Journal: eLife

Article Title: A nuclear-based quality control pathway for non-imported mitochondrial proteins

doi: 10.7554/eLife.61230

Figure Lengend Snippet: ( A ) 35 S-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 ) 35 S-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 ) 35 S-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.

Article Snippet: Membranes were blocked and probed in blocking buffer (1× PBS, 0.05% Tween 20, 5% non-fat dry milk) using the primary antibodies for GFP (1814460001, Sigma Millipore), HA (11583816001, Sigma Millipore), Ilv2 (gift from Dr. Agnieszka Chacinska, International Institute of Molecular and Cell Biology), Tom70, Tom20, Tim54, Tim50, Tim18 (gifts from Dr. Nikolaus Pfanner, University of Freiburg), OM45 (gift from Dr. Carla Koehlar, UCLA), Idh1 (Ap31099PU-N, ORIGENE) or Pgk1 (22C5D8, Abcam), and HRP-conjugated secondary antibodies (715-035-150, Jackson Immunoresearch).

Techniques: Labeling, Isolation, Western Blot, Expressing, Membrane, SDS Page, Autoradiography

Journal: eLife

Article Title: A nuclear-based quality control pathway for non-imported mitochondrial proteins

doi: 10.7554/eLife.61230

Figure Lengend Snippet:

Article Snippet: Membranes were blocked and probed in blocking buffer (1× PBS, 0.05% Tween 20, 5% non-fat dry milk) using the primary antibodies for GFP (1814460001, Sigma Millipore), HA (11583816001, Sigma Millipore), Ilv2 (gift from Dr. Agnieszka Chacinska, International Institute of Molecular and Cell Biology), Tom70, Tom20, Tim54, Tim50, Tim18 (gifts from Dr. Nikolaus Pfanner, University of Freiburg), OM45 (gift from Dr. Carla Koehlar, UCLA), Idh1 (Ap31099PU-N, ORIGENE) or Pgk1 (22C5D8, Abcam), and HRP-conjugated secondary antibodies (715-035-150, Jackson Immunoresearch).

Techniques: Recombinant, Plasmid Preparation, Generated, Protease Inhibitor, Staining, Cloning, Software