A functionally divergent hydrogenosomal peptidase with protomitochondrial ancestry

Matrix proteins of mitochondria, hydrogenosomes and mitosomes are typically targeted and translocated into their respective organelles using N-terminal presequences that are subsequently cleaved by a peptidase. Here we characterize a approximately 47 kDa metallopeptidase, from the hydrogenosome-bearing, unicellular eukaryote Trichomonas vaginalis, that contains the active site motif (HXXEHX(76)E) characteristic of the beta subunit of the mitochondrial processing peptidase (MPP) and localizes to hydrogenosomes. The purified recombinant protein, named hydrogenosomal processing peptidase (HPP), is capable of cleaving a hydrogenosomal presequence in vitro, in contrast to MPP which requires both an alpha and beta subunit for activity. T. vaginalis HPP forms an approximately 100 kDa homodimer in vitro and also exists in an approximately 100 kDa complex in vivo. Our phylogenetic analyses support a common origin for HPP and betaMPP and demonstrate that gene duplication gave rise to alphaMPP and betaMPP before the divergence of T. vaginalis and mitochondria-bearing lineages. These data, together with published analyses of MPPs and putative mitosomal processing peptidases, lead us to propose that the length of targeting presequences and the subunit composition of organellar processing peptidases evolved in concert. Specifically, longer mitochondrial presequences may have evolved to require an alpha/beta heterodimer for accurate cleavage, while shorter hydrogenosomal and mitosomal presequences did not.     

A Highlights from MBoC Selection: More than just a ticket canceller: the mitochondrial processing peptidase tailors complex precursor proteins at internal cleavage sites

Most mitochondrial proteins are synthesized as precursors that carry N-terminal presequences. After they are imported into mitochondria, these targeting signals are cleaved off by the mitochondrial processing peptidase (MPP). Using the mitochondrial tandem protein Arg5,6 as a model substrate, we demonstrate that MPP has an additional role in preprotein maturation, beyond the removal of presequences. Arg5,6 is synthesized as a polyprotein precursor that is imported into mitochondria and subsequently separated into two distinct enzymes. This internal processing is performed by MPP, which cleaves the Arg5,6 precursor at its N-terminus and at an internal site. The peculiar organization of Arg5,6 is conserved across fungi and reflects the polycistronic arginine operon in prokaryotes. MPP cleavage sites are also present in other mitochondrial fusion proteins from fungi, plants, and animals. Hence, besides its role as a “ticket canceller” for removal of presequences, MPP exhibits a second conserved activity as an internal processing peptidase for complex mitochondrial precursor proteins.     

Protein import into hydrogenosomes of Trichomonas vaginalis involves both N-terminal and internal targeting signals: a case study of thioredoxin reductases

The parabasalian flagellate Trichomonas vaginalis harbors mitochondrion-related and H₂-producing organelles of anaerobic ATP synthesis, called hydrogenosomes, which harbor oxygen-sensitive enzymes essential to its pyruvate metabolism. In the human urogenital tract, however, T. vaginalis is regularly exposed to low oxygen concentrations and therefore must possess antioxidant systems protecting the organellar environment against the detrimental effects of molecular oxygen and reactive oxygen species. We have identified two closely related hydrogenosomal thioredoxin reductases (TrxRs), the hitherto-missing component of a thioredoxin-linked hydrogenosomal antioxidant system. One of the two hydrogenosomal TrxR isoforms, TrxRh1, carried an N-terminal extension resembling known hydrogenosomal targeting signals. Expression of hemagglutinin-tagged TrxRh1 in transfected T. vaginalis cells revealed that its N-terminal extension was necessary to import the protein into the organelles. The second hydrogenosomal TrxR isoform, TrxRh2, had no N-terminal targeting signal but was nonetheless efficiently targeted to hydrogenosomes. N-terminal presequences from hydrogenosomal proteins with known processing sites, i.e., the alpha subunit of succinyl coenzyme A synthetase (SCSα) and pyruvate:ferredoxin oxidoreductase A, were investigated for their ability to direct mature TrxRh1 to hydrogenosomes. Neither presequence directed TrxRh1 to hydrogenosomes, indicating that neither extension is, by itself, sufficient for hydrogenosomal targeting. Moreover, SCSα lacking its N-terminal extension was efficiently imported into hydrogenosomes, indicating that this extension is not required for import of this major hydrogenosomal protein. The finding that some hydrogenosomal enzymes require N-terminal signals for import but that in others the N-terminal extension is not necessary for targeting indicates the presence of additional targeting signals within the mature subunits of several hydrogenosome-localized proteins.     

