The ubiquitin-mediated proteasomal degradation of misfolded proteins is generally thought to require Hsp70 chaperones, particularly Ssa1 and Ssa2 in yeast. This study reveals that Ssa1/Ssa2 are involved in the degradation of misfolded proteins in the yeast nucleus, but their degree of involvement varies depending on the misfolded nuclear protein.

Stress is ubiquitous to life and can irreparably damage essential biomolecules and organelles in cells. To survive, organisms must sense and adapt to stressful conditions. One highly conserved adaptive stress response is through the post-translational modification of proteins by the small ubiquitin-like modifier (SUMO). Here, we examine the effects of acute ethanol stress on protein sumoylation in the budding yeast Saccharomyces cerevisiae . We found that cells exhibit a transient sumoylation response after acute exposure to ≤ 7.5% ethanol. By contrast, the sumoylation response becomes chronic at 10% ethanol exposure. Mass spectrometry analyses identified 18 proteins that are sumoylated after acute ethanol exposure, with 15 known to associate with chromatin. Upon further analysis, we found that the chromatin structural proteins Smc5 and Smc6 undergo ethanol-induced sumoylation that depends on the activity of the E3 SUMO ligase Mms21. Using cell-cycle arrest assays, we observed that Smc5 and Smc6 ethanol-induced sumoylation occurs during G1 and G2/M phases but not S phase. Acute ethanol exposure also resulted in the formation of Rad52 foci at levels comparable to Rad52 foci formation after exposure to the DNA alkylating agent methyl methanesulfonate (MMS). MMS exposure is known to induce the intra-S phase DNA damage checkpoint via Rad53 phosphorylation, but ethanol exposure did not induce Rad53 phosphorylation. Ethanol abrogated the effect of MMS on Rad53 phosphorylation when added simultaneously. From these studies, we propose that acute ethanol exposure induces a change in chromatin leading to sumoylation of specific chromatin-structural proteins.

Cellular homeostasis depends on robust protein quality control (PQC) pathways that discern misfolded proteins from functional ones in the cell. One major branch of PQC involves the controlled degradation of misfolded proteins by the ubiquitin-proteasome system. Here ubiquitin ligases must recognize and bind to misfolded proteins with sufficient energy to form a complex and with an adequate half-life to achieve poly-ubiquitin chain formation, the signal for protein degradation, prior to its dissociation from the ligase. It is not well understood how PQC ubiquitin ligases accomplish these tasks. Employing a fully reconstituted enzyme and substrate system to perform quantitative biochemical experiments, we demonstrate that the yeast PQC ubiquitin ligase San1 contains multiple substrate binding sites along its polypeptide chain that appear to display specificity for unique misfolded proteins. The results are consistent with a model where these substrate binding sites enable San1 to bind to misfolded substrates avidly, resulting in high affinity ubiquitin ligase-substrate complexes.

Stress is a ubiquitous part of life that disrupts cellular function and, if unresolved, can irreparably damage essential biomolecules and organelles. All organisms can experience stress in the form of unfavorable environmental conditions including exposure to extreme temperatures, hypoxia, reactive oxygen species, alcohol, or shifts in osmolarity. To survive, organisms must sense these changes then react and adapt. One highly conserved adaptive response to stress is through protein sumoylation, which is a post-translational modification by the small ubiquitin-like modifier (SUMO) protein. In this study, we examine the effects of acute ethanol stress on protein sumoylation in the budding yeast Saccharomyces cerevisiae. Although ethanol induces protein sumoylation, the targets and roles of sumoylation are largely unknown. Here, we found that cells exhibit a transient sumoylation response after exposure of cells to ≤ 7.5% volume/volume ethanol. The response peaks at 15 minutes and resolves by 60 minutes, indicating that cells have an adaptive response to low concentrations of ethanol. By contrast, the sumoylation response becomes chronic at 10% ethanol stress with no resolution by 60 minutes. To identify key targets of ethanol-induced sumoylation, we performed mass spectrometry analyses and identified 18 proteins with increased sumoylation after acute ethanol exposure, with 15 identified as known chromatin-associated proteins. Two of these proteins are the chromatin structural proteins Smc5 and Smc6, which are sumoylated by the activity of the SUMO ligase Mms21. Ethanol-induced Smc5/6 sumoylation occurs during G1 and G2M phases of the cell cycle but is abrogated during S phase despite the fact that other proteins are sumoylated during this phase. Acute ethanol exposure leads to formation of Rad52 foci indicating DNA damage similar to that observed with the addition of methyl methanesulfonate (MMS), which is an alkylating agent that damages DNA. Whereas MMS exposure induces the intra-S phase DNA damage checkpoint as observed by Rad53 phosphorylation, ethanol exposure does not induce the intra-S phase checkpoint and prevents Rad53 phosphorylation when added with MMS. From these results, we propose that ethanol induces a structural change in chromatin, possibly through DNA damage, and this causes sumoylation of conserved chromatin-associated proteins, including Smc5 and Smc6.