Parallel and comparative analysis of the proteome and transcriptome of sorbic acid-stressed Saccharomyces cerevisiae.

Exposure of Saccharomyces cerevisiae to 0.9 mM sorbic acid at pH 4.5 resulted in the upregulation of 10 proteins; Hsp42, Atp2, Hsp26, Ssa1 or Ssa2, Ssb1 or Ssb2, Ssc1, Ssa4, Ach1, Zwf1 and Tdh1; and the downregulation of three proteins; Ade16, Adh3 and Eno2. In parallel, of 6144 ORFs, 94 (1.53%) showed greater than a 1.4-fold increase in transcript level after exposure to sorbic acid and five of these were increased greater than two-fold; MFA1, AGA2, HSP26, SIP18 and YDR533C. Similarly, of 6144 ORFs, 72 (1.17%) showed greater than a 1.4-fold decrease in transcript level and only one of these, PCK1, was decreased greater than two-fold Functional categories of genes that were induced by sorbic acid stress included cell stress (particularly oxidative stress), transposon function, mating response and energy generation. We found that proteomic analysis yielded distinct information from transcript analysis. Only the upregulation of Hsp26 was detected by both methods. Subsequently, we demonstrated that a deletion mutant of Hsp26 was sensitive to sorbic acid. Thus, the induction of Hsp26, which occurs during adaptation to sorbic acid, confers resistance to the inhibitory effects of this compound.

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.

Low external pH induces HOG1-dependent changes in the organization of the Saccharomyces cerevisiae cell wall.

Low environmental pH strongly affected the organization of the Saccharomyces cerevisiae cell wall, resulting in rapidly induced resistance to beta1,3-glucanase. At a molecular level, we found that a considerable amount of Cwp1p became anchored through a novel type of linkage for glycosylphosphatidylinositol (GPI)-dependent cell wall proteins, namely an alkali-labile linkage to beta1,3-glucan. This novel type of modification for Cwp1p did not require the presence of a GPI-derived structure connecting the protein with beta1,6-glucan. In addition, we found high levels of Cwp1p, which was double-anchored through both the novel alkali-sensitive bond to beta1,3-glucan and the alkali-resistant GPI-derived linkage to beta1,6-glucan. Further cell wall analyses demonstrated that Pir2p/Hsp150 and possibly other Pir cell wall proteins, which were already known to be linked to the beta1,3-glucan framework by an alkali-sensitive linkage, were also more efficiently retained in the cell wall at pH 3.5 than at pH 5.5. Consequently, the alkali-sensitive type of linkage of cell wall proteins to beta1,3-glucan was induced by low pH. The low pH-induced alterations in yeast cell wall architecture were demonstrated to be dependent on a functional HOG1 gene, but not on the Slt2p-mediated MAP kinase pathway. Consistent with this observation, DNA microarray studies revealed transcriptional induction of many known high-osmolarity glycerol (HOG) pathway-dependent genes, including four cell wall-related genes, namely CWP1, HOR7, SPI1 and YGP1.

A new strategy for inhibition of the spoilage yeasts Saccharomyces cerevisiae and Zygosaccharomyces bailii based on combination of a membrane-active peptide with an oligosaccharide that leads to an impaired glycosylphosphatidylinositol (GPI)-dependent yeast wall protein layer.

Glycosylphosphatidylinositol (GPI)-dependent cell wall proteins in yeast are connected to the beta-1,3-glucan network via a beta-1,6-glucan moiety. Addition of gentiobiose or beta-1,6-glucan oligomers to growing cells affected the construction of a normal layer of GPI-dependent cell wall proteins at the outer rim of the Saccharomyces cerevisiae cell wall. Treated S. cerevisiae cells secreted significant amounts of cell wall protein 2, were much more sensitive to the lytic action of zymolyase 20T and displayed a marked increase in sensitivity to the small amphipathic antimicrobial peptide MB-21. Similar results in terms of sensitization of yeast cells to the antimicrobial peptide were obtained with the notorious food spoilage yeast Zygosaccharomyces bailii. Our results indicate that treating cells with a membrane-perturbing compound together with compounds that lead to an impaired construction of a normal GPI-dependent yeast wall protein layer represents an effective strategy to prevent the growth of major food spoilage yeasts.

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.

Homology modeling of an immunoglobulin-like domain in the Saccharomyces cerevisiae adhesion protein alpha-agglutinin.

The Saccharomyces cerevisiae adhesion protein alpha-agglutinin is expressed by cells of alpha mating type. On the basis of sequence similarities, alpha-agglutinin has been proposed to contain variable-type immunoglobulin-like (IgV) domains. The low level of sequence similarity to IgV domains of known structure made homology modeling using standard sequence-based alignment algorithms impossible. We have therefore developed a secondary structure-based method that allowed homology modeling of alpha-aggulutinin domain III, the domain most similar to IgV domains. The model was assessed and where necessary refined to accommodate information obtained by biochemical and molecular genetic approaches, including the positions of a disulfide bond, glycosylation sites, and proteolytic sites. The model successfully predicted surface exposure of glycosylation and proteolytic sites, as well as identifying residues essential for binding activity. One side of the domain was predicted to be covered by carbohydrate residues. Surface accessibility and volume packing analyses showed that the regions of the model that have greatest sequence dissimilarity from the IgV consensus sequence are poorly structured in the biophysical sense. Nonetheless, the utility of the model suggests that these alignment and testing techniques should be of general use for building and testing of models of proteins that share limited sequence similarity with known structures.

