Pir Proteins of Saccharomyces cerevisiae Are Attached to β-1,3-Glucan by a New Protein-Carbohydrate Linkage

Abstract

A family of covalently linked cell wall proteins of Saccharomyces cerevisiae, called Pir proteins, are characterized by up to 10 conserved repeating units. Ccw5/Pir4p contains only one complete repeating sequence and its deletion caused a release of the protein into the medium. The exchange of each of three glutamines (Gln69, Gln74, Gln76) as well as one aspartic acid (Asp72) within the repeating unit leads to a loss of the protein from the cell wall. Amino acid sequencing revealed that only Gln74 is modified. Release of the protein with mild alkali, changed Gln74 to to glutamic acid, suggesting that Gln74 is involved in the linkage. Analysis by mass spectrometry showed that 5 hexoses are attached to Gln/Glu74. Sugar analysis revealed glucose as the only constituent. It is suggested that Pir proteins form novel, alkali labile ester linkages between the γ-carboxyl group of glutamic acids, arising from specific glutamines, with hydroxyl groups of glucoses of β-1,3-glucan chains. This transglutaminase-type reaction could take place extracellularly and would energetically proceed on the account of amido group elimination. A family of covalently linked cell wall proteins of Saccharomyces cerevisiae, called Pir proteins, are characterized by up to 10 conserved repeating units. Ccw5/Pir4p contains only one complete repeating sequence and its deletion caused a release of the protein into the medium. The exchange of each of three glutamines (Gln69, Gln74, Gln76) as well as one aspartic acid (Asp72) within the repeating unit leads to a loss of the protein from the cell wall. Amino acid sequencing revealed that only Gln74 is modified. Release of the protein with mild alkali, changed Gln74 to to glutamic acid, suggesting that Gln74 is involved in the linkage. Analysis by mass spectrometry showed that 5 hexoses are attached to Gln/Glu74. Sugar analysis revealed glucose as the only constituent. It is suggested that Pir proteins form novel, alkali labile ester linkages between the γ-carboxyl group of glutamic acids, arising from specific glutamines, with hydroxyl groups of glucoses of β-1,3-glucan chains. This transglutaminase-type reaction could take place extracellularly and would energetically proceed on the account of amido group elimination. Fungal cell walls are rigid albeit dynamic, complex structures, withstanding intracellular osmotic pressures of more than 10 bar (1Eamus D.G. Jennings D.H. Ayres P. Body L. Water, Fungi and Plants. Cambridge University Press, Cambridge, UK1986: 27-47Google Scholar). In Saccharomyces cerevisiae the cell wall amounts to about 20% of the total dry weight of the cell. Besides a small amount of chitin, it mainly consists of β-glucans and mannoproteins. The actual protein content comes to about 10% of the wall by weight (2Orlean P. Pringle J.R. Broach J.R. Jones E.W. The Molecular and Cellular Biology of the Yeast Saccharomyces. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 229-362Google Scholar). Almost all the proteins are highly O- and N-mannosylated (2Orlean P. Pringle J.R. Broach J.R. Jones E.W. The Molecular and Cellular Biology of the Yeast Saccharomyces. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 229-362Google Scholar, 3Ballou C.E. Strathern J.N. Jones E.W. Broach J.R. The Molecular Biology of the Yeast Saccharomyces. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1982: 335-360Google Scholar, 4De Groot P.W. Ram A.F. Klis F.M. Fungal Genet. Biol. 2005; 42: 657-675Crossref PubMed Scopus (231) Google Scholar, 5Tanner W. Lehle L. Biochim. Biophys. Acta. 1987; 906: 81-99Crossref PubMed Scopus (252) Google Scholar). Cell wall studies in the past ten to fifteen years have focused on the various cell wall proteins, since the information concerning their function is expected to help understand the biogenesis as well as the dynamic rebuilding and dissolving of this highly intricate, extracellular organelle during budding and mating. By now, more than 30 cell wall proteins have been identified (6Cappellaro C. Mrsa V. Tanner W. J. Bacteriol. 1998; 180: 5030-5037Crossref PubMed Google Scholar, 7Molina M. Gil C. Pla J. Arroyo J. Nombela C. Microsc. Res. Tech. 2000; 51: 601-612Crossref PubMed Scopus (24) Google Scholar, 8Mrsa V. Seidl T. Gentzsch M. Tanner W. Yeast. 