No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Sequence analysis of rat mitochondrial intermediate peptidase: Similarity to zinc metallopeptidases and to a putative yeast homologue
Abstract
Proteolytic removal of amino-terminal octapeptides from mitochondrial intermediate proteins is a required step for a subgroup of nuclear-encoded mitochondrial precursors and is specifically catalyzed by mitochondrial intermediate peptidase (MIP). We recently reported the purification of MIP from rat liver and showed that the enzyme is a monomer of 75 kDa. We now report the sequence of a full-length rat MIP cDNA. This cDNA codes for a protein of 710 amino acids, including an amino-terminal mitochondrial leader peptide of 33 residues. The region surrounding the mature MIP amino terminus shows a cleavage site typically recognized by the general mitochondrial processing peptidase (MPP). In vitro synthesized MIP precursor is cleaved to mature MIP by purified MPP, and thus MIP is not required for its own proteolytic maturation. Comparison of the deduced MIP sequence with other sequences in the GenBank data base reveals two important similarities. The first is to a sequence encoding a putative MIP homologue in the recently reported sequence of yeast chromosome III. The putative yeast protein is predicted to be 712 amino acids long and includes a putative 23-residue mitochondrial leader peptide also with a MPP processing site. It shows 47% similarity and 24% identity to rat MIP. The second similarity is to members of a subfamily of metallopeptidases that includes rat metalloendopeptidase EC 3.4.24.15 and two bacterial proteases, oligopeptidase A and dipeptidyl carboxypeptidase. A region of greater than 50% similarity over 400 residues between MIP and these proteins is centered around the sequence motif HEXXH, typical of zinc metallopeptidases.
... Chapter 323) was probably responsible for both cleavages, studies of rat liver mitochondria showed that the intermediate-processing activity had requirements different from those exhibited by MPP [6]. Subsequently, MIP was purified to homogeneity from rat liver mitochondrial matrix [7], and a full-length RMIP cDNA was isolated [8]. s0020 Activity and Specificity p0065 MIP is involved in the two-step processing of mitochondrial protein precursors which contain the motif Arg-Xaa-(Phe/Leu/Ile)-Xaa-Xaa-(Ser/Thr/Gly)-(Xaa) 4 k at the C-terminus of their leader peptide. ...
... 95% purity [19]. s0035 Biological Properties p0115 MIP is encoded in the nucleus and initially synthesized in the cytoplasm as a large precursor molecule carrying a mitochondrial leader peptide; upon import into the mitochondrion, the precursor is processed to the mature form by MPP [8]. In both rat and yeast mitochondria, the active enzyme is a soluble monomer localized to the matrix compartment. ...
... to both the cytoplasm and the mitochondria with 24% identity and 47% similarity to YMIP. Although the PRD1 gene product had initially been proposed as the yeast ortholog of mammalian MIP [8], a prd1 knockout mutant showed normal mitochondrial function [31] and normal processing of octapeptide-containing precursors to the mature form [21]. As a double prd1Àmip1 mutant did not present a more severe phenotype than a single mip1 mutant, it was concluded that YMIP and yscD do not have overlapping biologic roles. ...
This chapter examines the structural chemistry and the preparation of mitochondrial intermediate peptidase (MIP). MIP denotes cleavage of intermediate-sized mitochondrial proteins to the mature form. MIP activity can be measured by incubation of in vitro translated octapeptide-containing precursors with isolated mitochondria. Under such conditions, the cleavage catalyzed by MIP is at the end of a mitochondrial protein import reaction which also involves outer membrane receptors, outer and inner membrane translocation complexes, molecular chaperones and MPP. If the precursor is incubated directly with mitochondrial matrix or purified enzyme, initial cleavage by MPP is required to observe processing of the intermediate by MIP. This requirement is circumvented when MIP activity is determined using an intermediate protein as the substrate. Intermediates that are translated in vitro from a methionine artificially placed at the octapeptide N-terminus can be processed to the mature form by MIP independent of the presence of MPP. Native RMIP has been purified 2250-fold from rat liver mitochondrial matrix with a final yield of about 2%. Expression of recombinant enzyme has been achieved in S. cerevisiae.
... The resulting intermediate-size proteins are then processed to mature subunits by MIP, which specifically cleaves off the octapeptides (25). MIP has been purified to homogeneity from rat liver mitochondrial matrix (28), and a full-length cDNA has been isolated (26). Sequence analysis revealed that rat MIP (RMIP) is structurally related to a putative metallopeptidase predicted from the sequence of gene YCL57w of yeast chromosome III (34). ...
... Sequence analysis revealed that rat MIP (RMIP) is structurally related to a putative metallopeptidase predicted from the sequence of gene YCL57w of yeast chromosome III (34). Because a typical mitochondrial targeting signal was found at the amino terminus of this protein sequence, we proposed that YCL57w might encode a yeast homolog of RMIP (26). ...
... Cloning of MIPI by library screening. A K DASH yeast genomic library (Stratagene) was screened by use of a random primer-labeled (Boehringer Mannheim) full-length RMIP cDNA probe (26). Hybridization of Hybond-N nylon membrane filters (Amersham Corp.) was carried out in 6x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate, pH 7.0)-5x Denhardt's solution (lx Denhardt's solution is 0.02% polyvinylpyrrolidone-0.02% Ficoll-0.02% ...
Cleavage of amino-terminal octapeptides, F/L/IXXS/T/GXXXX, by mitochondrial intermediate peptidase (MIP) is typical of many mitochondrial precursor proteins imported to the matrix and the inner membrane. We previously described the molecular characterization of rat liver MIP (RMIP) and indicated a putative homolog in the sequence predicted from gene YCL57w of yeast chromosome III. A new yeast gene, MIP1, has now been isolated by screening a Saccharomyces cerevisiae genomic library with an RMIP cDNA probe. MIP1 predicts a protein of 772 amino acids (YMIP), which is 54% similar and 31% identical to RMIP and includes a putative 37-residue mitochondrial leader peptide. RMIP and YMIP contain the sequence LFHEMGHAM HSMLGRT, which includes a zinc-binding motif, HEXXH, while the predicted YCL57w protein contains a comparable sequence with a lower degree of homology. No obvious biochemical phenotype was observed in a chromosomally disrupted ycl57w mutant. In contrast, a mip1 mutant was unable to grow on nonfermentable substrates, while a mip1 ycl57w double disruption did not result in a more severe phenotype. The mip1 mutant exhibited defects of complexes III and IV of the respiratory chain, caused by failure to carry out the second MIP-catalyzed cleavage of the nuclear-encoded precursors for cytochrome oxidase subunit IV (CoxIV) and the iron-sulfur protein (Fe-S) of the bc1 complex to mature proteins. In vivo, intermediate-size CoxIV was accumulated in the mitochondrial matrix, while intermediate-size Fe-S was targeted to the inner membrane. Moreover, mip1 mitochondrial fractions failed to carry out maturation of the human ornithine transcarbamylase intermediate (iOTC), specifically cleaved by RMIP. A CEN plasmid-encoded YMIP protein restored normal MIP activity along with respiratory competence. Thus, YMIP is a functional homolog of RMIP and represents a new component of the yeast mitochondrial import machinery.
... In addition the sequence of proteinase yscD has significant similarity with the amino acid sequence of mammalian thimet oligopeptidase (EP 24.15 ; Pierotti et al., 1990;McKie et al., 1993). In addition, similarities with other peptidases, such as rat mitochondria1 intermediate peptidase (Isaya et al., 1992), oligopeptidase A and dipeptidyl carboxypeptidase (Hamilton and Miller, 1992) from Salmonella typhimurium were found. In Fig. 3 we aligned the sequences of yeast proteinase yscD with the corrected amino acid sequence of mammalian thimet oligopeptidase (EP 24.15;Pierotti et al., 1990, as corrected by McKie et al., 1993 and with a member of the bacterial enzymes, oligopeptidase A. Proteinase yscD shows 34.8% identity and 55.0% similarity (Dayhoff et al., 1978) with the sequence of EP 24.15. ...
... In this region we find the HEXXH motif containing the two zinc-binding histidine residues. This motif is also found in rat mitochondria1 intermediate peptidase (Isaya et al., 1992), Salmonella dipeptidyl carboxypeptidase (Hamilton and Miller, 1992) and the putative E. coli prlc gene product . Based on X-ray diffraction analysis of thermolysin (Matthews et al., 1972) and Bacillus cereus neutral protease a glutamic acid residue has been identified as third ligand of the zinc-binding domain in the active site of metallopeptidases. ...
... References: EP 24.15 (Pierotti et al., 1990;McKie et al., 1993), OPDA , DCP (Hamilton and Miller, 1992). MIP (Isaya et al., 1992). cal level. ...
The yeast PRD1 gene, encoding proteinase yscD, was cloned by complementation of the prd1-6 point mutation. Sequencing of the gene revealed an open reading frame of 2.136 kb, encoding a protein of 712 amino acids with a calculated molecular mass of 81.8 kDa. The sequence HEGLG beginning at residue 501 represents the HEXXH motif, unique for the zinc metallo-peptidases. Sequence comparison revealed complete identity of the proteinase yscD gene with a recently published open reading frame of yeast chromosome III. We found 34.8% identity between proteinase yscD and rat metalloendopeptidase (thimet oligopeptidase, EP 24.15). Proteinase yscD hydrolyzes several chromogenic and fluorogenic peptides that are substrates of thimet oligopeptidase. N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoic acid, a compound designed as specific inhibitor of EP 24.15, is also a strong inhibitor of the yeast enzyme. Proteinase yscD is a nonvacuolar enzyme. 3-5% of the total enzyme activity can be detected in the intermembrane space of mitochondria. In a mutant carrying a deletion of the PRD1 gene no proteinase yscD activity is detectable in the cytoplasm and in mitochondria of these cells. They do not show any grossly altered phenotype but exhibit a decrease in the intracellular degradation of peptides. This suggests a function of proteinase yscD in the late stages of protein degradation.
