SUMMARY Identifying mutations that stabilize proteins is challenging because most substitutions are destabilizing. In addition to being of immense practical utility, the ability to evolve protein stability in vivo may indicate how evolution has formed today's protein sequences. Here we describe a genetic selection that directly links the in vivo stability of proteins to antibiotic resistance. It allows the identification of stabilizing mutations within proteins. The large majority of mutants selected for improved antibiotic resistance are stabilized both thermodynamically and kinetically, indicating that similar principles govern stability in vivo and in vitro. The approach requires no prior structural or functional knowledge and allows selection for stability without a need to maintain function. Mutations that enhance thermodynamic stability of the protein Im7 map overwhelmingly to surface residues involved in binding to colicin E7, implying that evolutionary pressures that drive Im7-E7 complex formation may have compromised the stability of the isolated Im7 protein.

Cdc48 (also known as p97), a conserved chaperone-like ATPase, plays a strategic role in the ubiquitin system. Empowered by ATP-driven conformational changes, Cdc48 acts as a segregase by dislodging ubiquitylated proteins from their environment. Ufd1, a known co-factor of Cdc48, also binds SUMO (ref. 6), but whether SUMOylated proteins are subject to the segregase activity of Cdc48 as well and what these substrates are remains unknown. Here we show that Cdc48 with its co-factor Ufd1 is SUMO-targeted to proteins involved in DNA double-strand break repair. Cdc48 associates with SUMOylated Rad52, a factor that assembles the Rad51 recombinase on chromatin. By acting on the Rad52-Rad51 complex, Cdc48 curbs their physical interaction and displaces the proteins from DNA. Genetically interfering with SUMO-targeting or segregase activity leads to an increase in spontaneous recombination rates, accompanied by aberrant in vivo Rad51 foci formation in yeast and mammalian cells. Our data thus suggest that SUMO-targeted Cdc48 restricts the recombinase Rad51 by counterbalancing the activity of Rad52. We propose that Cdc48, through its ability to associate with co-factors that have affinities for ubiquitin and SUMO, connects the two modification pathways for protein degradation or other regulatory purposes.

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Chromatin is a highly regulated environment, and protein association with chromatin is often controlled by post‐translational modifications and the corresponding enzymatic machinery. Specifically, SUMO ‐targeted ubiquitin ligases ( STU bLs) have emerged as key players in nuclear quality control, genome maintenance, and transcription. However, how STU bLs select specific substrates among myriads of SUMO ylated proteins on chromatin remains unclear. Here, we reveal a remarkable co‐localization of the budding yeast STU bL Slx5/Slx8 and ubiquitin at seven genomic loci that we term “ubiquitin hotspots”. Ubiquitylation at these sites depends on Slx5/Slx8 and protein turnover on the Cdc48 segregase. We identify the transcription factor‐like Ymr111c/Euc1 to associate with these sites and to be a critical determinant of ubiquitylation. Euc1 specifically targets Slx5/Slx8 to ubiquitin hotspots via bipartite binding of Slx5 that involves the Slx5 SUMO ‐interacting motifs and an additional, novel substrate recognition domain. Interestingly, the Euc1‐ubiquitin hotspot pathway acts redundantly with chromatin modifiers of the H2A.Z and Rpd3L pathways in specific stress responses. Thus, our data suggest that STU bL‐dependent ubiquitin hotspots shape chromatin during stress adaptation.