Let’s make it clear: Systematic exploration of mitochondrial DNA- and RNA-protein complexes by complexome profiling

Abstract

Complexome profiling (CP) is a powerful tool for systematic investigation of protein interactors that has been primarily applied to study the composition and dynamics of mitochondrial protein complexes. Here, we further optimised this method to extend its application to survey mitochondrial DNA- and RNA-interacting protein complexes. We established that high-resolution clear native gel electrophoresis (hrCNE) is a better alternative to preserve DNA- and RNA-protein interactions that are otherwise disrupted when samples are separated by the widely used blue native gel electrophoresis (BNE). In combination with enzymatic digestion of DNA, our CP approach improved the identification of a wide range of protein interactors of the mitochondrial gene expression system without compromising the detection of other multi-protein complexes. The utility of this approach was particularly demonstrated by analysing the complexome changes in human mitochondria with impaired gene expression after transient, chemically-induced mtDNA depletion. Effects of RNase on mitochondrial protein complexes were also evaluated and discussed. Overall, our adaptations significantly improved the identification of mitochondrial DNA- and RNA-protein interactions by CP, thereby unlocking the comprehensive analysis of a near-complete mitochondrial complexome in a single experiment.

Uncharacterized protein C17orf80 – a novel interactor of human mitochondrial nucleoids

“…Furthermore, the signal of DNAJA3 in C17orf80 KO cells was 2-fold lower than in the control, and slightly increased in ddC-treated samples ( H ES = 1.24, 1.5-fold increase after 72 h). The two proteins also displayed a clear comigration in the complexome profile of EtBr-treated cells, as has recently been published by our group ( Potter et al, 2023 preprint) ( Fig. S7D ).…”