Oxidative protein import into mitochondria of human cells

The respiratory chain of mitochondria produces the main amount of the cellular ATP. It harbors five membrane-embedded multi-protein complexes. To maintain the function of this machinery most of its proteins have to be imported into mitochondria after synthesis in the cytosol. Some of these proteins do not contain a common mitochondrial targeting sequence but instead are imported by an oxidative folding mechanism. Examples of such proteins are so called twin-CX3C and twin-CX9C proteins that contain four conserved cysteines in a helix-loop-helix motif. After the translocation into the intermembrane space of mitochondria (IMS) two intramolecular disulfide bonds are formed between these cysteines which contribute to the correct folding and the retention of the proteins. The machinery that facilitates oxidation is composed of two main components, Mia40 and ALR. Both are essential disease-related proteins that reside in the IMS. Mia40 is an oxidoreductase that interacts with the substrate proteins and oxidizes them. Because Mia40 is reduced after this reaction it is subsequently regenerated by the sulfhydryl oxidase ALR. In our experiments we focus on the dynamics of the substrate oxidation that is driven by Mia40. We thereby analyze the in vivo kinetics of oxidative folding and import in human tissue culture cells.