Solution structure of the ubiquitin-binding domain in Swa2p from Saccharomyces cerevisiae

The SWA2/AUX1 gene has been proposed to encode the Saccharomyces cerevisiae ortholog of mammalian auxilin. Swa2p is required for clathrin assembly/dissassembly in vivo, thereby implicating it in intracellular protein and lipid trafficking. While investigating the 287-residue N-terminal region of Swa2p, we found a single stably folded domain between residues 140 and 180. Using binding assays and structural analysis, we established this to be a ubiquitin-associated (UBA) domain, unidentified by bioinformatics of the yeast genome. We determined the solution structure of this Swa2p domain and found a characteristic three-helix UBA fold. Comparisons of structures of known UBA folds reveal that the position of the third helix is quite variable. This helix in Swa2p UBA contains a bulkier tyrosine in place of smaller residues found in other UBAs and cannot pack as close to the second helix. The molecular surface of Swa2p UBA has a mostly negative potential, with a single hydrophobic surface patch found also in the UBA domains of human protein, HHR23A. The presence of a UBA domain implicates Swa2p in novel roles involving ubiquitin and ubiquitinated substrates. We propose that Swa2p is a multifunctional protein capable of recognizing several proteins through its protein-protein recognition domains.     

Solution structure of SWI1 AT-rich interaction domain from Saccharomyces cerevisiae and its nonspecific binding to DNA

SWI1 is a subunit of the SWI/SNF complex involved in chromatin remodeling. It contains an AT-rich interaction domain (ARID) which has the potential DNA binding activity. In this study, we determined the solution structure of the SWI1 ARID domain from Saccharomyces cerevisiae by nuclear magnetic resonance spectroscopy. Yeast SWI1 ARID domain is composed of seven alpha helices, six of which are conserved among the ARID family. In addition, the DNA-binding activity of the SWI1 ARID domain was confirmed by chemical shift perturbation assay. Similar to its human homolog, the yeast SWI1 ARID domain binds DNA nonspecifically.     

Solution structure of Rap1 BRCT domain from Saccharomyces cerevisiae reveals a novel fold

Rap1 (repressor-activator protein 1) from Saccharomyces cerevisiae, containing a BRCT domain at its N-terminus, is a multifunctional protein that controls telomere function, silencing, and the activation of glycolytic and ribosomal protein genes. In this work, we determined the solution structure of Rap1 BRCT domain, which contains three β-strands and three α-helices. Structural comparison indicated that Rap1 BRCT domain adopts a global fold similar to other BRCT domains, implying some common structural aspects of BRCT domain family. On the other hand, Rap1 BRCT domain displays structural characteristics significantly different from other BRCT domains in that Rap1 BRCT domain adopts a rather flexible conformation with less secondary structure elements, revealing a novel fold of the BRCT domain family.     

Solution structure of the orphan PABC domain from Saccharomyces cerevisiae poly(A)-binding protein

We have determined the solution structure of the PABC domain from Saccharomyces cerevisiae Pab1p and mapped its peptide-binding site. PABC domains are peptide binding domains found in poly(A)-binding proteins (PABP) and are a subset of HECT-family E3 ubiquitin ligases (also known as hyperplastic discs proteins (HYDs)). In mammals, the PABC domain of PABP functions to recruit several different translation factors to the mRNA poly(A) tail. PABC domains are highly conserved, with high specificity for peptide sequences of roughly 12 residues with conserved alanine, phenylalanine, and proline residues at positions 7, 10, and 12. Compared with human PABP, the yeast PABC domain is missing the first alpha helix, contains two extra amino acids between helices 2 and 3, and has a strongly bent C-terminal helix. These give rise to unique peptide binding specificity wherein yeast PABC binds peptides from Paip2 and RF3 but not Paip1. Mapping of the peptide-binding site reveals that the bend in the C-terminal helix disrupts binding interactions with the N terminus of peptide ligands and leads to greatly reduced binding affinity for the peptides tested. No high affinity or natural binding partners from S. cerevisiae could be identified by sequence analysis of known PABC ligands. Comparison of the three known PABC structures shows that the features responsible for peptide binding are highly conserved and responsible for the distinct but overlapping binding specificities.     

