Abstract
The cell wall of yeast contains proteins that are covalently bound to the glycan network. These cell wall proteins (CWPs) mediate cell-cell interactions and may be involved in cell wall biosynthesis. Using tandem mass spectrometry, we have identified 19 covalently bound CWPs of Saccharomyces cerevisiae. Twelve of them are shown for the first time to be covalently incorporated into the cell wall. The identified proteins include 12 predicted glycosylphosphatidylinositol-modified CWPs, all four members of the Pir protein family, and three additional proteins (Scw4p, Scw10p, and Tos1p) that are, like Pir proteins, connected to the cell wall glycan network via an alkali-sensitive linkage. However, Scw4p, Scw10p, and Tos1p do not contain internal repeat sequences shown to be essential for Pir protein incorporation and may represent a separate class of CWPs. Strikingly, seven of the identified proteins (Gas1p, Gas3p, Gas5p, Crh1p, Utr2p, Scw4p, and Scw10p) are classified as glycoside hydrolases. Phenotypic analysis of deletion mutants lacking the corresponding CWP-encoding genes indicated that most of them have altered cell wall properties, which reinforces the importance of the identified proteins for proper cell wall formation. In particular, gas1Δ and ecm33Δ were highly sensitive to Calcofluor White and high temperature, whereas gas1Δ, scw4Δ, and tos1Δ were highly resistant to incubation with β-1,3-glucanase. The CWP identification method developed here relies on directly generating tryptic peptides from isolated cell walls and is independent of the nature of the covalent linkages between CWPs and cell wall glycans. Therefore, it will probably be equally effective in many other fungi.
Highlights
The cell wall of yeast contains proteins that are covalently bound to the glycan network
We show that cell walls of S. cerevisiae, like C. albicans, contain multiple covalently bound glycoside hydrolases and that many of the identified cell wall proteins (CWPs) are required for normal cell wall formation
CWPs Are Efficiently Digested by Proteases While Being Linked to the Glucan Network—Previously, we have identified covalently bound CWPs of C. albicans by releasing specific classes of CWPs using biochemical methods, followed by proteolytic digestion of the liberated protein pools and tandem mass spectrometry [13]
Summary
Strains and Cell Culture—Wild-type strain FY833 (MATa his3⌬300 ura leu2⌬1 lys2⌬202 trp1⌬63) was grown in YPD (1% (w/v) yeast extract, 2% (w/v) bactopeptone, and 2% (w/v) glucose) and harvested at A600 ϭ 2. Protein Extraction and Fractionation—GPI-modified CWPs were released by incubating the cell walls in undiluted HF-pyridine (SigmaAldrich) at 0 °C for 3 h. Sample Preparation for Mass Spectrometric Analysis—Freeze-dried cell walls (4 mg) were resuspended in a solution containing 100 mM NH4HCO3 and 10 mM dithiothreitol and incubated for 1 h at 56 °C. Cell walls, unfractionated protein pools, and excised protein bands in 50 mM NH4HCO3 were incubated overnight at 37 °C with sequencing grade trypsin (Roche Applied Science) or at 25 °C with endoprotease Glu-C (Sigma), using a CWP/enzyme ratio of 50:1. Immunoblot Analysis—Freeze-dried undigested cell walls and proteolytically treated cell wall residues were incubated with recombinant endo--1,6-glucanase (ProZyme, San Leandro, CA) to release GPI-modified proteins and with cold NaOH to release Pir proteins, as previously described [28]. The effect of -1,3-glucanase treatment was followed by measuring the decrease of A600 in time and expressed as the percentage of the A600 at t0
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