Genetics of a-agglutunin function in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae cell adhesion protein a-agglutinin is composed of an anchorage subunit (Aga1p) and an adhesion subunit (Aga2p). Although functional a-agglutinin is expressed only by a cells, previous results indicated that AGA1 RNA is expressed in both a and alpha cells after pheromone induction. Expression of the Aga2p adhesion subunit in alpha cells allowed a-agglutinability, indicating that alpha cells express the a-agglutinin anchorage subunit, although no role for Aga1p in alpha cells has been identified. Most of the a-specific agglutination-defective mutants isolated previously were defective in AGA1; a single mutant (La199) was a candidate for an aga2 mutant. Expression of AGA2 under PGK control allowed secretion of active Aga2p from control strains but did not complement the La199 agglutination defect or allow secretion of Aga2p from La199, suggesting that the La199 mutation might identify a new gene required for a-agglutinin function. However, the La199 agglutination defect showed tight linkage to aga2::URA3 and did not complement aga2::URA3 in a/a diploids. The aga2 gene cloned from La199 was nonfunctional and contained an ochre mutation. The inability of pPGK-AGA2 to express functional Aga2p in La199 was shown to result from an additional mutation(s) that reduces expression of plasmid-borne genes. AGA2 was mapped to the left arm of chromosome VII approximately 28 cM from the centromere.

Is there a role for GPIs in yeast cell-wall assembly?

Glycosylphosphatidylinositol (GPI) membrane anchors are essential for the integration of yeast cell adhesion proteins into the cell wall, but mature cell-wall proteins are unlikely to be attached directly to the membrane. We thus propose that GPI-anchored glycoprotein forms are intermediates in a process that crosslinks the major components of the cell wall by transglycosylation. This mechanism may be critical for both the biosynthesis and overall architecture of the cell wall.

Studies on the functionality of fibrinogen during rt-PA therapy: results of three different methods of fibrinogen determination.

The conversion of soluble fibrinogen to insoluble fibrin is a crucial event in the process of haemostasis. In the present study three different methods were used for the determination of plasma fibrinogen levels in 12 patients with acute myocardial infarction during rt-PA therapy. Two methods were based on fibrin formation and thus measured 'functional' fibrinogen: a clotting rate assay according to the Clauss method and a photometric polymerization rate assay. Thirdly, fibrinogen antigen was measured with a new enzyme immunoassay (EIA) for immunologically intact fibrinogen. The levels of functional and intact fibrinogen were strikingly different: 4.1 g/l (Clauss) and 3.6 g/l (photometric) vs 2.6 g/l (EIA) at baseline, 2.2 g/l (54% of preinfusion value) and 2.1 g/l (59%) vs 2.1 g/l (81%) at 90 min, 5.7 g/l (146%) and 4.5 g/l (130%) vs 2.7 g/l (106%) at 72 h. The rebound phenomenon exhibited by functional fibrinogen (130-150%) was not observed for levels of intact fibrinogen. A ratio was calculated of functional (Clauss method) to intact fibrinogen (EIA) for each individual patient and each time point. The mean ratio (n = 12 patients) was 1.6 at baseline, 1.0 at 90 min, and increased markedly between 8 and 24 h to a maximum of 2.1 (P less than 0.01). Our data indicate that the 'functionality', i.e. the clotting rate per unit concentration of circulating fibrinogen, changed during acute myocardial infarction and subsequent thrombolytic therapy. This is in keeping with data from literature that the relative amount of fast clotting high molecular weight (HMW) fibrinogen of total fibrinogen was increased in the initial phase of acute myocardial infarction.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibodies directed against the sexual binding site of Chlamydomonas eugametos gametes.

Monoclonal antibodies were raised against the mt- sexual agglutinin of Chlamydomonas eugametos gametes. Those that blocked the agglutination site were selected. They were divided into two classes dependent upon whether they gave a weak (class A) or clear positive (class B) reaction with mt- flagellar membranes in an ELISA and an indirect immunofluorescence test using glutaraldehyde-fixed mt- gametes. Class A antibodies were shown to be specific for the agglutinin in an extract of mt- gametes, based on results from immunoblotting, immunoprecipitation, affinity chromatography, and the absence of a reaction with nonagglutinable cells. Surprisingly, class A mAbs also recognized two mt+ glycoproteins, one of which is the mt+ agglutinin. Class B antibodies were shown to bind to several glycoproteins in both mt- and mt+ gametes, including the mt- agglutinin. Fab fragments from class A mAbs blocked the sexual agglutination process, but those from class B did not, even though the parent antibody did. We conclude that the class A epitope lies in or close to the agglutination site of the mt- agglutinin, whereas the class B epitope lies elsewhere on the molecule. We also conclude that the mt- agglutinin is the only component on the mt- flagellar surface directly involved in agglutination. Class A mAbs were found to elicit several reactions displayed by the mt+ agglutinin. They bound to the mt- agglutinin on gamete flagella and induced most of the reactions typical of sexual agglutination, with the exception of flagellar tip activation. None of these reactions was induced by Fab fragments. High concentrations of class A mAbs completely repressed the sexual competence of live mt- gametes, but low concentrations stimulated cell fusion.