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Stanična stijenka kvasca Saccharomyces cerevisiae je ekstracelularna organela građena od polisaharida i proteina koja stanici pruža oblik, čvrstoću, mehaničku zaštitu, osmotsku stabilnost, ali služi i u komunikaciji s drugim stanicama. Proteini sudjeluju u održavanju strukture stanične stijenke i komunikaciji stanica s okolinom, a osim prema funkciji razlikuju se prema građi i načinu vezanja u staničnu stijenku. Poznato je da se Ccw5 u staničnu stijenku veže esterskom vezom između β-1,3-glukana i ostatka glutamina unutar tzv. repetitivne sekvence ovog proteina. Međutim, objavljeno je nekoliko radova u kojima autori tvrde da se Ccw5 u stijenku kovalentno veže disulfidnim mostovima. Stoga je u ovom radu ispitana uloga disulfidnih mostova u ugradnji proteina Ccw5. Dobiveni rezultati su potvrdili da disulfidni mostovi nemaju ulogu u vezanju Ccw5, već da je on vezan esterski u stijenku, jer i nakon višestrukog tretmana sa SDS-om i/ili β-merkaptoetanolom u stijenci zaostaje kovalentno vezana frakcija ovog proteina koja se ekstrahira pomoću NaOH. Saccharomyces cerevisiae cell wall is an extracellular organelle built from polysaccharides and proteins, which gives the cell its shape, firmness, protection, osmotic stability and participates in communication with other cells. Proteins maintain the structure of cell wall and are used for communication with surroundings. They vary in function and in the type of bond by which they are linked to the cell wall. It is known that Ccw5 is linked to the cell wall by ester linkage between glutamine from repeated sequence of protein and β-1, 3-glucan. However, several articles were published stating that Ccw5 is linked to the cell wall covalently by disulphide bonds. Therefore, the aim of this work was to examine the role of disulphide bonds in incorporation of Ccw5. Obtained results confirmed that disulphide bonds don't have a role in linking of Ccw5, and that Ccw5 is linked to the cell wall by ester linkage, because even after multiple treatment of cell walls with SDS and/or β-mercaptoethanol, covalently linked fraction of Ccw5 remains linked to cell wall and is extracted by NaOH.

Proteini stanične stijenke kvasca Sacharomyces cerevisiae se u staničnu stijenku mogu vezati putem dva mehanizma: kovalentno i nekovalentno. Nekovalentno vezani proteini se iz stanične stijenke izoliraju tretmanom sa vrućim SDS-om, a kovalentno vezani se mogu izolirati pomoću enzima glukanaza (tzv. GPI-proteini) ili ekstrakcijom pomoću 30 mM NaOH (tzv. Pir-proteini). Protein Scw4p je specifičan po tome što se u staničnu stijenku veže putem oba mehanizma tj. kovalentno i nekovalentno. Kovalentno vezani Scw4p izolira se iz stijenki prekonoćnom inkubacijom u 30 mM NaOH na 40C kao i proteini Pir porodice. Pir proteini su vezani na β-1,3-glukan esterskom vezom između hidroksilne grupe glukoze i γ-karboksilne grupe određenog ostatka glutaminske kiseline smještenog unutar ponavljajuće sekvence specifične za ovu porodicu proteina. Budući da unutar gena SCW4 takva sekvenca nije prisutna, mehanizam vezanja Scw4p u stijenku je nepoznat. Unutar gena SCW4 je međutim pronađena sekvenca slična PIR ponavljajućem motivu za koju se stoga pretpostavlja da bi mogla biti zaslužna za kovalentno vezanje Scw4p u staničnu stijenku kvasca. Kako bi se ta pretpostavka provjerila u ovom radu je koristeći PCR metodu „megapočetnice“ ta sekvenca deletirana iz gena SCW4. Yeast cell wall proteins can be linked to the Saccharomyces cerevisiae cell wall by two different mechanisms: covalently and non-covalently. Non-covalently linked proteins can be isolated from cell wall by treatment with hot SDS, and covalently linked proteins can be isolated with glucanase (GPI-proteins) or with 30mM NaOH (Pir-proteins). Protein Scw4p is specific because it can be linked to the cell wall both covalently and non-covalently. Covalently linked Scw4p is isolated by overnight extraction in 30mM NaOH on +40C like proteins of Pir family. Pir proteins are linked to β-1,3-glukan by ester bound between hydroxyl group of glucose and γ-carbonyl group of specific glutamic acid residue, placed in repetitive sequence specific for proteins of this family. Since that sequence is not present in SCW4 gene the mechanism of Scw4p covalent linkage to the cell wall is unknown. However, in the SCW4 gene sequence quite similar to PIR repetitive motive was found, that might be involved in covalently bounding of Scw4p in yeast cell wall. In order to check this assumption deletion of that sequence from the SCW4 gene was performed using „megaprimer“ PCR method.

