Two distinct mechanisms operate in the reactivation of heat-denatured proteins by the mitochondrial Hsp70/Mdj1p/Yge1p chaperone system.
The yeast mitochondrial Hsp70, Ssc1p, functions as a molecular chaperone with its partner proteins, Mdj1p (DnaJ homologue) and Yge1p (GrpE homologue). We have purified a mature form of Ssc1p from yeast mitochondria and those of Mdj1p and Yge1p from Escherichia coli overexpresser cells. With these purified components of the mitochondrial ... Hsp70 chaperone system, we have succeeded in reconstituting their chaperone functions in the protection of firefly luciferase against thermal damage in vitro. Heat-denatured luciferase is prevented from irreversible aggregation and is maintained in a refolding-competent state by Ssc1p and/or Mdj1p at 42 degreesC. Luciferase denatured at 42 degreesC is actively reactivated by Ssc1p, Mdj1p and/or Yge1p after lowering the temperature to 25 degreesC. The reactivation process of heat-denatured luciferase shows two-phase kinetics. The slow refolding process requires either Ssc1p or Mdj1p at 42 degreesC but the presence of Ssc1p, Mdj1p and Yge1p, and ATP hydrolysis, is essential at 25 degreesC. The slow refolding of luciferase involves multiple rounds of formation and dissociation of the complex between luciferase and Mdj1p/Ssc1p. On the other hand, the fast refolding process is most enhanced when luciferase is incubated with Ssc1p alone at 42 degreesC, and it requires neither the assistance of Mdj1p and Yge1p nor ATP hydrolysis. We have observed a similar two-pathway reactivation of heat-denatured luciferase by the bacterial Hsp70 and the yeast cytosolic Hsp70 systems.
Mesh Terms:
Adenosine Triphosphate, Bacterial Proteins, Calcium-Transporting ATPases, Cell-Free System, Cytosol, Escherichia coli Proteins, Fungal Proteins, Genes, Reporter, HSP40 Heat-Shock Proteins, HSP70 Heat-Shock Proteins, Heat-Shock Proteins, Hot Temperature, Kinetics, Luciferases, Macromolecular Substances, Membrane Proteins, Membrane Transport Proteins, Mitochondria, Mitochondrial Membrane Transport Proteins, Models, Biological, Molecular Chaperones, Protein Conformation, Protein Denaturation, Protein Folding, Recombinant Fusion Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins
Adenosine Triphosphate, Bacterial Proteins, Calcium-Transporting ATPases, Cell-Free System, Cytosol, Escherichia coli Proteins, Fungal Proteins, Genes, Reporter, HSP40 Heat-Shock Proteins, HSP70 Heat-Shock Proteins, Heat-Shock Proteins, Hot Temperature, Kinetics, Luciferases, Macromolecular Substances, Membrane Proteins, Membrane Transport Proteins, Mitochondria, Mitochondrial Membrane Transport Proteins, Models, Biological, Molecular Chaperones, Protein Conformation, Protein Denaturation, Protein Folding, Recombinant Fusion Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins
J. Mol. Biol.
Date: Feb. 19, 1999
PubMed ID: 9973563
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