... Currently, there are two known matrix proteases that exclusively cleave mitochondrial presequences after an initial cleavage by MPP. One of these proteases (MIP) has been known for about 20 years [14,[73][74][75][76][77][78][79], and shown to cleave an octapeptide from the MPP generated N-terminus in the substrate proteins, reason that motivated the current designation of Octapeptidyl aminopeptidase 1 (Oct1). Oct1 (MEROPS M3 family) is a soluble mitochondrial matrix protein containing a conserved Zn-binding motif (HEXXH) required for activity [76,77]. ...
... Following an analysis of determined N-termini from both yeast and mammalian mitochondrial proteins, most substrates of Oct1 were shown to contain R in the -10 position from the mature protein (MPP recognition site), a bulky hydrophobic residue (F, L or I) at the -8 position and S/T at positions -5, -6 and -7 [74,79,80]. An interesting exception to this consensus sequence is the uncharacterized yeast protein Imo32, which contains a -10C residue, implying that in this case a -10R is not required for MPP cleavage [79]. ...
Most of the mitochondrial and chloroplastic proteins are nuclear encoded and synthesized in the cytosol as precursor proteins with N-terminal extensions called targeting peptides. Targeting peptides function as organellar import signals, they are recognized by the import receptors and route precursors through the protein translocons across the organellar membranes. After the fulfilled function, targeting peptides are proteolytically cleaved off inside the organelles by different processing peptidases. The processing of mitochondrial precursors is catalyzed in the matrix by the Mitochondrial Processing Peptidase, MPP, the Mitochondrial Intermediate Peptidase, MIP (recently called Octapeptidyl aminopeptidase 1, Oct1) and the Intermediate cleaving peptidase of 55kDa, Icp55. Furthermore, different inner membrane peptidases (Inner Membrane Proteases, IMPs, Atp23, rhomboids and AAA proteases) catalyze additional processing functions, resulting in intra-mitochondrial sorting of proteins, the targeting to the intermembrane space or in the assembly of proteins into inner membrane complexes. Chloroplast targeting peptides are cleaved off in the stroma by the Stromal Processing Peptidase, SPP. If the protein is further translocated to the thylakoid lumen, an additional thylakoid-transfer sequence is removed by the Thylakoidal Processing Peptidase, TPP. Proper function of the D1 protein of Photosystem II reaction center requires its C-terminal processing by Carboxy-terminal processing protease, CtpA. Both in mitochondria and in chloroplasts, the cleaved targeting peptides are finally degraded by the Presequence Protease, PreP. The organellar proteases involved in precursor processing and targeting peptide degradation constitute themselves a quality control system ensuring the correct maturation and localization of proteins as well as assembly of protein complexes, contributing to sustenance of organelle functions. Dysfunctions of several mitochondrial processing proteases have been shown to be associated with human diseases. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.
... MPP usually consists of two subunits, alpha and beta, encoded by two separated genes (22). Oct1 and Icp55 are secondary processing peptidases functioning to cleave an octapeptide and one amino acid, respectively, from intermediates generated by MPP (23,24). In addition to proteases responsible for peptide processing, other proteases with chaperone-like properties in mitochondria, such as Pim1/LON and the mtClpXP complex, function to remove damaged proteins and thus help maintain mitochondrial protein homeostasis (25). ...
Significance
Stable expression of each component of the nitrogenase system in an active form is a prerequisite for engineering nitrogen fixation in eukaryotic cells. Mitochondria provide an oxygen-depleted environment for the expression of active nitrogenase in plants, but signal peptides are required to target nuclear encoded Nif proteins to this organelle. We demonstrate that one of the structural subunits of nitrogenase, NifD, is itself susceptible to cleavage by mitochondrial processing peptidases from a variety of plant origins, presenting a major challenge to engineering nitrogen fixation in mitochondria. To overcome this issue, we have engineered NifD variants that are resistant to cleavage and retain high levels of nitrogenase activity, thus providing a potential solution for engineering active MoFe protein in plants.
... MIP was identified in rats (rMIP) [5,7], humans (hMIP) [8], Saccharomyces cerevisiae (yMIP) [9] and Schizophillum commune (sMIP) [10]. In all cases, it was found as a soluble monomer of about 75 kDa with the canonical zinc ion ligand motif, HEXXH. ...
In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, and enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.
... As has been reviewed extensively elsewhere (Hartl and Neupert, 1990 ). nuclear-coded mitochondrial proteins are in most cases divested of their targeting signals by the action of the highly specific matrix and inner-membrane proteases (MPP, M P , IMP) during or shortly after impoh MPP, or more rarely MIp* (Isaya et al., 1992; Isaya et al., 1994). of the bipartite addressing signal of proteins destined for the intermembrane space. The MPP protease is a two-subunit enzyme, consisting of the catalytic subunit (encoded by the MAS2 gene) and a second protein that enhances activity (protease-enhancing protein, PEP, product of the MAS1 gene). ...
All proteins encoded by mitochondrial DNA (mtDNA) are dependent on proteins encoded by nuclear genes for their synthesis and function. Recent developments in the identification of these genes and the elucidation of the roles their products play at various stages of mitochondrial gene expression are covered in this review, which focuses mainly on work with the yeast Saccharomyces cerevisiae. The high degree of evolutionary conservation of many cellular processes between this yeast and higher eukaryotes, the ease with which mitochondrial biogenesis can be manipulated both genetically and physiologically, and the fact that it will be the first organism for which a complete genomic sequence will be available within the next 2 to 3 years makes it the organism of choice for drawing up an inventory of all nuclear genes involved in mitochondrial biogenesis and for the identification of their counterparts in other organisms.
... This change affects the deduced sequence for amino acids 577 to the C-terminus (Figure 1)The deduced amino acid sequence of (a) pig liver soluble angiotensirt 11-binding protein (Sugiura et al., 1992) is aligned with (b) that previously reported for rat testis thimet oligopeptidase (Pierotti et al., 1990) and (c) the revised sequence of rat thimet oligopeptidase described here. The sequences of other members of the family are (d) saccharolysin (EC 3.4.24.37, described as open reading frame YCL57w) (Oliver et al., 1992), (e) rat mitochondrial intermediate peptidase (Isaya et al., 1992), (f) Schizophyllum commune putative metalloendopeptidase (Giasson et at., 1989), (g) Escherichia co/ioligopeptidase A (ConUn et al., 1992), (h) Salmonella typhimuriumoligopeptidase A (), (i) E colidipeptidyl carboxypeptidase (S. Becker and R. Plapp, Swissprot database entry DCP_ECOLI), and (j) S typhimurium dipeptidyl carboxypeptidase (Hamilton and Miller, 1992). ...
The deduced amino acid sequence of pig liver soluble angiotensin II-binding protein [Sugiura, Hagiwara and Hirose (1992) J. Biol. Chem. 267, 18067-18072] is similar over most of its length to that reported for rat testis thimet oligopeptidase (EC 3.4.24.15) by Pierotti, Dong, Glucksman, Orlowski and Roberts [(1990) (Biochemistry 29, 10323-10329]. We have found that homogeneous rat testis thimet oligopeptidase binds angiotensin II with the same distinctive characteristics as the pig liver protein. Analysis of the nucleotide sequences reported for the two proteins pointed to the likelihood that sequencing errors had caused two segments of the amino acid sequence of the rat protein to be translated out of frame, and re-sequencing of selected parts of the clone (kindly provided by the previous authors) confirmed this. The revised deduced amino acid sequence of rat thimet oligopeptidase contains 687 residues, representing a protein of 78,308 Da, and is more closely related to those of the pig liver protein and other known homologues of thimet oligopeptidase than that described previously.
Limited proteolysis, called protein processing, is an essential post-translational mechanism that controls protein localization, activity, and in consequence, function. This process is prevalent for mitochondrial proteins, mainly synthesized as precursor proteins with N-terminal sequences (presequences) that act as targeting signals and are removed upon import into the organelle. Mitochondria have a distinct and highly conserved proteolytic system that includes proteases with sole function in presequence processing and proteases, which show diverse mitochondrial functions with limited proteolysis as an additional one. In virtually all mitochondria, the primary processing of N-terminal signals is catalyzed by the well-characterized mitochondrial processing peptidase (MPP). Subsequently, a second proteolytic cleavage occurs, leading to more stabilized residues at the newly formed N-terminus. Lately, mitochondrial proteases, intermediate cleavage peptidase 55 (ICP55) and octapeptidyl protease 1 (OCT1), involved in proteolytic cleavage after MPP and their substrates have been described in the plant, yeast, and mammalian mitochondria. Mitochondrial proteins can also be processed by removing a peptide from their N- or C-terminus as a maturation step during insertion into the membrane or as a regulatory mechanism in maintaining their function. This type of limited proteolysis is characteristic for processing proteases, such as IMP and rhomboid proteases, or the general mitochondrial quality control proteases ATP23, m-AAA, i-AAA, and OMA1. Identification of processing protease substrates and defining their consensus cleavage motifs is now possible with the help of large-scale quantitative mass spectrometry-based N-terminomics, such as combined fractional diagonal chromatography (COFRADIC), charge-based fractional diagonal chromatography (ChaFRADIC), or terminal amine isotopic labeling of substrates (TAILS). This review summarizes the current knowledge on the characterization of mitochondrial processing peptidases and selected N-terminomics techniques used to uncover protease substrates in the plant, yeast, and mammalian mitochondria.
