Highlights d IMS import by the disulfide relay proceeds via formation of a covalent intermediate d Import is driven by hydrophobic and disulfide-dependent interactions d The covalent intermediate is also formed by oxidationincompetent substrates d Redox quality control allows for IMS substrate release and proteasomal degradation

The intermembrane space (IMS) is a very small mitochondrial sub-compartment with critical relevance for many cellular processes. IMS proteins fulfil important functions in transport of proteins, lipids, metabolites and metal ions, in signalling, in metabolism and in defining the mitochondrial ultrastructure. Our understanding of the IMS proteome has become increasingly refined although we still lack information on the identity and function of many of its proteins. One characteristic of many IMS proteins are conserved cysteines. Different post-translational modifications of these cysteine residues can have critical roles in protein function, localization and/or stability. The close localization to different ROS-producing enzyme systems, a dedicated machinery for oxidative protein folding, and a unique equipment with antioxidative systems, render the careful balancing of the redox and modification states of the cysteine residues, a major challenge in the IMS. In this review, we discuss different functions of human IMS proteins, the involvement of cysteine residues in these functions, the consequences of cysteine modifications and the consequences of cysteine mutations or defects in the machinery for disulfide bond formation in terms of human health.

Plasticity of the proteome is critical to adapt to varying conditions. Control of mitochondrial protein import contributes to this plasticity. Here, we identified a pathway that regulates mitochondrial protein import by regulated N-terminal processing. We demonstrate that dipeptidyl peptidases 8/9 (DPP8/9) mediate the N-terminal processing of adenylate kinase 2 (AK2) en route to mitochondria. We show that AK2 is a substrate of the mitochondrial disulfide relay, thus lacking an N-terminal mitochondrial targeting sequence and undergoing comparatively slow import. DPP9-mediated processing of AK2 induces its rapid proteasomal degradation and prevents cytosolic accumulation of enzymatically active AK2. Besides AK2, we identify more than 100 mitochondrial proteins with putative DPP8/9 recognition sites and demonstrate that DPP8/9 influence the cellular levels of a number of these proteins. Collectively, we provide in this study a conceptual framework on how regulated cytosolic processing controls levels of mitochondrial proteins as well as their dual localization to mitochondria and other compartments.

The bacterial Rcs phosphorelay signals perturbations of the bacterial cell envelope to its response regulator RcsB, which regulates transcription of multiple loci related to motility, biofilm formation and various stress responses. RcsB is unique, as its set of target loci is modulated by interaction with auxiliary regulators including BglJ. The BglJ–RcsB heteromer is known to activate the HNS repressed leuO and bgl loci independent of RcsB phosphorylation. Here, we show that BglJ–RcsB activates the promoters of 10 additional loci (chiA, molR, sfsB, yecT, yqhG, ygiZ, yidL, ykiA, ynbA and ynjI). Furthermore, we mapped the BglJ–RcsB binding site at seven loci and propose a consensus sequence motif. The data suggest that activation by BglJ–RcsB is DNA phasing dependent at some loci, a feature reminiscent of canonical transcriptional activators, while at other loci BglJ–RcsB activation may be indirect by inhibition of HNS-mediated repression. In addition, we show that BglJ–RcsB activates transcription of bgl synergistically with CRP where it shifts the transcription start by 20 bp from a position typical for class I CRP-dependent promoters to a position typical for class II CRP-dependent promoters. Thus, BglJ–RcsB is a pleiotropic transcriptional activator that coordinates regulation with global regulators including CRP, LeuO and HNS.

Disulfide formation in the mitochondrial intermembrane space is an essential process catalyzed by a disulfide relay machinery. In mammalian cells, the key enzyme in this machinery is the oxidoreductase CHCHD4/Mia40. Here, we determined the in vivo CHCHD4 redox state, which is the major determinant of its cellular activity. We found that under basal conditions, endogenous CHCHD4 redox state in cultured cells and mouse tissues was predominantly oxidized, however, degrees of oxidation in different tissues varied from 70% to 90% oxidized. To test whether differences in the ratio between CHCHD4 and ALR might explain tissue-specific differences in the CHCHD4 redox state, we determined the molar ratio of both proteins in different mouse tissues. Surprisingly, ALR is superstoichiometric over CHCHD4 in most tissues. However, the levels of CHCHD4 and the ratio of ALR over CHCHD4 appear to correlate only weakly with the redox state, and although ALR is present in superstoichiometric amounts, it does not lead to fully oxidized CHCHD4.