L 35s methionine

The assay was performed as previously described^{52 (link),53 (link)}. Human ornithine transcarbamylase (OTC) precursor cDNA in pGEM-3Zf(+)-pOTC plasmid, which was kindly provided by M. Yano (Kumamoto University, Kumamoto, Japan), was transcribed and translated in vitro using the TNT-coupled reticulocyte lysate system (Promega) in the presence of l-[³⁵S]methionine (PerkinElmer). Following translation, [³⁵S]methionine-labeled pOTC was incubated with isolated mitochondria at 25 °C for the indicated times, and mitochondria containing imported OTC were collected by centrifugation (9,000g, 10 min) and subjected to SDS-PAGE. The radioactive polypeptides on the gel were visualized by fluorography with Amplify (GE Healthcare) followed by exposure to X-ray film. Cleaved mature OTC (mOTC), which represents the completion of import into the mitochondrial matrix and migrates faster than pOTC on SDS-PAGE, was quantified by ImageJ (NIH). The data are presented as the percentage of mOTC compared to input (total [³⁵S]pOTC amount added to each reaction) and scaled to mOTC in control mitochondria after the maximum reaction time (set equal to 1) unless otherwise specified. In the import assay with forebrain mitochondria, data are scaled to mOTC in control WT mitochondria at 60 min reaction time (set equal to 1).

Human C9orf72, NDUFA8, or NDUFS8 cDNA in the pGEM-7Zf(+) vector (Promega) was transcribed and translated in vitro using the TNT-coupled reticulocyte lysate system (Promega) in the presence of L-[³⁵S]methionine (PerkinElmer). Following translation, the mixture containing [³⁵S]methionine-labeled C9orf72, NDUFA8, or NDUFS8 protein was incubated with equal amounts of mitochondria that were freshly isolated from indicated cells at 25°C for 30 min. Unimported proteins were removed by adding proteinase K (10 μg/ml) at 4°C for 10 min. After adding PMSF (2 mM) to stop the digestion, mitochondria were collected by centrifugation at 10,000 g at 4°C for 10 min and solubilized in 1X SDS sample loading buffer. The imported C9orf72, NDUFA8, or NDUFS8 protein was analyzed by SDS-PAGE. To measure the CI assembly in vitro, following the mitochondrial importation of [³⁵S] NDUFS8, with or without 50 μM CCCP, and proteinase K treatment as mentioned above, the isolated mitochondria were solubilized by adding digitonin, and the intact CI was analyzed by BN-PAGE. The radioactive signals on the transferred membrane were visualized by exposure to X-ray film and quantified by Quantity One software.

The TNT T7 quick coupled transcription/translation system (Promega Corporation, Madison, WI) was used to synthesize L-[³⁵S]methionine (PerkinElmer, Waltham, MA) labeled MAGI-1 PDZ domains (PDZ1, 2, 3, and 5) according to the manufacturer's protocol and as previously described (Kolawole, Sharma et al. 2012 (link)). COS-7 cells were transfected with the FLAG-tagged CAR isoform plasmid and subjected to immunopreciptitation (IP). In vitro translated MAGI-1 PDZ domain proteins were mixed with CAR-IP lysates at 4°C for 2 hr, followed by washing, centrifugation and elution with 2X denaturing buffer. Labeled proteins were separated by SDS-PAGE and visualized either by autoradiography of dried gels or transferred to PVDF for autoradiography and WB.