The essential cysteines in the CIPC motif of the thioredoxin-like Trypanosoma brucei MICOS subunit TbMic20 do not form an intramolecular disulfide bridge in vivo.

The mitochondrial protein import machinery of trypanosomatids is highly divergent from that of the well-studied models such as baker's yeast. A notable example is that the central catalyst of the mitochondrial intermembrane space import and assembly pathway (MIA), named Mia40, is missing in trypanosomatids. Mia40 works in a two-step process. First it recognizes by direct binding reduced MIA substrate proteins and then catalyzes their oxidative folding to produce intramolecular disulfide bridges. It was recently proposed that a thioredoxin-like subunit of the trypanosomal mitochondrial contact site and cristae organizing system (MICOS) called TbMic20 may be the Mia40 replacement. Our study performed on procyclic stage of the parasite revealed that each of the two cysteines in TbMic20's active site is essential for the stability of MIA substrate proteins although they do not form a disulfide bridge in vivo. The two cysteines of Mia40's active site form an intramolecular disulfide bridge at steady state, which is a prerequisite for its oxidative folding of MIA substrates. Thus, we conclude that TbMic20 is unlikely to represent a bona fide Mia40 replacement and plays a still unresolved role in the stability and/or import of MIA substrates in trypanosomatids. Despite this, the effect of TbMic20 depletion and mutation indicates that the trypanosomal MICOS complex still plays a vital role in the maturation and/or stability of proteins imported by the MIA pathway.

The highly diverged trypanosomal MICOS complex is organized in a nonessential integral membrane and an essential peripheral module.

The mitochondrial contact site and cristae organization system (MICOS) mediates the formation of cristae, invaginations in the mitochondrial inner membrane. The highly diverged MICOS complex of the parasitic protist Trypanosoma brucei consists of nine subunits. Except for two Mic10-like and a Mic60-like protein, all subunits are specific for kinetoplastids. Here, we determined on a proteome-wide scale how ablation of individual MICOS subunits affects the levels of the other subunits. The results reveal co-regulation of TbMic10-1, TbMic10-2, TbMic16 and TbMic60, suggesting that these nonessential, integral inner membrane proteins form an interdependent network. Moreover, the ablation of TbMic34 and TbMic32 reveals another network consisting of the essential, intermembrane space-localized TbMic20, TbMic32, TbMic34 and TbMic40, all of which are peripherally associated with the inner membrane. The downregulation of TbMic20, TbMic32 and TbMic34 also interferes with mitochondrial protein import and reduces the size of the TbMic10-containing complexes. Thus, the diverged MICOS of trypanosomes contains two subcomplexes: a nonessential membrane-integrated one, organized around the conserved Mic10 and Mic60, that mediates cristae formation, and an essential membrane-peripheral one consisting of four kinetoplastid-specific subunits, that is required for import of intermembrane space proteins.

The Diverged Trypanosome MICOS Complex as a Hub for Mitochondrial Cristae Shaping and Protein Import.

The mitochondrial contact site and cristae organization system (MICOS) is a multiprotein complex responsible for cristae formation. Even though cristae are found in all mitochondria capable of oxidative phosphorylation, only Mic10 and Mic60 appear to be conserved throughout eukaryotes. The remaining 4 or 5 known MICOS subunits are specific to the supergroup Opisthokonta, which includes yeast and mammals that are the only organisms in which this complex has been analyzed experimentally. We have isolated the MICOS from Trypanosoma brucei, a member of the supergroup Excavata that is profoundly diverged from opisthokonts. We show that it is required for the maintenance of the unique discoidal cristae that typify excavates, such as euglenids and kinetoplastids, the latter of which include trypanosomes. The trypanosome MICOS consists of 9 subunits, most of which are essential for normal growth. Unlike in opisthokonts, it contains two distinct Mic10 orthologs and an unconventional putative Mic60 that lacks a mitofilin domain. Interestingly, one of the essential trypanosomatid-specific MICOS subunits called TbMic20 is a thioredoxin-like protein that appears to be involved in import of intermembrane space proteins, including respiratory chain complex assembly factors. This result points to trypanosome MICOS coordinating cristae shaping and population of its membrane with proteins involved in respiration, the latter via the catalytic activity of TbMic20. Thus, trypanosome MICOS allows us to define which of its features are conserved in all eukaryotes and decipher those that represent lineage-specific adaptations.

Trypanosomatid mitochondrial RNA editing: dramatically complex transcript repertoires revealed with a dedicated mapping tool.

RNA editing by targeted insertion and deletion of uridine is crucial to generate translatable mRNAs from the cryptogenes of the mitochondrial genome of kinetoplastids. This type of editing consists of a stepwise cascade of reactions generally proceeding from 3' to 5' on a transcript, resulting in a population of partially edited as well as pre-edited and completely edited molecules for each mitochondrial cryptogene of these protozoans. Often, the number of uridines inserted and deleted exceed the number of nucleotides that are genome-encoded. Thus, analysis of kinetoplastid mitochondrial transcriptomes has proven frustratingly complex. Here we present our analysis of Leptomonas pyrrhocoris mitochondrial cDNA deep sequencing reads using T-Aligner, our new tool which allows comprehensive characterization of RNA editing, not relying on targeted transcript amplification and on prior knowledge of final edited products. T-Aligner implements a pipeline of read mapping, visualization of all editing states and their coverage, and assembly of canonical and alternative translatable mRNAs. We also assess T-Aligner functionality on a more challenging deep sequencing read input from Trypanosoma cruzi. The analysis reveals that transcripts of cryptogenes of both species undergo very complex editing that includes the formation of alternative open reading frames and whole categories of truncated editing products.