The MIA40 pathway is a novel import pathway in mitochondria specific for cysteine-rich proteins of the intermembrane space (IMS). The newly synthesised precursors are trapped in the IMS by a disulfide relay mechanism that involves introduction of disulfides from the sulfhydryl oxidase Erv1 to the redox-regulated import receptor Mia40 and then on to the substrate. This thiol–disulfide exchange mechanism is essential for the import and oxidative folding of the incoming cysteine-rich substrate proteins. In this chapter we will describe the experimental methods that have been developed in order to study and characterise disulfide-trapped intermediates in yeast mitochondria.
