Protein turnover is an essential cellular process needed for health and survival. It is a way to ensure proper function of proteins, regulate cellular mechanisms and prevent the accumulation of the aberrant proteins. Here, we focus on the turnover of the mammalian molecular chaperones Hsp70 and Hsp90. Many proteins are dependent upon these chaperones for proper folding and maturation. Numerous kinases and transcription factors, for example, are ‘clients’ of Hsp70 and Hsp90.1
Hsp70 is a monomer with two domains: an N-terminal ATPase domain and a C-terminal client protein binding domain.2
Hsp90 functions as a dimer and contains three domains: an N-terminal ATPase domain, a middle domain, and a C-terminal dimerization domain.3
Through their C-terminal sequences, Hsp70 and Hsp90 bind the tetratricopeptide repeat 1 (TPR1) and TPR2A domains of Hsp70/Hsp90 organizing protein (HOP), respectively.4
An unfolded client protein first associates with Hsp70, where it is partially folded, and is then passed to Hsp90, where its maturation is completed.5
During the folding process, both chaperones undergo conformational changes which are coupled to their ATPase activities.
Hsp70 and Hsp90 turnover is dependent upon the ubiquitin-proteasome system. In this pathway, ubiquitin ligases attach the C-terminus of a ubiquitin molecule to typically a lysine side chain on a target protein via an isopeptide bond.6
In many cases a poly-ubiquitin chain, in which the C-terminus of one ubiquitin is linked to a lysine on another ubiquitin, is attached to a target protein. It is known that a target protein modified by a chain of four or more ubiquitin molecules linked via K48 is recognized by the proteasome and subsequently degraded.7
It has recently been demonstrated that poly-ubiquitin chains linked via other lysine residues may also target the modified protein for degradation.8
CHIP is the ubiquitin ligase which ubiquitinates Hsp70 and Hsp90. This enzyme contains an N-terminal TPR domain, a central α-helical domain, and a C-terminal U-box ubiquitin ligase domain.9,10
By associating, via its TPR domain, with the C-terminal sequences of Hsp70 and Hsp90, CHIP has been proposed to ubiquitinate the chaperones’ client proteins, and target them to the proteasome for degradation.11
CHIP is thus the quality control regulator of the folding pathway. We and others have previously shown that Hsp70 and Hsp90 are also ubiquitinated by CHIP in vitro
In addition, CHIP-mediated turnover of Hsp70 and Hsp90 has been studied extensively in vivo
Over-expression of CHIP leads to a decrease in Hsp70 levels; conversely an increase in Hsp70 was noted in the absence of CHIP. Furthermore, addition of exogenous CHIP to CHIP knock-out cells greatly decreased the half-life of both Hsp70 and Hsp90. Our previous data suggest that the cellular concentration of CHIP is fine-tuned relative to the concentrations of the chaperones, so that there is a constant, low level of CHIP-dependent Hsp70 and Hsp90 ubiquitination and consequent proteasome-mediated degradation.12
Here, we further investigate the mechanism of Hsp70 and Hsp90 degradation by identifying the lysine residues on the chaperones ubiquitinated by CHIP and mapping these residues on homology models of Hsp70 and Hsp90 to find any structural regions where the ubiquitination sites cluster. We also determine that CHIP forms poly-ubiquitin chains on Hsp70 and Hsp90 and identify the types of ubiquitin-ubiquitin linkages in these chains.