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

Figure 5. The mitochondrial targeting sequence (MTS) is required for non-imported precursor toxicity and quality control.

(A) Schematic of full-length GFP-tagged Ilv2 (FL), mitochondrial targeting sequence deleted (ΔMTS) GFP-tagged Ilv2, and MTSIlv2 GFP only (MTS). (B) Tom70-mCherry yeast expressing endogenous Ilv2-GFP ± the indicated Ilv2 variant ± FCCP. Nucleus stained with DAPI. Arrows = nuclear-associated foci. Bars = 2 μm. (C, D) Quantification of cells with diffuse Ilv2 nuclear localization (C) or Ilv2 nuclear foci (D) from (B). For (C, D), N > 99 cells per replicate, error bars = SEM of three replicates. (E) Western blot of strains expressing indicated Ilv2-GFP variants ± FCCP. Pgk1 = loading control. (F) Fivefold serial dilutions of WT and E3 KO strains expressing endogenous Ilv2-GFP (endo) ± mild overexpression of the indicated Ilv2-GFP variants on SD-His ± FCCP agar plates. (G) Western blot of strains expressing indicated Cox15-GFP or Lat1-GFP variants, respectively, ± FCCP. Pgk1 = loading control. (H) Fivefold serial dilutions of WT strains expressing endogenous Cox15-GFP or Lat1-GFP (endo) ± mild overexpression of the indicated variants on SD-His ± FCCP agar plates. P = precursor and M = mature.

Figure 5.

Figure 5—figure supplement 1. The MTS is required for non-imported precursor toxicity and degradation.

Figure 5—figure supplement 1.

(A) Fivefold serial dilutions of strains expressing endogenous Ilv2-GFP (endo) ± mild overexpression of the indicated Ilv2-GFP variants or untagged Ilv2 variants on SD-His ± FCCP agar plates. (B) Tom70-mCherry yeast expressing endogenous Dld2-GFP ± the indicated Dld2 plasmid-expressed variants ± FCCP. Nucleus stained with DAPI. Arrows = nuclear associated foci. Bars = 2 μm. (C, D) Quantification of cells with diffuse Dld2-GFP nuclear localization (C) or Dld2-GFP nuclear foci (D) from (B). For (C and D), N > 99 cells per replicate, error bars = SEM of three replicates. (E) Western blot of strains expressing indicated Dld2-GFP variants ± FCCP. Pgk1 = loading control. (F) Fivefold serial dilutions of WT and E3 KO strains expressing endogenous Dld2-GFP (endo) ± mild overexpression of the indicated Dld2-GFP variants on SD-His ± FCCP agar plates.