Figure 6. MTS-mediated toxicity of Ilv2 is not entirely linked to mitochondrial import clogging.

(A) ³⁵S-labeled Su9-DHFR was imported into mitochondria isolated from yeast overexpressing the indicated Ilv2-GFP variants. (B) Western blot showing protein levels in mitochondria isolated from yeast overexpressing the indicated Ilv2-GFP variants. (C) ³⁵S-labeled Su9-DHFR was imported into mitochondria isolated from yeast expressing endogenous Ilv2-GFP ± FCCP. (D) Western blot showing protein levels in mitochondria isolated from yeast expressing endogenous Ilv2-GFP ± FCCP. (E) Yeast expressing Idh1-GFP and Tom70-mCherry in WT or Ilv2 (untagged) overexpressing strains ± FCCP. Bars = 2 μm. (F) Quantification of (E). (G) ³⁵S-labeled Su9-DHFR was imported into mitochondria isolated from yeast expressing endogenous Ilv2-GFP or Ilv2-NES-GFP ± FCCP. (H) Western blot showing protein levels in mitochondria isolated from yeast expressing endogenous Ilv2-GFP or Ilv2-NES-GFP. For (A), (C), and (G), a mixture of antimycin A, oligomycin, and valinomycin (AVO) was used to dissipate the membrane potential. Non-imported proteins were proteolytically removed with proteinase K and the import was analyzed by SDS–PAGE and autoradiography. P = precursor and M = mature.