Solution structure of the DNA-binding domain of RPA from Saccharomyces cerevisiae and its interaction with single-stranded DNA and SV40 T antigen

Replication protein A (RPA) is a three-subunit complex with multiple roles in DNA metabolism. DNA-binding domain A in the large subunit of human RPA (hRPA70A) binds to single-stranded DNA (ssDNA) and is responsible for the species-specific RPA–T antigen (T-ag) interaction required for Simian virus 40 replication. Although Saccharomyces cerevisiae RPA70A (scRPA70A) shares high sequence homology with hRPA70A, the two are not functionally equivalent. To elucidate the similarities and differences between these two homologous proteins, we determined the solution structure of scRPA70A, which closely resembled the structure of hRPA70A. The structure of ssDNA-bound scRPA70A, as simulated by residual dipolar coupling-based homology modeling, suggested that the positioning of the ssDNA is the same for scRPA70A and hRPA70A, although the conformational changes that occur in the two proteins upon ssDNA binding are not identical. NMR titrations of hRPA70A with T-ag showed that the T-ag binding surface is separate from the ssDNA-binding region and is more neutral than the corresponding part of scRPA70A. These differences might account for the species-specific nature of the hRPA70A–T-ag interaction. Our results provide insight into how these two homologous RPA proteins can exhibit functional differences, but still both retain their ability to bind ssDNA.     

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.     

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.     

Solution phase synthesis of Saccharomyces cerevisiae a-mating factor and its analogs

The solution phase synthesis of the Saccharomyces cerevisiae a-mating factor and nonfarnesylated and nonmethylated a-factor analogs are reported. The a-factor, a lipopeptide with the sequence Tyr-Ile-Ile-Lys-Gly-Val-Phe-Trp-Asp-Pro-Ala-Cys(S-Farnesyl)OCH3 was synthesized by the condensation of the amine terminal protected decapeptide with the carboxyl terminal farnesylated dipeptide using benzotriazol-l-yloxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP reagent) as the coupling agent. The synthesis of the decapeptide involved 5 + 5 fragment coupling with the BOP reagent and the successful application of 9-fluorenylmethyl ester(OFm) and 9-fluorenylmethoxycarbonyl(Fmoc) groups for the protection of Asp and Lys side chains and Tyr alpha-amine and of phenacyl esters (OPa) for alpha-carboxyl protection. The OFm and Fmoc groups tolerated repeated couplings and were completely stable to zinc powder in acetic acid, a condition under which the OPa group was removed. The synthesis of the nonfarnesylated alpha-factor was accomplished by the coupling of the decapeptide with tetrapeptide (Ala-CysOCH3)2 followed by the deprotection of the OFm and Fmoc groups with piperidine and the cleavage of the disulfide bond with zinc powder in acetic acid. The nonmethylated a-factor was prepared by 10 + 2 fragment coupling using OFm protection of the dipeptide carboxyl group followed by removal of all protecting groups with piperidine. Attempts to saponify a-factor were not successful. The synthetic nonfarnesylated and nonmethylated a-mating pheromones were 100-1000 times less active than the a-factor, indicating that although the methyl ester and the farnesyl group are not essential for biological activity, they are necessary for high potency.     

Four novel suppressors of gic1 gic2 and their roles in cytokinesis and polarized cell growth in Saccharomyces cerevisiae

Gic1 and Gic2 are two Cdc42/Rac interactive binding (CRIB) domain-containing effectors of Cdc42-GTPase that promote polarized cell growth in S. cerevisiae. To identify novel genes that functionally interact with Gic1 and Gic2, we screened for high-copy suppressors of a gic1 gic2 temperature-sensitive strain. We identified two pairs of structurally related genes, SKG6-TOS2 and VHS2-MLF3. These genes have been implicated in polarized cell growth, but their functions have not previously been characterized. We found that overproduction of Skg6 and Tos2 in wild-type cells causes aberrant localization of Cdc3 septin and actin structures as well as defective recruitment of Hof1 and impaired formation of the septum at the mother-bud neck. These data suggest a negative regulatory function for Skg6 and Tos2 in cytokinesis. Consistent with this model, deletion of SKG6 suppresses the growth defects associated with loss of HOF1, a positive regulator of cytokinesis. Our analysis of the second pair of gic1 gic2 suppressors, VHS2 and MLF3, suggests that they regulate polarization of the actin cytoskeleton and cell growth and function in a pathway distinct from and parallel to GIC1 and GIC2.     

Calmodulin concentrates at regions of cell growth in Saccharomyces cerevisiae

Calmodulin was localized in Saccharomyces cerevisiae by indirect immunofluorescence using affinity-purified polyclonal antibodies. Calmodulin displays an asymmetric distribution that changes during the cell cycle. In unbudded cells, calmodulin concentrates at the presumptive site of bud formation approximately 10 min before bud emergence. In small budded cells, calmodulin accumulates throughout the bud. As the bud grows, calmodulin concentrates at the tip, then disperses, and finally concentrates in the neck region before cytokinesis. An identical staining pattern is observed when wild-type calmodulin is replaced with mutant forms of calmodulin impaired in binding Ca2+. Thus, the localization of calmodulin does not depend on its ability to bind Ca2+ with a high affinity. Double labeling of yeast cells with affinity-purified anti-calmodulin antibody and rhodamine-conjugated phalloidin indicates that calmodulin and actin concentrate in overlapping regions during the cell cycle. Furthermore, disrupting calmodulin function using a temperature-sensitive calmodulin mutant delocalizes actin, and act1-4 mutants contain a random calmodulin distribution. Thus, calmodulin and actin distributions are interdependent. Finally, calmodulin localizes to the shmoo tip in cells treated with alpha-factor. This distribution, at sites of cell growth, implicates calmodulin in polarized cell growth in yeast.     

Effect of 2-deoxyglucose on cell wall formation in Saccharomyces cerevisiae and its relation to cell growth inhibition

The growth inhibition and the lysis of Saccharomyces cerevisiae caused by 2-deoxy-d-glucose (2-DG) were shown to be a consequence of unbalanced cellular growth and division. The lysis, but not the repression of growth and osmotic fragility of cells, could be suppressed by the addition of mannitol as an osmotic stabilizer. This result, as well as the morphological changes observed in the cells and changes in the chemical composition of the cell walls, showed that S. cerevisiae grown in the presence of 2-DG formed weakened cell walls responsible for the osmotic fragility. Evidence is presented for the first time demonstrating the incorporation of 2-DG into yeast cell wall material. Other data suggest that the inhibition of yeast growth by 2-DG results from an interference of phosphorylated metabolites of 2-DG with metabolic processes of glucose and mannose involved in the synthesis of structural cell wall polysaccharides.     

Effect of growth rate and substrate limitation on the composition and structure of the cell wall of Saccharomyces cerevisiae

1. A study was made of the composition and structure of walls isolated from yeast grown in continuous culture at different rates, under three conditions of glucose limitation in which the concentrations of glucose and ammonium sulphate in the medium and the oxygen-transfer rate in the culture were varied, and one condition of NH(4) (+) limitation. 2. The contents of total glucan and total mannan in the walls were relatively little affected by the growth rate under any of the four sets of conditions. The phosphorus and protein contents of walls from yeast grown under each of the four conditions increased as the growth rate was decreased. Walls from yeast grown under NH(4) (+) limitation contained only half as much protein as walls from cells grown under glucose limitation. The proportion of lipid was greatest in walls from yeast grown under NH(4) (+) limitation. 3. A procedure was devised for fractionating isolated walls, based on the ease with which the glucan and mannan were extracted with water and with hot and cold 6% (w/v) potassium hydroxide solution. The percentage of glucan, mannan, protein and phosphorus in each of the fractions was affected by the rate of growth and by the nature of the substrate limitation. 4. The beta-fructofuranosidase activities of yeast grown under glucose limitation increased as the growth rate was lowered, but decreased at very low growth rates. The effects at low growth rates were probably due to repression of enzyme synthesis by residual glucose in the culture filtrate. The beta-fructofuranosidase activities of yeast grown under NH(4) (+) limitation were much lower than those from yeast grown under any of the conditions of glucose limitation. 5. Yeast cells grown at any of the rates under NH(4) (+) limitation were longer and thinner than those grown at the same rate under any of the conditions of glucose limitation. Mean cell volumes were dependent on growth rate but not on the nature of the substrate limitation. 6. Electron micrographs of thin sections of isolated walls showed that cells grown under NH(4) (+) limitation had a more porous structure than those from cells grown under any of the conditions of glucose limitation.     

Transport-limited fermentation and growth of Saccharomyces cerevisiae and its competitive inhibition

Summary The anaerobic glucose uptake (at 20°, pH 3.5) by resting cells of Saccharomyces cerevisiae followed unidirectional Michaelis-Menten kinetics and was competitively inhibited by l-sorbose; K _m and K _i were respectively 5.6×10^-4 m and 1.8×10^-1 m; V _max was 6.5×10^-8 moles mg^-1 min^-1. The aerobic uptake of glucose by resting yeast was also inhibited by l-sorbose but did not follow unidirectional Michaelis-Menten kinetics. Glucose-limited growth in the chemostat of a respiration-deficient mutant of S. cerevisiae was competitively inhibited by l-sorbose. As predicted by theory for transport-limited growth in the chemostat (van Uden, 1967) the steady state glucose concentrations were linear functions of the l-sorbose concentrations with different slopes at different dilution rates; K _m and K _i were respectively 7.2×10^-4 m and 1.8×10^-1 m. It is concluded that glucose transport was the rate-limiting step of anaerobic fermentation of S. cerevisiae and of growth of the mutant and that l-sorbose is a competitive inhibitor of active glucose transport in this yeast. The latter conclusion is accommodated in the transport model of van Steveninck and Rothstein (1965).