Cleavage of amino-terminal octapeptides, F/L/IXXS/T/GXXXX, by mitochondrial intermediate peptidase (MIP) is typical of many mitochondrial precursor proteins imported to the matrix and the inner membrane. We previously described the molecular characterization of rat liver MIP (RMIP) and indicated a putative homolog in the sequence predicted from gene YCL57w of yeast chromosome III. A new yeast gene, MIP1, has now been isolated by screening a Saccharomyces cerevisiae genomic library with an RMIP cDNA probe. MIP1 predicts a protein of 772 amino acids (YMIP), which is 54% similar and 31% identical to RMIP and includes a putative 37-residue mitochondrial leader peptide. RMIP and YMIP contain the sequence LFHEMGHAM HSMLGRT, which includes a zinc-binding motif, HEXXH, while the predicted YCL57w protein contains a comparable sequence with a lower degree of homology. No obvious biochemical phenotype was observed in a chromosomally disrupted ycl57w mutant. In contrast, a mip1 mutant was unable to grow on nonfermentable substrates, while a mip1 ycl57w double disruption did not result in a more severe phenotype. The mip1 mutant exhibited defects of complexes III and IV of the respiratory chain, caused by failure to carry out the second MIP-catalyzed cleavage of the nuclear-encoded precursors for cytochrome oxidase subunit IV (CoxIV) and the iron-sulfur protein (Fe-S) of the bc1 complex to mature proteins. In vivo, intermediate-size CoxIV was accumulated in the mitochondrial matrix, while intermediate-size Fe-S was targeted to the inner membrane. Moreover, mip1 mitochondrial fractions failed to carry out maturation of the human ornithine transcarbamylase intermediate (iOTC), specifically cleaved by RMIP. A CEN plasmid-encoded YMIP protein restored normal MIP activity along with respiratory competence. Thus, YMIP is a functional homolog of RMIP and represents a new component of the yeast mitochondrial import machinery.
The endosymbiotic origin of the mitochondrion and the subsequent transfer of its genome to the host nucleus has resulted in intricate mechanisms of regulating mitochondrial biogenesis and protein content. The majority of mitochondrial
proteins are nuclear encoded and synthesized in the cytosol, thus requiring specialized and dedicated machinery for the correct targeting import and sorting of its proteome. Most proteins targeted to the mitochondria utilize N-terminal targeting signals called presequences that are cleaved upon import. This cleavage is carried out by
a variety of peptidases, generating free peptides that can be detrimental to organellar and cellular activity. Research over the last few decades has elucidated a range of mitochondrial peptidases that are involved in the initial removal of the targeting signal and its sequential degradation, allowing for the recovery of single amino acids. The significance of these processing pathways goes beyond presequence degradation after protein import, whereby the deletion of processing peptidases induces plant stress responses, compromises mitochondrial respiratory capability, and alters overall plant growth and development. Here, we review the multitude of plant mitochondrial peptidases that are known to be involved in protein import and processing of targeting signals to detail how their activities can affect organellar protein homeostasis and overall plant growth.
In the past few years, information about primary and tertiary structures of proteins has revealed the enormous diversity and abundance of metalloproteases, and has led to a new understanding of the number and variety of distinct families that make up the general class of metalloproteases [1–4]. It has become clear that most, if not all, of the metalloproteases contain zinc at their active sites, and employ this metal in catalysis. The majority of the characterized metalloen-dopeptidases contain the zinc-binding consensus sequence HEXXH in a region of α-helical secondary structure; the two conserved histidines are ligands to the zinc ion via their imidazole side chains. The amino acids designated as X are uncharged and usually hydrophobic. An additional Zn ligand is supplied by a distant Glu side chain in a subgroup of metalloendopeptidas-es called gluzincins [4]. Examples of gluzincins are thermolysin (EC 3.4.24.27) and neprilysin (EC 3.4.24.11). For the metzincins, another subgroup of metalloendopeptidases, the third Zn ligand is contributed by the last histidine residue in the extended consensus sequence HEXXHXXGFXH. Examples of metzincins are astacin (EC 3 4.24.21) and interstitial collagenase (EC 3.4.24.7). A water molecule involved in catalysis is the fourth zinc ligand in these enzymes. Inverzincins, such as insulysin (EC 3.4.24.56) possess the inverted zinc binding sequence HXXEH, with a downstream Glu residue implicated as a third zinc ligand. Catalysis by zinc metallopeptidases does not involve intermediates covalently bound to protein groups, as in the serine or cysteine proteinases, but rather a zinc-bound water molecule attacks the substrate carbonyl carbon, with hydrolysis assisted by a general base. The general base in the protein, however, has not yet been unambiguously identified for many zinc metallopeptidases. It has been proposed that the conserved Glu residue of the HEXXH consensus sequence performs this function in thermolysin and other related metalloendopeptidases, but recent studies have provided evidence that the catalytic base in thermolysin may actually be a histidine residue, and a tyrosine side chain has been implicated as the general base for serralysins and astacins. A reevaluation of the catalytic mechanisms of the various classes of metalloendopeptidases may be necessary to resolve the controversy and advance our understanding of how they function.
This chapter discusses the proteolytic processing of mitochondrial precursor proteins. It is suggested that pattern of proteolytic maturation of imported mitochondrial proteins involves a hierarchy of cleavages by a limited number of mitochondrial peptidases. Most cleaved precursors, whether ultimately destined for the matrix, the inner membrane, or the intermembrane space, are acted on by mitochondrial processing peptidase (MPP) in its role as the general mitochondrial peptidase. A major subset of these, eventually localizing to either the matrix or the inner membrane, is cleaved specifically by mitochondrial intermediate peptidase only after MPP has exposed a suitable octapeptide at the amino-terminus of the intermediate. A few others, targeted to the intermembrane space (or that face of the inner membrane) by exposed sequences reminiscent of bacterial signal peptides, are cleaved by a localized protease, inner membrane peptidase, after the second targeting step is complete. In all cases examined, the proteolytic steps are not required for transport, but serve to generate mature amino-termini that permit protein folding, membrane insertion, and/or macromolecular complex assembly to produce the active enzymes or functional structures of mitochondria.
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 P1́ substrate positions: Ser=Gln>Thr at P1 and Ser>Thr at P1́. Non-polar residues were frequent at the substrate P3, P2, P2́and P3́ 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 majority of more than 1000 proteins present in mitochondria are imported from nuclear-encoded, cytosolically synthesized precursor proteins. This impressive feat of transport and sorting is achieved by the combined action of targeting signals on mitochondrial proteins and the mitochondrial protein import apparatus. The mitochondrial protein import apparatus is composed of a number of multi-subunit protein complexes that recognize, translocate, and assemble mitochondrial proteins into functional complexes. While the core subunits involved in mitochondrial protein import are well conserved across wide phylogenetic gaps, the accessory subunits of these complexes differ in identity and/or function when plants are compared with Saccharomyces cerevisiae (yeast), the model system for mitochondrial protein import. These differences include distinct protein import receptors in plants, different mechanistic operation of the intermembrane protein import system, the location and activity of peptidases, the function of inner-membrane translocases in linking the outer and inner membrane, and the association/regulation of mitochondrial protein import complexes with components of the respiratory chain. Additionally, plant mitochondria share proteins with plastids, i.e. dual-targeted proteins. Also, the developmental and cell-specific nature of mitochondrial biogenesis is an aspect not observed in single-celled systems that is readily apparent in studies in plants. This means that plants provide a valuable model system to study the various regulatory processes associated with protein import and mitochondrial biogenesis.
This chapter discusses the conservative sorting of intermembrane space (IMS) targeted proteins. Experimental evidence exists to support the conservative sorting hypothesis. This model proposes that IMS-targeted proteins embark on the general mitochondrial import pathway crossing the outer membrane (OM) and inner membrane (IM), where they emerge into the matrix. Upon exposure to the matrix, the sorting signal is recognized by a putative signal binding protein. The preprotein then embarks on a translocation event back across the IM, a process postulated to resemble the export of proteins in bacteria across the periplasmic membrane. Initiation of this export step is thought to occur prior to completion ofthe import of carboxy-terminal portions of the preproteins through the import machinery. Thus, it is highly plausible that, if of sufficient length, such IMS-targeted proteins can span both the OM and IM import machineries while being translocated across the IM during its export step. IM spanning intermediates have been identified and have been shown to be looping through the matrix by two independent approaches.
The current picture of the mitochondrial targeting peptide displayed in this chapter against a background of what is known about two other kinds of related sorting signals, namely secretory signal peptides and chloroplast transit peptides. Picture of the mitochondria1 matrix-targeting signal is based on the concept of the positively charged amphiphilic α-helix. This model is supported by a substantial body of evidence: statistical, biophysical, and genetical. The amphililic helix is required not only for correct targeting, but apparently also for correct cleavage, where it might serve to roughly position the cleavage enzyme in relation to the rather loosely defined cleavage site pattern. It is still unclear how the targeting peptide is recognized. Biophysical studies have demonstrated that targeting peptides have strong lipid-interacting properties, suggesting that direct protein-lipid interaction may be important at some stage. Both cytoplasmic and mitochondrial receptors that bind targeting peptides have been identified, and it is recently suggested that mitochondrial hsp70 also binds to targeting peptides as they emerge in to the matrix space.
This chapter discusses the strategies for characterizing, cloning, and expressing soluble endopeptidases. The cloning of the complementary DNA encoding an endopeptidase becomes a crucial step in explicating the role that the peptidase plays in nervous system function. Ultimately, elucidating the function and structure of one such protease can aid in understanding the regulation of neuropeptide function by these enzymes as a class. The peptidases can be targeted for pharmacological intervention through the use of specifically designed modulatory ligands, either agonistic or antagonistic. Examples using this rationale involve the development of inhibitors of the human immunodeficiency virus aspartic protease as a treatment for human immunodeficiency virus, inhibitors of angiotensin-converting enzyme, such as captopril, to treat hypertension, and inhibitors of enkephalinase as a treatment for congestive heart failure and as a nonaddictive analgesic.
This chapter examines the structural chemistry and the preparation of mitochondrial intermediate peptidase (MIP). MIP denotes cleavage of intermediate-sized mitochondrial proteins to the mature form. MIP activity can be measured by incubation of in vitro translated octapeptide-containing precursors with isolated mitochondria. Under such conditions, the cleavage catalyzed by MIP is at the end of a mitochondrial protein import reaction which also involves outer membrane receptors, outer and inner membrane translocation complexes, molecular chaperones and MPP. If the precursor is incubated directly with mitochondrial matrix or purified enzyme, initial cleavage by MPP is required to observe processing of the intermediate by MIP. This requirement is circumvented when MIP activity is determined using an intermediate protein as the substrate. Intermediates that are translated in vitro from a methionine artificially placed at the octapeptide N-terminus can be processed to the mature form by MIP independent of the presence of MPP. Native RMIP has been purified 2250-fold from rat liver mitochondrial matrix with a final yield of about 2%. Expression of recombinant enzyme has been achieved in S. cerevisiae .
