Fig. 1: Jac1^sfGFP localizes to mitochondria and accumulates in a TOM-TIM23 supercomplex.

a Design of Jac1^sfGFP supercomplex forming protein and its schematic representation within the TOM-TIM23 supercomplex. b STED super-resolution microscopy of yeast cells expressing Jac1^sfGFP. Cells were labeled with antibodies against GFP (green) and Tom40 (magenta) or Tim23 (magenta). Representative images from three biological replicates are depicted. c Increasing amounts of purified Jac1^sfGFP were imported into wild-type mitochondria. Mitochondria were solubilized and supercomplex formation was monitored by BN-PAGE followed by immunoblotting using indicated antibodies. Four independent experiments were carried out. d Indicated amounts of purified Jac1^sfGFP were imported for 30 min into 50 µg mitochondria. Mitochondria were briefly washed and [³⁵S]-labeled F1β was imported for 15 min. The reaction was stopped by the addition of AVO followed by PK (Proteinase K) treatment. Samples were analyzed by SDS-PAGE and digital autoradiography. The imported precursor was quantified as percent of the 0 µM protein sample with PK treatment (100% control) (lane 3). Results are presented as mean ± SEM, n = 4. e After import, solubilized mitochondria with or without imported Jac1^sfGFP were subjected to glycerol gradient centrifugation. Fractions were analyzed by SDS-PAGE and immunoblotting. The signal intensity of supercomplex subunits were quantified and normalized as % of the total signal. ps: presequence, aa: amino acid, sfGFP: superfolder GFP, TOM: translocase of the outer mitochondrial membrane, TIM23: presequence translocase of the inner mitochondrial membrane, PAM: presequence translocase-associated motor complex, OMM: outer mitochondrial membrane, IMS: intermembrane space, IMM: inner mitochondrial membrane, p: precursor, m: mature (processed) form, AVO: a mixture of Antimycin A, Valinomycin Oligomycin.