In Escherichia coli, the molecular chaperones (DnaK, DnaJ, and GrpE) are essential for the rapid degradation of certain proteins. To see if chaperones are involved more generally in proteolysis, we studied the degradation of a short-lived fusion protein, CRAG, which associates with DnaK and GroEL in vivo. Its rapid degradation requires ATP and ClpP, the proteolytic subunit of protease Ti (Clp). However, this process is not reduced in strains lacking the complementary ATPase subunit, ClpA, or its homologs, ClpB and ClpX. At 37 degrees C, but not at 42 degrees C, protease La also contributes partially to CRAG degradation. Nevertheless, CRAG is not degraded in cell-free extracts or upon incubation with ClpP or protease La. We tested whether the chaperones associated with CRAG might be involved in its degradation. CRAG breakdown was accelerated 2-3-fold in strains with high levels of heat-shock proteins (hsps), i.e. in those that overproduce the hsp transcription factor (sigma 32) or carry a dnaK deletion. A similar stimulation of proteolysis was observed in cells overproducing GroEL or both GroEL and GroES; in these cells, more CRAG was associated with GroEL than in the wild type. In a temperature-sensitive groEL44 mutant at the nonpermissive temperature, CRAG breakdown was accelerated, and more CRAG was found complexed with GroEL. However, in a temperature-sensitive groES mutant, CRAG was completely stable at the nonpermissive temperature and accumulated bound to GroEL. These findings indicate that the association of CRAG with GroEL is a rate-limiting step in CRAG degradation, which also requires a subsequent action of GroES. We propose that if the hsp60/hsp10 chaperonins fail to catalyze the proper folding of a protein, they can facilitate its rapid degradation.