Mitochondrial Intermediate Peptidase Research Articles

Analysis of mitochondrial targeting signal cleavage and protein processing by mass spectrometry.

The majority of mitochondrial proteins are encoded in the nucleus, synthesized in the cytosol and imported into mitochondria mediated by an N-terminal mitochondrial targeting sequences (MTS). After import, the MTS is cleaved off by the mitochondrial processing peptidase (MPP) and subsets of the imported proteins are further processed by the aminopeptidase intermediate cleaving peptidase 55 (ICP55), the mitochondrial intermediate peptidase (MIP), octapeptidyl aminopeptidase 1 (Oct1) or other proteolytic enzymes. Mutations that impair the mitochondrial processing machinery or mitochondrial protein degradation result in rare but severe human diseases. In addition, aging and various stress conditions are associated with altered proteolysis of mitochondrial proteins. Enrichment of protein terminal peptides in combination with mass spectrometry-based identification and quantification has become the method of choice to study proteolytic processing. Here, we describe an updated step-by-step protocol for the enrichment of N-terminal peptides by Hypersensitive Undecanal-mediated Enrichment of N-Terminal peptides (HUNTER). We describe analysis of mass spectrometry data acquired for HUNTER samples and present a suite of dedicated Python and R scripts for HUNTER quality control, classification of the enriched peptides, annotation of mitochondrial processing sites and quantitative evaluation. The scripts are freely available at https://github.com/FabianStockert/mito_annotation.

Effect of mitochondrial quantity and quality controls in white adipose tissue on healthy lifespan: Essential roles of GH/IGF-1-independent pathways in caloric restriction-mediated metabolic remodeling.

Long-term caloric restriction is a conventional and reproducible dietary intervention to improve whole body metabolism, suppress age-related pathophysiology, and extend lifespan. The beneficial actions of caloric restriction are widely accepted to be regulated in both growth hormone/insulin-like growth factor 1-dependent and -independent manners. Although growth hormone/insulin-like growth factor 1-dependent regulatory mechanisms are well described, those occurring independent of growth hormone/insulin-like growth factor 1 are poorly understood. In this review, we focus on molecular mechanisms of caloric restriction regulated in a growth hormone/insulin-like growth factor 1-independent manner. Caloric restriction increases mitochondrial quantity and improves mitochondrial quality by activating an axis involving sterol regulatory element binding protein-c/peroxisome proliferator-activated receptor γ coactivator-1α/mitochondrial intermediate peptidase in a growth hormone/insulin-like growth factor 1-independent manner, particularly in white adipose tissue. Fibroblast growth factor 21 is also involved in this axis. Moreover, the axis may be regulated by lower leptin signaling. Thus, caloric restriction appears to induce beneficial actions partially by regulating mitochondrial quantity and quality in white adipose tissue in a growth hormone/insulin-like growth factor 1-independent manner.

Mitochondrial Protein Homeostasis and Cardiomyopathy.

Human mitochondrial disorders impact tissues with high energetic demands and can be associated with cardiac muscle disease (cardiomyopathy) and early mortality. However, the mechanistic link between mitochondrial disease and the development of cardiomyopathy is frequently unclear. In addition, there is often marked phenotypic heterogeneity between patients, even between those with the same genetic variant, which is also not well understood. Several of the mitochondrial cardiomyopathies are related to defects in the maintenance of mitochondrial protein homeostasis, or proteostasis. This essential process involves the importing, sorting, folding and degradation of preproteins into fully functional mature structures inside mitochondria. Disrupted mitochondrial proteostasis interferes with mitochondrial energetics and ATP production, which can directly impact cardiac function. An inability to maintain proteostasis can result in mitochondrial dysfunction and subsequent mitophagy or even apoptosis. We review the known mitochondrial diseases that have been associated with cardiomyopathy and which arise from mutations in genes that are important for mitochondrial proteostasis. Genes discussed include DnaJ heat shock protein family member C19 (DNAJC19), mitochondrial import inner membrane translocase subunit TIM16 (MAGMAS), translocase of the inner mitochondrial membrane 50 (TIMM50), mitochondrial intermediate peptidase (MIPEP), X-prolyl-aminopeptidase 3 (XPNPEP3), HtraA serine peptidase 2 (HTRA2), caseinolytic mitochondrial peptidase chaperone subunit B (CLPB) and heat shock 60-kD protein 1 (HSPD1). The identification and description of disorders with a shared mechanism of disease may provide further insights into the disease process and assist with the identification of potential therapeutics.

