Sko1 is constitutively sumoylated at Lys 567.

(A) List of yeast and human bZIP motif-containing proteins that have been identified as putative SUMO targets in published proteomics studies. (B) Lysates from yeast strains expressing 6xHA-tagged Sko1 from its chromosomal locus, or a strain with no HA-tagged proteins (No tag), were examined by HA IP and immunoblot analysis with HA and SUMO antibodies. Inputs represent approximately 5–10% of the IPed material. (C) A yeast strain was generated with a chromosomal sko1 K567R mutation (Sko1-MT.HA) and used for IP-immunoblot analysis, as in (B), alongside No tag and Sko1-WT.HA controls. (D) Schematic of the Sko1 protein indicating bZIP DNA binding domain, regions targeted by Hog1 and PKA kinases, region required for nuclear entry [54], and the primary site of sumoylation, Lys 567, which is found within a SUMO consensus motif, VKSE. (E) IP-immunoblot analysis of lysates from the SKO1-WT.HA strain grown in SC medium (“Untreated”), or SC medium treated for 20 min with 0.5 μg/mL sulfometuron methyl (SM, amino acid starvation), 0.4 M NaCl (osmotic stress), 1 mM H2O2 (oxidative stress), or incubated at 37°C (temperature stress), as in (B). (F) Spot assay comparing growth of indicated strains in various types of osmotic stress. Approximately 10,000 cells were spotted on the left-most position and serial five-fold dilutions were spotted on adjacent spots to the right in each panel. Media plates were imaged after two days. Asterisks (*) indicate position of the major (mono-) sumoylated form of Sko1 in each immunoblot.