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. 2021 Mar 18;10:e61230. doi: 10.7554/eLife.61230

Figure 3. Nuclear protein quality control degrades non-imported mitochondrial proteins.

(A) Western blot of yeast expressing Ilv2-GFP ± FCCP ± MG-132. (B) Western blot of yeast expressing Ilv2-GFP ± FCCP in wild-type (WT) and E3 KO strains. (C) Western blots showing cycloheximide (CHX) chase of Ilv2-GFP in WT and E3 KO strains in the presence of FCCP. (D) Western blot showing ubiquitylation of immunoprecipitated Ilv2-GFP ± FCCP in WT and E3 KO strains. Pgk1 = loading control. E3 KO = san1Δ ubr1Δ doa10Δ. P = precursor and M = mature.

Figure 3.

Figure 3—figure supplement 1. Nuclear protein quality control promotes non-imported mitochondrial protein degradation.

Figure 3—figure supplement 1.

(A) Western blot of yeast expressing Ilv2-HA ± FCCP ± MG-132. (B, C) Western blots of yeast expressing the Ilv2-HA (B) or endogenous Ilv2 (C) ± FCCP in WT and E3 KO strains. (D) Western blots of yeast expressing Ilv2-GFP ± FCCP in WT and the indicated mutant yeast strains. (E, F) Western blots showing the CHX chase of Ilv2-HA (E) or endogenous Ilv2 (F) in WT and E3 KO strains in the presence of FCCP. (G) Western blots of yeast expressing the Lat1-GFP ± FCCP in WT and E3 KO strains. P = precursor form, M = mature form. Pgk1 = loading control.
Figure 3—figure supplement 2. Nuclear protein quality control promotes non-imported mitochondrial protein degradation.

Figure 3—figure supplement 2.

(A) Yeast expressing the indicated GFP and mCherry tagged mitochondrial proteins ± FCCP. (B) Quantification of (A). N > 99 cells per replicate, error bars = SEM of three replicates. (C) Tom70-mCherry wild-type (WT) and tetp-TOM40 (TOM40 KD) yeast expressing the indicated GFP-tagged mitochondrial proteins in the presence of doxycycline. (A, C) Nucleus stained with DAPI, Arrows = nucleus. Bar = 2 μm. (D) Western blots of yeast strains expressing indicated GFP-tagged mitochondrial proteins ± FCCP ± MG-132. (E) Western blots of yeast expressing the indicated GFP-tagged mitochondrial proteins ± FCCP in WT and E3 KO strains. (F) Western blot of yeast expressing indicated GFP-tagged mitochondrial proteins ± FCCP in WT and the indicated mutant yeast strains. Pgk1 = loading control. E3 KO = san1Δ ubr1Δ doa10Δ. P = precursor and M = mature.