1997; 13: 1145-1154Crossref PubMed Scopus (190) Google Scholar, 9Yin Q.Y. de Groot P.W. Dekker H.L. de Jong L. Klis F.M. de Koster C.G. J. Biol. Chem. 2005; 280: 20894-20901Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Some of them are solubilized from the cell wall by SDS under reducing conditions and are therefore called soluble cell wall proteins (10Valentin E. Herrero W. Pastor J.F.I. Sentandreu R. J. Gen. Microbiol. 1984; 130: 1419-1428Google Scholar), however, most of them are covalently attached to the glucan layer and can be released from SDS-extracted cell walls by β-glucanases (11Kopecka M. Phaff H.J. Fleet G.H. J. Cell Biol. 1974; 62: 66-76Crossref PubMed Scopus (70) Google Scholar, 12Montijn R.C. van Rinsum J. van Schagen F.A. Klis F.M. J. Biol. Chem. 1994; 269: 19338-19342Abstract Full Text PDF PubMed Google Scholar). Two modes of covalent linkage can be distinguished. In the first case the cell wall protein is synthesized as GPI 5The abbreviations used are: GPI, glycosylphosphatidylinositol; HA, hemagglutinin; HPLC, high performance liquid chromatography; PTH, phenylthiohydantoin; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; HPAEC, high performance anion exchange chromatography. (glycosylphosphatidylinositol) modified intermediate. These are trans-mannosylated by hydrolyzing the oligomannosyl moiety of the GPI lipid anchor and by a subsequent transfer to β-1,6-glucan (12Montijn R.C. van Rinsum J. van Schagen F.A. Klis F.M. J. Biol. Chem. 1994; 269: 19338-19342Abstract Full Text PDF PubMed Google Scholar, 13Kollar R. Reinhold B.B. Petrakova E. Yeh H.J. Ashwell G. Drgonova J. Kapteyn J.C. Klis F.M. Cabib E. J. Biol. Chem. 1997; 272: 17762-17775Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 14Lipke P.N. Ovalle R. J. Bacteriol. 1998; 180: 3735-3740Crossref PubMed Google Scholar, 15Lu C.F. Montijn R.C. Brown J.L. Klis F. Kurjan J. Bussey H. Lipke P.N. J. Cell Biol. 1995; 128: 333-340Crossref PubMed Scopus (188) Google Scholar, 16Fujii T. Shimoi H. Iimura Y. Biochim. Biophys. Acta. 1999; 1427: 133-144Crossref PubMed Scopus (65) Google Scholar). This group of covalently attached cell wall proteins, called GPI-Cwp, are released from the wall both by β-1,6- and by β-1,3-glucanases, due to the attachment of GPI-Cwps to β-1,3-glucan via a short β-1,6-glucan bridge (4De Groot P.W. Ram A.F. Klis F.M. Fungal Genet. Biol. 2005; 42: 657-675Crossref PubMed Scopus (231) Google Scholar, 12Montijn R.C. van Rinsum J. van Schagen F.A. Klis F.M. J. Biol. Chem. 1994; 269: 19338-19342Abstract Full Text PDF PubMed Google Scholar). The second group of covalently linked cell wall proteins are the so-called Pir proteins, named originally according to three genes coding for putative proteins with internal repeats (Pir) (17Toh-e A. Yasunaga S. Nisogi H. Tanaka K. Oguchi T. Matsui Y. Yeast. 1993; 9: 481-494Crossref PubMed Scopus (100) Google Scholar) and subsequently identified as covalently linked cell wall proteins Ccw1, -2, -3, and -4p (8Mrsa V. Seidl T. Gentzsch M. Tanner W. Yeast. 1997; 13: 1145-1154Crossref PubMed Scopus (190) Google Scholar, 18Yun D.J. Zhao Y. Pardo J.M. Narasimhan M.L. Damsz B. Lee H. Abad L.R. D'Urzo M.P. Hasegawa P.M. Bressan R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7082-7087Crossref PubMed Scopus (123) Google Scholar, 19Moukadiri I. Jaafar L. Zueco J. J. Bacteriol. 1999; 181: 4741-4745Crossref PubMed Google Scholar). For S. cerevisiae five PIR genes are known (Table 1) coding for proteins with varying numbers of repetitive units (1 to 10). Pir proteins do not contain a C-terminal GPI-addition signal, they are all processed by Kex2 protease, and they are released from intact cells by very mild alkaline treatment (30 mm NaOH, 12 h, 4 °C) (8Mrsa V. Seidl T. Gentzsch M. Tanner W. Yeast. 1997; 13: 1145-1154Crossref PubMed Scopus (190) Google Scholar). Pir proteins are attached directly to β-1,3-glucan (20Kapteyn J.C. Van Den Ende H. Klis F.M. Biochim. Biophys. Acta. 1999; 1426: 373-383Crossref PubMed Scopus (318) Google Scholar) and can therefore be released from S. cerevisiae by the corresponding β-1,3-glucanase, not however by β-1,6-glucanase (4De Groot P.W. Ram A.F. Klis F.M. Fungal Genet. Biol. 2005; 42: 657-675Crossref PubMed Scopus (231) Google Scholar). The actual alkali labile linkage between the protein moiety and β-1,3-glucan is not known.TABLE 1The Pir proteins of S. cerevisiae (proteins with internal repeats)Gene locusGene nameNumber of repeatsYKL164cPIR1/CCW68YJL159wPIR2/CCW7/HSP14010YKL163wPIR3/CCW88YJL158cPIR4/CCW5/CIS31(-2)YJL160cPIR54 Open table in a new tab Pir proteins known to be involved in various biological phenomena are not essential for viability; a quadruple mutant, however, possesses a severe growth phenotype (21Mrsa V. Tanner W. Yeast. 