title: Alpha-complemented beta-galactosidase. An in vivo model substrate for the molecular chaperone heat-shock protein 90 in yeast.
abstract: Intracistronic complementation of N-terminally truncated beta-galactosidase mutants such as M15 by coexpressed alpha-peptide was originally discovered in Escherichia coli and exploited for plasmid cloning as the well-known blue-white screening method. We show here that alpha-complementation also works in the budding yeast Saccharomyces cerevisiae, and that it can be used as a simple nonselective enzymatic marker for a variety of in vivo studies, for example, on the role of molecular chaperones in protein folding and assembly. To be able to induce alpha-complementation post-translationally, we have constructed a hormone-inducible alpha-peptide by fusion of the DNA encoding the alpha-peptide to that of to the hormone binding domain of the estrogen receptor. The accumulation of both subunits, the alpha-peptide and M15, is severely compromised when they are expressed separately, presumably because their hydrophobic surfaces remain exposed. Moreover, alpha-complementation is defective in a strain of S. cerevisiae carrying a point mutant of the molecular chaperone heat-shock protein 90. Heat-shock protein 90, which coprecipitates with M15, might be required in vivo to prevent the degradation of unassembled M15 and to hold it in an interaction-competent conformation.
authors.affiliation: Départment de Biologie Cellulaire, Université de Genève, Switzerland.
authors.forename: T
authors.initials: T
authors.lastname: Abbas-Terki
authors.forename: D
authors.initials: D
authors.lastname: Picard
mesh_terms: D015153:Blotting, Western; D003513:Cycloheximide / Q000494:pharmacology; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism; D008956:Models, Chemical; D018832:Molecular Chaperones / Q000737:chemistry*; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D001616:beta-Galactosidase / Q000737:chemistry*
chemical_list: C074510:HSP82 protein, S cerevisiae; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D010455:Peptides; D011500:Protein Synthesis Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D003513:Cycloheximide; D001616:beta-Galactosidase