Identification and functional analysis of ULP2 SUMO protease mutants of the stress-tolerant yeast Kluyveromyces marxianus.

K. marxianus ( Km ), a stress-tolerant yeast, is closely related to S. cerevisiae ( Sc ). Unlike Sc , Km yeast grows robustly in the presence of proteotoxic stressors, including temperatures up to 45°C and oxidizing agents. Sumoylation, the posttranslational modification with SUMO, plays an important role in the response to proteotoxic stress in the mesophilic yeast Sc . Therefore, we tested the possibility that perturbation of SUMO dynamics may affect Km 's remarkable stress tolerance. Km yeast expresses variants of functionally conserved SUMO pathway genes, and we previously found that the Km SUMO protease KmUlp1 is considerably more tolerant to proteotoxic insults than ScUlp1 (Peek et al., 2018). Here, we tested the possibility that disruption of the non-essential SUMO protease KmUlp2, an isopeptidase related to Ulp1, may negatively affect Km 's remarkable stress tolerance. To this end, we used a Km -specific CRISPR/Cas9 system to generate Kmulp2 truncation mutants in vivo . We then compare the growth properties of these Kmulp2 mutants and their respective WT controls to the Sculp2 knockout mutant after exposure to various cellular stressors. Overall, we find that Kmulp2 mutants recapitulate major phenotypes associated with the Sculp2 mutant. This includes sensitivity to the ribonucleotide reductase inhibitor hydroxyurea and the accumulation of SUMO chains. Our study shows that KmUlp2 is required for Km 's response to hydroxyurea-induced DNA damage stress. Furthermore, alterations in SUMO dynamics by Ulp2 truncations do not grossly affect the temperature tolerance of K. m. yeast cells.