1999; 15: 813-820Crossref PubMed Scopus (97) Google Scholar). Pir2/Ccw7/Hsp150p is strongly induced by heat stress and nitrogen starvation (22Russo P. Kalkkinen N. Sareneva H. Paakkola J. Makarow M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3671-3675Crossref PubMed Scopus (90) Google Scholar, 23Russo P. Simonen M. Uimari A. Teesalu T. Makarow M. Mol. Gen. Genet. 1993; 239: 273-280Crossref PubMed Scopus (41) Google Scholar). Pir proteins 1 through 3 have been shown to cause resistance to an antifungal protein of tobacco (18Yun D.J. Zhao Y. Pardo J.M. Narasimhan M.L. Damsz B. Lee H. Abad L.R. D'Urzo M.P. Hasegawa P.M. Bressan R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7082-7087Crossref PubMed Scopus (123) Google Scholar). Special functional importance of Pir proteins has also to be deduced from the fact that PIR1–3 are the most highly regulated genes in the cell cycle; the expression score of PIR1 amounts to 16 with the activity peaking in M/G1, i.e. during cell separation (24Spellman P.T. Sherlock G. Zhang M.Q. Iyer V.R. Anders K. Eisen M.B. Brown P.O. Botstein D. Futcher B. Mol. Biol. Cell. 1998; 9: 3273-3297Crossref PubMed Scopus (3900) Google Scholar). In this report we characterize the linkage between Ccw5/Pir4p and β-1,3-glucan. Evidence will be presented that the amido group of a specific glutamine residue within the repetitive sequence is deamidated and a carboxyl ester with β-1,3-linked glucose is formed. Yeast Strains and Genetic Methods—The following isogenic yeast strains were used: SEY6210 (MATα ura 3–52 leu2–3,112 his3-Δ200 trp1-Δ901 lys2–801 suc2-Δ9), VMA5 (MATα ura3–52 leu2–3,112 his3-Δ200 trp1-Δ901 lys2–801 suc2-Δ9 ccw5Δ). Cells were grown at 30 °C in standard yeast media either YEPD (1% Bacto yeast extract, 2% Bacto peptone, 2% dextrose) or YNBD (0.67% yeast nitrogen base, 0.08% complete amino acid supplement mixture (from Bio 101, Inc.), 2% dextrose). Transformation into yeast and Eschericia coli was carried out using standard techniques. Plasmid Constructions—Standard molecular biology techniques were used for all plasmid constructions. The correct sequence of PCR-amplified products was verified by DNA sequencing. The sequences of primers used in this study are available on request. CCW5 was amplified from yeast genomic DNA. The amplified gene was inserted into the SmaI site of vector YEp351. To introduce a hemagglutinin (HA) epitope at the C terminus of CCW5 a NotI site was first created, and the fragment was inserted into the SalI restriction site at the very end of CCW5. Then, six HA epitopes were inserted into the NotI site in frame with CCW5 and verified by Western blot analysis. CCW5—plasmids containing N-terminal deletions, pCCW5-(Δ65–82)-HA and pCCW5-(Δ83–95)-HA, respectively (see Fig. 2) were generated by two-step PCR technology. Site-directed mutagenesis to exchange specific amino acids in pCCW5-HA was performed by the two step PCR method to give pCCW5-(Q69A)-HA, pCCW5-(D72N)-HA, pCCW5-(Q74A)-HA, and pCCW5-(Q76A)-HA, respectively. The correct sequences have been confirmed by sequencing. Plasmid pCCW5-ZZ contains two C-terminal protein A epitopes in frame with CCW5 inserted into the vector pRS425. In plasmid pCCW5-(S79M)-ZZ amino acid Ser79 has been replaced by Met as described above for the exchanges in the plasmid pCCW5HA. Isolation and Purification of Pir4/Ccw5-ZZ—SEY6210, harboring pCCW5-ZZ, was grown in YNBD without leucine to an A600 of 3. Cells (A600 2000) were harvested and washed in 50 ml 0.1 m potassium phosphate buffer (pH 8), resuspended in 5 ml of ice-cold 0.1 m potassium phosphate buffer, containing the protease inhibitors Nα-p-tosyl-l-lysine chloromethyl ketone (0.1 mg/ml), l-1-tosylamido-2-phenylethyl chloromethyl ketone (1 (1 and each 1 The cells were for with in a in of ml by Cell walls were by at for 5 The cell wall were washed with ml of potassium phosphate Cell walls were for 10 at °C in ml of SDS buffer, at washed with ml of and with 50 mm (pH and cell walls were resuspended in ml of 50 mm (pH with protease inhibitors (see and with units of for 3 at an units were and for The was at for The was and to mm with protease inhibitors (see and