TUMOUR necrosis factor-αa (TNF-α) is a potent
pro-inflammatory agent produced primarily by activated monocytes and
macrophages1. TNF-α is synthesized as a precursor
protein of Mr 26,000 (26K) which is processed to a secreted
17K mature form by cleavage of an Ala-Val bond between residues 76-77.
The enzyme(s) responsible for processing pro-TNF-α has yet to be
identified. Here, we describe the capacity of a metalloproteinase
inhibitor, GI129471, to block TNF-α secretion both in vitro and in
vivo. The inhibition is specific to TNF-α the production of other
secreted cytokines, such as the interleukins IL-1β, IL-2, or IL-6,
is not inhibited. The mechanism of inhibition occurs at a
post-translational step in TNF-α production. Our data suggest that
TNF-α processing is mediated by a unique Zn2+
endopeptidase which is inhibited by GI 129471 and would represent a
novel target for therapeutic intervention in TNF-α associated
pathologies.
The presence of plastids in plant cells requires a higher level of precursor recognition by the mitochondrial protein import
apparatus than in nonplant organisms. Although the plant presequences display the overall features observed in yeast and mammals,
they are generally longer and more hydrophilic. Most of them are highly organelle specific, but some have ambiguous targeting
specificity delive-ring a protein to both mitochondria and chloroplasts. Many components of plant protein import apparatus
appear different to that in yeast and mammalian systems. The three outer membrane mitochondrial proteins characterized to
play role as receptors in plants – Tom20, OM64, and metaxin – are plant specific. However, the channel forming units of the
TOM and SAM complexes, Tom40 and Sam50, respectively, are orthologous to these components in yeast. While components of the
MIA and TIM complexes also display high levels of orthology, functional studies indicate divergences in function and mechanism.
Differences exist also in terms of intraorganellar localization of proteolytic events, e.g., the location of the mitochondrial
processing peptidase, MPP, involved in removing targeting signals is different, whereas the function and location of the presequence
protease, PreP, degrading targeting peptides, is well conserved. Overall, although the protein import machinery of mitochondria
from all organisms appears to have coopted and uses the channel forming subunits from the endosymbiont that gave rise to mitochondria,
there is a greater diversity in plant components in comparison to those from nonplant species.
KeywordsTargeting peptides-Dual targeting-Mitochondrial protein import-Import machinery-TOM-TIM-Precursor processing-Presequence degradation-PreP
The mitochondrial genome encodes just a small number of subunits of the respiratory chain. All the other mitochondrial proteins are encoded in the nucleus and produced in the cytosol. Various enzymes participate in the activation and intramitochondrial transport of imported proteins. To finally take their place in the various mitochondrial compartments, the targeting signals of imported proteins have to be cleaved by mitochondrial processing peptidases. Mitochondria must also be able to eliminate peptides that are internally synthesized in excess, as well as those that are improperly assembled, and those with abnormal conformation caused by mutation or oxidative damage. Damaged mitochondrial proteins can be removed in two ways: either through lysosomal autophagy, that can account for at most 25–30% of the biochemically estimated rates of average mitochondrial catabolism; or through an intramitochondrial proteinolytic pathway. Mitochondrial proteases have been extensively studied in yeast, but evidence in recent years has demonstrated the existence of similar systems in mammalian cells, and has pointed to the possible importance of mitochondrial proteolytic enzymes in human diseases and ageing. A number of mitochondrial diseases have been identified whose mechanisms involve proteolytic dysfunction. Similar mechanisms probably play a role in diminished resistance to oxidative stress, and in the aging process. In this paper we review current knowledge of mammalian mitochondrial proteolysis, under normal conditions and in several disease states, and we propose an etiological classification of human diseases characterized by a decline or loss of function of mitochondrial proteolytic enzymes.
The chromosomal loci of expressed genes provide useful information for a candidate gene approach to the genes responsible for genetic diseases. A large set of randomly isolated cDNAs catalogued by partial sequencing can serve as a resource for accessing and isolating these disease genes. Using fluorescence in situ hybridization, we examined the chromosomal loci of 217 human keratinocyte-derived cDNAs, with independent novel sequence tags at the 3′ end region. Among them, we determined the loci of 50 cDNAs. Single-pass sequencing of these from the 5′ ends indicated that 39 cDNAs still can be produced for new genes. These cDNAs with identified chromosomal loci are powerful tools that can be used to help elucidate the genes responsible for hereditary skin disorders.
Most mitochondrial proteins are encoded by the nucleus and synthesized in the cytoplasm. Protein sorting within mitochondria depends on the precise coordination of several components of the import machinery. In this review we will discuss how precursor proteins are targeted to mitochondria, the energetics of import, intramito-chondrial sorting, the requirement of cytosolic and matrix localized chaperones, as well as the import channels themselves.
Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder that affects individuals who are carriers of small CGG premutation expansions in the fragile X mental retardation 1 (FMR1) gene. Mitochondrial dysfunction was observed as an incipient pathological process occurring in individuals who do not display overt features of FXTAS (1). Fibroblasts from premutation carriers had lower oxidative phosphorylation capacity (35% of controls) and Complex IV activity (45%), and higher precursor-to-mature ratios (P:M) of nDNA-encoded mitochondrial proteins (3.1-fold). However, fibroblasts from carriers with FXTAS symptoms presented higher FMR1 mRNA expression (3-fold) and lower Complex V (38%) and aconitase activities (43%). Higher P:M of ATPase β-subunit (ATPB) and frataxin were also observed in cortex from patients that died with FXTAS symptoms. Biochemical findings observed in FXTAS cells (lower mature frataxin, lower Complex IV and aconitase activities) along with common phenotypic traits shared by Friedreich's ataxia and FXTAS carriers (e.g. gait ataxia, loss of coordination) are consistent with a defective iron homeostasis in both diseases. Higher P:M, and lower ZnT6 and mature frataxin protein expression suggested defective zinc and iron metabolism arising from altered ZnT protein expression, which in turn impairs the activity of mitochondrial Zn-dependent proteases, critical for the import and processing of cytosolic precursors, such as frataxin. In support of this hypothesis, Zn-treated fibroblasts showed a significant recovery of ATPB P:M, ATPase activity and doubling time, whereas Zn and desferrioxamine extended these recoveries and rescued Complex IV activity.
Mitochondrial intermediate peptidase (MIP) is a component of the mitochondrial protein import machinery required for maturation of nuclear-encoded precursor proteins targeted to the mitochondrial matrix or inner membrane. We previously characterized this enzyme in rat (RMIP) and Saccharomyces cerevisiae (YMIP) and showed that MIP activity is essential for mitochondrial function in yeast. We have now defined the structure of a new MIP homologue (SMIP) from the basidiomycete fungus Schizophyllum commune. SMIP includes 4 exons of 523, 486, 660, and 629 bp separated by 3 short introns. The predicted SMIP, YMIP, and RMIP sequences share 31-37% identity and 54-57% similarity over 700 amino acids. When SMIP and RMIP were expressed in a yeast mip1 delta mutant, they were both able to rescue the respiratory-deficient phenotype caused by genetic inactivation of YMIP, indicating that the function of this enzyme is conserved in eukaryotes. Moreover, the MIP sequences show 20-24% identity and 40-47% similarity to a family of oligopeptidases from bacteria, yeast, and mammals. MIP and these proteins are characterized by a highly conserved motif, F-H-E-X-G-H-(X)2-H-(X)12-G-(X)5-D-(X)2-E-X-P-S-(X)3-E-X, centered around a zinc-binding site and appear to represent a new family of genes associated with proteolytic processing in the mitochondrial and cytosolic compartments.
Protein sorting signals provide good examples of peptides that can be studied both from a chemical and a biochemical perspective. Their simple designs and low degree of sequence conservation suggest that they are involved in rather non-specific peptide-lipid interactions, yet their ability to discriminate efficiently between the import machineries of different subcellular compartments rather points to the importance of peptide-receptor interactions. The study of protein sorting signals thus invites a cross-disciplinary approach.
Mitochondria import most of their proteins from the cytosol. A multi-subunit machinery accomplishes the translocation of precursor polypeptides into and across the two mitochondrial membranes. Within recent years more than 20 different proteins have been identified which are involved in mitochondrial protein import. This review summarizes the successful genetic and biochemical approaches that led to the identification of these transport and folding components. The identification and functional characterization of the components can be seen as a paradigm for the molecular analysis of a complex biological process by a combination of biochemical and genetic procedures.
Most mitochondrial precursor proteins are processed to the mature form in one step by mitochondrial processing peptidase (MPP),
while a subset of precursors destined for the matrix or the inner membrane are cleaved sequentially by MPP and mitochondrial
intermediate peptidase (MIP). We showed previously that yeast MIP (YMIP) is required for mitochondrial function in Saccharomyces cerevisiae. To further define the role played by two-step processing in mitochondrial biogenesis, we have now characterized the natural
substrates of YMIP. A total of 133 known yeast mitochondrial precursors were collected from the literature and analyzed for
the presence of the motif RX()(F/L/I)XX(T/S/G)XXXX(), typical of precursors cleaved by MPP and MIP. We found characteristic MIP cleavage sites in two distinct sets of proteins:
respiratory components, including subunits of the electron transport chain and tricarboxylic acid cycle enzymes, and components
of the mitochondrial genetic machinery, including ribosomal proteins, translation factors, and proteins required for mitochondrial
DNA metabolism. Representative precursors from both sets were cleaved to predominantly mature form by mitochondrial matrix
or intact mitochondria from wild-type yeast. In contrast, intermediate-size forms were accumulated upon incubation of the
precursors with matrix from mip1Δ yeast or intact mitochondria from mip1 yeast, indicating that YMIP is necessary for maturation of these proteins. Consistent with the fact that some of these substrates
are essential for the maintenance of mitochondrial protein synthesis and mitochondrial DNA replication, mip1Δ yeast undergoes loss of functional mitochondrial genomes.