Variants in the MIPEP gene presenting with complex neurological phenotype without cardiomyopathy, impair OXPHOS protein maturation and lead to a reduced OXPHOS abundance in patient cells

Most mitochondrial proteins are synthesized in the cytosol and targeted to mitochondria via N-terminal mitochondrial targeting signals (MTS) that are proteolytically removed upon import. Sometimes, MTS removal is followed by a cleavage of an octapeptide by the mitochondrial intermediate peptidase (MIP), encoded by the MIPEP gene. Previously, MIPEP variants were linked to four cases of multisystemic disorder presenting with cardiomyopathy, developmental delay, hypotonia and infantile lethality. We report here a patient carrying compound heterozygous MIPEP variants—one was not previously linked to mitochondrial disease—who did not have cardiomyopathy and who is alive at the age of 20 years. This patient had developmental delay, global hypotonia, mild optic neuropathy and mild ataxia. Functional characterization of patient fibroblasts and HEK293FT cells carrying MIPEP hypomorphic alleles demonstrated that deficient MIP activity was linked to impaired post-import processing of subunits from four of the five OXPHOS complexes and decreased abundance and activity of some of these complexes in human cells possibly underlying the development of mitochondrial disease. Thus, our work expands the genetic and clinical spectrum of MIPEP-linked disease and establishes MIP as an important regulator of OXPHOS biogenesis and function in human cells.

Maturation of Mitochondrially Targeted Prx V Involves a Second Cleavage by Mitochondrial Intermediate Peptidase That Is Sensitive to Inhibition by H2O2.

Prx V mRNA contains two in-frame AUG codons, producing a long (L-Prx V) and short form of Prx V (S-Prx V), and mouse L-Prx V is expressed as a precursor protein containing a 49-amino acid N-terminal mitochondria targeting sequence. Here, we show that the N-terminal 41-residue sequence of L-Prx V is cleaved by mitochondrial processing peptidase (MPP) in the mitochondrial matrix to produce an intermediate Prx V (I-Prx V) with a destabilizing phenylalanine at its N-terminus, and further, that the next 8-residue sequence is cleaved by mitochondrial intermediate peptidase (MIP) to convert I-Prx V to a stabilized mature form that is identical to S-Prx V. Further, we show that when mitochondrial H2O2 levels are increased in HeLa cells using rotenone, in several mouse tissues by deleting Prx III, and in the adrenal gland by deleting Srx or by exposing mice to immobilized stress, I-Prx V accumulates transiently and mature S-Prx V levels decrease in mitochondria over time. These findings support the view that MIP is inhibited by H2O2, resulting in the accumulation and subsequent degradation of I-Prx V, identifying a role for redox mediated regulation of Prx V proteolytic maturation and expression in mitochondria.

Functional coupling of presequence processing and degradation in human mitochondria.

