A mitochondrion-free eukaryote contains proteins capable of import into an exogenous mitochondrion-related organelle.

The endobiotic flagellate Monocercomonoides exilis is the only known eukaryote to have lost mitochondria and all its associated proteins in its evolutionary past. This final stage of the mitochondrial evolutionary pathway may serve as a model to explain events at their very beginning such as the initiation of protein import. We have assessed the capability of proteins from this eukaryote to enter emerging mitochondria using a specifically designed in vitro assay. Hydrogenosomes (reduced mitochondria) of Trichomonas vaginalis were incubated with a soluble protein pool derived from a cytosolic fraction of M. exilis, and proteins entering hydrogenosomes were subsequently detected by mass spectrometry. The assay detected 19 specifically and reproducibly imported proteins, and in 14 cases the import was confirmed by the overexpression of their tagged version in T. vaginalis. In most cases, only a small portion of the signal reached the hydrogenosomes, suggesting specific but inefficient transport. Most of these proteins represent enzymes of carbon metabolism, and none exhibited clear signatures of proteins targeted to hydrogenosomes or mitochondria, which is consistent with their inefficient import. The observed phenomenon may resemble a primaeval type of protein import which might play a role in the establishment of the organelle and shaping of its proteome in the initial stages of endosymbiosis.

Reduced mitochondria provide an essential function for the cytosolic methionine cycle.

The loss of mitochondria in oxymonad protists has been associated with the redirection of the essential Fe-S cluster assembly to the cytosol. Yet as our knowledge of diverse free-living protists broadens, the list of functions of their mitochondrial-related organelles (MROs) expands. We revealed another such function in the closest oxymonad relative, Paratrimastix pyriformis, after we solved the proteome of its MRO with high accuracy, using localization of organelle proteins by isotope tagging (LOPIT). The newly assigned enzymes connect to the glycine cleavage system (GCS) and produce folate derivatives with one-carbon units and formate. These are likely to be used by the cytosolic methionine cycle involved in S-adenosyl methionine recycling. The data provide consistency with the presence of the GCS in MROs of free-living species and its absence in most endobionts, which typically lose the methionine cycle and, in the case of oxymonads, the mitochondria.

Analysis of diverse eukaryotes suggests the existence of an ancestral mitochondrial apparatus derived from the bacterial type II secretion system.

The type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion.