Characterization of recombinant forms of the yeast Gas1 protein and identification of residues essential for glucanosyltransferase activity and folding.
Gas1p is a glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of Saccharomyces cerevisiae and is a representative of Family GH72 of glycosidases/transglycosidases, which also includes proteins from human fungal pathogens. Gas1p, Phr1-2p from Candida albicans and Gel1p from Aspergillus fumigatus have been shown to be beta-(1,3)-glucanosyltransferases required for proper cell wall assembly and morphogenesis. Gas1p is organized into three modules: a catalytic domain; a cys-rich domain; and a highly O-glycosylated serine-rich region. In order to provide an experimental system for the biochemical and structural analysis of Gas1p, we expressed soluble forms in the methylotrophic yeast Pichia pastoris. Here we report that 48 h after induction with methanol, soluble Gas1p was produced at a yield of approximately 10 mg x L(-1) of medium, and this value was unaffected by the further removal of the serine-rich region or by fusion to a 6 x His tag. Purified soluble Gas1 protein showed beta-(1,3)-glucanosyltransferase activity that was abolished by replacement of the putative catalytic residues, E161 and E262, with glutamine. Spectral studies confirmed that the recombinant soluble Gas1 protein assumed a stable conformation in P. pastoris. Interestingly, thermal denaturation studies demonstrated that Gas1p is highly resistant to heat denaturation, and a complete refolding of the protein following heat treatment was observed. We also showed that Gas1p contains five intrachain disulphide bonds. The effects of the C74S, C103S and C265S substitutions in the membrane-bound Gas1p were analyzed in S. cerevisiae. The Gas1-C74S protein was totally unable to complement the phenotype of the gas1 null mutant. We found that C74 is an essential residue for the proper folding and maturation of Gas1p.
Carotti
Cristina
C
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Milano, Italy.
Ragni
Enrico
E
Palomares
Oscar
O
Fontaine
Thierry
T
Tedeschi
Gabriella
G
Rodríguez
Rosalía
R
Latgé
Jean Paul
JP
Vai
Marina
M
Popolo
Laura
L
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000032:analysis / Q000737:chemistry; D005656:Fungal Proteins / Q000378:metabolism*; D015870:Gene Expression; D005800:Genes, Fungal; D005973:Glutamine / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D012995:Solubility
D004220:Disulfides; D005656:Fungal Proteins; C066709:GAS1 protein, S cerevisiae; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D005973:Glutamine; D003545:Cysteine