Refining the Definition of Plant Mitochondrial Presequences through Analysis of Sorting Signals,N-Terminal Modifications,and Cleavage Motifs

Mitochondrial protein import is a complex multistep process from synthesis of proteins in the cytosol, recognition by receptors on the organelle surface, to translocation across one or both mitochondrial membranes and assembly after removal of the targeting signal, referred to as a presequence. In plants, import has to further discriminate between mitochondria and chloroplasts. In this study, we determined the precise cleavage sites in the presequences for Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) mitochondrial proteins using mass spectrometry by comparing the precursor sequences with experimental evidence of the amino-terminal peptide from mature proteins. We validated this method by assessments of false-positive rates and comparisons with previous available data using Edman degradation. In total, the cleavable presequences of 62 proteins from Arabidopsis and 52 proteins from rice mitochondria were determined. None of these proteins contained amino-terminal acetylation, in contrast to recent findings for chloroplast stromal proteins. Furthermore, the classical matrix glutamate dehydrogenase was detected with intact and amino-terminal acetylated sequences, indicating that it is imported into mitochondria without a cleavable targeting signal. Arabidopsis and rice mitochondrial presequences had similar isoelectric points, hydrophobicity, and the predicted ability to form an amphiphilic α-helix at the amino-terminal region of the presequence, but variations in length, amino acid composition, and cleavage motifs for mitochondrial processing peptidase were observed. A combination of lower hydrophobicity and start point of the amino-terminal α-helix in mitochondrial presequences in both Arabidopsis and rice distinguished them (98%) from Arabidopsis chloroplast stroma transit peptides. Both Arabidopsis and rice mitochondrial cleavage sites could be grouped into three classes, with conserved −3R (class II) and −2R (class I) or without any conserved (class III) arginines. Class II was dominant in both Arabidopsis and rice (55%–58%), but in rice sequences there was much less frequently a phenylalanine (F) in the −1 position of the cleavage site than in Arabidopsis sequences. Our data also suggest a novel cleavage motif of (F/Y)↓(S/A) in plant class III sequences.Plant mitochondria play a key role in energy production and metabolism that requires the import and assembly of at least 1,000 proteins. Protein import into mitochondria begins with synthesis of the precursor protein in the cytosol, followed by binding to various proteins in the cytosol, binding to receptors on the outer mitochondrial membrane, translocation across one or both mitochondrial membranes, removal of the targeting signal, termed a presequence, and intraorganellar sorting and assembly. A variety of studies have shown that there is no primary amino acid sequence conservation among presequences, but they do have a high proportion of positively charged residues and the capacity to form an amphiphilic α-helix (Roise et al., 1986; von Heijne, 1986). Many mitochondrial presequences have a loosely conserved motif near the cleavage site comprising an Arg residue at the −2 and/or −3 position (von Heijne et al., 1989; Schneider et al., 1998). This Arg has been experimentally shown to be an important recognition site for the mitochondrial processing peptidase (MPP; Arretz et al., 1994; Ogishima et al., 1995; Tanudji et al., 1999). MPP is a heterodimeric enzyme that contains two similar subunits: α-MPP is involved in binding precursor proteins and β-MPP catalyzes the cleavage of the presequence (Kitada et al., 1995; Luciano et al., 1997). In yeast and mammals, MPP is a soluble protein located in the matrix, but in plants, MPP is integrated into the inner membrane-bound cytochrome b/c₁ complex (Braun et al., 1992; Eriksson et al., 1994; Glaser and Dessi, 1999).The mechanism through which the targeting signal binds to a receptor protein has been revealed by NMR studies and the crystal structure of rat Tom20 (for translocase of the outer membrane) with a bound presequence (Abe et al., 2000; Saitoh et al., 2007). A dynamic binding model in which different hydrophobic residues in the presequence interact with Tom20 has been proposed. Thus, the presequence has mobility in the binding site via hydrophobic interactions, with several different binding states being possible. This model accounts for the ability of a single Tom20 in yeast to bind to a diverse array of presequences. Although plants contain a protein that is called Tom20 and that has a receptor function in mitochondrial import, it is not orthologous to yeast or mammalian Tom20 (Perry et al., 2006; Lister et al., 2007). However, the NMR structure of plant Tom20 reveals a similar hydrophobic binding pocket. This has been highlighted as a case of convergent evolution of a receptor that uses a similar mechanism of binding to