We have isolated by immunological screening of a ZAPII cDNA library constructed from rat brain mRNAs a cDNA clone encoding endopeptidase 3.4.24.16. The longest open reading
frame encodes a 704-amino acid protein with a theoretical molecular mass of 80,202 daltons and bears the consensus sequence
of the zinc metalloprotease family. The sequence exhibits a 60.2% homology with those of another zinc metallopeptidase, endopeptidase
3.4.24.15. Northern blot analysis reveals two mRNA species of about 3 and 5 kilobases in rat brain, ileum, kidney, and testis.
We have transiently transfected COS-7 cells with pcDNA containing the cloned cDNA and established the overexpression of a 70-75-kDa immunoreactive protein. This protein hydrolyzes
QFS, a quenched fluorimetric substrate of endopeptidase 3.4.24.16, and cleaves neurotensin at a single peptide bond, leading
to the formation of neurotensin(1, 2, 3, 4, 5, 6, 7, 8, 9, 10) and neurotensin (11, 12, 13). QFS and neurotensin hydrolysis are potently inhibited by the selective endopeptidase 3.4.24.16 dipeptide blocker Pro-Ile
and by dithiothreitol, while the enzymatic activity remains unaffected by phosphoramidon and captopril, the specific inhibitors
of endopeptidase 3.4.24.11 and angiotensin-converting enzyme, respectively. Altogether, these physicochemical, biochemical,
and immunological properties unambiguously identify endopeptidase 3.4.24.16 as the protein encoded by the isolated cDNA clone.
Lactococcus lactis possesses a complex proteolytic system which is essential for its growth in milk. We characterized one of the peptidases of this system, oligopeptidase PepF, together with its structural gene. PepF hydrolyzed peptides containing between 7 and 17 amino acids with a rather wide specificity. It was purified to homogeneity. The N-terminal sequences of PepF and of peptides resulting from tryptic digestion of PepF were determined and used to design degenerate oligonucleotides which served to amplify a DNA fragment internal to pepF. This fragment was used as a probe to screen a lactococcal genomic library in Escherichia coli and to clone the entire gene pepF. The gene coded for a 70 kDa protein and was located on a 55-kilobase lactose-protease plasmid. A motif His-Glu-X-X-His, characteristic of metallopeptidases was evidenced. Two regions of PepF were found similar, first to a stretch of 43 amino acids around the zinc-binding site of several other peptidases, second to a stretch of 33 amino acids well conserved among creatine and arginine kinases. Preliminary results suggest the presence of a second copy of pepF.
We have isolated a metallopeptidase from rat liver. The peptidase is primarily located in the mitochondrial intermembrane space, where it interacts non-covalently with the inner membrane. The enzyme hydrolyzes oligopeptides, the largest substrate molecule found being dynorphin A1-17; it has no action on proteins, and does not interact with alpha 2-macroglobulin, and can therefore be classified as an oligopeptidase. We term the enzyme oligopeptidase M. Oligopeptidase M acts similarly to thimet oligopeptidase (EC 3.4.24.15) on bradykinin and several other peptides, but hydrolyzes neurotensin exclusively at the -Pro+Tyr- bond (the symbol + is used to indicate a scissile peptide bond) rather than the -Arg+Arg- bond. The enzyme is inhibited by chelating agents and some thiol-blocking compounds, but differs from thimet oligopeptidase in not being activated by thiol compounds. The peptidase is inhibited by Pro-Ile, unlike thimet oligopeptidase, and the two enzymes are separable in chromatography on hydroxyapatite. The N-terminal amino acid sequence of rat mitochondrial oligopeptidase M contains 19 out of 20 residues identical with a segment of rabbit microsomal endopeptidase and 17 matching the corresponding segment of pig-soluble angiotensin II-binding protein. Moreover, the rat protein is recognized by a monoclonal antibody against rabbit soluble angiotensin II-binding protein, all of which is consistent with these proteins being species variants of a single protein that is a homologue of thimet oligopeptidase. The biochemical properties of the mitochondrial oligopeptidase leave us in no doubt that it is neurolysin (EC 3.4.24.16), for which no sequence has previously been reported, and which has not been thought to be mitochondrial.
Incompletely synthesized polypeptides in the mitochondrial inner membrane are subject to rapid proteolysis. We demonstrate that Yta10p, a mitochondrial homologue of a conserved family of putative ATPases in Saccharomyces cerevisiae, is essential for this proteolytic process. Yta10p-dependent degradation requires divalent metal ions and the hydrolysis of ATP. Yta10p is an integral protein of the inner mitochondrial membrane exposing the carboxy terminus to the mitochondrial matrix space. Based on the presence of consensus binding sites for ATP, and for divalent metal ions found in a number of metal dependent endopeptidases, a direct role of Yta10p in the proteolytic breakdown of membrane-associated polypeptides in mitochondria is suggested.
Tumour necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory agent produced primarily by activated monocytes and macrophages. TNF-alpha is synthesized as a precursor protein of M(r) 26,000 (26K) which is processed to a secreted 17K mature form by cleavage of an Ala-Val bond between residues 76-77. The enzyme(s) responsible for processing pro-TNF-alpha has yet to be identified. Here, we describe the capacity of a metalloproteinase inhibitor, GI 129471, to block TNF-alpha secretion both in vitro and in vivo. The inhibition is specific to TNF-alpha; the production of other secreted cytokines, such as the interleukins IL-1 beta, IL-2, or IL-6, is not inhibited. The mechanism of inhibition occurs at a post-translational step in TNF-alpha production. Our data suggest that TNF-alpha processing is mediated by a unique Zn2+ endopeptidase which is inhibited by GI 129471 and would represent a novel target for therapeutic intervention in TNF-alpha associated pathologies.
We have previously determined the amino acid sequence of porcine soluble angiotensin-binding protein (sABP) by cDNA cloning and sequencing. In this study, we have cloned a sABP homologue (PABH) from the same porcine cDNA libraries used for sABP cloning. PABH and sABP have 65% sequence identity. Sequence comparisons with other proteins revealed very high similarities between porcine PABH and rat thimet oligopeptidase (90%), and between porcine sABP and rabbit microsomal endopeptidase (93%). This suggests that PABH and thimet oligopeptidase are identical and that sABP and microsomal endopeptidase are also the same. Indeed, sABP was shown to have a peptidase activity that is sensitive to the metal-chelating agents EDTA and 1,10-phenanthroline; sABP was also sensitive to the thiol reagent p-chloromercuriphenylsulfonic acid. RNase-protection assays, using RNA preparations from various porcine tissues, indicated that thimet oligopeptidase mRNA is ubiquitously expressed whereas sABP mRNA is predominantly expressed in the liver, kidney and adrenal gland. This assay also revealed tissue-specific alternative splicing of the sABP-encoding message.
We previously identified an activity in the soluble fraction of the yeast Saccharomyces cerevisiae that is a candidate for catalyzing the proteolytic maturation of farnesylated-CXXX precursor polypeptides. We describe here a 1259-fold purification of this activity by chromatography on DEAE-cellulose, hydroxylapatite, phenyl-Sepharose, and Sephacryl S-200. Sodium dodecyl sulfate gel electrophoresis of this preparation demonstrated a single 68-kDa polypeptide chain. The experimentally determined N-terminal amino acid sequence was identical at all 20 positions with residues 28-47 of the deduced sequence of the S. cerevisiae YCL57w gene product. This analysis suggests that the YCL57w gene encodes this enzyme and that the initial translation product may contain a leader peptide. Its complete deduced amino acid sequence shows significant homology to a number of zinc metallopeptidases and is most closely related to rat metalloendopeptidase 24.15 (E.C. 3.4.24.15), an enzyme that preferentially cleaves after hydrophobic residues. Using the purified yeast enzyme, we show a unique cleavage site in the peptides bradykinin and beta-neoendorphin four residues from the C-terminus on the carboxyl side of a hydrophobic amino acid. The cleavage pattern for neurotensin revealed a major site three residues from the C-terminus also on the carboxyl side of a hydrophobic residue and a minor site four residues from the C-terminus of the peptide. This specificity is similar to that of rat endopeptidase 24.15 and may explain why the farnesylated peptide employed in our studies is a good substrate for the yeast enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Thimet oligopeptidase (EC 3.4.24.15) is a thiol-dependent metallo-endopeptidase also known as Pz-peptidase, collagenase-like peptidase, endooligopeptidase A, soluble metallo-endopeptidase and endopeptidase 24.15. The enzyme is closely related to the yeast proteinase yscD. Thimet oligopeptidase (M(r) 74000) is widely distributed in animals and plants. In rat liver it exists in a cytoplasmic and mitochondrial form; a membrane-bound form of the enzyme was discovered in rat brain. Thimet oligopeptidase hydrolyses small peptides but does not act on proteins. In rat brain thimet oligopeptidase is involved in the generation of enkephalins and inactivation of bioactive peptides and experiments with yeast provided good evidence that the enzyme is involved in the late stages of cytoplasmatic protein degradation.
The detergent extract of rabbit liver microsomes contains an endopeptidase (MEP) with substrate specificity for peptides containing Arg residues at the P1 and P4 positions in the cleavage site (Kawabata, S., and Davie, E. W. (1992) J. Biol. Chem. 267, 10331-10336). These sequences occur in many proproteins such as the vitamin K-dependent proproteins and prohormones. A cDNA coding for MEP has been obtained from three overlapping clones isolated from two rabbit liver lambda gt10 cDNA libraries. The longest open reading frame of the 3507-base pair cDNA codes for a protein of 704 amino acids, of which 406 residues were confirmed by amino acid sequence analysis. MEP contains a putative active site of -His-Glu-X-X-His-, which is typical of mammalian zinc metallopeptidases. Based on a hydropathy plot, MEP is a hydrophilic protein with no transmembrane domain and no NH2-terminal signal sequence. Amino acid sequence analysis identified Asn at the three potential N-glycosylation sites in the enzyme, indicating that MEP contains no N-linked sugar. MEP is homologous with rat testes metalloendopeptidase 24.15 (60% identity), rat mitochondrial intermediate peptidase (24% identity), Escherichia coli dipeptidyl carboxypeptidase (25% identity), and the open reading frame YCL57w present in yeast chromosome III (35% identity).
