Figure 4.

Hrd3 Antagonizes Hrd1's Suppressive Retrotranslocation Function

(A and B) Galactose-induced Hrd3 overexpression causes cell lethality in dfm1Δ suppressed cells. WT, hrd1Δ, and dfm1Δ suppressed cells overexpressing SUS-GFP (A) or Hmg2-GFP (B) and harboring either empty vector or GAL-driven Hrd3 were compared for growth by dilution assay. Each strain was spotted 5-fold dilutions on glucose-containing or galactose-containing plates to drive Hrd3 overexpression, and plates were incubated at 30°C.

(C) Hrd3 overexpression blocks restoration of ERAD-M retrotranslocation. WT and dfm1Δ cells overexpressing SUS-GFP were grown in the presence of glucose to turn off Hrd3 expression and passaged to suppression at the indicated number of times into fresh minimal media (P11). P11WT and dfm1Δ cells were then passaged into minimal media containing galactose as a sole carbon source to trigger Hrd3 overexpression (P12). Flow cytometry was used to assess SUS-GFP steady-state levels. Histograms of 10,000 cells are shown, with the number cells versus GFP fluorescence.

(D) Degradation of SUS was measured by CHX-chase assay in passaged suppressed dfm1Δ strains containing either empty vector or GAL-driven Hrd3. After CHX addition, cells were lysed at the indicated times, analyzed by SDS-PAGE, and immunoblotted for SUS with α-GFP. Band intensities were normalized to PGK1 loading control and quantified by ImageJ. t = 0 was taken as 100%, and data are represented as mean ± SEM from at least three experiments, ∗∗∗p < 0.001, Repeated measures ANOVA.