recognize presequences (Lister and Whelan, 2006). Although structural studies reveal the importance of hydrophobic residues for presequence binding, several studies on yeast, mammals, and plants reveal an important role for positively charged residues in presequences for import into mitochondria (Lister et al., 2005; Neupert and Herrmann, 2007). These positively charged residues may play a role in positioning the amphiphilic α-helix for binding to Tom20 and also in subsequent translocation into and across the pores forming proteins of the TOM and TIM (for translocase of the inner membrane) complexes. Movement of the presequence into and across a translocase is explained by the binding chain hypothesis (Pfanner and Geissler, 2001). According to this hypothesis, a presequence binds to higher affinity sites in the import apparatus until it is “trapped” on the inside of the inner membrane by a combination of electrostatic interactions, the net negative charge on the inside of the inner membrane, and binding to matrix-located HSP70 (Zhang and Glaser, 2002).In addition to the fact that plant Tom20s are not orthologous to other Tom20s, plant mitochondria also lack the other two receptor components that have been functionally characterized in yeast, namely Tom70 and Tom22 (Lister et al., 2007). Furthermore, mitochondrial and plastid targeting signals contain significant similarities in plants; thus, plant mitochondrial presequences have evolved to differentiate from the large number and abundant nature of plastid proteins requiring import from the cytosol (Macasev et al., 2000). This raises the question of how similar plant mitochondrial targeting signals are to those of yeast and how they are differentiated from plastid transit peptides. To adequately address these questions, a large number of presequences need to be assembled to define motifs that differentiate presequence classes. Traditionally, the N-terminal sequences of plant mitochondrial proteins have been obtained by Edman degradation either from purified mitochondrial protein complexes or in proteome studies (Braun and Schmitz, 1995; Jänsch et al., 1996; Millar et al., 1998, 1999; Kruft et al., 2001; Bardel et al., 2002). The presequences could only be obtained by comparison of these N-terminal sequences with the preprotein sequence deduced from full-length cDNA sequences, which were only available in a small number of cases. Glaser et al. (1998) presented a list of approximately 100 plant mitochondrial presequences; these were mainly derived from prediction and/or comparisons in homologous cDNA-derived protein sequences with a core set of 31 experimentally proven presequences for plant mitochondrial proteins. Later analysis of 58 experimentally proven plant mitochondrial presequences deposited in the Swiss-Prot database revealed two major classes containing an Arg residue at positions −2 and −3 and one class without any conserved Arg residues (Zhang et al., 2001; Zhang and Glaser, 2002). However, this data set relied on the sequences available at the time that were from a variety of plant species and contained redundant orthologs from similar proteins. This data set also clearly focused on dicot plants, as less than 20% of the sequences were from monocot species.In the chloroplast, N-terminal modification of chloroplast proteins has been shown to be important for protein viability (Pesaresi et al., 2003). N-terminal acetylation can be detected by high-resolution mass spectrometry (MS) through a change in mass of the N-terminal peptide. The recent systematic analysis of the Arabidopsis (Arabidopsis thaliana) chloroplast proteome revealed 47 stroma proteins with N-acetylated residues and 62 without N-acetylated residues (Zybailov et al., 2008). The detection of N-terminal and non-N-terminal acetylated proteins by identifications of semitryptic peptides also allowed analysis of the cleavage sites and potential motifs for cleavage by processing peptidases (Zybailov et al., 2008). However, no systematic experimental analysis of N-terminal modifications and potential cleavage sites of plant mitochondrial proteins has been carried out to date using such an MS approach.In this study, we have determined Arabidopsis and rice (Oryza sativa) mitochondrial protein-targeting presequences and cleavage sites using an MS approach after gel- or liquid chromatography (LC)-based separation and also identified a range of N-terminal modifications of mitochondrial proteins. Validation of this method was performed by false-positive analysis and comparison with previous results in Arabidopsis using an Edman degradation approach (Kruft et al., 2001). We compared the characteristics of the generated Arabidopsis and rice mitochondrial presequences and the cleavage site motifs. Comparison with experimentally proven yeast mitochondrial presequences and Arabidopsis plastid stroma transit peptides allowed consideration of some evolutionary questions and insights into the different signal-recognizing mechanism(s) used to distinguish between organelles.     