The yeast cell wall contains an unusually high number of different mannoproteins. The physiological role of most of them is unknown and gene disruptions leading to depletion of different proteins do not affect major functions of the wall. In this work the phenotype of different single and multiple cell wall protein mutants was observed at the level of individual cells. It was found that the lack of the non-covalently bound wall proteins Scw4p, Scw10p and Bgl2p increases the mortality of Saccharomyces cerevisiae cells grown exponentially under standard laboratory conditions, as assayed by methylene blue staining. Mutation of SCW11, however, suppressed the phenotype of scw4scw10, or scw4scw10bgl2, indicating that Scw4p, Scw10p and Bgl2p act synergistically while Scw11p has an activity antagonistic to that of the other three proteins. Mutants lacking major covalently bound proteins, either all four described Pir-proteins or the five most abundant glycosylphosphatidylinositol (GPI)-anchored proteins (Ccw12p, Ccw13p/Dan1p, Ccw14p/Icwp1p, Tip1p and Cwp1p), also had increased mortalities, the first somewhat more and the latter less than that of scw4scw10bgl2. In all cases the observed phenotype was suppressed by the addition of an osmotic stabilizer to the growth medium, indicating that cells died due to decreased osmotic stability. If cells were grown to stationary phase, Scw-mutants showed only slightly increased mortality, but mutants lacking Pir- or GPI-anchored proteins had significantly increased sensitivity, suggesting that their physiological function is primarily expressed in stationary-phase cells. In many cases structures consisting of a living ccw5ccw6ccw7ccw8 (multiple Pir-protein mutant) mother with two methylene blue-stained daughters could be seen.

During maturation of the Hungarian botrytized wine ˝Tokaji szamorodni˝ a buoyant film develops on the wine surface, which is similar to the flor or velum formed during aging of sherry wines. In both cases the yeast films play an important role in the formation of aroma compounds that are responsible for the typical organoleptic characters of these wines. Our aims were the characterization of cell wall proteins of film-forming and non-film forming (sedimenting) wine yeast strains and finding protein candidates that were playing role in the establishment of special cell surface structure. As the first step of research the patterns of biotinylated cell wall proteins extracted from film-forming and non-film forming Saccharomyces cerevisiae strains were compared. It was found that all of the tested 23 film-forming ˝Szamorodni˝ yeast strains had a decreased size of the Ccw7 protein, one of the members of the Pir-protein family. Sequencing of the encoding genes revealed that the strains were lacking three out of the eleven repeating sequences characteristic to this protein family. One of the film-forming strains contained CCW7 alleles of different length, what was generated by intragenic tandem duplication of a sequence containing two repetitive domains. Unlike the film-forming strains, sixteen non-film forming wine yeasts isolated from a different botrytized wine, ˝Tokaji Aszu˝ showed pronounced polymorphism of the CCW7 locus. It is highly probable that the modified Ccw7 protein contribute to the stress adaptation during film-formation.

In the Saccharomyces cerevisiae cell wall more than 20 different mannoproteins were evidenced. They are considered to play different roles in building, maintaining and modifying the wall itself during growth and different cell cycle events and they are important for interactions of cells with their surrounding. Yeasts have evolved three different ways of attaching proteins to cell wall glucan. Some proteins are bound to β -1, 3-glucan noncovalently (Scw – soluble cell wall proteins ; extracted by hot SDS), while others are attached covalently (Ccw – covalently linked cell wall proteins ; extracted by glucanases) through GPI-anchor and β -1, 6-glucan, or directly to β -1, 3-glucan by alkali labile ester linkage between the γ -carboxyl groups of glutamic acid and hydroxyl groups of glucoses (Pir – proteins with internal repeats ; extracted by NaOH). Disruption of genes coding for the Pir-proteins was performed to investigate their potential role. After disruption of all PIR genes, 67kDa protein still remained in NaOH extract. SCW4 disruption resulted in disappearance of a 67kDa band, indicating that Scw4p could also be covalently linked to the cell wall similar to Pir-proteins. Since it was reported previously that Scw4p was a noncovalently attached protein, this finding could be relevant for the role of Scw4p. In order to get insight in binding to glucan Scw4p was tagged and its localization in different mutants was studied. Besides, effects of different levels of synthesis of Scw4p, as well as its homologs Scw10p and Scw11p on yeast phenotype was studied to investigate their physiological roles.

