Fig. 2: Purification of TOM-TIM23 supercomplex.
From: Mapping protein interactions in the active TOM-TIM23 supercomplex
a Jac1sfGFP was imported into wild-type mitochondria and the TOM-TIM23 supercomplex isolated using a GFP nanobody. Samples were analyzed by SDS-PAGE and western blotting. Representative image from n > 3 biological replicates. b Preparative isolation of the TIM23 complex from HisS*Tim23 (HisSUMOstar-Tim23) mitochondria with or without accumulated Jac1sfGFP. After SUMO* protease-mediated elution, the TOM-TIM23 supercomplex was specifically purified with GFP nanobody (Nb). Samples were analyzed by SDS-PAGE, colloidal coomassie staining, and mass spectrometry. The experiment was repeated independently for n > 3. Plasmids containing ALFA- tagged c Tim23, Tim17, and Tim50 and e Tim50, Tim44, Pam16, and Pam18 were transformed into WT yeast. Mitochondria isolated from these cells were solubilized and subjected to ALFA immunoprecipitation. Total and elution fractions were analyzed by SDS-PAGE and immunoblotting. ALFA-tagged proteins (upper panel) were detected using an anti-ALFA Nanobody conjugated with HRP. d Mitochondria were isolated from Tim21FLAG expressing cells transformed with empty plasmid or plasmid encoding untagged Tim21 (TIM21WT). Following FLAG immunoisolation, samples were analyzed by SDS-PAGE and western blotting. For c−e, experiments were repeated for n = 3.
