RESUMO
The 18-kDa protein from Mycobacterium leprae is a major target for the immune response in leprosy. We have developed a system to express this antigen in yeast as a fusion protein with the C-terminal region of the yeast membrane protein GAS1, which would render the recombinant protein anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Cells lacking the GAS1 gene and transformed with the hybrid 18-kDa-GAS1 construct express a polypeptide that reacts with an 18-kDa-specific monoclonal antibody. In addition, these cells react with an alpha-CRD antibody after GPI-PLC treatment. The non-transformed cells are negative. These data indicate that our system may be suitable for the expression of foreign proteins in yeast in a GPI-anchored form.
Assuntos
Proteínas de Bactérias/genética , Glicosilfosfatidilinositóis/genética , Mycobacterium leprae/imunologia , Proteínas de Saccharomyces cerevisiae , Proteínas de Bactérias/imunologia , Proteínas Fúngicas/genética , Genes Fúngicos , Vetores Genéticos , Glicosilfosfatidilinositóis/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Mycobacterium leprae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/imunologiaRESUMO
The 18-kDa protein from Mycobacterium leprae is a major target for the immune response in leprosy. We have developed a system to express this antigen in yeast as a fusion protein with the C-terminal region of the yeast membrane protein GAS1, which would render the recombinant protein anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Cells lacking the GAS1 gene and transformed with the hybrid 18-kDa-GAS1 construct express a polypeptide that reacts with an 18-kDa-specific monoclonal antibody. In addition, these cells react with an alpha-CRD antibody after GPI-PLC treatment. The non-transformed cells are negative. These data indicate that our system may be suitable for the expression of foreign proteins in yeast in a GPI-anchored form
Assuntos
Glicosilfosfatidilinositóis/genética , Mycobacterium leprae/imunologia , Proteínas de Bactérias/genética , Genes Fúngicos , Vetores Genéticos , Glicosilfosfatidilinositóis/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Mycobacterium leprae/genética , Proteínas de Bactérias/imunologia , Proteínas Fúngicas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/imunologiaRESUMO
We have investigated the effects of an inhibitor of ceramide biosynthesis on the glycosylphosphatidylinositol (GPI)-anchoring and intracellular transport of the yeast Gas1 protein. No effect on anchor attachment was demonstrable, but a selective delay in transport from the endoplasmic reticulum to the Golgi complex was observed. The compound also blocked remodeling of GPI-anchors from their base-sensitive to base-resistant forms. A recessive mutation was found that caused resistance to the drug, restored transport of Gas1p, but did not restore ceramide biosynthesis in the presence of the inhibitor. Our results suggest that intracellular transport of GPI-anchored proteins is stimulated by new ceramide synthesis. The role of ceramide may be direct or may be through its use in the remodeling of GPI-anchored proteins other than Gas1p. The need for ceramide can be overcome in the mutant strain.
Assuntos
Ceramidas/metabolismo , Proteínas Fúngicas/metabolismo , Glicosilfosfatidilinositóis/química , Complexo de Golgi/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Ceramidas/fisiologia , Retículo Endoplasmático/metabolismoRESUMO
We have investigated the effects of an inhibitor of ceramide biosynthesis on the glycosylphosphatidylinositol (GPI)-anchoring and intracellular transport of the yeast Gas 1 protein. No effect on anchor attachment was demonstrable, but a selective delay in transport from the endoplasmic reticulum to the Golgi complex was observed. The compound also blocked remodeling of GPI-anchors from their base-sensitive to base-resistant forms. A recessive mutation was found that caused resistance to the drug, restored transport of Gas 1p, but did not restore ceramide biosynthesis in the presence of the inhibitor. Our results suggest that intracellular transport of GPI-anchored proteins is stimulated by new ceramide synthesis. The role of ceramide may be direct or may be through its use in the remodeling of GPI-anchored proteins other than Gas 1p. The need for ceramide can be overcome in the mutant strain