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. 2020 Aug 21;23(9):101493. doi: 10.1016/j.isci.2020.101493

Figure 5.

Figure 5

Hrd1's Ubiquitination Activity Is Dispensable for Suppressive ERAD-M Retrotranslocation

(A) Depiction of E3 ligase Hrd1, C399S-Hrd1, and hemi-Hrd1. C399S-Hrd1 is an inactive version of Hrd1 in which the essential cysteine is mutated in the RING finger. Hemi-Hrd1 is a truncated version Hrd1 only containing the transmembrane domain.

(B) Removal of Hrd3 allows RING-dead C399S and hemi-Hrd1 to support suppression and degradation of SUS-GFP when Dfm1 is absent. The indicated strains overexpressing SUS-GFP were passaged to suppression. Cells were passaged and SUS-GFP levels were analyzed by flow cytometry. Histograms of 10,000 cells are shown, with the number of cells versus GFP fluorescence.

(C) Overexpression of C399S-Hrd1 is sufficient for restoring retrotranslocation of SUS-GFP in dfm1Δhrd3Δ cells. Crude lysate was prepared from the indicated strains treated with vehicle or MG132 (25 μg/mL). Lysates were ultracentrifuged to discern ubiquitinated SUS-GFP that either has been retrotranslocated into the soluble fraction (S) or remained in the membrane (P). Following fractionation, SUS-GFP was immunoprecipitated from both fractions, resolved on 8% SDS-PAGE, and immunoblotted with α-GFP and α-Ubi.

(D) Strongly expressed hemi-Hrd1 is sufficient for restoring retrotranslocation of SUS-GFP in dfm1Δhrd3Δ cells. Same as (C) except dfm1Δhrd3Δ cells with empty vector or strongly expressed hemi-Hrd1 was used in the in vivo retrotranslocation assay.

(E) hemi-Hrd1 restores Cdc48 recruitment to ER membrane. Total cell lysate (T) from the indicated strains were separated into soluble cytosolic fraction (S) and pellet microsomal fraction (P) upon centrifugation at 14,000 × g. Each fraction was analyzed by SDS-PAGE and immunoblotted for Cdc48 with α-Cdc48 and PGK1 with α-PGK1. The graph shows the quantification of Cdc48 in the pellet fractions of the respective cells as measured from ImageJ. Data are represented as percentage of Cdc48 that is bound to pellet fraction and is shown as mean ± SEM from three independent experiments, ∗∗∗∗p < 0.0001, unpaired t test.