Crystal Structure and Activity Studies of the C11 Cysteine Peptidase from Parabacteroides merdae in the Human Gut Microbiome

Clan CD cysteine peptidases, a structurally related group of peptidases that include mammalian caspases, exhibit a wide range of important functions, along with a variety of specificities and activation mechanisms. However, for the clostripain family (denoted C11), little is currently known. Here, we describe the first crystal structure of a C11 protein from the human gut bacterium, Parabacteroides merdae (PmC11), determined to 1.7-Å resolution. PmC11 is a monomeric cysteine peptidase that comprises an extended caspase-like α/β/α sandwich and an unusual C-terminal domain. It shares core structural elements with clan CD cysteine peptidases but otherwise structurally differs from the other families in the clan. These studies also revealed a well ordered break in the polypeptide chain at Lys¹⁴⁷, resulting in a large conformational rearrangement close to the active site. Biochemical and kinetic analysis revealed Lys¹⁴⁷ to be an intramolecular processing site at which cleavage is required for full activation of the enzyme, suggesting an autoinhibitory mechanism for self-preservation. PmC11 has an acidic binding pocket and a preference for basic substrates, and accepts substrates with Arg and Lys in P1 and does not require Ca²⁺ for activity. Collectively, these data provide insights into the mechanism and activity of PmC11 and a detailed framework for studies on C11 peptidases from other phylogenetic kingdoms.     

The processing peptidase of yeast mitochondria: the two co-operating components MPP and PEP are structurally related.

Two proteins co-operate in the proteolytic cleavage of mitochondrial precursor proteins: the mitochondrial processing peptidase (MPP) and the processing enhancing protein (PEP). In order to understand the structure and function of this novel peptidase, we have isolated mutants of Saccharomyces cerevisiae which were temperature sensitive in the processing of mitochondrial precursor proteins. Here we report on the mif2 mutation which is deficient in MPP. Mitochondria from the mif2 mutant were able to import precursor proteins, but not to cleave the presequences. The MPP gene was isolated. MPP is a hydrophilic protein consisting of 482 amino acids. Notably, MPP exhibits remarkable sequence similarity to PEP. We speculate that PEP and MPP have a common origin and have evolved into two components with different but mutually complementing functions in processing of precursor proteins.     

Isolation, Characterization, and Expression of the Gene Encoding the β Subunit of the Mitochondrial Processing Peptidase from Blastocladiella emersonii

A 2.3-kb BamHI-KpnI fragment was isolated from a partial genomic library and shown by nucleotide sequence analysis to contain the entire coding region of the gene encoding the β subunit of the Blastocladiella mitochondrial processing peptidase (β-MPP). The predicted β-MPP protein has 465 amino acids and a calculated molecular mass of 50.8 kDa. S1 nuclease protection assays revealed an intron, 209 bp in size, interrupting the coding region between the putative signal sequence and the mature protein. Northern blot analysis showed that β-MPP mRNA levels decrease significantly during B. emersonii sporulation, reaching basal levels in the zoospore stage. The amount of β-MPP protein, determined in Western blots, unlike its mRNA, does not vary significantly throughout the fungal life cycle.     