The mitochondrial proteome is built and maintained mainly by import of nuclear-encoded precursor proteins. Most of these precursors use N-terminal presequences as targeting signals that are removed by mitochondrial matrix proteases. The essential mitochondrial processing protease MPP cleaves presequences after import into the organelle thereby enabling protein folding and functionality. The cleaved presequences are subsequently degraded by peptidases. While most of these processes have been discovered in yeast, characterization of the human enzymes is still scarce. As the matrix presequence peptidase PreP has been reported to play a role in Alzheimer's disease, analysis of impaired peptide turnover in human cells is of huge interest. Here, we report the characterization of HEK293T PreP knockout cells. Loss of PreP causes severe defects in oxidative phosphorylation and changes in nuclear expression of stress response marker genes. The mitochondrial defects upon lack of PreP result from the accumulation of presequence peptides that trigger feedback inhibition of MPP and accumulation of nonprocessed precursor proteins. Also, the mitochondrial intermediate peptidase MIP that cleaves eight residues from a subset of precursors after MPP processing is compromised upon loss of PreP suggesting that PreP also degrades MIP generated octapeptides. Investigation of the PrePR183Q patient mutation associated with neurological disorders revealed that the mutation destabilizes the protein making it susceptible to enhanced degradation and aggregation upon heat shock. Taken together, our data reveal a functional coupling between precursor processing by MPP and MIP and presequence degradation by PreP in human mitochondria that is crucial to maintain a functional organellar proteome.

Imipridone Anticancer Compounds Ectopically Activate the ClpP Protease and Represent a New Scaffold for Antibiotic Development.

Systematic genetic interaction profiles can reveal the mechanisms-of-action of bioactive compounds. The imipridone ONC201, which is currently in cancer clinical trials, has been ascribed a variety of different targets. To investigate the genetic dependencies of imipridone action, we screened a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) knockout library in the presence of either ONC201 or its more potent analog ONC212. Loss of the mitochondrial matrix protease CLPP or the mitochondrial intermediate peptidase MIPEP conferred strong resistance to both compounds. Biochemical and surrogate genetic assays showed that impridones directly activate CLPP and that MIPEP is necessary for proteolytic maturation of CLPP into a catalytically competent form. Quantitative proteomic analysis of cells treated with ONC212 revealed degradation of many mitochondrial as well as nonmitochondrial proteins. Prompted by the conservation of ClpP from bacteria to humans, we found that the imipridones also activate ClpP from Escherichia coli, Bacillus subtilis, and Staphylococcus aureus in biochemical and genetic assays. ONC212 and acyldepsipeptide-4 (ADEP4), a known activator of bacterial ClpP, caused similar proteome-wide degradation profiles in S. aureus ONC212 suppressed the proliferation of a number of Gram-positive (S. aureus, B. subtilis, and Enterococcus faecium) and Gram-negative species (E. coli and Neisseria gonorrhoeae). Moreover, ONC212 enhanced the ability of rifampin to eradicate antibiotic-tolerant S. aureus persister cells. These results reveal the genetic dependencies of imipridone action in human cells and identify the imipridone scaffold as a new entry point for antibiotic development.

Metabolic Alteration in Aging Process: Metabolic Remodeling in White Adipose Tissue by Caloric Restriction

Caloric restriction (CR) improves whole-body metabolism, suppresses various age-related pathophysiological changes, and extends lifespan. The beneficial actions of CR are regulated in growth hormone (GH)/insulin-like growth factor-1 (IGF-1) signal-dependent and -independent manners. To clarify the GH/IGF-1-independent mechanism, we compared gene expression profiles in white adipose tissue (WAT) between CR and GH/IGF-1 suppression, and found that CR upregulated sterol regulatory element-binding protein 1c (SREBP-1c) regulatory gene expression. To validate the impact of SREBP-1c as a beneficial mediator of CR, we compared the responses to CR between wild-type and SREBP-1c knockout (KO) mice. CR extended lifespan, upregulated gene expression involved in FA biosynthesis, activated mitochondrial biogenesis, and suppressed oxidative stress predominantly in WAT. In contrast, most of these findings were not observed in KO mice. Furthermore, SREBP-1c was implicated in CR-associated mitochondrial activation through upregulation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis. Sirtuin-3 (SIRT3) regulates mitochondrial quality and is also involved in the beneficial actions of CR. We observed that CR upregulated the mature form of SIRT3 protein and mitochondrial intermediate peptidase (MIPEP), a mitochondrial signal peptidase (MtSPase), in WAT. MIPEP cleaved precursor form of SIRT3 to mature form, and activated certain mitochondrial matrix proteins, suggesting that MIPEP might contribute to maintenance of mitochondrial quality during CR via SIRT3 activation. Taken together, CR induces SREBP-1c-dependent metabolic remodeling, including enhancement of FA biosynthesis and mitochondrial activation, via PGC-1α, and improvement of mitochondria quality via Mipep in WAT, resulting in beneficial actions.

