Mia40 and ALR substrates: initial steps in biogenesis and regulation

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The biogenesis and maintenance of mitochondria is fundamental for cellular viability. We characterize the biogenesis and function of mitochondrial intermembrane space (IMS) proteins which are crucial for example for the assembly of the respiratory chain, the induction of apoptosis and the homeostasis of calcium. While some IMS proteins are imported with the help of mitochondrial targeting sequences (MTS), most rely on conserved cysteines and the help of the IMS oxidoreductase Mia40 for import and folding. Mia40 and its sulfhydryl oxidase ALR are part of a recently discovered oxidative machinery that works different than the oxidation machinery in the endoplasmic reticulum. So far the mechanics of this machinery have been mainly elucidated in in vitro experiments and in yeast. However, we lack crucial insights into many fundamental aspects which include the regulation of the machinery, and the associated substrate networks and their regulation in mammalian cells. Moreover, the cytosolic processes prior to import of MTS-less Mia40 substrates remain unclear. This includes for example the identity of recognition signals in the substrates or how the substrates are guided to mitochondria.We recently solved the interactomes of human Mia40 and ALR and thereby revealed that their substrate spectrum is much broader and the structure of many of their substrates more complex than previously anticipated. Likewise, we found that substrates of Mia40 behave very different in the cytosol prior to mitochondrial import compared with MTS-containing substrates. Encouraged by these exciting new data we aim to next identify and characterize cytosolic interaction partners of Mia40 substrates that might be involved in stabilization, proof-reading and targeting of the substrates prior to substrate translocation into the IMS. We also aim to understand further functions associated with Mia40 and ALR by characterizing in mechanistic detail their interplay with selected proteins from their interactomes. Furthermore, we will extend our proteomic analyses to identifying Mia40 and ALR targets during different stress conditions to extend our insights on the dynamics of their interactomes.