Fig. 5.

Shp1 trimerization regulates Met30 dissociation during heavy metal stress. (A) shp1-∆sep mutants indicate cadmium sensitivity. Strains expressing endogenous ^12xMycMet30, Cdc48^RGS6H, Skp1, and Shp1^3xHA, or ∆SEP Shp1^3xHA, were cultured to logarithmic growth phase, cells were counted, and serial dilutions spotted onto YEPD plates supplemented with or without 50 µM CdCl₂. Plates were incubated for 2 d at 30 °C. (B) The SEP domain is necessary for Met4 activation during heavy metal stress. Whole-cell lysates (Totals) of strains shown in Fig. 5A were analyzed by Western blot using a Met4 antibody to follow the ubiquitylation and phosphorylation status of Met4. Tubulin was used as a loading control. (C) Decreased cadmium-induced gene expression in the absence of the SEP domain. RNA was extracted from samples shown in Fig. 5B. Expression of Met4 target genes MET25 and GSH1 was analyzed by RT-qPCR and normalized to 18S rRNA levels (n = 3). Data are presented as mean ± SD (t test *P < 0.1, **P < 0.05, ***P < 0.01). (D) Dissociation kinetics of Met30 from the SCF core ligase are decreased in shp1-∆sep mutants. Indicated strains were cultured at 30 °C in YEPD medium and treated with 100 µM CdCl₂, and samples were harvested after 20 and 40 min, respectively. ^12xMycMet30 was immunoprecipitated, and coprecipitated proteins were analyzed by Western blot. Results shown in B and D are representative blots from three independent experiments.