Approaches for the analysis of redox-dependent protein import into mitochondria of mammalian cells

Oxidation of cysteine residues in proteins can take place as part of an enzymatic reaction cycle, during oxidative protein folding or as a consequence of redox signalling or oxidative stress. Following changes in protein thiol redox states allows to investigate the mechanisms underlying thiol-disulphide redox processes. In this book chapter, we provide information and protocols on different methods for redox state determination with a focus on these processes in the context of oxidation-dependent protein import into the mitochondrial intermembrane space. These methods include assessing the cysteine redox state of mature proteins, methods to investigate oxidative protein folding in radioactive pulse chase assays and methods to follow specifically the formation of oxidative folding intermediates between oxidoreductases and substrates.