Mia40 and ALR substrates: initial steps in biogenesis and regulation

线粒体的生物发生和维持是细胞活力的基础。我们描述了线粒体膜间隙（IMS）蛋白的生物发生和功能，这些蛋白对呼吸链的组装、细胞凋亡的诱导和钙的体内平衡至关重要。虽然一些IMS蛋白在线粒体靶向序列（MTS）的帮助下导入，但大多数依赖于保守的半胱氨酸和IMS氧化还原酶Mia40的帮助导入和折叠。Mia40及其巯基氧化酶ALR是最近发现的一种氧化机制的一部分，这种氧化机制的工作原理与内质网中的氧化机制不同。到目前为止，这一机制的机制主要是在体外实验和酵母中阐明的。然而，我们缺乏对许多基本方面的关键见解，包括机制的调节，以及相关的底物网络及其在哺乳动物细胞中的调节。此外，进口MTS-less Mia40底物之前的细胞质过程仍不清楚。这包括例如底物中识别信号的身份或底物如何被引导到线粒体。我们最近解决了人类Mia40和ALR的相互作用组，从而揭示了它们的底物光谱比以前预期的更广泛，许多底物的结构比以前预期的更复杂。同样地，我们发现Mia40底物在线粒体导入之前与含有mts的底物在细胞质中的表现非常不同。受这些令人兴奋的新数据的鼓舞，我们的下一步目标是识别和表征Mia40底物的细胞质相互作用伙伴，这些伙伴可能参与底物转运到IMS之前底物的稳定、校对和靶向。我们还旨在通过表征Mia40和ALR与相互作用组中选定蛋白质的相互作用的机制细节，进一步了解与Mia40和ALR相关的功能。此外，我们将扩展我们的蛋白质组学分析，以确定不同应激条件下的Mia40和ALR靶点，以扩展我们对其相互作用组体动力学的见解。

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.