Exploring the role of disulfide bonds in the GPI-anchored Gas1 protein of Saccharomyces cerevisiae

Gas1 is an extracellular GPI-anchored enzyme of family GH72 active in cell wall remodelling. It is abundantly modified by N- and O- glycosylation and is rich in disulfide bonds (1). Gas1p has also been used as a model protein in studies on transport through the secretory pathway. The ER-precursor (100 kDa) is sequestered in Triton X-100-insoluble membranes and packaging in COPII-coated vesicles requires Emp24 (2). In this work we examined the role of three out of the seven disulfide bonds of Gas1p that were mapped by MS (1). Cys residues were replaced by Ser residues using site-directed mutagenesis. The effects were examined by testing the capability of mutant proteins to complement the defective phenotype of gas1 null mutants, Western blot, and pulse-chase experiments. C52 and C326 were found to be crucial for folding of Gas1p. The C52S and C326S mutant ER precursors (100-kDa) did not mature into the 115 kDa form and were degraded within 60 min of chase. Whereas wild type Gas1p is poorly solubilized by 1% Triton and requires boiling of the crude extract in 1% SDS in order to be detected by immunoprecipitation, this solubilization step was not necessary to detect the mutant proteins. This result indicates that proper folding is a prerequisite for association of Gas1 precursor with Triton X-100 insoluble structures in the ER. Beside the N-terminal GH72 signature domain, Gas1p and the homologous Gas2p contain the Cys-box, an extra domain of about 100 amino acids. Cys-box has four conserved disulfide bonds and low-similarity with a lectin domain (X8) of GH17 plant glucanases. Wild type and truncated forms devoid of GPI-and Cys Box (CB) but containing the same number of Cys residues were obtained. Wild type proteins were secreted. Gas1-delta CB was highly glycosylated and secreted but the protein was inactive whereas Gas2-delta CB, which is not glycosylated, was retained in the ER. Thus, Cys-Box is required for proper folding of the GH72 domain but glycosylation can promote its secretion. These results were examined on the basis of a homology-based molecular model built using the crystal structure of Gas2p, which is specifically expressed in sporulation and is required for spore wall formation (3, 4). 1. Popolo L. et al. J.Biol.Chem.(2008) 283: 18553-18565 2. Watanabe et al. Biochem. J. (2008) 414, 237-245 3. Hurtado-Guerreo R. et al J. Biol. Chem. (2009) 284: 8461-8469 4. Rolli E. et al. Mol. Biol Cell (2011) 22: 1585-1598