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Most mitochondrial proteins are synthesized in the cytosol and subsequently translocated as unfolded polypeptides into mitochondria. Cytosolic chaperones maintain precursor proteins in an import‐competent state. This post‐translational import reaction is under surveillance of the cytosolic ubiquitin‐proteasome system, which carries out several distinguishable activities. On the one hand, the proteasome degrades nonproductive protein precursors from the cytosol and nucleus, import intermediates that are stuck in mitochondrial translocases, and misfolded or damaged proteins from the outer membrane and the intermembrane space. These surveillance activities of the proteasome are essential for mitochondrial functionality, as well as cellular fitness and survival. On the other hand, the proteasome competes with mitochondria for nonimported cytosolic precursor proteins, which can compromise mitochondrial biogenesis. In order to balance the positive and negative effects of the cytosolic protein quality control system on mitochondria, mitochondrial import efficiency directly regulates the capacity of the proteasome via transcription factor Rpn4 in yeast and nuclear respiratory factor (Nrf) 1 and 2 in animal cells. In this review, we provide a thorough overview of how the proteasome regulates mitochondrial biogenesis.

Most mitochondrial proteins are synthesized as precursors that carry N-terminal presequences. After import into mitochondria, these targeting signals are cleaved off by the mitochondrial processing peptidase MPP. Using the mitochondrial tandem protein Arg5,6 as model substrate, we demonstrate that MPP has an additional role in preprotein maturation, beyond the removal of presequences. Arg5,6 is synthesized as a polyprotein precursor that is imported into mitochondria and subsequently separated into two distinct enzymes. This internal processing is performed by MPP, which cleaves the Arg5,6 precursor at its N-terminus and at an internal site. The peculiar organization of Arg5,6 is conserved across fungi and reflects the polycistronic arginine operon in prokaryotes. MPP cleavage sites are also present in other mitochondrial fusion proteins from fungi, plants and animals. Hence, besides its role as “ticket canceller” for removal of presequences, MPP exhibits a second, conserved activity as internal processing peptidase for complex mitochondrial precursor proteins.

Almost all mitochondrial proteins are synthesized in the cytosol and subsequently targeted to mitochondria. The accumulation of non-imported precursor proteins occurring upon mitochondrial dysfunction can challenge cellular protein homeostasis. Here we show that blocking protein translocation into mitochondria results in the accumulation of mitochondrial membrane proteins at the endoplasmic reticulum, thereby triggering the unfolded protein response (UPR-ER). Moreover, we find that mitochondrial membrane proteins are also routed to the ER under physiological conditions. The levels of ER-resident mitochondrial precursors is enhanced by import defects as well as metabolic stimuli that increase the expression of mitochondrial proteins. Under such conditions, the UPR-ER is crucial to maintain protein homeostasis and cellular fitness. We propose the ER serves as a physiological buffer zone for those mitochondrial precursors that cannot be immediately imported into mitochondria while engaging the UPRER to adjust the ER proteostasis capacity to the extent of precursor accumulation.

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