Matrix processing peptidase of mitochondria

The mitochondrial processing peptidase (MPP) and the processing enhancing protein (PEP) cooperate in the proteolytic cleavage of matrix targeting sequences from nuclear-encoded mitochondrial precursor proteins. We have determined the cDNA sequence of Neurospora MPP after expression cloning. MPP appears to contain two domains of approximately equal size which are separated by a loop-like sequence. Considerable structural similarity exists to the recently sequenced yeast MPP as well as to Neurospora and yeast PEP. Four cysteine residues are conserved in Neurospora and yeast MPP. Inactivation of MPP can be achieved by using sulfhydryl reagents. MPP (but not PEP) depends on the presence of divalent metal ions for activity. Both MPP and PEP are synthesized as precursors containing matrix targeting signals which are processed during import into mitochondria by the mature forms of MPP and PEP

Characterization of the mitochondrial processing peptidase of Neurospora crassa

The mitochondrial processing peptidase (MPP) of Neurospora crassa is constituted by an alpha- and a beta-subunit. We have purified alpha-MPP after expression in Escherichia coli while beta-MPP was purified from mitochondria. A fusion protein between precytochrome b2 and mouse dihydrofolate reductase was expressed in E. coli, and the purified protein was used as substrate for MPP. Both subunits of MPP are required for processing. MPP removes the matrix targeting signal of cytochrome b2 by a single cut, and the resulting presequence peptide is 31 amino acid residues in length. It acts as a competitive inhibitor of processing but has a approximately 30-fold lower affinity for MPP than the preprotein. Competition assays show that MPP recognizes the COOH- terminal portion of the presequence of cytochrome b2 rather than the NH2-terminal part which has the potential to form an amphiphilic helix. Substitution of arginine in position -2 of the matrix targeting sequence of cytochrome b2 prevents processing but not import of a chimeric precursor. Substitution of the tyrosyl residue in position +1 also prevents processing, indicating that MPP interacts with sequences COOH-terminal to the cleavage site. Non-cleavable preprotein is still recognized by MPP. Our data suggest that processing peptidase and import machinery recognize distinct structural elements in preproteins which, however, can be overlapping

Characterization of the bifunctional cytochrome c reductase-processing peptidase complex from potato mitochondria

In potato, cytochrome c reductase, a protein complex of the respiratory chain, exhibits processing activity toward mitochondrial precursor proteins. One of the two cooperating components of the processing peptidase was shown to be identical with subunit III of the complex. Here we report that two additional proteins of the complex (subunit I and II) share 40-50% sequence identity with the processing enhancing protein, the other component of the processing enzyme from fungi and mammals. Thus the composition and structure of the complex integrated processing peptidase seems to be different from its fungal and mammalian counterparts. Cytochrome c reductase from potato is extraordinarily stable, and separation of subunit III from the complex leads to aggregation of the remaining subcomplex and irreversible loss of processing activity. Expression of the three high molecular weight subunits of the complex allowed purification of each individual protein. Neither the individual subunits nor their combinations are active in in vitro processing assays suggesting that they may need the structural support of the complex for activity. In contrast to mitochondrial processing peptidases from other organisms, the purified potato enzyme is active in the presence of high salt (above 1 M NaCl) and works efficiently without addition of metal ions. These data indicate that potato cytochrome c reductase is a bifunctional protein complex with unique features. Possibly, there is a more general evolutionary relationship between cytochrome c reductases and mitochondrial processing peptidases than hitherto assumed

Transport of proteins into the various subcompartments of mitochondria

The import of proteins into mitochondria is an intricate process comprised of multiple steps. The first step involves the sorting of cytosolically synthesized precursor proteins to the mitochondrial surface. There precursor proteins are recognized by specific receptors which deliver them to the general import site present in the outer membrane. The second stage of import involves a series of complex intraorganelle sorting events which results in the delivery of the proteins to one of the four possible submitochondrial destinations, namely the outer and inner membranes, the matrix and intermembrane space. Here in this review, we discuss the current knowledge on these intramitochondrial sorting events. We especially focus on targetting of proteins to the intermembrane space. Sorting to the intermembrane space represents a particularly interesting situation, as at least three separate targetting pathways to this subcompartment are known to exist

Protein translocation across mitochondrial membranes

Protein translocation across biological membranes is of fundamental importance for the biogenesis of organelles and in protein secretion. We will give an overview of the recent achievements in the understanding of protein translocation across mitochondrial membranes(1-5). In particular we will focus on recently identified components of the mitochondrial import apparatus

Mdj1p, a novel chaperone of the DnaJ family, is involved in mitochondrial biogenesis and protein folding

Mdj1p, a novel member of the DnaJ family, is a heat shock protein that is associated with the inner membrane of mitochondria of Saccharomyces cerevisiae. Disruption of the MDJ1 gene resulted in a petite phenotype, loss of mitochondrial DNA, and inviability at 37°C. Import of precursor proteins was not affected by a lack of Mdj1p, but folding of newly imported proteins was markedly impaired. The efficiency of refolding of a tester protein, dihydrofolate reductase, was significantly reduced in mitochondria lacking Mdj1p after incubation at elevated temperature. We conclude that Mdj1p is an important mitochondrial chaperone that participates in the folding of newly imported proteins and in the protection of proteins against heat denaturation and aggregation

Reductive Evolution of the Mitochondrial Processing Peptidases of the Unicellular Parasites Trichomonas vaginalis and Giardia intestinalis

Mitochondrial processing peptidases are heterodimeric enzymes (α/βMPP) 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 α/β heterodimer. By contrast, and so far uniquely among eukaryotes, the Giardia processing peptidase functions as a monomer comprising a single βMPP-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 αMPP-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 α/βMPP 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

Genetic variability of the neogregarine apicystis bombi, an etiological agent of an emergent bumblebee disease

The worldwide spread of diseases is considered a major threat to biodiversity and a possible driver of the decline of pollinator populations, particularly when novel species or strains of parasites emerge. Previous studies have suggested that populations of introduced European honeybee (Apis mellifera) and bumblebee species (Bombus terrestris and Bombus ruderatus) in Argentina share the neogregarine parasite Apicystis bombi with the native bumblebee (Bombus dahlbomii). In this study we investigated whether A. bombi is acting as an emergent parasite in the non-native populations. Specifically, we asked whether A. bombi, recently identified in Argentina, was introduced by European, non-native bees. Using ITS1 and ITS2 to assess the parasite's intraspecific genetic variation in bees from Argentina and Europe, we found a largely unstructured parasite population, with only 15% of the genetic variation being explained by geographic location. The most abundant haplotype in Argentina (found in all 9 specimens of non-native species) was identical to the most abundant haplotype in Europe (found in 6 out of 8 specimens). Similarly, there was no evidence of structuring by host species, with this factor explaining only 17% of the genetic variation. Interestingly, parasites in native Bombus ephippiatus from Mexico were genetically distant from the Argentine and European samples, suggesting that sufficient variability does exist in the ITS region to identify continent-level genetic structure in the parasite. Thus, the data suggest that A. bombi from Argentina and Europe share a common, relatively recent origin. Although our data did not provide information on the direction of transfer, the absence of genetic structure across space and host species suggests that A. bombi may be acting as an emergent infectious disease across bee taxa and continents