Race-specific alterations in DNA methylation among middle-aged African Americans and Whites with metabolic syndrome

ABSTRACTMetabolic syndrome (MetS) is a cluster of cardiometabolic risk factors for all-cause mortality, cardiovascular disease, and cancer. Identifying epigenetic alterations associated with MetS in African Americans (AAs) and Whites may provide insight into genes that influence its differential health outcomes. We examined DNA methylation (DNAm) and performed an epigenome-wide association study (EWAS) of MetS among AAs and Whites with and without MetS. We assessed age, race and poverty status associated DNAm among AAs (n = 225) and White (n = 233) adults using NCEP-ATP III guidelines. Genome-wide DNAm measurement was assessed using Illumina Infinium Methylation EPIC BeadChip. Differentially methylated positions (DMPs) and differentially methylated regions (DMRs) were identified using dmpFinder and bumphunter. EWAS was performed using CpGassoc. We found significant DMPs associated with age, poverty status and MetS in each race. GSTT1(Glutathione S-Transferase Theta 1) was one of the top-hypermethylated genes and MIPEP (Mitochondrial Intermediate Peptidase) was one of the most hypomethylated genes when comparing AAs with and without MetS. PPP1R13L (Protein Phosphatase 1 Regulatory Subunit 13 Like) was the top hypermethylated and SCD (stearoyl-CoA desaturase-1) was one of the most hypomethylated genes for Whites with and without MetS. EWAS results showed that DNAm differences might contribute to MetS risk among Whites and AAs since different genes were identified in AAs and Whites. We replicated previously identified MetS associated genes and found that Thioredoxin-interacting protein (TXN1P) was statistically significantly differentially expressed only in Whites. Our results may be useful in further studies of genes underlying differences in MetS among AAs and Whites.

A Unique SUMO-Interacting Motif of Trx2 Is Critical for Its Mitochondrial Presequence Processing and Anti-oxidant Activity.

ObjectiveMitochondrial thioredoxin 2 (Trx2) is a vital mitochondrial redox protein that mediates normal protein thiol reduction and provides electrons to peroxiredoxin 3 (Prx3) to scavenge H2O2 in mitochondria. It has been widely reported that Trx2 deletion in cells or mice generates massive reactive oxygen species (ROS) which have been implicated in many pathological processes. On the contrary, how ROS regulate Trx2 processing and activity remains to be elucidated.Approach and ResultsHere we show that excess ROS induce endothelial cell senescence concomitant with an attenuation of Trx2 processing in which Trx2 presequence [i.e., mitochondrial targeting signal peptide (MTS)] is cleaved to generate a mature form. Mutation analyses indicate that Trx2 processing is mediated by mitochondrial processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP)-recognition sites within the MTS. Interestingly, a mutation at a SUMO- interacting motif (SIM), but not the catalytic sites within the mature Trx2 protein, completely blocks Trx2 processing with no effect on Trx2 mitochondrial targeting. Consistently, chemical inhibition of protein SUMOylation attenuates, while SUMOylation agonist promotes, Trx2 processing. Moreover, we identify the α–MPP subunit is a SUMOylated protein that potentially mediates Trx2-binding and cleavage. Furthermore, the unprocessed form of Trx2-SIM is unable to protect cells from both ROS generation and oxidative stress-induced cellular senescence.ConclusionOur study reveals that a unique SUMO-interacting motif of Trx2 is critical for its mitochondrial processing and subsequent anti-oxidant/antisenescence activities.

