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Alpha-complemented beta-galactosidase. An in vivo model substrate for the molecular chaperone heat-shock protein 90 in yeast

Abbas-Terki, Toufik
Published in European Journal of Biochemistry. 1999, vol. 266, no. 2, p. 517-23
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.
Keywords Blotting, WesternCycloheximide/pharmacologyGene Expression Regulation, FungalGenes, FungalGenetic Complementation TestHSP90 Heat-Shock Proteins/chemistry/metabolismHeat-Shock Proteins/metabolismModels, ChemicalMolecular Chaperones/chemistryMutationPeptides/chemistryPlasmids/metabolismPrecipitin TestsProtein BindingProtein FoldingProtein Structure, TertiaryProtein Synthesis Inhibitors/pharmacologySaccharomyces cerevisiae/enzymologySaccharomyces cerevisiae ProteinsBeta-Galactosidase/chemistry
PMID: 10561593
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ABBAS-TERKI, Toufik, PICARD, Didier. Alpha-complemented beta-galactosidase. An in vivo model substrate for the molecular chaperone heat-shock protein 90 in yeast. In: European Journal of Biochemistry, 1999, vol. 266, n° 2, p. 517-23. https://archive-ouverte.unige.ch/unige:4651

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Deposited on : 2009-12-10

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