Processing and Topology of the Yeast Mitochondrial Phosphatidylserine Decarboxylase 1

The inner mitochondrial membrane plays a crucial role in cellular lipid homeostasis through biosynthesis of the non-bilayer-forming lipids phosphatidylethanolamine and cardiolipin. In the yeast Saccharomyces cerevisiae, the majority of cellular phosphatidylethanolamine is synthesized by the mitochondrial phosphatidylserine decarboxylase 1 (Psd1). The biogenesis of Psd1 involves several processing steps. It was speculated that the Psd1 precursor is sorted into the inner membrane and is subsequently released into the intermembrane space by proteolytic removal of a hydrophobic sorting signal. However, components involved in the maturation of the Psd1 precursor have not been identified. We show that processing of Psd1 involves the action of the mitochondrial processing peptidase and Oct1 and an autocatalytic cleavage at a highly conserved LGST motif yielding the α- and β-subunit of the enzyme. The Psd1 β-subunit (Psd1β) forms the membrane anchor, which binds the intermembrane space-localized α-subunit (Psd1α). Deletion of a transmembrane segment in the β-subunit results in mislocalization of Psd1 and reduced enzymatic activity. Surprisingly, autocatalytic cleavage does not depend on proper localization to the inner mitochondrial membrane. In summary, membrane integration of Psd1 is crucial for its functionality and for maintenance of mitochondrial lipid homeostasis.     

Cardiolipin, a lipid found in mitochondria, hydrogenosomes and bacteria was not detected in Giardia lamblia

Giardia lamblia is a protozoan parasite with many characteristics common among eukaryotic cells, but lacking other features found in most eukaryotes. Cardiolipin is a phospholipid located exclusively in energy transducing membranes and it was identified in mitochondria, bacteria, hydrogenosomes and chloroplasts. In eukaryotes, cardiolipin is the only lipid that is synthesized in the mitochondria. Biochemical procedures (TLC, HPLC) and fluorescent tools (NAO) were applied in order to search for cardiolipin in G. lamblia. In addition, BLAST searches were used to find homologs of enzymes that participate in the cardiolipin synthesis. Cardiolipin synthase was searched in the Giardia genome, using Saccharomyces cerevisiae and Mycoplasma penetrans sequences as bait. However, a good match to G. lamblia related proteins was not found. Here we show that mitosomes of G. lamblia apparently do not contain cardiolipin, which raises the discussion for its endosymbiotic origin and for the previous proposal that Giardia mitosomes are modified mitochondria.     

The Pre-Endosymbiont Hypothesis: A New Perspective on the Origin and Evolution of Mitochondria

Mitochondrial DNA (mtDNA) is unquestionably the remnant of an α-proteobacterial genome, yet only ∼10%–20% of mitochondrial proteins are demonstrably α-proteobacterial in origin (the “α-proteobacterial component,” or APC). The evolutionary ancestry of the non-α-proteobacterial component (NPC) is obscure and not adequately accounted for in current models of mitochondrial origin. I propose that in the host cell that accommodated an α-proteobacterial endosymbiont, much of the NPC was already present, in the form of a membrane-bound metabolic organelle (the premitochondrion) that compartmentalized many of the non-energy-generating functions of the contemporary mitochondrion. I suggest that this organelle also possessed a protein import system and various ion and small-molecule transporters. In such a scenario, an α-proteobacterial endosymbiont could have been converted relatively directly and rapidly into an energy-generating organelle that incorporated the extant metabolic functions of the premitochondrion. This model (the “pre-endosymbiont hypothesis”) effectively represents a synthesis of previous, contending mitochondrial origin hypotheses, with the bulk of the mitochondrial proteome (much of the NPC) having an endogenous origin and the minority component (the APC) having a xenogenous origin.Considering the central role played in all eukaryotic cells by mitochondria or mitochondrion-related organelles (MROs, such as hydrogenosomes and mitosomes) (Hjort et al. 2010; Shiflett and Johnson 2010; Müller et al. 2012), the question of the origin and subsequent evolution of the mitochondrion has long captivated and challenged biologists. In a recent article in this series (Gray 2012), I discussed in detail several aspects of mitochondrial evolution, focusing particularly on how well the accumulating molecular data can be accommodated in current models of mitochondrial origin. In this context, the origin and evolution of the mitochondrial proteome, as opposed to the origin and evolution of the mitochondrial genome, were examined from the perspective of comparative mitochondrial proteomics. Somewhat disconcertingly, as more data have become available, we find ourselves considerably less certain about key aspects of how mitochondria originated than we were (or thought we were) several decades ago.Here, I summarize key points discussed in more detail in the previous article before presenting a novel perspective on how the mitochondrion might have originated. The new model proposed here, which represents a synthesis of both endogenous (“origin from within”) and xenogenous (“origin from outside”) modes, is advanced in an attempt to account for the inability of a purely endosymbiotic model, whose strongest support has come from studies of the mitochondrial genome, to adequately accommodate data on the mitochondrial proteome.     