Offspring analysis using two cleaved amplified polymorphic sequence (CAPS) markers reveals amphithallism in the edible mushroom Agaricus sinodeliciosus

ABSTRACTAgaricus sinodeliciosus is an edible wild mushroom known in northwest China. It belongs to Agaricus section Bivelares that includes several popular cultivated species, such as A. bisporus, the button mushroom. The life cycle of the latter species has been described as amphithallic because both homokaryotic (n) and heterokaryotic (n+n) spores are produced that lead to heterothallic and pseudohomothallic life cycles, respectively. The type of life cycle can impact population structures and breeding strategies. The main objective of this study was to identify the different categories of spores produced by A. sinodeliciosus. Using either a morphological approach based on the number of sterigmata per basidium or a genetic approach based on the genotypes of the progeny at two loci, the proportion of heterokaryotic spores was estimated at 6% and 15%, respectively. Two codominant markers were chosen from the mitochondrial intermediate peptidase gene (MIP) and the nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS) region. Genotypic analysis and mating tests confirmed that in A. sinodeliciosus, MIP is centromere-linked and tightly linked to the mating type locus as in A. bisporus and A. subrufescens. We conclude that A. sinodeliciosus has a unifactorial system of sexual incompatibility and an amphithallic life cycle that is predominantly heterothallic, and that its pseudohomothallism follows a nonrandom model with nonsister postmeiotic nuclei paired in the same spore, which give rise to a potentially fertile heterokaryon. This method of using two informative markers is reliable not only in selecting the homokaryotic offspring but also in classifying the homokaryons in two breeding stocks according to their mating type alleles.

Genome sequence of the cauliflower mushroom Sparassis crispa (Hanabiratake) and its association with beneficial usage

Sparassis crispa (Hanabiratake) is a widely used medicinal mushroom in traditional Chinese medicine because it contains materials with pharmacological activity. Here, we report its 39.0-Mb genome, encoding 13,157 predicted genes, obtained using next-generation sequencing along with RNA-seq mapping data. A phylogenetic analysis by comparison with 25 other fungal genomes revealed that S. crispa diverged from Postia placenta, a brown-rot fungus, 94 million years ago. Several features specific to the genome were found, including the A-mating type locus with the predicted genes for HD1 and HD2 heterodomain transcription factors, the mitochondrial intermediate peptidase (MIP), and the B-mating type locus with seven potential pheromone receptor genes and three potential pheromone precursor genes. To evaluate the benefits of the extract and chemicals from S. crispa, we adopted two approaches: (1) characterization of carbohydrate-active enzyme (CAZyme) genes and β-glucan synthase genes and the clusters of genes for the synthesis of second metabolites, such as terpenes, indoles and polyketides, and (2) identification of estrogenic activity in its mycelial extract. Two potential β-glucan synthase genes, ScrFKS1 and ScrFKS2, corresponding to types I and II, respectively, characteristic of Agaricomycetes mushrooms, were newly identified by the search for regions homologous to the reported features of β-glucan synthase genes; both contained the characteristic transmembrane regions and the regions homologous to the catalytic domain of the yeast β-glucan synthase gene FKS1. Rapid estrogenic cell-signaling and DNA microarray-based transcriptome analyses revealed the presence of a new category of chemicals with estrogenic activity, silent estrogens, in the extract. The elucidation of the S. crispa genome and its genes will expand the potential of this organism for medicinal and pharmacological purposes.

Trypanosomal mitochondrial intermediate peptidase does not behave as a classical mitochondrial processing peptidase.

Upon their translocation into the mitochondrial matrix, the N-terminal pre-sequence of nuclear-encoded proteins undergoes cleavage by mitochondrial processing peptidases. Some proteins require more than a single processing step, which involves several peptidases. Down-regulation of the putative Trypanosoma brucei mitochondrial intermediate peptidase (MIP) homolog by RNAi renders the cells unable to grow after 48 hours of induction. Ablation of MIP results in the accumulation of the precursor of the trypanosomatid-specific trCOIV protein, the largest nuclear-encoded subunit of the cytochrome c oxidase complex in this flagellate. However, the trCOIV precursor of the same size accumulates also in trypanosomes in which either alpha or beta subunits of the mitochondrial processing peptidase (MPP) have been depleted. Using a chimeric protein that consists of the N-terminal sequence of a putative subunit of respiratory complex I fused to a yellow fluorescent protein, we assessed the accumulation of the precursor protein in trypanosomes, in which RNAi was induced against the alpha or beta subunits of MPP or MIP. The observed accumulation of precursors indicates MIP depletion affects the activity of the cannonical MPP, or at least one of its subunits.

