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.     

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.     

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.     

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 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.     

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 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.     

Kinetic evidence for a critical rate of protein synthesis in the Saccharomyces cerevisiae yeast cell cycle

The kinetics of cell cycle initiation were measured at pH 2.7 for cells that had been arrested at the "start" step of cell division with the polypeptide pheromone alpha-factor. Cell cycle initiation was induced by the removal of alpha-factor. The rate at which cells completed start was identical to the rate of subsequent bud emergence. After short times of prearrest with alpha-factor (e.g. 5.2 h), the kinetics of bud emergence were biphasic, indicative of two subpopulations of cells that differed by greater than 10-fold in their rates of cell cycle initiation. The subpopulation that exhibited a slow rate of cell cycle initiation is comprised of cells that resided in G1 prior to start at the time of removal of alpha-factor, whereas the subpopulation that initiated the cell cycle rapidly is comprised of cells that had reached and become blocked at start. A critical concentration of cycloheximide was found to reintroduce slow budding cells into a population of 100% fast budding cells, suggesting that the two subpopulations differ with respect to attainment of a critical rate of protein synthesis that is necessary for the performance of start. Cycloheximide and an increase in the time of prearrest with alpha-factor had opposite effects on both the partitioning of cells between the two subpopulations and the net rate of protein synthesis per cell, consistent with this conclusion. Cell cycle initiation by the subpopulation of fast budding cells required protein synthesis even though the critical rate of protein synthesis had been achieved during arrest. It is concluded that alpha-factor inhibits the synthesis of and/or inactivates specific proteins that are required for the performance of start, but alpha-factor does not prevent attainment of the critical rate of protein synthesis.     

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.     

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.     

Sexual agglutination in Saccharomyces cerevisiae.

Treatment of either mating type of Saccharomyces cerevisiae with the appropriate sex pheromone increased cell-cell binding in a modified cocentrifugation assay. Constitutive agglutination of haploids was qualitatively similar to pheromone-induced agglutination. Regardless of exposure to pheromone, agglutinable combinations of cells exhibited maximal binding across similar ranges of ionic strength, pH, and temperature. Binding of all combinations was inhibited by 8 M urea, 1 M pyridine, or 0.05% sodium dodecyl sulfate. From alpha-cells we solubilized and partially purified an inhibitor of a-cell agglutinability. This inhibitor reversibly masked all a-cell adhesion sites and inactivated pheromone-treated and control cells with similar kinetics. The inhibitor behaved as a homogeneous species in heat inactivation experiments. Based on these results, we proposed a model for pheromone effects on agglutination in S. cerevisiae.     

Qualitative and quantitative interactions of lectins with untreated and neuraminidase-treated normal, wild-type, and temperature-sensitive polyoma-transformed fibroblasts.

The lectin receptors of confluently grown hamster BHK, wild type polyoma virus transformed PyBHK, and temperature-sensitive polyoma transformed ts3-PyBHK fibroblasts were investigated using cell agglutination, quantitative (125I)lectin binding, and ferritin-lectin labeling. PyBHK and permissively grown ts3-PyBHK cells agglutinated more strongly with Ricinus communis I agglutinin (RCA-I)compared to BHK and nonpermissively grown ts3-PyBHK, although saturation binding of (125I)RCA-I to these cells at 4 degrees resulted in a twofold difference in lectin-binding sites on BHK and nonpermissively grown ts3-PyBHK cells (1.0-1.3 x 10 7 sites/cell) compared to PyBHK and permissively grown ts3-PyBHK (0.4-0.6 x 10 7 sites/cell). These cells bound equivalent amounts of (125I)concanavalin A (0.8-1 x 10 7 sites/cell) and (125I)wheat germ agglutinin (1-2.2 x 10 7 sites/cell). Under these binding conditions little endocytosis occurred, as judged by the subsequent release of greater than 90% cell-bound (125I)RCA-I by the RCA-I inhibitor lactose and localization of ferritin-RCA-I exclusively to the extracellular plasma membrane surface. However, if the binding is performed at 22 degrees, only 50% of the bound lectin can be removed by lactose, and ferritin-RCA-I is localized inside the cell within endocytotic vesicles. The relative mobility of RCA-I receptors was examined on ts3-PyBHK cells by the ability of ferritin-RCA-I to induce clustering of its receptors at 22 degrees. RCA-I receptors on permissively grown ts3-PyBHK cells appeared to be more mobile than on nonpermissively grown cells. BHK and PyBHK cells were treated with neuraminidase, and the resulting enzyme-treated cells were assayed for lectin agglutinability and quantitative binding of RCA-I, concanavalin A, and wheat germ agglutinin. Neuraminidase treatment resulted in decreased concanavalin A and wheat germ agglutinability and a slight increase in RCA-I agglutinability. The enzyme-treated BHK and PyBHK cells bound less (125I)wheat germ agglutinin (2.8 x 10 6 and 2.2 x 10 6 sites/cell, respectively) and 2.5 and 6.2 times more (125I)RCA-I (2.5-3 x 10 7) and 3.5-4 x 10 7 sites/per cell, respectively). There was no change in the number of concanavalin A binding sites after neuraminidase treatment. The increase in RCA-I binding sites approximated the decrease in wheat germ agglutinin binding sites indicating that the predominant penultimate oligosaccharide residue to sialic acid on these cells is D-Gal.     

