than Aga2p expressed alone. Circular dichroism of the complex revealed a mixed alpha/beta structure, whereas Aga2p alone had no periodic secondary structure. A 30-residue Cys-rich Aga1p fragment was partially active in stabilization of Aga2p activity. Mutation of either or both Aga2p cysteine residues eliminated stabilization of Aga2p. Thus the roles of Aga1p include both cell wall anchorage and cysteine-dependent conformational restriction of the binding subunit Aga2p. Mutagenesis of AGA2 identified only C-terminal residues of Aga2p as being essential for binding activity. Aga2p residues 45-72 are similar to sequences in soybean Nod genes, and include residues implicated in interactions with both Aga1p (including Cys(68)) and alpha-agglutinin.     

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.     

Pheromone induction of agglutination in Saccharomyces cerevisiae a cells.

a-Agglutinin, the cell surface sexual agglutinin of yeast a cells, was assayed by its ability to bind its complementary agglutinin, alpha-agglutinin. The specific binding of 125I-alpha-agglutinin to a cells treated with the sex pheromone alpha-factor was 2 to 2.5 times that of binding to a cells not treated with alpha-factor. Competition with unlabeled alpha-agglutinin revealed that the increased binding was due to increased cell surface expression of a-agglutinin, with no apparent change in the binding constant. The increase in site number was similar to the increase in cellular agglutinability. Increased expression of a-agglutinin followed the same kinetics as the increase in cellular agglutinability, with a 10-min lag followed by a 15- to 20-min response time. Induction kinetics were similar in cells in phases G1 and G2 of the cell cycle. Maximal expression levels were similar in cells treated with excess pheromone and in cells exposed to pheromone after destruction of constitutively expressed a-agglutinin.     

Alpha mating type-specific expression of mutations leading to constitutive agglutinability in Saccharomyces cerevisiae.

Two mutants of Saccharomyces cerevisiae have been isolated and characterized. The mutants were constitutively agglutinable at 36 degrees C, the temperature at which wild-type cells agglutinate only after induction by mating pheromone. The mutant cells had other properties specific for the normal alpha cell type, i.e., conjugation with a cells, response to a mating pheromone, and production of alpha mating pheromone. The two mutations, cag1 and cag2, were recessive and expressed only in alpha cells. cag1 is linked very closely to the MAT locus, but cag2 is unlinked to the MAT locus. These cag mutations complemented ste3-1. These results indicate that CAG genes are novel alpha-specific genes involved in the regulation of sex agglutinin synthesis.     

Cell-Cell Recognition in Saccharomyces cerevisiae: Regulation of Mating-Specific Adhesion

Mating-specific adhesion between haploid yeast cells of opposite mating type (a and alpha) was studied by using a quantitative agar plate assay. Washed a and alpha cells that had not previously been exposed to their respective opposite mating type ("naive" cells) adhered relatively weakly. In water, only 5 to 10% of the a cells stuck tightly enough to alpha cells to give rise subsequently to diploid clones on the assay plates. Under optimum conditions (pH 6 to 7, at least 0.1 M Nacl or 0.01 M Mg(2+)), there was about 20% adhesion. Nevertheless, this weak binding defined a mating type-specific interaction because, even under optimum conditions, the homologous interactions (a with a and alpha with alpha) yielded only 3 to 5% cohesion. In contrast to these results, washed cells that had been preincubated in the cell-free culture medium of their opposite mating type ("preconditioned" cells) adhered quite strongly. The degree of adhesion between preconditioned cells (40 to 50%) was essentially unaffected by extremes of ionic strength, pH, and temperature and by the absence of divalent cation. This strong interaction was also mating type specific since cohesion between preconditioned cells of like mating type was only about 5%. The increase in agglutinability was obtained if only the a cells were preconditioned and could be induced by highly purified preparations of natural or synthetically prepared alpha-factor, an oligopeptide pheromone released by the alpha cells. The appearance of increased adhesiveness was blocked by an inhibitor of RNA synthesis and by an inhibitor of protein synthesis, but not by an inhibitor of polysaccharide synthesis. Adhesion between preconditioned cells could be inhibited by pretreatment with functionally univalent succinylated concanavalin A or with extracts from preconditioned cells of the opposite mating type. These results confirm in a quantitative manner that the recognition between conjugating cells of S. cerevisiae is a developmentally regulated event that is under the control of the mating type locus.     