Substrate specificity of mitochondrial intermediate peptidase analysed by a support-bound peptide library

The substrate specificity of recombinant human mitochondrial intermediate peptidase (hMIP) using a synthetic support-bound FRET peptide library is presented. The collected fluorescent beads, which contained the hydrolysed peptides generated by hMIP, were sequenced by Edman degradation. The results showed that this peptidase presents a remarkable preference for polar uncharged residues at P1 and P1′ substrate positions: Ser=Gln>Thr at P1 and Ser>Thr at P1′. Non-polar residues were frequent at the substrate P3, P2, P2′ and P3′ positions. Analysis of the predicted MIP processing sites in imported mitochondrial matrix proteins shows these cleavages indeed occur between polar uncharged residues. Previous analysis of these processing sites indicated the importance of positions far from the MIP cleavage site, namely the presence of a hydrophobic residue (Phe or Leu) at P8 and a polar uncharged residue (Ser or Thr) at P5. To evaluate this, additional kinetic analyses were carried out, using fluorogenic substrates synthesized based on the processing sites attributed to MIP. The results described here underscore the importance of the P1 and P1′ substrate positions for the hydrolytic activity of hMIP. The information presented in this work will help in the design of new substrate-based inhibitors for this peptidase.

The genetic structure of the A mating-type locus of Lentinula edodes

The Shiitake mushroom, Lentinula edodes (Berk.) Pegler is a tetrapolar basidiomycete with two unlinked mating-type loci, commonly called the A and B loci. Identifying the mating-types in shiitake is important for enhancing the breeding and cultivation of this economically-important edible mushroom. Here, we identified the A mating-type locus from the first draft genome sequence of L. edodes and characterized multiple alleles from different monokaryotic strains. Two intron-length polymorphism markers were developed to facilitate rapid molecular determination of A mating-type. L. edodes sequences were compared with those of known tetrapolar and bipolar basidiomycete species. The A mating-type genes are conserved at the homeodomain region across the order Agaricales. However, we observed unique genomic organization of the locus in L. edodes which exhibits atypical gene order and multiple repetitive elements around its A locus. To our knowledge, this is the first known exception among Homobasidiomycetes, in which the mitochondrial intermediate peptidase (mip) gene is not closely linked to A locus.

Polyporales genomes reveal the genetic architecture underlying tetrapolar and bipolar mating systems

The process of mating in Basidiomycota is regulated by homeodomain-encoding genes (HD) and pheromones and G protein-coupled pheromone receptor genes (P/R). Whether these genes are actually involved in determining mating type distinguishes mating systems that are considered tetrapolar (two locus) from bipolar (one locus). Polyporales are a diverse group of wood-decay basidiomycetes displaying high variability in mating and decay systems. Many of the bipolar species appear to be brown-rot fungi, and it has been hypothesized that there is a functional basis for this correlation. Here we characterize mating genes in recently sequenced Polyporales and other Agaricomycete genomes. All Agaricomycete genomes encode HD and pheromone receptor genes regardless of whether they are bipolar or tetrapolar. The HD genes are organized into a MAT-HD locus with a high degree of gene order conservation among neighboring genes, with the gene encoding mitochondrial intermediate peptidase consistently syntenic but no linkage to the P/R genes. To have a complete dataset of species with known mating systems we determined that Wolfiporia cocos appears to be bipolar, using the criterion that DNA polymorphism of MAT genes should be extreme. Testing the correlation of mating and decay systems while controlling for phylogenetic relatedness failed to identify a statistical association, likely due to the small number of taxa employed. Using a phylogenetic analysis of Ste3 proteins, we identified clades of sequences that contain no known mating type-specific receptors and therefore might have evolved novel functions. The data are consistent with multiple origins of bipolarity within the Agaricomycetes and Polyporales, although the alternative hypothesis that tetrapolarity and bipolarity are reversible states needs better testing.