to ml of according to the and in m (pH containing m and The was by for 4 at 4 °C and into a and washed three with 5 and two with 5 mm (pH was with m (pH and from For of from the this was and containing protease and subsequently to as described Analysis of from cell wall and and by was with ml of 30 mm at 4 The was in through a and by high performance anion exchange with on a using a and a from 16 mm to mm in mm within For were and the and 3 (see Fig. were The was on and used for analysis. for β-1,3-linked and and for and by 1 m acid for 1 To the of the and were with 4 m acid and 4 for 4 at under in and by on a with 16 mm as Isolation and Analysis of was performed at 4 °C in acid containing 5 The of the was under a of the residue was with and The were by using a and identified by of was using a with of the acids according to the were with a using acid as a of the Amino of the to study a of the repetitive sequences of Pir proteins, we on the This protein has only one repetitive sequence of the in all Pir proteins, in a to the first one 5 of the 12 conserved amino acids are L. A. E. Sentandreu R. Yeast. PubMed Scopus Google Scholar, V. D. M. Tanner W. M. Molecular of Fungal Cell Scholar), a deletion of the repetitive sequence acids in the release of to the medium. In Fig. is presented that the conserved repetitive to attached to the cell wall in a withstanding with SDS under reducing The following sequence acids is for covalent cell wall in the of the is not is due to the fact that the site to the deletion is not used in this as confirmed by N-terminal sequencing. is to a also in the in is due to the of the In a amount of with the cell wall and is released as also into the (8Mrsa V. Seidl T. Gentzsch M. Tanner W. Yeast. 1997; 13: 1145-1154Crossref PubMed Scopus (190) Google Scholar). This is not with of the Pir proteins, all have repeating units. The alkaline of the linkage of Pir proteins suggested that they be attached to via a amino the and in the repetitive as well as the were replaced by the attachment of to the cell wall was not not the each exchange of the three glutamine within the repetitive sequence as well as with and to a complete loss of cell The exchange of an residue of the repetitive sequence This all 4 amino acids are for the protein to cell at most one could form the actual information concerning the amino acid involved in the linkage was by sequencing was released from cell walls by either or mild alkali shown in Fig. sequencing the protein released by revealed that the expected acid for in was the of a modified amino acid not by the standard however, in the protein released by alkali, was a glutamic acid at This that Gln74 is the amino acid and that it is deamidated during the attachment to β-1,3-glucan. In with this is the fact that to the contain a glutamine at that A well known reaction using glutamine as is the reaction C. I. J. 2005; 272: PubMed Scopus Google Scholar). The the group of the with an group of and proteins in this D. M. 1994; PubMed Scopus Google Scholar). a of reaction for to the cell one would have to that β-1,3-glucan contains for in Fig. In one would have to that the is alkali of a glutamine to β-1,3-glucan via the release of the amido group is suggested in Fig. The in this case would be an ester between the γ-carboxyl group from glutamine and an group of a residue within the β-1,3-glucan chains. this would be on the account of the and an ester would be of the to of the the of the amino acid at of we used a C-terminal protein in Ser79 was replaced with a residue by site mutagenesis This the of with a C-terminal by treatment of the protein by of the and separation of the released acids for and that amino acids are modified and under the separation conditions for standard amino that all hydroxyl amino acids of are M. Mrsa V. I. R. S. Tanner W. PubMed Scopus Google Scholar), we can that and are the of Gln74 To the of this an of was with mild alkali, in of A and were in a glutamic acid showed in with the for the intact as described this treatment was to the of Gln74 not to the from and mass spectrometry we information about the of the mass spectrometry of showed a with a of and one with with a mass than 1 in Fig. to the molecular mass of with at the C-terminal end and 12 respectively. In the at and with by mass most to the a C-terminal of by of the mild alkali treatment the at and its and only the mass not with the by this is that and are by