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

Figure 4. Non-imported mitochondrial precursors are toxic and localize to nuclear-associated foci when clearance is impaired.

(A) Fivefold serial dilutions of WT and E3 KO strains on YPAD ± FCCP agar plates. (B) WT and E3 KO yeast expressing Ilv2-GFP and Tom70-mCherry ± FCCP. Nucleus stained with DAPI. Arrows = nuclear associated foci. Bar = 2 μm. (C) Quantification of (B). N > 99 cells per replicate, error bars = SEM of three replicates. (D) Quantification of average pixel intensity of Ilv2-GFP nuclear foci from (B). N = 20 cells, error bars = SD, p-value=0.0005. (E) Fivefold serial dilutions of WT and E3 KO strains expressing endogenous Ilv2-GFP (endo) ± mild overexpression of full-length Ilv2-GFP (FL) from pRS413-Ilv2-GFP on SD-His ± FCCP agar plates. (F) Yeast strain expressing WT Ilv2-GFP or Ilv2-NES-GFP and Tom70-mCherry ± FCCP. Nucleus stained with DAPI. Arrows = nucleus. Bar = 2 μm. (G) Quantification of (F). (H) Western blot of yeast expressing Ilv2-GFP or Ilv2-NES-GFP ± FCCP. (I) Fivefold serial dilutions of endogenously tagged WT Ilv2-GFP and Ilv2-NES-GFP strains on YPAD ± FCCP agar plates. P = precursor and M = mature.

Figure 4.

Figure 4—figure supplement 1. Impaired clearance of non-imported mitochondrial proteins targets them to nuclear-associated foci.

Figure 4—figure supplement 1.

(A) Fivefold serial dilutions of WT and the indicated mutant strains on YPAD ± FCCP agar plates. (B, C) WT and E3 KO (san1Δ ubr1Δ doa10Δ) yeast expressing Dld1-GFP or Dld2-GFP and Tom70-mCherry ± FCCP. Nucleus stained with DAPI, arrows = nuclear associated foci, and bar = 2 μm. (D, E) Quantification of (B) and (C), respectively. N > 99 cells per replicate, error bars = SEM of three replicates.