Yeast cell wall contains proteins that are noncovalently (Scw-proteins), or covalently (Ccw-proteins) bound to beta-1, 3-glucan, the latter either through GPI-anchors and beta-1, 6-glucan, or by alkali labile ester linkages between gamma-carboxyl groups of glutamic acid and hydroxyl groups of glucoses (Pir-proteins, extracted from the cell wall by mild alkali). Disruption of all four genes coding for Pir-proetins revealed that 67 kDa protein still remained in the NaOH extract. Disruption of the SCW4, a gene coding for one of most abundant SCW proteins resulted in the disappearance of the band, indicating that Scw4p was partly also covalently linked to the cell wall by an apparently new, so far unobserved alkali sensitive linkage. Structural analysis revealed that N-terminal part of the protein is required for anchoring in the wall. Both the absence and the increased concentration of Scw proteins resulted in undesirable changes in the wall and in the increase in cell mortality, particulary upon entering the stationary growth conditions. Possibilities for application of the knowledge gained on different mechanisms for incorporation of wall proteins in heterologous expression of proteins in yeast and their ˝genetic immobilisation˝ will be discussed.

The cell wall of yeast Saccharomyces cerevisiae contains more than 20 different manoproteins. They are considerd to play different roles in building, maintaining and modifying the wall itself when different cell cycle events and they are important for interactions of cells with their surrounding. Yeasts have evolved three different ways of attaching proteins to cell wall glucan. Some proteins are bound to β -1, 3-glucan noncovalently (Scw – soluble cell wall proteins ; extracted by hot SDS), while others are attached covalently (Ccw – covalently linked cell wall proteins ; extracted by glucanases) through GPI-anchor and β -1, 6-glucan, or directly to β -1, 3-glucan by alkali labile ester linkage between the γ -carboxyl groups of glutamic acid and hydroxyl groups of glucoses (Pir – proteins with internal repeats ; extracted by NaOH). Disruption of genes coding for the Pir-proteins was performed to investigate their potential role. After disruption of all PIR genes, 67kDa protein still remained in NaOH extract. SCW4 disruption resulted in disappearance of a 67kDa band from the extract, indicating that Scw4p could also be covalently linked to the cell wall simillar to Pir-proteins. Since it was reported previously that Scw4p was a noncovalently attached protein, this finding can be relevant for the role of Scw4p. In order to investigate the role of the Scw4p in the construction of the cell wall, yeast was transformed with a high copy number plasmid containing SCW4. In order to get further insight in the binding mechanism, a novel, simple binding assay for Pir family proteins was developed in which externally added Ccw5p/Pir4p was bound to different cell wall mutant strains. It has been shown that pir, as well as scw4 and scw10 mutants can bind externally added Ccw5p to their cell wall. Further, we investigated the conditions under which these proteins were attached to cell wall. The presence of EDTA blocked the binding of Ccw5p, indicating the cation dependence of the reaction. Both wild type and mutant cells showed enhanced binding in 0.6 M KCl. It was also shown that the native conformation of Ccw5p is required for its binding.

Saccharomyces cerevisiae cell wall has a bilayered structure. The inner part consists mainly of a network of linear ß-1, 3-glucan molecules, and the outer layer is composed mostly of mannoproteins. Yeasts have evolved three different ways of attaching proteins to the polysaccharide network. Some proteins are bound to ß-1, 3-glucan noncovalently (Scw – soluble cell wall proteins ; extracted by hot SDS), while others are attached covalently (Ccw – covalently linked cell wall proteins ; extracted by glucanases) through glycosylphosphatidyl inositol (GPI) anchor and ß-1, 6-glucan, or directly to ß-1, 3-glucan by an alkali labile ester linkage between the γ -carboxyl groups of glutamic acid and hydroxyl groups of glucoses (Pir – proteins with internal repeats ; extracted by NaOH). The exact physiological roles for most of the cell wall proteins are still unknown. A multiple disruption of genes coding for the Pir family proteins (ccw5 ccw6 ccw7 ccw8) was performed in order to investigate their potential role. After disruption of the genes coding for all Pir family members, 67kDa protein band still remained in the NaOH extract. Disruption of SCW4 resulted in apparent disappearance of the 67kDa band, indicating that Scw4p could also be covalently linked to the cell wall in a similar manner like Pir proteins. In order to investigate the role of the Scw4p in the construction of the cell wall, wilde type yeast was transformed with a high copy number plasmid containing SCW4 gene. Phenotypes of this strain as well as scw4, ccw5ccw6ccw7ccw8 and ccw5ccw6ccw7ccw8scw4 were examined. In order to get further insight in the binding mechanism a novel, simple binding assay for Pir family proteins, was developed. It has been shown that pir, as well as scw4 and scw10 mutant cells, can bind externally added Ccw5p to their cell wall. A study of appropriate binding conditions revealed the requirement of the native conformation of Ccw5p. The presence of EDTA blocked the binding of Ccw5p, indicating the cation dependence of the reaction. Both wild type and mutant cells showed enhanced binding in 0.6 M KCl. Further experiments should provide answers to question regarding exact conditions required, as well as reveal if the binding occurs autocatalytically or require a particular enzyme in the wall.