Interaction of sialoglycoproteins with wheat germ agglutinin-sepharose of varying ratio of lectin to Sepharose. Use for the purification of mucin glycoproteins from membrane extracts

The influence of varying the amount of wheat germ agglutinin immobilized on Sepharose beads on the binding of glycoproteins to these beads was investigated. A series of wheat germ agglutinin-Sepharose gels containing between 0.10 and 10.0 mg of lectin/ml of gel was prepared, and the actual lectin content was established by acid hydrolysis of the gel followed by analysis of glycine, a major amino acid in wheat germ agglutinin. Affinity chromatography of labeled glycoproteins indicated that glycophorin bound to all the wheat germ agglutinin-Sepharose preparations. Fetuin, ovomucoid, and alpha 1-acid glycoprotein bound not at all or very poorly to gels with a low content of wheat germ agglutinin (less than 0.95 mg/ml). The specific binding of these glycoproteins increased with increasing lectin content on the gels, and on gels of high content (greater than 3 mg/ml) the binding was virtually quantitative. On chromatographing a mixture of glycophorin, alpha 1-acid glycoprotein, fetuin, and ovomucoid on wheat germ agglutinin-Sepharose, containing 0.08 mg of lectin/ml of gel, glycophorin was selectively retained on the gel. It was possible to purify glycophorin from an extract of human erythrocyte membranes in one step by chromatography on the above gel. By using the series of gels, it was demonstrated that Morris hepatoma 7777 membranes contained at least 4-fold more sialoglycoproteins which bound to low density wheat germ agglutinin-Sepharose compared to rat liver membranes. These hepatoma sialoglycoproteins were isolated, purified, and partially characterized as having a high proportion of O-linked sialyloligosaccharides. Our studies illustrate the use of low density wheat germ agglutinin-Sepharose gels both for the detection and for easy isolation of mucin-type glycoproteins from crude extracts of cells or membranes.     

Genetically controlled self-aggregation of cell-surface-engineered yeast responding to glucose concentration

We constructed an arming (cell-surface-engineered) yeast displaying two types of agglutinin (modified a-agglutinin and alpha-agglutinin) on the cell surface, with agglutination being independent of both mating type and pheromones. The modified a-agglutinin was artificially prepared by the fusion of the genes encoding Aga1p and Aga2p. The modified a-agglutinin could induce agglutination of cells displaying Agalpha1p (alpha-agglutinin). The upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL), active at a low glucose concentration, was used as the promoter to express the modified a-agglutinin- and alpha-agglutinin-encoding genes. The arming yeast displaying both agglutinins agglutinated and sedimented in response to decreased glucose concentration. When the glucose concentration was high, the arming yeast grew normally. In the late log phase, when the glucose concentration became very low, agglutination occurred suddenly and drastically and yeast cells sedimented completely. Sedimentation was confirmed by weighing the aggregated cells after filtration of the broth. Strains in which aggregation can be genetically controlled can be used in industrial processes in which the separation of yeast cells from the supernatant is necessary.     

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.     

Glycosyl phosphatidylinositol-dependent cross-linking of alpha- agglutinin and beta 1,6-glucan in the Saccharomyces cerevisiae cell wall

The cell adhesion protein alpha-agglutinin is bound to the outer surface of the Saccharomyces cerevisiae cell wall and mediates cell- cell contact in mating. alpha-Agglutinin is modified by addition of a glycosyl phosphatidylinositol (GPI) anchor as it traverses the secretory pathway. The presence of a GPI anchor is essential for cross- linking into the wall, but the fatty acid and inositol components of the anchor are lost before cell wall association (Lu, C.-F., J. Kurjan, and P. N. Lipke, 1994. A pathway for cell wall anchorage of Saccharomyces cerevisiae alpha-agglutinin. Mol. Cell. Biol. 14:4825- 4833). Cell wall association of alpha-agglutinin was accompanied by an increase in size and a gain in reactivity to antibodies directed against beta 1,6-glucan. Several kre mutants, which have defects in synthesis of cell wall beta 1,6-glucan, had reduced molecular size of cell wall alpha-agglutinin. These findings demonstrate that the cell wall form of alpha-agglutinin is covalently associated with beta 1,6- glucan. The alpha-agglutinin biosynthetic precursors did not react with antibody to beta 1,6-glucan, and the sizes of these forms were unaffected in kre mutants. A COOH-terminal truncated form of alpha- agglutinin, which is not GPI anchored and is secreted into the medium, did not react with the anti-beta 1,6-glucan. We propose that extracellular cross-linkage to beta 1,6-glucan mediates covalent association of alpha-agglutinin with the cell wall in a manner that is dependent on prior addition of a GPI anchor to alpha-agglutinin.     