Morphogenic effects of alpha-factor on Saccharomyces cerevisiae a cells.

Saccharomyces cerevisiae mating type a cells enlarged and elongated when exposed to alpha-factor, a sex pheromone produced by mating-type alpha cells. This morphogensis required exogenous-D-glucose, nitrogen, and phosphate, and cells in exponential phase responded better than stationary-phase cells. Morphogenesis was blocked by cycloheximide and by inhibitors of cell wall biosynthesis such as 2-deoxy-D-glucose, 2-deoxy-2-fluoro-D-glucose, and 2-deoxy-2-fluoro-D-mannose, but not by polyoxin D. One to two hours after addition of pheromone, a cells became more susceptible to lysis by glucanases, a change that was dependendent on the dose of alpha-factor and was blocked by drugs that block morphogenesis. On the other hand, treatment with alpha-factor did not increase susceptibility to attack by trypsin, subtilisin, or exo-alpha-mannanase. Radioactive label, incorporated into cell wall polysaccharides during treatment with alpha-factor, was not secreted into the medium during morphogenesis. Analysis of the labeled wall polymers showed that alpha-factor-treated cells contain more glucan and less mannan than control cells, and that the mannan of treated cells contains an increased proportion of shorter side chains and unsubstituted backbone mannose units. Thin-section electron microscopy of treated cells revealed that the cell wall possesses a diffuse outer layer in the extension and is thinner at the tip.     

Functional expression of the yeast alpha-factor receptor in Xenopus oocytes

The STE2 gene of the yeast Saccharomyces cerevisiae encodes a 431-residue polypeptide that has been shown by chemical cross-linking and genetic studies to be a component of the receptor for the peptide mating pheromone, alpha-factor. To demonstrate directly that the ligand binding site of the alpha-factor receptor is comprised solely of the STE2 gene product, the STE2 protein was expressed in Xenopus oocytes. Oocytes microinjected with synthetic STE2 mRNA displayed specific surface binding for 35S-labeled alpha-factor (up to 40 sites/micron2/ng RNA). Oocytes injected with either STE2 antisense RNA or heterologous receptor mRNA (nicotinic acetylcholine receptor alpha, beta, gamma, and delta subunit mRNAs) showed no binding activity (indistinguishable from uninjected control oocytes). The apparent KD (7 nM) of the alpha-factor binding sites expressed on the oocyte surface, determined by competition binding studies, agreed with the values reported for intact yeast cells and yeast plasma membrane fractions. These findings demonstrate that the STE2 gene product is the only yeast polypeptide required for biogenesis of a functional alpha-factor receptor. Electrophysiological measurements indicated that the membrane conductance of oocytes injected with STE2 mRNA, or with both STE2 and GPA1 (encoding a yeast G protein alpha-subunit) mRNAs, did not change and was not affected by pheromone binding. Thus, the alpha-factor receptor, like mammalian G protein-coupled receptors, apparently lacks activity as an intrinsic or ligand-gated ion channel. This report is the first instance in which a membrane-bound receptor from a unicellular eukaryote has been expressed in a vertebrate cell.     

Quantitation of alpha-factor internalization and response during the Saccharomyces cerevisiae cell cycle.

The alpha-factor pheromone binds to specific cell surface receptors on Saccharomyces cerevisiae a cells. The pheromone is then internalized, and cell surface receptors are down-regulated. At the same time, a signal is transmitted that causes changes in gene expression and cell cycle arrest. We show that the ability of cells to internalize alpha-factor is constant throughout the cell cycle, a cells are also able to respond to pheromone throughout the cycle even though there is cell cycle modulation of the expression of two pheromone-inducible genes, FUS1 and STE2. Both of these genes are expressed less efficiently near or just after the START point of the cell cycle in response to alpha-factor. For STE2, the basal level of expression is modulated in the same manner.     

Localization and signaling of G(beta) subunit Ste4p are controlled by a-factor receptor and the a-specific protein Asg7p

Haploid yeast cells initiate pheromone signaling upon the binding of pheromone to its receptor and activation of the coupled G protein. A regulatory process termed receptor inhibition blocks pheromone signaling when the a-factor receptor is inappropriately expressed in MATa cells. Receptor inhibition blocks signaling by inhibiting the activity of the G protein beta subunit, Ste4p. To investigate how Ste4p activity is inhibited, its subcellular location was examined. In wild-type cells, alpha-factor treatment resulted in localization of Ste4p to the plasma membrane of mating projections. In cells expressing the a-factor receptor, alpha-factor treatment resulted in localization of Ste4p away from the plasma membrane to an internal compartment. An altered version of Ste4p that is largely insensitive to receptor inhibition retained its association with the membrane in cells expressing the a-factor receptor. The inhibitory function of the a-factor receptor required ASG7, an a-specific gene of previously unknown function. ASG7 RNA was induced by pheromone, consistent with increased inhibition as the pheromone response progresses. The a-factor receptor inhibited signaling in its liganded state, demonstrating that the receptor can block the signal that it initiates. ASG7 was required for the altered localization of Ste4p that occurs during receptor inhibition, and the subcellular location of Asg7p was consistent with its having a direct effect on Ste4p localization. These results demonstrate that Asg7p mediates a regulatory process that blocks signaling from a G protein beta subunit and causes its relocalization within the cell.