Processing of the dual targeted precursor protein of glutathione reductase in mitochondria and chloroplasts

Pea glutathione reductase (GR) is dually targeted to mitochondria and chloroplasts by means of an N-terminal signal peptide of 60 amino acid residues. After import, the signal peptide is cleaved off by the mitochondrial processing peptidase (MPP) in mitochondria and by the stromal processing peptidase (SPP) in chloroplasts. Here, we have investigated determinants for processing of the dual targeting signal peptide of GR by MPP and SPP to examine if there is separate or universal information recognised by both processing peptidases. Removal of 30 N-terminal amino acid residues of the signal peptide (GRDelta1-30) greatly stimulated processing activity by both MPP and SPP, whereas constructs with a deletion of an additional ten amino acid residues (GRDelta1-40) and deletion of 22 amino acid residues in the middle of the GR signal sequence (GRDelta30-52) could be cleaved by SPP but not by MPP. Numerous single mutations of amino acid residues in proximity of the cleavage site did not affect processing by SPP, whereas mutations within two amino acid residues on either side of the processing site had inhibitory effect on processing by MPP with a nearly complete inhibition for mutations at position -1. Mutation of positively charged residues in the C-terminal half of the GR targeting peptide inhibited processing by MPP but not by SPP. An inhibitory effect on SPP was detected only when double and triple mutations were introduced upstream of the cleavage site. These results indicate that: (i) recognition of processing site on a dual targeted GR precursor differs between MPP and SPP; (ii) the GR targeting signal has similar determinants for processing by MPP as signals targeting only to mitochondria; and (iii) processing by SPP shows a low level of sensitivity to single mutations on targeting peptide and likely involves recognition of the physiochemical properties of the sequence in the vicinity of cleavage rather than a requirement for specific amino acid residues.     

Hydrolysis of Sequenced β-Casein Peptides Provides New Insight into Peptidase Activity from Thermophilic Lactic Acid Bacteria and Highlights Intrinsic Resistance of Phosphopeptides

The peptidases of thermophilic lactic acid bacteria have a key role in the proteolysis of Swiss cheeses during warm room ripening. To compare their peptidase activities toward a dairy substrate, a tryptic/chymotryptic hydrolysate of purified β-casein was used. Thirty-four peptides from 3 to 35 amino acids, including three phosphorylated peptides, constitute the β-casein hydrolysate, as shown by tandem mass spectrometry. Cell extracts prepared from Lactobacillus helveticus ITG LH1, ITG LH77, and CNRZ 32, Lactobacillus delbrueckii subsp. lactis ITG LL14 and ITG LL51, L. delbrueckii subsp. bulgaricus CNRZ 397 and NCDO 1489, and Streptococcus thermophilus CNRZ 385, CIP 102303, and TA 060 were standardized in protein. The peptidase activities were assessed with the β-casein hydrolysate as the substrate at pH 5.5 and 24°C (conditions of warm room ripening) by (i) free amino acid release, (ii) reverse-phase chromatography, and (iii) identification of undigested peptides by mass spectrometry. Regardless of strain, L. helveticus was the most efficient in hydrolyzing β-casein peptides. Interestingly, cell extracts of S. thermophilus were not able to release a significant level of free proline from the β-casein hydrolysate, which was consistent with the identification of numerous dipeptides containing proline. With the three lactic acid bacteria tested, the phosphorylated peptides remained undigested or weakly hydrolyzed indicating their high intrinsic resistance to peptidase activities. Finally, several sets of peptides differing by a single amino acid in a C-terminal position revealed the presence of at least one carboxypeptidase in the cell extracts of these species.     

