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 gamma-carboxyl group of glutamic acids, arising from specific glutamines, with hydroxyl groups of glucoses of beta-1,3-glucan chains. This transglutaminase-type reaction could take place extracellularly and would energetically proceed on the account of amido group elimination.
Highlights
More than 30 cell wall proteins have been identified (6 –9)
Identification of the Amino Acid of the Pir4/Ccw5 Protein That Is Linked to -1,3-Glucan—To study a possible role of the repetitive sequences of proteins with internal repeats (Pir) proteins, we concentrated on the Pir4p/Ccw5p/Cis3p
The analysis reported shows that the Ccw5/Pir4 protein is attached to the cell wall via glutamine residue 74 within the repetitive sequence QIGDGQ74VQ
Summary
Yeast Strains and Genetic Methods—The following isogenic yeast strains were used: SEY6210 (MAT␣ ura 3–52 leu112 his3-⌬200 trp1-⌬901 lys 801 suc2-⌬9), VMA5 (MAT␣ ura leu112 his3-⌬200 trp1-⌬901 lys 801 suc2-⌬9 ccw5⌬). 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 PCRamplified 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.
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