Yeasts have evolved three different ways regarding the glucan attached proteins of cell wall. Some proteins are bound noncovalently to the β -1, 3-glucan, while others are attached covalently to the β -1, 6-glucan through a GPI-anchor, or directly by alkali labile ester linkage between the γ -carboxyl group of glutamic acid and the hydroxyl group of glucose (named as Pir – proteins). Unusual versatility of the cell wall proteins implies different roles and reactions taking place in the wall. Different mutants lacking individual wall proteins or groups of proteins were constructed and their phenotypes were studied. After disruption of all the PIR genes (CCW5, CCW6, CCW7 and CCW8), a 67kDa protein still remained in NaOH extract. SCW4 disruption in the ccw5ccw6ccw7ccw8 mutant, however, resulted in disappearance of the 67kDa band from the extract, indicating that Scw4p could also be covalently linked to the cell wall, similarly to the Pir-proteins. Since it was reported previously that Scw4p was a noncovalently attached protein, this finding can be relevant for the role of Scw4p. A mild treatment of yeast cells with β -glucanase induced a drastic decrease in the cell surface hydrophobicity indicating that it is at least to a great extent due to mannoproteins of the outer layer of the cell wall. This phenomenon together with the obvious assumption that the outermost located mannoproteins play crucial role in the cell-cell interaction during film or floc formation motivated us to screen for the differences in cell wall protein composition between the wine yeast strains, which form a film (flor) on the wine surface and those unable to do so in order to get a better insight into the intermolecular interactions during cell attachment.

Saccharomyces cerevisiae cell wall has a bilayered structure. The inner part consists mainly of a network of linear ß-1, 3-glucan molecules, whereas the outer layer is composed mostly of mannoproteins. Yeasts have evolved three different ways of attaching proteins to the polysaccharide network. Some proteins are bound noncovalently while the others are covalently attached through glycosylphosphatidyl inositol (GPI) anchor and ß-1, 6-glucan, or directly to ß-1, 3-glucan (Pir – proteins with internal repeats). Pir proteins are soluble in mild alkali and do not have GPI anchor. Since these proteins are highly glycosylated their deletion changes the polysaccharide composition of the cell walls. Five PIR genes with varying number of repeating units (1-11) were identified, and the repeating unit seems to be responsible for the formation of an ester bond between the γ -carboxyl groups of glutamic acid and hydroxyl groups of glucoses. In order to get further insight in the binding mechanism, a novel, simple binding assay for Pir family proteins was developed in which externally added Ccw5p/Pir4p was bound to different cell wall mutant strains. It has been shown that pir, as well as scw4 and scw10 mutants can bind externally added Ccw5p to their cell wall. Further, we investigated the conditions under which these proteins were attached to cell wall. The presence of EDTA blocked the binding of Ccw5p, indicating the cation dependence of the reaction. Both wild type and mutant cells showed enhanced binding in 0.6 M KCl. It was also shown that the native conformation of Ccw5p is required for its binding. A multiple disruption of genes coding for the Pir family proteins (ccw5 ccw6 ccw7 ccw8) was performed in order to investigate their potential role in the cell wall biogenesis. After disruption of all Pir family genes, 67kDa protein band still remained in the NaOH extract. It was eliminated by the disruption of SCW4, indicating that Scw4p could also be covalently linked to the cell wall by an alkali sensitive linkage.

Saccharomyces cerevisiae cell wall contains more than 20 different mannoproteins. Some of them are noncovalently bound to the wall structural polysaccharides and can be extracted by hot SDS (Scw proteins-soluble cell wall proteins). The second group of proteins is covalently linked to the wall (Ccw proteins-covalently linked cell wall proteins) and can be divided into those extractable by NaOH, or by glucanases, respectively. Possible role of NaOH extractable proteins (members of the Pir protein family Ccw5p/Pir4p, Ccw6p/Pir1p, Ccw7p/Pir2p and Ccw8p/Pir3p) in the cell wall is still unknown. It has been suggested that Pir proteins get covalently linked enzimatically to cell walls by a transglutaminase-type of reaction. In order to get further insight in their binding mechanism capability of cells for binding Ccw5p protein added externally to growth medium was monitored. Successive mutations of PIR genes led to increased capability of cells to bind Ccw5p from growth medium and, similar to other phenotypes observed previously, it showed cummulative effect. The presence of EDTA in growth medium blocked the binding of Ccw5p, indicating that reaction is dependent on the presence of divalent cations. Both wild type and ccw mutant cells showed higher capacity for binding Ccw5p under increased osmotic pressure indicating that reaction is under control of the HOG pathway. Results observed by monitoring viability of ccw mutants showed increased fraction of dead cells in multiple ccw mutants (about 8% in exponentially, and about 25% in stationary phase), compared to wild type (less than 1%). Number of dead cells was significantly reduced in both growth phases when cells were grown in 1M sorbitol, indicating that increased mortality is caused by osmotic instability of mutant cells. These two results are in good correlation and indicate that Pir proteins are involved in osmotic stabilization of yeast cell wall. On the other hand some other shock conditions (addition of cell wall synthesis inhibitors Calcoflour White or Congo Red) do not affect capability of cells to bind Ccw5p, showing that increased binding level of Pir proteins is not generally caused by shock.

Yeasts communicate with their environment through their cell wall composed of a ß-1, 3-glucan network to which some chitin and a number of mannoporteins are attached. Some of the cell wall proteins are linked to the glucan backbone noncovalently, and can be extracted by SDS or DTT. Covalently linked wall proteins can be devided according to their localisation mechanism in two groups. The first group can be extracted from purified cell walls using different glucanase preparations. Identification of nine proteins of this group has shown that they all posses C-terminal signal for the attachment of the GPI-anchor. The second group of proteins was obtained by an extraction of walls with 30 mM NaOH. Four proteins of this group were identified by N-terminal sequencing and they were found to be members of the same, so called PIR protein family having several common structural features. All four proteins contain characteristic repetetive sequence at the N-terminal end, they are extensively O-glycosylated and do not posses the signal for the addition of GPI-anchor. Therefore, they must be incorporated into the cell wall by another molecular mechanism. To investigate which part of Pir proteins is responsible for their attachment to the cell wall, Ccw5p was used as a model. The protein was tagged with the haemagglutinin tag at the C-terminal end, and different deletions of the CCW5 gene were analysed. It was found that the protein lacking one repetitive unit was still attached to the cell wall but if both copies were deleted, the protein was secreted into the medium. This strongly suggests that Pir proteins are attached to the carbohydrate network of the wall via their repetitive sequence.

