Scientific article

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

Published inEuropean journal of biochemistry, vol. 266, no. 2, p. 517-523
Publication date1999

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.

  • Blotting, Western
  • Cycloheximide/pharmacology
  • Gene Expression Regulation, Fungal
  • Genes, Fungal
  • Genetic Complementation Test
  • HSP90 Heat-Shock Proteins/chemistry/metabolism
  • Heat-Shock Proteins/metabolism
  • Models, Chemical
  • Molecular Chaperones/chemistry
  • Mutation
  • Peptides/chemistry
  • Plasmids/metabolism
  • Precipitin Tests
  • Protein Binding
  • Protein Folding
  • Protein Structure, Tertiary
  • Protein Synthesis Inhibitors/pharmacology
  • Saccharomyces cerevisiae/enzymology
  • Saccharomyces cerevisiae Proteins
  • Beta-Galactosidase/chemistry
Citation (ISO format)
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. 517–523. doi: 10.1046/j.1432-1327.1999.00881.x
Main files (1)
Article (Published version)
ISSN of the journal0014-2956

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