Raw microscopy data of split-GFP and immunofluorescence experiments

Mitochondria critically rely on protein import and its tight regulation. Here, we found that the complex I assembly factor NDUFAF8 follows a two-step import pathway linking IMS and matrix import systems. A weak targeting sequence drives TIM23-dependent NDUFAF8 matrix import, and en route allows exposure to the IMS disulphide relay which oxidizes NDUFAF8. Import is closely surveyed by proteases: YME1L prevents accumulation of excess NDUFAF8 in the IMS, while CLPP degrades reduced NDUFAF8 in the matrix. Therefore, NDUFAF8 can only fulfil its function in complex I biogenesis if both oxidation in the IMS and subsequent matrix import work efficiently. We propose that the two-step import pathway for NDUFAF8 allows to integrate the activity of matrix complex I biogenesis pathways with the activity of the mitochondrial disulphide relay system in the IMS. Such coordination might not be limited to NDUFAF8 as we identified further proteins that can follow such a two-step import pathway. The raw mi..., For the image acquisition the microscope LSM 980 with Airyscan 2 and multiplex from Carl Zeiss Microscopy was used with a Plan-Apochromat 63x/1,4 Oil DIC objective and the GaAsP-PMT, Multi-Alkali-PMT detector. The cells were imaged at room temperature with oil as imaging medium.The following fluorochromes were used: GFP, Mitotracker CMXRos and Alexa Fluor488. Images were displayed using the acquisition software ZEN 3.3. and were processed using the software OMERO.insight. ,