Diversity and reductive evolution of mitochondria among microbial eukaryotes

All extant eukaryotes are now considered to possess mitochondria in one form or another. Many parasites or anaerobic protists have highly reduced versions of mitochondria, which have generally lost their genome and the capacity to generate ATP through oxidative phosphorylation. These organelles have been called hydrogenosomes, when they make hydrogen, or remnant mitochondria or mitosomes when their functions were cryptic. More recently, organelles with features blurring the distinction between mitochondria, hydrogenosomes and mitosomes have been identified. These organelles have retained a mitochondrial genome and include the mitochondrial-like organelle of Blastocystis and the hydrogenosome of the anaerobic ciliate Nyctotherus. Studying eukaryotic diversity from the perspective of their mitochondrial variants has yielded important insights into eukaryote molecular cell biology and evolution. These investigations are contributing to understanding the essential functions of mitochondria, defined in the broadest sense, and the limits to which reductive evolution can proceed while maintaining a viable organelle.     

Mitochondrial processing peptidases

Three peptidases are responsible for the proteolytic processing of both nuclearly and mitochondrially encoded precursor polypeptides targeted to the various subcompartments of the mitochondria. Mitochondrial processing peptidase (MPP) cleaves the vast majority of mitochondrial proteins, while inner membrane peptidase (IMP) and mitochondrial intermediate peptidase (MIP) process specific subsets of precursor polypeptides. All three enzymes are structurally and functionally conserved across species, and their human homologues begin to be recognized as potential players in mitochondrial disease.     

Mitochondrial protein turnover: role of the precursor intermediate peptidase Oct1 in protein stabilization

Most mitochondrial proteins are encoded in the nucleus as precursor proteins and carry N-terminal presequences for import into the organelle. The vast majority of presequences are proteolytically removed by the mitochondrial processing peptidase (MPP) localized in the matrix. A subset of precursors with a characteristic amino acid motif is additionally processed by the mitochondrial intermediate peptidase (MIP) octapeptidyl aminopeptidase 1 (Oct1), which removes an octapeptide from the N-terminus of the precursor intermediate. However, the function of this second cleavage step is elusive. In this paper, we report the identification of a novel Oct1 substrate protein with an unusual cleavage motif. Inspection of the Oct1 substrates revealed that the N-termini of the intermediates typically carry a destabilizing amino acid residue according to the N-end rule of protein degradation, whereas mature proteins carry stabilizing N-terminal residues. We compared the stability of intermediate and mature forms of Oct1 substrate proteins in organello and in vivo and found that Oct1 cleavage increases the half-life of its substrate proteins, most likely by removing destabilizing amino acids at the intermediate's N-terminus. Thus Oct1 converts unstable precursor intermediates generated by MPP into stable mature proteins.     

Neprilysin Impedes Islet Amyloid Formation by Inhibition of Fibril Formation Rather Than Peptide Degradation