A review of the proteinaceous machinery involved in protein sorting pathways and protein folding and assembly in mitochondria and peroxisomes is presented. After considering the various sorting pathways and targeting signals of mitochondrial and peroxisomal proteins, we make a comparative dissection of the protein factors involved in: i) the stabilization of cytosolic precursor proteins in a translocation competent conformation; ii) the membrane import apparatus of mitochondria and peroxisomes; iii) the processing of mitochondrial precursor proteins, and the eventual processing of certain peroxisomal precursor, in the interior of the organelles; and iv) the requirement of molecular chaperones for appropriate folding and assembly of imported proteins in the matrix of both organelles. Those aspects of mitochondrial biogenesis that have developed rapidly during the last few years, such as the requirement of molecular chaperones, are stressed in order to stimulate further parallel investigations aimed to understand the origin, biochemistry, molecular biology and pathology of peroxisomes. In this regard, a brief review of findings from our group and others is presented in which the role of the F1-ATPase alpha-subunit is pointed out as a molecular chaperone of mitochondria and chloroplasts. In addition, data are presented that could question our previous indication that the immunoreactive protein found in the rat liver peroxisomes is due to the presence of the F1-ATPase alpha-subunit.
The mitochondrial intermediate peptidase (MIP) cleaves characteristic octapeptides, (F/L/I)XX(T/S/ G)XXXX(decreases), from the N-terminus of many imported mitochondrial proteins. This leader peptidase is activated by divalent cations and inactivated by thiol-blocking agents, properties which are typical of metallo- and cysteine-proteases, respectively. To elucidate the mechanism of action of MIP, we analyzed by site-directed mutagenesis the functional role of a putative zinc-binding domain (F-H-E-X-G-H-(X)2-H-(X)12-G-(X)5-D-(X)2-E-X-P-S-(X)3-E) and two cysteine residues (C131 and C581), which are highly conserved in evolutionarily distant MIP sequences. We show that two histidines and a glutamic acid in the H-E-X-G-H motif and a glutamic acid 25 residues from the second histidine are essential for MIP function in vivo. In contrast, C131 and C581 are important for protein stability but are not required for activity in vivo or in vitro. These findings are consistent with MIP being a metallopeptidase.
cDNA clones encoding subunit VII of the Neurospora crassa bc1 complex (ubiquinol:cytochrome-c oxidoreductase), which is homologous with subunit VIII of the complex from yeast (encoded by QCR8), were identified on the basis of functional complementation of a yeast QCR8 deletion strain. The clones contain an open reading frame encoding a protein with a calculated molecular mass of 11.8 kDa. The N-terminal eight residues of the amino acid sequence deduced from the cDNA clones are absent from the mature protein, as revealed by direct sequencing of the isolated protein. To investigate the potential role of the N-terminal octapeptide in mitochondrial targeting, constructs were made encoding the precursor and the mature form of subunit VII from Neurospora. Incubation of isolated mitochondria with the two proteins revealed that the N-terminal extension of the precursor is removed on import. However, the presequence does not encode information for targeting, as the proteins encoded by both constructs can be imported into isolated mitochondria with equal efficiency. In contrast, the octapeptide seems to have functional importance: the defect in the yeast qcr8-null mutant is not complemented on transformation with the construct encoding mature subunit VII from N. crassa in a single-copy plasmid. We therefore speculate that the N-terminal extension plays a role in intramitochondrial sorting of N. crassa subunit VII. This is supported by the fact that the subunit VII precursor is processed by a protease other than the general mitochondrial processing peptidase. Interestingly, the presequence of N. crassa subunit VII has an amino acid composition similar to the octapeptides cleaved off by the mitochondrial intermediate peptidase.
Precursor proteins destined for the mitochondrial matrix traverse inner and outer organelle membranes in an extended conformation. Translocation events are therefore integrally coupled to the processes of protein unfolding in the cytosol and protein refolding in the matrix. To successfully import proteins from the cytoplasm into mitochondria, cells have recruited a variety of molecular chaperone systems and folding catalysts. Within the organelles, mitochondrial Hsp70 (mt-Hsp70) is a major player in this process and exerts multiple functions. First, mt-Hsp70 binds together with cohort proteins to incoming polypeptide chains, thus conferring unidirectionality on the translocation process, and then assists in their refolding. A subset of imported proteins requires additional assistance by chaperonins of the Hsp60/Hsp10 family. Protein folding occurs within the cavity of these cylindrical complexes. A productive interaction of precursor proteins with molecular chaperones in the matrix is not only crucial for correct refolding and assembly, but also for processing of presequences, intramitochondrial sorting, and degradation of proteins. This review focuses on the role of mt-Hsp70 and Hsp60/Hsp10 in protein folding in the mitochondrial matrix and discusses recent findings on their molecular mechanism of action.
Endopeptidase 24.16 or mitochondrial oligopeptidase, abbreviated here as EP 24.16 (MOP), is a thiol- and metal-dependent oligopeptidase that is found in multiple intracellular compartments in mammalian cells. From an analysis of the corresponding gene, we found that the distribution of the enzyme to appropriate subcellular locations is achieved by the use of alternative sites for the initiation of transcription. The pig EP 24.16 (MOP) gene spans over 100 kilobases and is organized into 16 exons. The core protein sequence is encoded by exons 5-16 which match perfectly with exons 2-13 of the gene for endopeptidase 24.15, another member of the thimet oligopeptidase family. These two sets of 11 exons share the same splice sites, suggesting a common ancestor. Multiple species of mRNA for EP 24.16 (MOP) were detected by the 5'-rapid amplification of cDNA ends and they were shown to have been generated from a single gene by alternative choices of sites for the initiation of transcription and splicing. Two types of transcript were prepared, corresponding to transcription from distal and proximal sites. Their expression in vitro in COS-1 cells indicated that they encoded two isoforms (long and short) which differed only at their amino termini: the long form contained a cleavable mitochondrial targeting sequence and was directed to mitochondria; the short form, lacking such a signal sequence, remained in the cytosol. The complex structure of the EP 24.16 (MOP) gene thus allows, by alternative promoter usage, a fine transcriptional regulation of coordinate expression, in the different subcellular compartments, of the two isoforms arising from a single gene.
Aspergillus fumigatus produces an 82 kDa intracellular metalloproteinase that hydrolyses the Pz-peptide, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg, a typical substrate of members of the thimet oligopeptidase family which is ubiquitously distributed across animal species. The A. fumigatus mepB gene encoding this 82 kDa metalloproteinase was cloned and sequenced. Analysis of the deduced amino acid sequence of mepB showed that the MepB protein is a cytosolic zinc metalloproteinase of the thimet oligopeptidase family (M3) and as such is probably involved in the intracellular degradation of small peptides. An A. fumigatus mutant that lacks the MepB Pz-peptidolytic activity was constructed by gene disruption at the mepB locus. Analysis of this mutant did not reveal any detectable phenotype.
The entire DNA sequence of chromosome III of the yeast Saccharomyces cerevisiae has been determined. This is the first complete sequence analysis of an entire chromosome from any organism. The 315-kilobase sequence reveals 182 open reading frames for proteins longer than 100 amino acids, of which 37 correspond to known genes and 29 more show some similarity to sequences in databases. Of 55 new open reading frames analysed by gene disruption, three are essential genes; of 42 non-essential genes that were tested, 14 show some discernible effect on phenotype and the remaining 28 have no overt function.
The opdA gene (formerly called optA) of Salmonella typhimurium encodes a metallopeptidase, oligopeptidase A (OpdA), first recognized by its ability to cleave and allow utilization of N-acetyl-L-Ala4 (E. R. Vimr, L. Green, and C. G. Miller, J. Bacteriol. 153:1259-1265, 1983). Derivatives of pBR328 carrying the opdA gene were isolated and shown to express oligopeptidase activity at levels approximately 100-fold higher than that of the wild type. These plasmids complemented all of the phenotypes associated with opdA mutations (failure to use N-acetyl-L-Ala4, defective phage P22 development, and diminished endopeptidase activity). The opdA region of one of these plasmids (pCM127) was defined by insertions of Tn1000 (gamma delta), and these insertions were used as priming sites to determine the nucleotide sequence of a 2,843-bp segment of the insert DNA. This region contained an open reading frame coding for a 680-amino-acid protein, the N terminus of which agreed with that determined for purified OpdA. This open reading frame contained both a sequence motif typical of Zn2+ metalloproteases and a putative sigma 32 promoter. However, no induction was detected upon temperature shift by using a beta-galactosidase operon fusion. The predicted OpdA sequence showed similarity to dipeptidyl carboxypeptidase, the product of the S. typhimurium gene dcp, and to rat metallopeptidase EC 3.4.24.15., which is involved in peptide hormone processing.
We have shown previously that cleavage of a number of precursors by the mitochondrial processing peptidase (MPP) requires an intermediate octapeptide (FXXSXXXX) between the MPP cleavage site and the mature protein amino terminus. We show now that these octapeptides, present at the amino termini of the intermediates, direct recognition of these substrates by the mitochondrial intermediate peptidase (MIP), leading to formation of mature proteins. Synthetic peptides, corresponding to the intermediate octapeptides of human ornithine transcarbamylase (OTC) and of Neurospora cytochrome c reductase Fe/S subunit (Fe/S), inhibit the processing activity of purified rat liver MIP in vitro, without affecting MPP activity; this indicates that the octapeptides can be recognized by MIP independent of the presence of the corresponding mature proteins and interact with a site that is crucial for MIP activity. MIP activity is not inhibited by a peptide lacking the amino-terminal hydrophobic residue, while substitution of such a residue by a polar amino acid causes a 10-fold reduction in the efficiency of MIP inhibition. To analyze the requirements for removal of the octapeptide from the intermediate proteins by MIP, artificial intermediates were synthesized and subjected to in vitro processing by purified MIP. The octapeptide can be cleaved by MIP only when the amino-terminal hydrophobic residue is also the amino terminus of the intermediate. Further, when the OTC octapeptide is joined to the mature amino terminus of another twice-cleaved precursor (pFe/S; rat malate dehydrogenase, pMDH), the chimeric intermediate is cleaved by MIP to the corresponding mature-sized protein. When the OTC octapeptide is joined to the mature amino terminus of a once-cleaved precursor (yeast F1-beta-ATPase, pF1-beta), however, this intermediate is not cleaved by MIP; rather, it is processed by MPP to mature-sized F1-beta. Therefore, amino-terminal octapeptides can be cleaved by MIP only within the structural context of twice-cleaved precursors.
