The E3 ubiquitin ligase CHIP (C-terminus of Hsc70-interacting protein) is 
believed to be a central player in the cellular triage decision, as it links the 
molecular chaperones Hsp70/Hsc70 and Hsp90 to the ubiquitin proteasomal 
degradation pathway. To better understand the decision process, we determined 
the affinity of CHIP for Hsp70 and Hsp90 using isothermal titration calorimetry. 
We analyzed the influence of CHIP on the ATPase cycles of both chaperones in the 
presence of co-chaperones and a substrate, and determined the ubiquitination 
efficacy of CHIP in the presence of the chaperones. We found that CHIP has a 
sixfold higher affinity for Hsp90 compared with Hsc70. CHIP had no influence on 
ADP dissociation or ATP association, but reduced the Hsp70 cochaperone 
Hdj1-stimulated single-turnover ATPase rates of Hsc70 and Hsp70. CHIP did not 
influence the ATPase cycle of Hsp90 in the absence of co-chaperones or in the 
presence of the Hsp90 cochaperones Aha1 or p23. Polyubiquitination of 
heat-denatured luciferase and the native substrate p53 was much more efficient 
in the presence of Hsc70 and Hdj1 than in the presence of Hsp90, indicating that 
CHIP preferentially ubiquitinates Hsp70-bound substrates.