Deposition of islet amyloid polypeptide (IAPP) as islet amyloid in type 2 diabetes contributes to loss of β-cell function and mass, yet the mechanism for its occurrence is unclear. Neprilysin is a metallopeptidase known to degrade amyloid in Alzheimer disease. We previously demonstrated neprilysin to be present in pancreatic islets and now sought to determine whether it plays a role in degrading islet amyloid. We used an in vitro model where cultured human IAPP (hIAPP) transgenic mouse islets develop amyloid and thereby have increased β-cell apoptosis. Islet neprilysin activity was inhibited or up-regulated using a specific inhibitor or adenovirus encoding neprilysin, respectively. Following neprilysin inhibition, islet amyloid deposition and β-cell apoptosis increased by 54 and 75%, respectively, whereas when neprilysin was up-regulated islet amyloid deposition and β-cell apoptosis both decreased by 79%. To determine if neprilysin modulated amyloid deposition by cleaving hIAPP, analysis of hIAPP incubated with neprilysin was performed by mass spectrometry, which failed to demonstrate neprilysin-induced cleavage. Rather, neprilysin may act by reducing hIAPP fibrillogenesis, which we showed to be the case by fluorescence-based thioflavin T binding studies and electron microscopy. In summary, neprilysin decreases islet amyloid deposition by inhibiting hIAPP fibril formation, rather than degrading hIAPP. These findings suggest that targeting the role of neprilysin in IAPP fibril assembly, in addition to IAPP cleavage by other peptidases, may provide a novel approach to reduce and/or prevent islet amyloid deposition in type 2 diabetes.     

Solid Phase Synthesis of Mitochondrial Triphenylphosphonium-Vitamin E Metabolite Using a Lysine Linker for Reversal of Oxidative Stress

Mitochondrial targeting of antioxidants has been an area of interest due to the mitochondria''s role in producing and metabolizing reactive oxygen species. Antioxidants, especially vitamin E (α-tocopherol), have been conjugated to lipophilic cations to increase their mitochondrial targeting. Synthetic vitamin E analogues have also been produced as an alternative to α-tocopherol. In this paper, we investigated the mitochondrial targeting of a vitamin E metabolite, 2,5,7,8-tetramethyl-2-(2′-carboxyethyl)-6-hydroxychroman (α-CEHC), which is similar in structure to vitamin E analogues. We report a fast and efficient method to conjugate the water-soluble metabolite, α-CEHC, to triphenylphosphonium cation via a lysine linker using solid phase synthesis. The efficacy of the final product (MitoCEHC) to lower oxidative stress was tested in bovine aortic endothelial cells. In addition the ability of MitoCEHC to target the mitochondria was examined in type 2 diabetes db/db mice. The results showed mitochondrial accumulation in vivo and oxidative stress decrease in vitro.     

N-Terminal Presequence-Independent Import of Phosphofructokinase into Hydrogenosomes of Trichomonas vaginalis

Mitochondrial evolution entailed the origin of protein import machinery that allows nuclear-encoded proteins to be targeted to the organelle, as well as the origin of cleavable N-terminal targeting sequences (NTS) that allow efficient sorting and import of matrix proteins. In hydrogenosomes and mitosomes, reduced forms of mitochondria with reduced proteomes, NTS-independent targeting of matrix proteins is known. Here, we studied the cellular localization of two glycolytic enzymes in the anaerobic pathogen Trichomonas vaginalis: PP_i-dependent phosphofructokinase (TvPP_i-PFK), which is the main glycolytic PFK activity of the protist, and ATP-dependent PFK (TvATP-PFK), the function of which is less clear. TvPP_i-PFK was detected predominantly in the cytosol, as expected, while all four TvATP-PFK paralogues were imported into T. vaginalis hydrogenosomes, although none of them possesses an NTS. The heterologous expression of TvATP-PFK in Saccharomyces cerevisiae revealed an intrinsic capability of the protein to be recognized and imported into yeast mitochondria, whereas yeast ATP-PFK resides in the cytosol. TvATP-PFK consists of only a catalytic domain, similarly to “short” bacterial enzymes, while ScATP-PFK includes an N-terminal extension, a catalytic domain, and a C-terminal regulatory domain. Expression of the catalytic domain of ScATP-PFK and short Escherichia coli ATP-PFK in T. vaginalis resulted in their partial delivery to hydrogenosomes. These results indicate that TvATP-PFK and the homologous ATP-PFKs possess internal structural targeting information that is recognized by the hydrogenosomal import machinery. From an evolutionary perspective, the predisposition of ancient ATP-PFK to be recognized and imported into hydrogenosomes might be a relict from the early phases of organelle evolution.