AG alpha 1 is the structural gene for the Saccharomyces cerevisiae alpha-agglutinin, a cell surface glycoprotein involved in cell-cell interactions during mating.

We have cloned the alpha-agglutinin structural gene, AG alpha 1, by the isolation of alpha-specific agglutination-defective mutants, followed by isolation of a complementing plasmid. Independently isolated alpha-specific agglutination-defective mutations were in a single complementation group, consistent with biochemical results indicating that the alpha-agglutinin is composed of a single polypeptide. Mapping results suggested that the complementation group identified by these mutants is allelic to the ag alpha 1 mutation identified previously. Expression of AG alpha 1 RNA was alpha specific and inducible by a-factor. Sequences similar to the consensus sequences for positive control by MAT alpha 1 and pheromone induction were found upstream of the AG alpha 1 initiation codon. The AG alpha 1 gene could encode a 650-amino-acid protein with a putative signal sequence, 12 possible N-glycosylation sites, and a high proportion of serine and threonine residues, all of which are features expected for the alpha-agglutinin sequence. Disruption of the AG alpha 1 gene resulted in failure to express alpha-agglutinin and loss of cellular agglutinability in alpha cells. An Escherichia coli fusion protein containing 229 amino acids of the AG alpha 1 sequence was recognized by an anti-alpha-agglutinin antibody. In addition, the ability of this antibody to inhibit agglutination was prevented by this fusion protein. These results indicate that AG alpha 1 encodes alpha-agglutinin. Features of the AG alpha 1 gene product suggest that the amino-terminal half of the protein contains the a-agglutinin binding domain and that the carboxy-terminal half contains a cell surface localization domain, possibly including a glycosyl phosphatidylinositol anchor.     

Roles of sexual cell agglutination in yeast mass mating

The agalpha1 mutant MAT alpha cells specifically lack the cell surface alpha-type sexual agglutination substance, which is also called alpha-agglutinin. Because the mutant cells (MATalpha agalpha1) can not form aggregates with MATa cells, MATalpha agalpha1 cells are unable to mate with MATa cells when they are co-inoculated in a liquid medium, and the mating is attenuated on solid medium. The attenuated mating ability shown in the previous studies gave us a vague idea about a physiological function of the sexual agglutinability. In order to solve the question, mating behavior of MATalpha agalpha1 cells was investigated here under conditions where the contact between MATa and MAT alpha cells is assisted by physical methods. A synthetic mutation agalpha1::URA3 was constructed and used as well as agalpha1-1 for this study to ensure the genetic defect. When a mixture of MATa and MAT alpha cells was kept on filter membrane placed on relatively dry agar medium, even agalpha1::URA3 mutant cells mated as efficiently as the wild type (AGalpha1) cells did. On filter membrane placed on moist agar medium, agalpha1 mutants mated 10-fold less efficiently than wild type cells did. The mutant cells mated 10000-time less efficiently than the wild type cells in a pellet formed by brief low speed centrifugation. In contrast, the wild type MATalpha cells mated well under all conditions tested. Under the pellet condition, a mixture of MATa and MATalpha AG alpha1 cells formed an extended and cotton-like pellet while a mixture of MATa and MATalpha agalpha1 cells formed a compact and tight pellet. These results suggest that sexual cell agglutination contributes not only to cell contact between MATa and MAT alpha cells thereby stabilizing a-alpha cell pairs, but also to construction of a uniquely organized ultra structure favorable for zygote formation and subsequent growth of diploid cells. The mating specific extended pellet formation was observed also in 4 pairs of a and alpha strains in ascosporogenous yeast genera Hansenula and Pichia.     

