The IMS domain is critical for the degradation of Tom22 and Om45. (A) The predicted secondary structures of the IMS domain of Tom22 and its mutant (Tom22ΔH) using the YASPIN program. (B–D) Schematic illustration (B) and turnover rate analysis (C and D) of the full-length and truncated forms of Tom22-HA. (E and F) Schematic illustration (E) and turnover rate analysis (F) of the indicated mutants. (G and H) The WT and indicated mutant strains were grown in ethanol and glycerol (YPEG) media at 30°C to log phase and then treated with CHX at 40°C. Cell lysates were analyzed by Western blotting with anti-Om45 and anti-Por1 antibodies (G). The Om45/Por1 ratio was measured by ImageJ software and plotted in H. (I and J) The WT and indicated mutant strains were grown in YPD media at 30°C to log phase and then treated with CHX at 37°C. Cell lysates were analyzed by Western blotting with anti-Tom22 and anti-Por1 antibodies (I). The Tom22/Por1 ratio (J) was analyzed as in H. (K and L) The Tom22ΔH mutant was treated and analyzed as in I and J. Data values represent means and SD from three independent experiments. Data were analyzed by two-way ANOVA followed by Bonferroni’s post hoc tests. ***, P < 0.001. Molecular masses are shown in kilodaltons. (M) WT or yme1Δ cells harboring empty vectors (V) or the indicated overexpression 2μ plasmids were grown in glucose media to log phase and then spotted on glucose (YPD) or ethanol and glycerol (YPEG) plates in a 10-fold serial dilution and then were incubated for 2–5 d at the indicated temperature.
