Selective degradation of non-native proteins by cytoplasmic quality control (CQC) mechanisms is at the heart of many misfolded protein disorders. The first line of defense is the molecular chaperone network that binds to misfolded proteins and prevents them from engaging in deleterious interactions with the surrounding environment. Degradation by the ubiquitin proteasome system is a main avenue for eliminating these misfolded proteins. How the chaperone network and the ubiquitination machinery are working together in the cytoplasm to accomplish this feat is only beginning to be understood. These studies have discovered two ubiquitin ligases that are responsible for ubiquitinating and promoting the degradation of cytoplasmic misfolded proteins. These two ligases are Ubr1 and San1. Ubr1 is the first cytoplasmic ligase to be discovered to carry out quality control in this compartment in S. cerevisiae. San1 is a nuclear quality control ligase, and its discovered role in CQC highlights a previously unknown pathway for degrading cytoplasmic misfolded proteins. Both ubiquitin ligases function in a chaperone dependent manner. San1 requires chaperones for transport of misfolded proteins to the nucleus where it is believed it then ubiquitinates its substrates directly. Ubr1 more directly utilizes the chaperones for to facilitate ubiquitination. The chaperone networks of Hsp70 and Hsp90 were required for degradation and ubiquitination of CQC substrates. The Hsp70 cochaperones, Ydj1 and Sse1, were found to also be required for elimination of CQC substrates; however, other cochaperones previously found to be involved in CQC were not. This suggests that the framework for CQC is built upon the Hsp70 and Hsp90 machines, and the cochaperones modulate their activity by providing substrate specificity and recruitment of certain UPS factors to promote degradation. Additionally, Ubr1 is able to ubiquitinate insoluble CQC substrates, but requires the activity of Hsp70, Ydj1 and Sse1 to do so; indicating that chaperone function is not merely relegated to keeping misfolded proteins soluble, but is needed for presentation of misfolded substrate to the ubiquitin ligase. The E3 ligase Ubr1 is highly conserved across eukaryotes, from yeast to humans, and the knowledge gained of its role in chaperone dependent CQC will impact future studies of misfolded protein diseases

Chaperones can mediate both protein folding and degradation. This process is referred to as protein triage, which demands study to reveal mechanisms of quality control for both basic scientific and translational purposes. In yeast, many misfolded proteins undergo chaperone-dependent ubiquitination by the action of the E3 ligases Ubr1 and San1, allowing detailed study of protein triage. In cells, both HSP70 and HSP90 mediated substrate ubiquitination, and the canonical ATP cycle was required for HSP70's role: we have found that ATP hydrolysis by HSP70, the nucleotide exchange activity of Sse1, and the action of J-proteins are all needed for Ubr1-mediated quality control. To discern whether chaperones were directly involved in Ubr1-mediated ubiquitination, we developed a bead-based assay with covalently immobilized but releasable misfolded protein to obviate possible chaperone effects on substrate physical state or transport. In this in vitro assay, only HSP70 was required, along with its ATPase cycle and relevant cochaperones, for Ubr1-mediated ubiquitination. The requirement for the HSP70 ATP cycle in ubiquitination suggests a possible model of triage in which efficiently folded proteins are spared, while slow-folding or nonfolding proteins are iteratively tagged with ubiquitin for subsequent degradation.