Formation in vitro of complexes between an abnormal fusion protein and the heat shock proteins from Escherichia coli and yeast mitochondria.
Heat shock proteins (HSPs) of the Hsp70 and GroEL families associate with a variety of cell proteins in vivo. However, the formation of such complexes has not been systematically studied. A 31-kDa fusion protein (CRAG), which contains 12 residues of cro repressor, truncated protein A, and 14 residues of beta-galactosidase, when expressed in Escherichia coli, was found in complexes with DnaK, GrpE, protease La, and GroEL. When an E. coli extract not containing CRAG was applied to an affinity column containing CRAG, DnaK, GroEL, and GrpE were selectively bound. These HSPs did not bind to a normal protein A column. DnaK, GrpE, and the fraction of GroEL could be eluted from the CRAG column with ATP but not with a nonhydrolyzable ATP analog. The ATP-dependent release of DnaK and GroEL also required Mg2+, but GrpE dissociated with ATP alone. The binding and release of DnaK and GroEL were independent events, but the binding of GrpE required DnaK. Inactivation of DnaJ, GrpE, and GroES did not affect the association or dissociation of DnaK or GroEL from CRAG. The DnaK and GrpE proteins could be eluted with 10(-6) M ATP, but 10(-4) M was required for GroEL release. This approach allows a one-step purification of these proteins from E. coli and also the isolation of the DnaK and GroEL homologs from yeast mitochondria. Competition experiments with oligopeptide fragments of CRAG showed that DnaK and GroEL interact with different sites on CRAG and that the cro-derived domain of CRAG contains the DnaK-binding site.
Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Massachusetts 02115.
M Y
MY
Sherman
A L
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Goldberg
D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005838:Genotype; D006360:Heat-Shock Proteins / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D010641:Phenotype; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D013205:Staphylococcal Protein A; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D001616:beta-Galactosidase