Mitochondria provide numerous essential functions for cells and their dysfunction leads to a variety of diseases. Thus, obtaining a complete mitochondrial proteome should be a crucial step toward understanding the roles of mitochondria. Many mitochondrial proteins have been identified experimentally but a complete list is not yet available. To fill this gap, methods to computationally predict mitochondrial proteins from amino acid sequence have been developed and are widely used, but unfortunately, their accuracy is far from perfect. Here we describe MitoFates, an improved prediction method for cleavable N-terminal mitochondrial targeting signals (presequences) and their cleavage sites. MitoFates introduces novel sequence features including positively charged amphiphilicity, presequence motifs, and position weight matrices modeling the presequence cleavage sites. These features are combined with classical ones such as amino acid composition and physico-chemical properties as input to a standard support vector machine classifier. On independent test data, MitoFates attains better performance than existing predictors in both detection of presequences and in predicting their cleavage sites. We used MitoFates to look for undiscovered mitochondrial proteins from 42,217 human proteins (including isoforms such as alternative splicing or translation initiation variants). MitoFates predicts 1167 genes to have at least one isoform with a presequence. Five-hundred and eighty of these genes were not annotated as mitochondrial in either UniProt or Gene Ontology. Interestingly, these include candidate regulators of parkin translocation to damaged mitochondria, and also many genes with known disease mutations, suggesting that careful investigation of MitoFates predictions may be helpful in elucidating the role of mitochondria in health and disease. MitoFates is open source with a convenient web server publicly available.

Mitochondria fulfill central functions in cellular energetics, metabolism and signaling. The outer membrane TOM40 complex imports virtually all mitochondrial proteins, however, its architecture and the molecular mechanisms of preprotein translocation are unknown. We mapped the active translocator with resolution down to single amino acid residues, discovering distinct transport paths for hydrophilic and hydrophobic preproteins through the Tom40 channel. An N-terminal segment of Tom40 passes from the cytosol through the channel interior to recruit intermembrane space chaperones that guide the transfer of hydrophobic preproteins. The translocator possesses an intricate architecture with three Tom40 β-barrel channels sandwiched 2 between a central α-helical Tom22 receptor cluster and external regulatory Tom proteins. The preprotein-translocating trimeric complex is in exchange with a dimeric isoform that is crucial for assembly of new TOM40 complexes. The dynamic coupling of α-helical receptors, β-barrel channels and chaperones generates a versatile machinery that manages transport of ~1,000 different proteins into mitochondria.One Sentence Summary: Architecture of the mitochondrial TOM40 entry gate identifies preprotein paths and the blueprint for its assembly.Main Text: Mitochondria are essential organelles in eukaryotic cells. They are pivotal for cellular ATP production, numerous metabolic pathways and regulatory processes, and programmed cell death. During evolution of eukaryotes, most genes for mitochondrial proteins were transferred to the nucleus. The proteins are synthesized as preproteins in the cytosol and imported back into mitochondria. Different classes of preproteins have been identified that either contain N-terminal targeting sequences (presequences) or internal targeting information in the mature part (1-3). The protein translocator of the outer membrane (TOM40 complex) functions as the main entry gate of mitochondria (1-3). Most of the >1,000 different mitochondrial proteins are imported by the TOM40 complex, followed by transfer to distinct intramitochondrial machineries specialized for individual classes of preproteins. Whereas the structurally known membrane protein complexes consist of either α-helical or β-barrel proteins, the TOM40 complex is composed of both α-helical and β-barrel integral membrane proteins. The complex consists of the channel-forming β-barrel protein Tom40 and six other subunits each containing single α-helical transmembrane (TM) segments: the receptor proteins Tom20, Tom22 and Tom70, and the small regulatory subunits Tom5, Tom6 and Tom7 (1-3). Tom40, Tom22 and the small Tom proteins form the TOM40 core complex, whereas Tom20 and Tom70 are more loosely associated with the complex. The molecular architecture of the complex has not been elucidated. It is thus unknown how α-helical and β-barrel membrane proteins can be combined into a functional complex and how diverse classes of preproteins can be transported by the same transmembrane channel.To define the archite...

We propose LongQC as an easy and automated quality control tool for genomic datasets generated by third generation sequencing (TGS) technologies such as Oxford Nanopore technologies (ONT) and SMRT sequencing from Pacific Bioscience (PacBio). Key statistics were optimized for long read data, and LongQC covers all major TGS platforms. LongQC processes and visualizes those statistics automatically and quickly.

Protein transport systems are fundamentally important for maintaining mitochondrial function. Nevertheless, mitochondrial protein translocases such as the kinetoplastid ATOM complex have recently been shown to vary in eukaryotic lineages. Various evolutionary hypotheses have been formulated to explain this diversity. To resolve any contradiction, estimating the primitive state and clarifying changes from that state are necessary. Here, we present more likely primitive models of mitochondrial translocases, specifically the translocase of the outer membrane (TOM) and translocase of the inner membrane (TIM) complexes, using scrutinized phylogenetic profiles. We then analyzed the translocases’ evolution in eukaryotic lineages. Based on those results, we propose a novel evolutionary scenario for diversification of the mitochondrial transport system. Our results indicate that presequence transport machinery was mostly established in the last eukaryotic common ancestor, and that primitive translocases already had a pathway for transporting presequence-containing proteins. Moreover, secondary changes including convergent and migrational gains of a presequence receptor in TOM and TIM complexes, respectively, likely resulted from constrained evolution. The nature of a targeting signal can constrain alteration to the protein transport complex.

Entamoeba histolytica, an anaerobic intestinal parasite causing dysentery and extra-intestinal abscesses in humans, possesses highly reduced and divergent mitochondrion-related organelles (MROs) called mitosomes. This organelle lacks many features associated with canonical aerobic mitochondria and even other MROs such as hydrogenosomes. The Entamoeba mitosome has been found to have a compartmentalized sulfate activation pathway, which was recently implicated to have a role in amebic stage conversion. It also features a unique shuttle system via Tom60, which delivers proteins from the cytosol to the mitosome. In addition, only Entamoeba mitosomes possess a novel subclass of β-barrel outer membrane protein called MBOMP30. With the discoveries of such unique features of mitosomes of Entamoeba, there still remain a number of significant unanswered issues pertaining to this organelle. Particularly, the present understanding of the inner mitosomal membrane of Entamoeba is extremely limited. So far, only a few homologs for transporters of various substrates have been confirmed, while the components of the protein translocation complexes appear to be absent or are yet to be discovered. Employing a similar strategy as in our previous work, we collaborated to screen and discover mitosomal membrane proteins. Using a specialized prediction pipeline, we searched for proteins possessing α-helical transmembrane domains, which are unique to E. histolytica mitosomes. From the prediction algorithm, 25 proteins emerged as candidates, two of which were initially observed to be localized to the mitosomes. Further screening and analysis of the predicted proteins may provide clues to answer key questions on mitosomal evolution, biogenesis, dynamics, and biochemical processes.