Knockdown of BRAWNIN minimally affect mitochondrial complex III assembly in human cells.

BRAWNIN was found as a mitochondrial respiratory complex III (CIII) assembly factor. Here, we showed that the deletion rather than knockdown of BRAWNIN impaired the assembly of CIII. BRAWNIN levels were affected by nutritional stress and negatively associated with AMPK activation. Although the BRAWNIN knockout via CRISPR/Cas9 led to decreased complex III levels, both biochemical and functional studies of oxidative phosphorylation system (OXPHOS) complexes revealed that knockdown of BRAWNIN neither affected mitochondrial respiration nor impaired the integrity of OXPHOS complexes I-V. Transcriptomic and proteomic profiling further confirmed that the BRAWNIN knockdown had a minimal effect on mitochondrial function. Moreover, only a small proportion of BRAWNIN interacted with the subunits of the OXPHOS complexes, which might be difficult to detect via co-immunoprecipitation and mass spectrometry. Finally, our findings also indicated that although only a minimal amount of BRAWNIN was required for CIII assembly, metabolic analyses revealed that it may fine-tune the pyruvate metabolism route in mitochondria.