The mitochondrial proteome comprises ~1000 (yeast)–1500 (human) different proteins, which are distributed into four different subcompartments. The sublocalization of these proteins within the organelle in most cases remains poorly defined. Here we describe an integrated approach combining stable isotope labeling, various protein enrichment and extraction strategies and quantitative mass spectrometry to produce a quantitative map of submitochondrial protein distribution in S. cerevisiae. This quantitative landscape enables a proteome-wide classification of 986 proteins into soluble, peripheral, and integral mitochondrial membrane proteins, and the assignment of 818 proteins into the four subcompartments: outer membrane, inner membrane, intermembrane space, or matrix. We also identified 206 proteins that were not previously annotated as localized to mitochondria. Furthermore, the protease Prd1, misannotated as intermembrane space protein, could be re-assigned and characterized as a presequence peptide degrading enzyme in the matrix.

Mitochondrial disorders causing neurodegeneration in childhood are genetically heterogeneous, and the underlying genetic etiology remains unknown in many affected individuals. We identified biallelic variants in PMPCB in individuals of four families including one family with two affected siblings with neurodegeneration and cerebellar atrophy. PMPCB encodes the catalytic subunit of the essential mitochondrial processing protease (MPP), which is required for maturation of the majority of mitochondrial precursor proteins. Mitochondria isolated from two fibroblast cell lines and induced pluripotent stem cells derived from one affected individual and differentiated neuroepithelial stem cells showed reduced PMPCB levels and accumulation of the processing intermediate of frataxin, a sensitive substrate for MPP dysfunction. Introduction of the identified PMPCB variants into the homologous S. cerevisiae Mas1 protein resulted in a severe growth and MPP processing defect leading to the accumulation of mitochondrial precursor proteins and early impairment of the biogenesis of iron-sulfur clusters, which are indispensable for a broad range of crucial cellular functions. Analysis of biopsy materials of an affected individual revealed changes and decreased activity in iron-sulfur cluster-containing respiratory chain complexes and dysfunction of mitochondrial and cytosolic Fe-S cluster-dependent enzymes. We conclude that biallelic mutations in PMPCB cause defects in MPP proteolytic activity leading to dysregulation of iron-sulfur cluster biogenesis and triggering a complex neurological phenotype of neurodegeneration in early childhood.

SummaryThe mitochondrial proteome is built mainly by import of nuclear-encoded precursors, which are targeted mostly by cleavable presequences. Presequence processing upon import is essential for proteostasis and survival, but the consequences of dysfunctional protein maturation are unknown. We find that impaired presequence processing causes accumulation of precursors inside mitochondria that form aggregates, which escape degradation and unexpectedly do not cause cell death. Instead, cells survive via activation of a mitochondrial unfolded protein response (mtUPR)-like pathway that is triggered very early after precursor accumulation. In contrast to classical stress pathways, this immediate response maintains mitochondrial protein import, membrane potential, and translation through translocation of the nuclear HMG-box transcription factor Rox1 to mitochondria. Rox1 binds mtDNA and performs a TFAM-like function pivotal for transcription and translation. Induction of early mtUPR provides a reversible stress model to mechanistically dissect the initial steps in mtUPR pathways with the stressTFAM Rox1 as the first line of defense.

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Ste23 is a novel mitochondrial protease located in the matrix. Ste23 degrades presequence peptides derived from imported preproteins. Impaired peptide degradation leads to inhibited presequence-processing machinery and accumulation of immature preproteins. Thus efficient peptide turnover is crucial for mitochondrial proteostasis.