The entire DNA sequence of chromosome III of the yeast Saccharomyces cerevisiae has been determined. This is the first complete sequence analysis of an entire chromosome from any organism. The 315-kilobase sequence reveals 182 open reading frames for proteins longer than 100 amino acids, of which 37 correspond to known genes and 29 more show some similarity to sequences in databases. Of 55 new open reading frames analysed by gene disruption, three are essential genes; of 42 non-essential genes that were tested, 14 show some discernible effect on phenotype and the remaining 28 have no overt function.
Many precursors of mitochondrial proteins are processed in two successive steps by independent matrix peptidases (MPP and MIP), whereas others are cleaved in a single step by MPP alone. To explain this dichotomy, we have constructed deletions of all or part of the octapeptide characteristic of a twice cleaved precursor (human ornithine transcarbamylase [pOTC]), have exchanged leader peptide sequences between once-cleaved (human methylmalonyl-CoA mutase [pMUT]; yeast F1ATPase beta-subunit [pF1 beta]) and twice-cleaved (pOTC; rat malate dehydrogenase (pMDH); Neurospora ubiquinol-cytochrome c reductase iron-sulfur subunit [pFe/S]) precursors, and have incubated these proteins with purified MPP and MIP. When the octapeptide of pOTC was deleted, or when the entire leader peptide of a once-cleaved precursor (pMUT or pF1 beta) was joined to the mature amino terminus of a twice-cleaved precursor (pOTC or pFe/S), no cleavage was produced by either protease. Cleavage of these constructs by MPP was restored by re-inserting as few as two amino-terminal residues of the octapeptide or of the mature amino terminus of a once-cleaved precursor. We conclude that the mature amino terminus of a twice-cleaved precursor is structurally incompatible with cleavage by MPP; such proteins have evolved octapeptides cleaved by MIP to overcome this incompatibility.
Several precursors transported from the cytoplasm to the intermembrane space of yeast mitochondria are first cleaved by the MAS-encoded protease in the matrix space and then by additional proteases that have not been characterized. We have now developed a specific assay for one of these other proteases. The enzyme is an integral protein of the inner membrane; it requires divalent cations and acidic phospholipid for activity, and is defective in yeast mutant pet ts2858 which accumulates an incompletely processed cytochrome b2 precursor. The protease contains a 21.4 kd subunit whose C-terminal part is exposed on the outer face of the inner membrane. An antibody against this polypeptide inhibits the activity of the protease. As overproduction of the polypeptide does not increase the activity of the protease in mitochondria, the enzyme may be a hetero-oligomer. This 'inner membrane protease I' shares several key features with the leader peptidase of Escherichia coli and the signal peptidase of the endoplasmic reticulum.
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.
The yeast gene for the Rieske iron-sulfur protein of the cytochrome b.c1 complex was subcloned into the expression vector, pSP64, then transcribed and translated in vitro in a reticulocyte lysate in the presence of [35S]methionine. Import studies in vitro of the newly synthesized precursor form of the iron-sulfur protein into isolated yeast mitochondria revealed that the precursor form of the iron-sulfur protein is processed into the mature form via an intermediate form. After the import reaction at 18 or 27 degrees C, treatment of mitochondria with exogenous protease indicated that both intermediate and mature forms had been internalized into mitochondria where they were resistant to digestion by external protease. Import and processing of the iron-sulfur protein into mitochondria also occurred at temperatures ranging from 2 to 27 degrees C in a temperature-dependent manner. Processing of the precursor form to the intermediate form appeared to be less sensitive to temperature than the processing of the intermediate form to the mature form. Moreover, at temperatures of 12 degrees C or lower, the mature form produced was completely digested by exogenous protease suggesting that it was assembled incorrectly in the membrane and not assembled into the b.c1 complex. The successive disappearance of first the mature form and then the intermediate form of the iron-sulfur protein by increasing concentrations of the metal chelators, EDTA and o-phenanthroline, suggested that two different proteases requiring divalent metal ions are involved in the two-step processing of the presequence of the iron-sulfur protein. Furthermore, mitoplasts containing only the matrix/inner membrane fraction were able to import and process the precursor form of the iron-sulfur protein indicating that both proteolytic processing events occur in the matrix/inner membrane fraction.
Endopeptidase 24.15, a metalloendopeptidase (EC 3.4.24.15) with an Mr of about 70,000, was purified to homogeneity from rat testes. The enzyme cleaves preferentially bonds on the carboxyl side of hydrophobic amino acids. Secondary enzyme-substrate interactions at sites removed from the scissile bond are indicated by the finding that a hydrophobic or bulky residue in the P3' position greatly contributes to substrate binding and catalytic efficiency. The isolated enzyme is inhibited by metal chelators and by thiols. Loss of enzymic activity after dialysis against EDTA can be restored by low concentrations of Zn2+ and Co2+ ions. The rate of reaction of the Co2+ enzyme with a synthetic substrate was higher than that of the Zn2+ enzyme. These results are consistent with the classification of the enzyme as a metalloendopeptidase. N-Carboxymethyl peptides that fulfil the binding requirements of the substrate recognition site of the enzyme act as potent competitive inhibitors. Biologically active peptides such as luteinizing hormone-releasing hormone, bradykinin and neurotensin are cleaved at sites consistent with the specificity of the enzyme deduced from studies with synthetic peptides. Dynorphin A (1-8)-peptide, beta-neoendorphin, metorphamide, and Metenkephalin-Arg6-Gly7-Leu8 are rapidly converted to the corresponding enkephalins. The testis enzyme is catalytically and immunologically closely related to the previously identified brain enzyme.
We have purified the metalloprotease which is localized in the soluble matrix space of Saccharomyces cerevisiae mitochondria and cleaves the amino-terminal matrix-targeting sequences from imported mitochondrial precursor proteins. The enzyme consists of two loosely associated non-identical subunits of mol. wt 48,000 and 51,000, respectively. Attempts to separate the two subunits from each other caused loss of activity. The smaller subunit had been identified as the product of the nuclear MAS1 gene (Witte et al., 1988). The larger subunit is now identified as the product of the nuclear MAS2 gene.
Cyclophilin (cyclosporin A-binding protein) has a dual localization in the mitochondria and in the cytosol of Neurospora crassa. The two forms are encoded by a single gene which is transcribed into mRNAs having different lengths and 5' termini (approximately 1 and 0.8 kilobases). The shorter mRNA specifies the cytosolic protein consisting of 179 amino acids. The longer mRNA is translated into a precursor polypeptide with an amino-terminal extension of 44 amino acids which is cleaved in two steps upon entry into the mitochondrial matrix. Neurospora cyclophilin shows about 60% sequence homology to human and bovine cyclophilins.
Yeast cytochrome c oxidase subunit IV (an imported mitochondrial protein) is made as a larger precursor with a transient pre-sequence of 25 amino acids. If this pre-sequence is fused to the amino terminus of mouse dihydrofolate reductase (a cytosolic protein) the resulting fusion protein is imported into the matrix space, and cleaved to a smaller size, by isolated yeast mitochondria. We have now fused progressively shorter amino-terminal segments of the subunit IV pre-sequence to dihydrofolate reductase and tested each fusion protein for import into the matrix space and cleavage by the matrix-located processing protease. The first 12 amino acids of the subunit IV pre-sequence were sufficient to direct dihydrofolate reductase into the mitochondrial matrix, both in vitro and in vivo. However, import of the corresponding fusion protein into the matrix was no longer accompanied by proteolytic processing. Fusion proteins containing fewer than nine amino-terminal residues from the subunit IV pre-piece were not imported into isolated mitochondria. The information for transporting attached mouse dihydrofolate reductase into mitochondria is thus contained within the first 12 amino acids of the subunit IV pre-sequence.
The mitochondrial matrix enzyme malate dehydrogenase (MDH) is synthesized on cytoplasmic polysomes as a larger precursor (pMDH) with an NH2-terminal leader peptide of 24 amino acids. Import of in vitro synthesized MDH into mitochondria results in formation of the mature-sized subunit. We report here that the conversion of pMDH to mMDH occurs via two distinct cleavage events within the leader peptide. First, pMDH is cleaved to an intermediate form (iMDH) of MDH. Conversion of the precursor to the intermediate form is catalyzed by a protease localized to the mitochondrial matrix. The cleavage of pMDH to iMDH involves the removal of 15 amino acids from the NH2 terminus of the pMDH leader peptide. The iMDH is subsequently cleaved, also by a matrix protease, to mature MDH in a reaction which is O-phenanthroline-sensitive. Cleavage to iMDH and to mature MDH occurs prior to completion of translocation of the MDH polypeptide chain into the mitochondrial matrix.
We have previously described a yeast mutant (mas1) that accumulates mitochondrial precursor proteins at high temperature and is deficient in the activity of a matrix-localized protease which cleaves presequences from mitochondrial precursor proteins. We have now cloned and sequenced the wild-type MAS1 gene and found that it encodes a subunit of the mitochondrial processing protease, that it is essential for cell viability and that the protein product participates in its own cleavage during import into mitochondria. The MAS1 protein is thus the first genetically defined component of the mitochondrial protein import pathway.
The mas2 mutant of Saccharomyces cerevisiae is temperature sensitive for import of proteins into mitochondria. To identify the lesion in this mutant, we have cloned and sequenced the wild-type MAS2 gene and determined the intracellular location of its protein product. MAS2 encodes an essential 53-kd protein that is located in the mitochondrial matrix and is homologous to the MAS1 protein, a previously identified subunit of the protease that cleaves presequences from mitochondrial precursor proteins. The activity of this enzyme is temperature sensitive in mas2 cells. Together with the results of the accompanying study these results show that MAS2 and MAS1 encode the two subunits of the processing protease.