S. cerevisiae cell wall is composed of a beta-1, 3-gucan network to which more then 20 different mannoproteins are attached (1). Besides, small amounts of chitin and beta-1, 6-glucan are also found. Some of the cell wall proteins are linked to the glucan backbone noncovalently or solely via S-S links, and can be extracted from the walls by SDS or DTT (2). Six proteins of this group have been indentified so far and it has been found that they are all either hydrolytic enzymes, or at least are homologous to known yeast glucanases (2). Covalently linked cell wall proteins can be divided according to their attachment in two groups. The first group represents proteins, which can be extracted from purified cell walls depleted of SDS-extractable proteins, using different glucanase preparations such as zymolyase, or laminarinase. Identification of nine proteins of this group has shown that they all posses a C-terminal signal for the attachment of the GPI-anchor (3, 4, 5, 6). It has, therefore, been postulated that these proteins get plasma membrane anchored through GPI, and are then translocated, by a so far unknown mechanism, to beta-1, 6-glucan, which serves as a link to the beta-1, 3-glucan network of the cell wall (7). The second group of covalently linked cell wall proteins was obtained from walls after SDS extraction with 30 mM NaOH (8, 9). Four proteins of this group were identified by N-terminal sequencing and they were designated Ccw5p to Ccw8p (Ccw for Covalently linked cell wall). It has been found that Ccw6p, Ccw7p and Ccw8p were products of PIR1, PIR2 and PIR3 genes for which it has previously been reported to contain a characteristic repetitive sequence (PIR for Proteins with internal repeats, 10). The four proteins belong to the same family sharing a high degree of identity and having several common structural features. All are proteolytically processed by the Kex2p protease so that the repetitive sequence occupies the N-terminal part of mature proteins. The number of repetitive units ranges from 2 in Ccw5p, to 9 in Ccw7p. Besides, all four proteins are extensively O-glycosylated and do not posses the signal for the addition of a GPI-anchor. The latter indicates that there must be another molecular mechanism for the incorporation of proteins into the cell wall, different from the one suggested for proteins extracted by glucanases. Therefore, the aim of this investigation was to get an insight into the way of attachment of this group of proteins to the cell wall. Since members of the Pir family are generally not found in glucanase wall extracts they may be attached to chitin in the wall, rather than to glucan. Therefore, proteins were isolated from purified cell walls by chitinase. Three distinct electrophoretic protein bands at 47, 67 and 117 kDa were obtained in the extract. The same bands were, however obteined from the walls of the mutant lacking all four proteins of the Pir family, indicating that chitinase-extractable proteins were not Pir-proteins. The same bands were also obtained from cell walls of the mutant lacking five major glucanase-extractable proteins i.e. Ccw12p, Ccw13p, Cwp1p, Icwp1p and Tip1p, suggesting that the three proteins were so far unidentified. To find out which part of Pir proteins is responsible for their attachment to the cell wall, Ccw5p was used as a model. The protein was tagged with the haemagglutinin tag at the C-trminal end, and deletions of different parts of the CCW5 gene were analysed. It was found taht the protein lacking one repetitive unit was attached to the cell wall as was the wild type. If both copies of the repetitive sequence were deleted, the protein was secreted into the medium. This strongly suggests that Pir proteins are attached to the carbohydrate network of the wall, most probably to beta-1, 3-glucan, via their repetitive sequence and that the single copy of this sequence is sufficient for the effective binding.The study of the linker which connects Pir proteins to wall carbohydrates is a subject of current investigation and the results obtained so far indicate that it most probably does not contain phosphate.

It has been shown that the PMT gene family is responsible for protein-O-mannosylation in S. cerevisiae and that certain triple pmt disruptions are lethal (1). Other multiple disruptions could be saved, if the cells were osmotically stabilized indicating that protein O-glycosylation is required for cell awll integrity. To understand this phenomenon, it was decided to analyzed and characterized the complete set of yeast cell wall proteins. Using non-permeant biotinylation reagent more than 20 cell surface proteins could be specifically labelled (2). One group of proteins could be extarcted by SDS under reducing conditions, other proteins were covalently linked within the wall and could be devided in those extarctable by glucanases and those which could be released by 30 mM NaOH. After purification and N-terminal sequencing, nine new cell wall components have been identified. Among them were four NaOH extractable proteins, which formed a new family of Pir-proteins (proteins with internal repeats). In order to investigate the role of covalently linked, NaOH-extractable cell wall proteins, genes encoding the four Pir-proteins Ccw5p, Ccw6p, Ccw7p and Ccw8p were disrupted and the phenotype of the mutants optained was examened (3). Besides, three new identified noncovalently linked cell wall proteins were found to be related to glucanases. Single knockouts of this three potential glucanases did not result in dramatic phenotypes, however multiple ones partly did (4). Evidence has been obtained that almost all of these cell wall proteins are O-glycosylated.

The Saccharomyces cerevisiae cell wall contains more than 20 identified mannoproteins. Some of them can be released from the wall by hot SDS/mercaptoethanol treatment and are, therefore, considered as disulphide-linked or non-covalently attached to wall structural components. A number of covalently linked cell wall proteins are released after SDS extraction. They can be divided into these extractable by glucanases and those which can be released with 30 mM NaOH. The SDS-extractable proteins either possess enzymatic activities or are homologues of enzymes, mainly glucanases. Nothing is known, however, about the function of covalently linked proteins. In order to investigate the role of NaOH-extractable cell wall proteins, genes encoding all four identified members of this family of Pir proteins, CCW5, CCW6, CCW7 and CCW8, were disrupted and the phenotype of the mutants obtained was examined. They grew somewhat more slowly, were larger and irregularly shaped, and showed pronounced susceptibility to cell wall synthesis inhibitors like Calcofluor white and Congo red. In addition, the triple and the quadruple deletants had a decreased mating ability. All these properties were more obvious the more of these genes were disrupted, indicating that probably all members of this protein family are at least functionally equivalent in the cell wall.