a total of and 5 hexoses are attached to the amino acid in the of the mass spectrometry analysis was than 1 are with attachment of the 5 hexoses to and not analysis by was not due to during the of the could only be in and not in the not To the attached to we it from the SDS-extracted cell walls by a the protein was also from the medium. were with mild alkali, and the released was by can be from the shown in Fig. the cell to three were from protein from the medium. The three with and and from the the three were only glucose and a amount of however, has been the amount to at 20% in the case of the and to in the case of the It has to be that is directly linked to β-1,3-glucan. A attached to the protein be by The treatment with 30 mm caused of in the For mass spectrometry the was with conditions that not cause of the as with a release the covalently protein from yeast cell we used an treatment with 30 mm at 4 as shown in Fig. the linkage is more at 10 mm more than of the protein is released from the cell wall in at 4 and at 30 mm are for complete A linkage with the of the γ-carboxyl group of glutamic acid, the of linkage that would form in a is known to be more than a linkage. To the of the wall with that of an we the alkali of 30 mm than 2% of the were not This in is as for the ester linkage an an ester was available to the of the corresponding in The analysis that the protein is attached to the cell wall via glutamine residue within the repetitive sequence This is conserved in all Pir The also that the linkage at Gln74 is alkali labile and that the the glutamine has been to a glutamic This to the of a transglutaminase-type an amino a containing was as of the β-1,3-glucan attached to the and since gene a can be identified in the S. cerevisiae it is that an ester linkage between the γ-carboxyl of a arising from and the hydroxyl is formed. an ester would be expected to be alkali the of S. cerevisiae cell wall is well known since is known about the the various are linked and of the for the corresponding have been of the is to β-1,3-glucan for a reaction has been R. Petrakova E. Ashwell G. P.W. Cabib E. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). molecular analysis of a yeast cell wall complex has been carried out in R. Reinhold B.B. Petrakova E. Yeh H.J. Ashwell G. Drgonova J. Kapteyn J.C. Klis F.M. Cabib E. J. Biol. Chem. 1997; 272: 17762-17775Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar). this and from of the and T. Shimoi H. Iimura Y. Biochim. Biophys. Acta. 1999; 1427: 133-144Crossref PubMed Scopus (65) Google Scholar), it was that the GPI proteins are linked to β-1,6-glucan via one or of the GPI the linkage was presented that with its reducing end also be covalently to a cell wall proteins of the GPI be in the cell wall either due to their to β-1,3-glucan or to and in either case a short β-1,6-glucan will a The various for covalent have to be The linkages between cell wall described give to a very complex and highly This however, a covalently as for the of The covalently the resistance to high internal could in the yeast cell for cell wall proteins in in cell walls is well is for protein The Pir proteins with up two 10 repetitive sequence (see 1) could be for a the repetitive unit a to β-1,3-glucan as shown in the and since we can that this is the case for each repetitive the Pir proteins could up to 10 β-1,3-glucan chains. The yeast in PIR genes a in as well as a changed (21Mrsa V. Tanner W. Yeast. 1999; 15: 813-820Crossref PubMed Scopus (97) Google Scholar). also a to cell wall the that Pir proteins an in cell wall their deletion would be expected to in a to the gene expression analysis (24Spellman P.T. Sherlock G. Zhang M.Q. Iyer V.R. Anders K. Eisen M.B. Brown P.O. Botstein D. Futcher B. Mol. Biol. Cell. 1998; 9: 3273-3297Crossref PubMed Scopus (3900) Google Scholar), the of Pir proteins during This that the Pir proteins are to both the synthesized walls and that and are at the has been presented T. T. Y. Y. Cell. 2005; PubMed Scopus Google Scholar). A is the of the is the an of an reaction would not a since the of would be for the of the ester linkage. The reaction new not however, a concerning the gene and gene A would be that the repetitive sequences of the Pir proteins the of their attachment to the β-1,3-glucan. This could the essential of glutamine and an one in the of the Gln74, to the glucan The of a is under the of and and A. and S. from for