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.
The mitochondrial matrix enzyme ornithine transcarbamylase (OTC) is synthesized on cytoplasmic polyribosomes as a precursor (pOTC) with an NH2-terminal extension of 32 amino acids. We report here that rat pOTC synthesized in vitro is internalized and cleaved by isolated rat liver mitochondria in two, temporally separate steps. In the first step, which is dependent upon an intact mitochondrial membrane potential, pOTC is translocated into mitochondria and cleaved by a matrix protease to a product designated iOTC, intermediate in size between pOTC and mature OTC. This product is in a trypsin-protected mitochondrial location. The same intermediate-sized OTC is produced in vivo in frog oocytes injected with in vitro-synthesized pOTC. The proteolytic processing of pOTC to iOTC involves the removal of 24 amino acids from the NH2 terminus of the precursor and utilizes a cleavage site two residues away from a critical arginine residue at position 23. In a second cleavage step, also catalyzed by a matrix protease, iOTC is converted to mature OTC by removal of the remaining eight residues of leader sequence. To define the critical regions in the OTC leader peptide required for these events, we have synthesized OTC precursors with alterations in the leader. Substitution of either an acidic (aspartate) or a "helix-breaking" (glycine) amino acid residue for arginine 23 of the leader inhibits formation of both iOTC and OTC, without affecting translocation. These mutant precursors are cleaved at an otherwise cryptic cleavage site between residues 16 and 17 of the leader. Interestingly, this cleavage occurs at a site two residues away from an arginine at position 15. The data indicate that conversion of pOTC to mature OTC proceeds via the formation of a third discrete species: an intermediate-sized OTC. The data suggest further that, in the rat pOTC leader, the essential elements required for translocation differ from those necessary for correct cleavage to either iOTC or mature OTC.
The mitochondrial matrix enzyme ornithine transcarbamylase (OTC) is synthesized on cytoplasmic polyribosomes as a precursor (pOTC) with an NH2-terminal extension of 32 amino acids. We report here that rat pOTC synthesized in vitro is internalized and cleaved by isolated rat liver mitochondria in two, temporally separate steps. In the first step, which is dependent upon an intact mitochondrial membrane potential, pOTC is translocated into mitochondria and cleaved by a matrix protease to a product designated iOTC, intermediate in size between pOTC and mature OTC. This product is in a trypsin-protected mitochondrial location. The same intermediate-sized OTC is produced in vivo in frog oocytes injected with in vitro-synthesized pOTC. The proteolytic processing of pOTC to iOTC involves the removal of 24 amino acids from the NH2 terminus of the precursor and utilizes a cleavage site two residues away from a critical arginine residue at position 23. In a second cleavage step, also catalyzed by a matrix protease, iOTC is converted to mature OTC by removal of the remaining eight residues of leader sequence. To define the critical regions in the OTC leader peptide required for these events, we have synthesized OTC precursors with alterations in the leader. Substitution of either an acidic (aspartate) or a "helix-breaking" (glycine) amino acid residue for arginine 23 of the leader inhibits formation of both iOTC and OTC, without affecting translocation. These mutant precursors are cleaved at an otherwise cryptic cleavage site between residues 16 and 17 of the leader. Interestingly, this cleavage occurs at a site two residues away from an arginine at position 15. The data indicate that conversion of pOTC to mature OTC proceeds via the formation of a third discrete species: an intermediate-sized OTC. The data suggest further that, in the rat pOTC leader, the essential elements required for translocation differ from those necessary for correct cleavage to either iOTC or mature OTC.
Many precursors of mitochondrial proteins are processed in two successive steps by independent matrix peptidases (MPP and MIP), whereas others are cleaved in a single step by MPP alone. To explain this dichotomy, we have constructed deletions of all or part of the octapeptide characteristic of a twice cleaved precursor (human ornithine transcarbamylase [pOTC]), have exchanged leader peptide sequences between once-cleaved (human methylmalonyl-CoA mutase [pMUT]; yeast F1ATPase beta-subunit [pF1 beta]) and twice-cleaved (pOTC; rat malate dehydrogenase (pMDH); Neurospora ubiquinol-cytochrome c reductase iron-sulfur subunit [pFe/S]) precursors, and have incubated these proteins with purified MPP and MIP. When the octapeptide of pOTC was deleted, or when the entire leader peptide of a once-cleaved precursor (pMUT or pF1 beta) was joined to the mature amino terminus of a twice-cleaved precursor (pOTC or pFe/S), no cleavage was produced by either protease. Cleavage of these constructs by MPP was restored by re-inserting as few as two amino-terminal residues of the octapeptide or of the mature amino terminus of a once-cleaved precursor. We conclude that the mature amino terminus of a twice-cleaved precursor is structurally incompatible with cleavage by MPP; such proteins have evolved octapeptides cleaved by MIP to overcome this incompatibility.
The role of nucleoside triphosphates (NTPs) in mitochondrial protein import was investigated with the precursors of N. crassa ADP/ATP carrier, F1-ATPase subunit β, F0-ATPase subunit 9, and fusion proteins between subunit 9 and mouse dihydrofolate reductase. NTPs were necessary for the initial interaction of precursors with the mitochondria and for the completion of translocation of precursors from the mitochondrial surface into the mitochondria. Higher levels of NTPs were required for the latter reactions as compared with the early stages of import. Import of precursors having identical presequences but different mature protein parts required different levels of NTPs. The sensitivity of precursors in reticulocyte lysate to proteases was decreased by removal of NTPs and increased by their readdition. We suggest that the hydrolysis of NTPs is involved in modulating the folding state of precursors in the cytosol, thereby conferring import competence.
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
Plasmids carrying the Salmonella typhimurium dcp gene were isolated from a pBR328 library of Salmonella chromosomal DNA by screening for complementation of a peptide utilization defect conferred by a dcp mutation. Strains carrying these plasmids overproduced dipeptidyl carboxypeptidase approximately 50-fold. The nucleotide sequence of a 2.8-kb region of one of these plasmids contained an open reading frame coding for a protein of 77,269 Da, in agreement with the 80-kDa size for dipeptidyl carboxypeptidase (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration). The N-terminal amino acid sequence of dipeptidyl carboxypeptidase purified from an overproducer strain agreed with that predicted by the nucleotide sequence. Northern (RNA) blot data indicated that dcp is not cotranscribed with other genes, and primer extension analysis showed the start of transcription to be 22 bases upstream of the translational start. The amino acid sequence of dcp was not similar to that of a mammalian dipeptidyl carboxypeptidase, angiotensin I-converting enzyme, but showed striking similarities to the amino acid sequence of another S. typhimurium peptidase encoded by the opdA (formerly optA) gene.
Only five mitochondrial proteins are known to be essential for viability of the yeast Saccharomyces cerevisiae; all of them are key components of the mitochondrial protein import system. Other components of this system are not essential for life; they include functionally redundant import receptors on the mitochondrial surface and enzymes acting upon only a few precursor proteins.
A critical step in the import of nuclear-encoded precursor proteins into mitochondria involves proteolytic cleavage of their amino-terminal leader peptides by processing proteases found in the mitochondrial matrix. We report here the characterization of the general matrix processing protease from rat liver mitochondria. The final enzyme preparation consisted of two polypeptides, a catalytically active 55-kDa subunit and a 52-kDa one. To deduce the complete primary structure of the 55-kDa subunit, we first sequenced its mature amino terminus and several tryptic peptides derived from the pure protein. Next, using mixed oligonucleotide primers that had sequences based on two of these peptides, we synthesized a partial cDNA probe by selective amplification of liver RNA with the polymerase chain reaction. The amplified probe was then used to obtain a nearly full-length clone from a rat liver cDNA library. This cDNA codes for 508 amino acid residues, including 16 residues of an amino-terminal leader peptide, the cleavage site of which is located two polypeptide bonds downstream from an arginine residue. The mature portion has a predicted molecular mass of 55.2 kDa; it shows 36% identity with the mitochondrial processing peptidases of Saccharomyces cerevisiae and Neurospora crassa. A conserved structural feature is a putative, negatively charged alpha-helix, located in the amino-terminal half of the subunit; this element might be important for the recognition of positively charged leader peptides characteristic of mitochondrial precursor proteins.
Although mitochondrial targeting peptides lack a common consensus sequence, a certain bias in the positional distribution of amino acids has recently been found. These patterns seem to be associated with cleavage of the precursor proteins by matrix processing proteases. We have extended the previous studies and found new sequence motifs that are conserved within subgroups of mitochondrial targeting peptides. These motifs have certain common themes, indicating that they are associated with cleavage by one single protease. Two of the conserved patterns have a high predictive value, but even for sequences that do not possess these patterns, a fairly accurate prediction of the cleavage site is shown to be possible. We also suggest that a well-conserved RXY decreases (S/A) pattern may be used to engineer efficiently recognized cleavage sites into uncleaved or artificial mitochondrial targeting peptides.
Most mitochondrial proteins are synthesized as precursor proteins on cytosolic polysomes and are subsequently imported into mitochondria. Many precursors carry amino-terminal presequences which contain information for their targeting to mitochondria. In several cases, targeting and sorting information is also contained in non-amino-terminal portions of the precursor protein. Nucleoside triphosphates are required to keep precursors in an import-competent (unfolded) conformation. The precursors bind to specific receptor proteins on the mitochondrial surface and interact with a general insertion protein (GIP) in the outer membrane. The initial interaction of the precursor with the inner membrane requires the mitochondrial membrane potential (delta psi) and occurs at contact sites between outer and inner membranes. Completion of translocation into the inner membrane or matrix is independent of delta psi. The presequences are cleaved off by the processing peptidase in the mitochondrial matrix. In several cases, a second proteolytic processing event is performed in either the matrix or in the intermembrane space. Other modifications can occur such as the addition of prosthetic groups (e.g., heme or Fe/S clusters). Some precursors of proteins of the intermembrane space or the outer surface of the inner membrane are retranslocated from the matrix space across the inner membrane to their functional destination ('conservative sorting'). Finally, many proteins are assembled in multi-subunit complexes. