Mitochondrial processing peptidase (MPP) consists of α and β subunits that catalyze the cleavage of N-terminal mitochondrial-targeting sequences (N-MTSs) and deliver preproteins to the mitochondria. In plants, both MPP subunits are associated with the respiratory complex bc1, which has been proposed to represent an ancestral form. Subsequent duplication of MPP subunits resulted in separate sets of genes encoding soluble MPP in the matrix and core proteins (cp1 and cp2) of the membrane-embedded bc1 complex. As only α-MPP was duplicated in Neurospora, its single β-MPP functions in both MPP and bc1 complexes. Herein, we investigated the MPP/core protein family and N-MTSs in the kinetoplastid Trypanosoma brucei, which is often considered one of the most ancient eukaryotes. Analysis of N-MTSs predicted in 336 mitochondrial proteins showed that trypanosomal N-MTSs were comparable with N-MTSs from other organisms. N-MTS cleavage is mediated by a standard heterodimeric MPP, which is present in the matrix of procyclic and bloodstream trypanosomes, and its expression is essential for the parasite. Distinct Genes encode cp1 and cp2, and in the bloodstream forms the expression of cp1 is downregulated along with the bc1 complex. Phylogenetic analysis revealed that all eukaryotic lineages include members with a Neurospora-type MPP/core protein family, whereas cp1 evolved independently in metazoans, some fungi and kinetoplastids. Evolution of cp1 allowed the independent regulation of respiration and protein import, which is essential for the procyclic and bloodstream forms of T. brucei. These results indicate that T. brucei possesses a highly derived MPP/core protein family that likely evolved in response to its complex life cycle and does not appear to have an ancient character proposed earlier for this eukaryote.

• Keywords
• bc1 complex, evolution, mitochondrial processing peptidase, mitochondrial targeting sequence, trypanosome,
• MeSH
• Eukaryota genetics   MeSH
• Phylogeny MeSH
• Metalloendopeptidases genetics   MeSH
• Mitochondrial Proteins genetics   metabolism   MeSH
• Mitochondria genetics   MeSH
• Evolution, Molecular MeSH
• Mitochondrial Processing Peptidase MeSH
• Amino Acid Sequence MeSH
• Base Sequence MeSH
• Trypanosoma brucei brucei genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Metalloendopeptidases MeSH
• Mitochondrial Proteins MeSH

An all atomic, non-restrained molecular dynamics (MD) simulation in explicit water was used to study in detail the structural features of the highly conserved glycine-rich loop (GRL) of the α-subunit of the yeast mitochondrial processing peptidase (MPP) and its importance for the tertiary and quaternary conformation of MPP. Wild-type and GRL-deleted MPP structures were studied using non-restrained MD simulations, both in the presence and the absence of a substrate in the peptidase active site. Targeted MD simulations were employed to study the mechanism of substrate translocation from the GRL to the active site. We demonstrate that the natural conformational flexibility of the GRL is crucial for the substrate translocation process from outside the enzyme towards the MPP active site. We show that the α-helical conformation of the substrate is important not only during its initial interaction with MPP (i.e. substrate recognition), but also later, at least during the first third of the substrate translocation trajectory. Further, we show that the substrate remains in contact with the GRL during the whole first half of the translocation trajectory and that hydrophobic interactions play a major role. Finally, we conclude that the GRL acts as a precisely balanced structural element, holding the MPP subunits in a partially closed conformation regardless the presence or absence of a substrate in the active site.

• MeSH
• Time Factors MeSH
• Glycine chemistry   MeSH
• Catalytic Domain MeSH
• Metalloendopeptidases chemistry   metabolism   MeSH
• Mitochondrial Processing Peptidase MeSH
• Protein Subunits chemistry   metabolism   MeSH
• Saccharomyces cerevisiae enzymology   MeSH
• Protein Structure, Secondary MeSH
• Molecular Dynamics Simulation MeSH
• Substrate Specificity MeSH
• Computational Biology * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Glycine MeSH
• Metalloendopeptidases MeSH
• Protein Subunits MeSH

Mitochondrial processing peptidases are heterodimeric enzymes (alpha/betaMPP) that play an essential role in mitochondrial biogenesis by recognizing and cleaving the targeting presequences of nuclear-encoded mitochondrial proteins. The two subunits are paralogues that probably evolved by duplication of a gene for a monomeric metallopeptidase from the endosymbiotic ancestor of mitochondria. Here, we characterize the MPP-like proteins from two important human parasites that contain highly reduced versions of mitochondria, the mitosomes of Giardia intestinalis and the hydrogenosomes of Trichomonas vaginalis. Our biochemical characterization of recombinant proteins showed that, contrary to a recent report, the Trichomonas processing peptidase functions efficiently as an alpha/beta heterodimer. By contrast, and so far uniquely among eukaryotes, the Giardia processing peptidase functions as a monomer comprising a single betaMPP-like catalytic subunit. The structure and surface charge distribution of the Giardia processing peptidase predicted from a 3-D protein model appear to have co-evolved with the properties of Giardia mitosomal targeting sequences, which, unlike classic mitochondrial targeting signals, are typically short and impoverished in positively charged residues. The majority of hydrogenosomal presequences resemble those of mitosomes, but longer, positively charged mitochondrial-type presequences were also identified, consistent with the retention of the Trichomonas alphaMPP-like subunit. Our computational and experimental/functional analyses reveal that the divergent processing peptidases of Giardia mitosomes and Trichomonas hydrogenosomes evolved from the same ancestral heterodimeric alpha/betaMPP metallopeptidase as did the classic mitochondrial enzyme. The unique monomeric structure of the Giardia enzyme, and the co-evolving properties of the Giardia enzyme and substrate, provide a compelling example of the power of reductive evolution to shape parasite biology.

• MeSH
• Down-Regulation genetics   MeSH
• Phylogeny MeSH
• Gene Dosage MeSH
• Giardia lamblia genetics   metabolism   ultrastructure   MeSH
• Glycine chemistry   genetics   physiology   MeSH
• Metalloendopeptidases chemistry   genetics   metabolism   MeSH
• Mitochondria metabolism   MeSH
• Evolution, Molecular * MeSH
• Mitochondrial Processing Peptidase MeSH
• Protein Multimerization MeSH
• Organelles metabolism   MeSH
• Protein Subunits genetics   MeSH
• Protein Processing, Post-Translational genetics   MeSH
• Proline-Rich Protein Domains genetics   physiology   MeSH
• Amino Acid Sequence MeSH
• Protein Transport MeSH
• Trichomonas vaginalis genetics   metabolism   ultrastructure   MeSH
• Hydrogen metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Glycine MeSH
• Metalloendopeptidases MeSH
• Protein Subunits MeSH
• Hydrogen MeSH