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. 2016 Jul 19;113(31):E4467–E4475. doi: 10.1073/pnas.1605497113

Fig. 2.

Fig. 2.

pATOM36 is required for assembly but not for insertion of ATOM subunits. (A) In vitro insertion assay of the indicated 35S-Met–labeled OM proteins using mitochondria isolated from the uninduced (–Tet) and induced (+Tet, 2 d) procyclic pATOM36-RNAi cell line. Incubation time is indicated at the top. After alkaline carbonate extraction, the integral membrane proteins were separated by SDS/PAGE and analyzed by autoradiography. Sections of the Coomassie-stained gels serve as loading controls. (B) Assembly of 35S-Met–labeled ATOM46 into the ATOM complex is monitored by BN-PAGE followed by autoradiography. Incubation time is indicated at the top. A section of the Coomassie-stained gel serves as a loading control. (C) pATOM36 transiently interacts with ATOM46. 35S-labeled ATOM46 was incubated for 15 min with mitochondria isolated from wild-type cell (427 wt) and from a cell line that exclusively expresses C-terminally HA-tagged pATOM36. Subsequently mitochondria were solubilized and subjected to IP using anti-HA antiserum. Ten percent of the input (In), 100% of the eluate (IP), and 100% of the flow-through (FT) fractions were analyzed by SDS/PAGE. 35S-Met–ATOM46 and ATOM40, which serves as a control, were detected by autoradiography and immunoblots, respectively.