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The AAA-ATPase Msp1 extracts mislocalized outer membrane proteins and thus contributes to mitochondrial proteostasis. Using pull down experiments we show that trypanosomal Msp1 localizes to both glycosomes and the mitochondrial outer membrane, where it forms a stable complex with four outer membrane proteins. The trypanosome-specific pATOM36 mediates complex assembly of a-helically anchored mitochondrial outer membrane proteins such as protein translocase subunits. Inhibition of their assembly triggers a pathway that results in the proteasomal digestion of unassembled substrates. Using inducible single, double and triple RNAi cell lines combined with proteomic analyses we demonstrate that not only Msp1 but also the trypanosomal homolog of the AAA-ATPase VCP are implicated in this quality control pathway. Moreover, in the absence of VCP three out of the four Msp1-interacting mitochondrial proteins are required for efficient proteasomal digestion of pATOM36 substrates suggesting they act in concert with Msp1. pATOM36 is a functional analogue of the yeast MIM complex and possibly of human MTCH2 suggesting that similar mitochondrial quality control pathways linked to Msp1 might also exist in yeast and humans.

Consistent with other eukaryotes, theTrypanosoma bruceimitochondrial genome encodes mainly hydrophobic core subunits of the oxidative phosphorylation system. These proteins must be co-translationally inserted into the inner mitochondrial membrane and are synthesized by the highly divergent trypanosomal mitoribosomes, which have a much higher protein to RNA ratio than any other ribosome. Here, we show that the trypanosomal ortholog of the mitoribosome receptor Mba1 (TbMba1) is essential for normal growth of procyclic trypanosomes but redundant in the bloodstream form, which lacks an oxidative phosphorylation system. Proteomic analyses of TbMba1-depleted mitochondria from procyclic cells revealed reduced levels of many components of the oxidative phosphorylation system, most of which belong to the cytochrome c oxidase (Cox) complex, three subunits of which are mitochondrially encoded. However, the integrity of the mitoribosome and its interaction with the inner membrane were not affected. Pulldown experiments showed that TbMba1 forms a dynamic interaction network that includes the trypanosomal Mdm38/Letm1 ortholog and a trypanosome-specific factor that stabilizes the CoxI and CoxII mRNAs. In summary, our study suggests that the function of Mba1 in the biogenesis of membrane subunits of OXPHOS complexes is conserved among yeast, mammalian, and trypanosomes, which belong to two eukaryotic supergroups.

The mitochondrial F1Fo ATP synthase of Trypanosoma brucei has been studied in detail. Whereas its F1 moiety is relatively highly conserved in structure and composition, the same is not the case for the Fo part and the peripheral stalk. A core subunit of the latter, the normally conserved subunit b, could not be identified in trypanosomes suggesting that it might be absent. Here we have identified a 17 kDa mitochondrial protein of the inner membrane that is essential for normal growth, efficient oxidative phosphorylation and membrane potential maintenance. Pulldown experiments and native PAGE analysis indicate that the protein is associated with the F1Fo ATP synthase. Its ablation reduces the levels of Fo subunits, but not those of F1, and disturbs the cell cycle. HHpred analysis showed that the protein has structural similarities to subunit b of other species, indicating that the Fo part of the trypanosomal ATP synthase does contain a highly diverged subunit b. Thus, the Fo part of the trypanosomal ATPase synthase may be more widely conserved than initially thought.

Mitochondrial protein import in the parasitic protozoan Trypanosoma brucei is mediated by the atypical outer membrane translocase, ATOM. It consists of seven subunits including ATOM69, the import receptor for hydrophobic proteins. Ablation of ATOM69, but not of any other subunit, triggers a unique quality control pathway resulting in the proteasomal degradation of non-imported mitochondrial proteins. The process requires a protein of unknown function, an E3 ubiquitin ligase and the ubiquitin-like protein (TbUbL1), which all are recruited to the mitochondrion upon ATOM69 depletion. TbUbL1 is a nuclear protein, a fraction of which is released to the cytosol upon triggering of the pathway. Nuclear release is essential as cytosolic TbUbL1 can bind mislocalised mitochondrial proteins and likely transfers them to the proteasome. Mitochondrial quality control has previously been studied in yeast and metazoans. Finding such a pathway in the highly diverged trypanosomes suggests such pathways are an obligate feature of all mitochondria.

The universally conserved P-loop ATPase Ola1 is implicated in various cellular stress response pathways, as well as in cancer and tumor progression. However, Ola1p functions are divergent between species and the involved mechanisms are only poorly understood. Here, we studied the role of Ola1p in the heat shock response of the yeast Saccharomyces cerevisiae using a combination of quantitative and pulse labeling-based proteomics approaches, in vitro studies and cell-based assays. Our data show that when heat stress is applied to cells lacking Ola1p, the expression of stress-protective proteins is enhanced. During heat stress Ola1p associates with detergent-resistant protein aggregates and rapidly forms assemblies that localize to stress granules. The assembly of Ola1p was also observed in vitro using purified protein and conditions, which resembled those in living cells. We show that loss of Ola1p results in increased protein ubiquitination of detergent-insoluble aggregates recovered from heat-shocked cells. When subsequently cells lacking Ola1p were relieved from heat stress, reinitiation of translation was delayed, whereas, at the same time, de novo synthesis of central factors required for protein refolding and the clearance of aggregates was enhanced when compared to wildtype cells. The combined data suggest that upon acute heat stress, Ola1p is involved in the stabilization of misfolded proteins, which become sequestered in cytoplasmic stress granules. This function of Ola1p enables cells to resume translation in a timely manner as soon as heat stress is relieved.

Mitochondria cannot form de novo but require mechanisms that mediate their inheritance to daughter cells. The parasitic protozoanTrypanosoma bruceihas a single mitochondrion with a single-unit genome that is physically connected across the mitochondrial membranes to the basal body of the flagellum. This connection, termed tripartite attachment complex (TAC), is essential for the segregation of the replicated mitochondrial genomes prior to cytokinesis. Here we identify a protein complex consisting of three integral mitochondrial outer membrane proteins - TAC60, TAC42 and TAC40 - which are essential subunits of the TAC. TAC60 contains separable mitochondrial import and TAC-sorting signals and its biogenesis depends on the main outer membrane protein translocase. TAC40 is a member of the mitochondrial porin family, whereas TAC42 represents a novel class of mitochondrial outer membrane β-barrel proteins. Consequently TAC40 and TAC42 contain C-terminal β-signals. Thus in trypanosomes the highly conserved β-barrel protein assembly machinery plays a major role in the biogenesis of its unique mitochondrial genome segregation system.