Extensive Trans-Specific Polymorphism at the Mating Type Locus of the Root Decay Fungus Heterobasidion

Incompatibility systems in which individuals bearing identical alleles reject each other favor the maintenance of a diversity of alleles. Mushroom mating type loci (MAT) encode for dozens or hundreds of incompatibility alleles whose loss from the population is greatly restricted through negative frequency selection, leading to a system of alleles with highly divergent sequences. Here, we use DNA sequences of homeodomain (HD) encoding genes at the MAT locus of five closely related species of the root rot basidiomycete Heterobasidion annosum sensu lato to show that the extended coalescence time of MAT alleles greatly predates speciation in the group, contrasting loci outside of MAT that show allele divergences largely consistent with the species phylogeny with those of MAT, which show rampant trans-species polymorphism. We observe a roughly 6-fold greater genealogical depth and polymorphism of MAT compared with non-MAT that argues for the maintenance of balanced polymorphism for a minimum duration of 24 My based on a molecular-clock calibrated species phylogeny. As with other basidiomycete HD genes, balancing selection appears to be concentrated at the specificity-determining region in the N-terminus of the protein based on identification of codons under selection and the absence of recombination within the region. However, the elevated polymorphism extends into the nonspecificity determining regions as well as a neighboring non-MAT gene, the mitochondrial intermediate peptidase (MIP). In doing so, increased divergence should decrease recombination among alleles and as a by-product create incompatibilities in the functional domains not involved in allele recognition but in regulating sexual development.

Sequence and comparative analysis of theMIPgene in Chinese straw mushroom,Volvariella volvacea

The mitochondrial intermediate peptidase (MIP) gene is conserved in fungi. It is linked closely with the mating-type A (mtA) gene. In this study, a fragment of the MIP gene in Volvariella volvacea (Bull. ex Fr.) Singer was first cloned by homologue-based cloning technology. Subsequently, the entire MIP DNA sequence (PYd21-MIP) was obtained after the fragment was compared with the genomic data through BLAST analysis. The PYd21-MIP sequence appeared to be homologous with the MIP gene in other fungi. Phylogenetic analysis of PYd21-MIP and other MIP sequences from diverse fungi agreed with the current organism phylogeny. Analysis of protein domains by InterProScan software and motif searching demonstrated that PYd21-MIP encodes a homologous MIP protein. These data support the hypothesis that the PYd21-MIP protein is a Hog-MIP protein homologue from V. volvacea.

γ-Secretase-regulated Proteolysis of the Notch Receptor by Mitochondrial Intermediate Peptidase

Notch is a transmembrane receptor that controls a diverse array of cellular processes including cell proliferation, differentiation, survival, and migration. The cellular outcome of Notch signaling is dependent on extracellular and intracellular signals, but the complexities of its regulation are not well understood. Canonical Notch signaling involves ligand association that triggers sequential and regulated proteolysis of Notch at several sites. Ligand-dependent proteolysis at the S2 site removes the bulk of the extracellular domain of Notch. Subsequent γ-secretase-mediated intramembrane proteolysis of the remaining membrane-tethered Notch fragment at the S3 site produces a nuclear-destined Notch intracellular domain (NICD). Here we show that following γ-secretase cleavage, Notch is proteolyzed at a novel S5 site. We have identified this S5 site to be eight amino acids downstream of the S3 site. Biochemical fractionation and purification resulted in the identification of the S5 site protease as the mitochondrial intermediate peptidase (MIPEP). Expression of the MIPEP-cleaved NICD (ΔNICD) results in a decrease in cell viability and mitochondria membrane potential. The sequential and regulated proteolysis by γ-secretase and MIPEP suggests a new means by which Notch function can be modulated.

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.