Cristina Carotti, Enrico Ragni, Oscar Palomares, Thierry Fontaine, Gabriella Tedeschi, Rosalía Rodríguez, Jean Paul Latgé, Marina Vai, Laura Popolo
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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.</p>","PeriodicalId":11817,"journal":{"name":"European journal of biochemistry","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2004-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/j.1432-1033.2004.04297.x","citationCount":"64","resultStr":"{\"title\":\"Characterization of recombinant forms of the yeast Gas1 protein and identification of residues essential for glucanosyltransferase activity and folding.\",\"authors\":\"Cristina Carotti, Enrico Ragni, Oscar Palomares, Thierry Fontaine, Gabriella Tedeschi, Rosalía Rodríguez, Jean Paul Latgé, Marina Vai, Laura Popolo\",\"doi\":\"10.1111/j.1432-1033.2004.04297.x\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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. 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引用次数: 64
摘要
Gas1p是酿酒酵母(Saccharomyces cerevisiae)的糖基磷脂酰肌醇锚定质膜糖蛋白,是糖苷酶/转糖苷酶家族GH72的代表,该家族还包括来自人类真菌病原体的蛋白质。来自白色念珠菌的Gas1p、Phr1-2p和来自烟曲霉的Gel1p已被证明是正常细胞壁组装和形态发生所必需的β -(1,3)-葡聚糖基转移酶。Gas1p被组织成三个模块:催化结构域;富含蔬菜的地区;和一个高度o糖基化的富含丝氨酸的区域。为了给Gas1p的生化和结构分析提供实验系统,我们在甲基营养酵母毕赤酵母中表达了Gas1p的可溶性形式。在这里,我们报告了用甲醇诱导48小时后,以大约10 mg x L(-1)的培养基产量产生可溶性Gas1p,并且该值不受进一步去除富丝氨酸区域或融合到6 x His标签的影响。纯化的可溶性Gas1蛋白显示β -(1,3)-葡聚糖基转移酶活性,该活性通过用谷氨酰胺取代推定的催化残基E161和E262而被消除。光谱研究证实,重组可溶性Gas1蛋白在巴氏酵母中具有稳定的构象。有趣的是,热变性研究表明,Gas1p对热变性具有高度的抗性,并且在热处理后观察到蛋白质完全重新折叠。我们还发现Gas1p含有5个链内二硫键。分析了C74S、C103S和C265S在酿酒酵母膜结合的Gas1p中的取代作用。gas1 - c74s蛋白完全不能补充gas1零突变体的表型。我们发现C74是Gas1p正确折叠和成熟所必需的残基。
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.