Cell surface specific immunoglobulin inhibits alpha factor mediated morphogenesis in Saccharomyces cerevisiae

Immunoglobulins raised from Saccharomyces cerevisiae a and alpha mating type cell envelope preparations inhibited alpha factor mediated morphogenesis of the a cell without inhibiting normal cell division. The Ig responsible for this inhibition was absorbed to both a and alpha whole cells and heat-killed cells, indicating that the immunoglobulin binding sites were exposed on the cell surface and not mating type specific. Additionally, alpha factor mediated cell cycle arrest was not affected by the immunoglobulin preparations, implying that the immunoglobulin was not preventing alpha factor from binding to its receptor.     

Identification of a ligand-binding site in an immunoglobulin fold domain of the Saccharomyces cerevisiae adhesion protein alpha-agglutinin.

The Saccharomyces cerevisiae adhesion protein alpha-agglutinin (Ag alpha 1p) is expressed by alpha cells and binds to the complementary a-agglutinin expressed by a cells. The N-terminal half of alpha-agglutinin is sufficient for ligand binding and has been proposed to contain an immunoglobulin (Ig) fold domain. Based on a structural homology model for this domain and a previously identified critical residue (His292), we made Ag alpha 1p mutations in three discontinuous patches of the domain that are predicted to be in close proximity to His292 in the model. Residues in each of the three patches were identified that are important for activity and therefore define a putative ligand binding site, whereas mutations in distant loops had no effect on activity. This putative binding site is on a different surface of the Ig fold than the defined binding sites of immunoglobulins and other members of the Ig superfamily. Comparison of protein interaction sites by structural and mutational analysis has indicated that the area of surface contact is larger than the functional binding site identified by mutagenesis. The putative alpha-agglutinin binding site is therefore likely to identify residues that contribute to the functional binding site within a larger area that contacts a-agglutinin.     

Purification and characterization of the human brain insulin receptor

The insulin receptor from human brain cortex was purified by a combination monoclonal antibody affinity column and a wheat germ agglutinin column. This purified receptor preparation exhibited major protein bands of apparent Mr = 135,000 and 95,000, molecular weights comparable to those for the alpha and beta subunits of the purified human placental and rat liver receptors. A minor protein band of apparent Mr = 120,000 was also observed in the brain receptor preparation. Crosslinking of 125I-insulin to all three receptor preparations was found to preferentially label a protein of apparent Mr = 135,000. In contrast, cross-linking of 125I-labeled insulin-like growth factor I to the brain preparation preferentially labeled the protein of apparent Mr = 120,000. The purified brain insulin receptor was found to be identical with the placental insulin receptor in the amount of neuraminidase-sensitive sialic acid and reaction with three monoclonal antibodies to the beta subunit of the placental receptor. In contrast, a monoclonal antibody to the insulin binding site recognized the placental receptor approximately 300 times better than the brain receptor. These results indicate that the brain insulin receptor differs from the receptor in other tissues and suggests that this difference is not simply due to the amount of sialic acid on the receptor.     

Schistosoma mansoni: radiometric assay of lectin binding specificities of the major egg glycoprotein and its carbohydrate-rich fragment

The binding by lectins of the Schistosoma mansoni major egg glycoprotein and of a carbohydrate-rich fragment which is serologically cross-reactive with it was studied. The major egg glycoprotein was purified from a crude soluble egg antigen by a succession of affinity chromatography procedures on concanavalin A-sepharose and by ion-exchange chromatography. The carbohydrate-rich fragment was isolated by ultrafiltration of the crude glycoprotein fraction initially obtained from the crude soluble egg antigens. The major egg glycoprotein and the carbohydrate-rich fragment contain 77 and 92.5% carbohydrate, respectively. When radioiodinated and run on SDS-polyacrylamide gel electrophoresis, each of them exhibited a single peak with respective Rf values of 0.33 and 1.0, and their respective molecular weights were 70K and 10-13K. The binding of the radioiodinated major egg glycoprotein and the carbohydrate-rich fragment by peanut agglutinin, Ricinus communis agglutinin-60, wheat germ agglutinin, and lotus agglutinin was studied by double diffusion in agar, and by a radiometric solid-phase assay in which the lectins were used to coat microtiter plates. The latter assay was employed to determine the specificity of the binding by inhibition with the specific sugars. Both the major egg glycoprotein and the carbohydrate-rich fragment bound specifically to concanavalin A columns as indicated by their isolation procedure. They also bound specifically to peanut agglutinin, R. communis agglutinin 60, and lotus agglutinin, while binding by wheat germ agglutinin appeared not to be specific.(ABSTRACT TRUNCATED AT 250 WORDS)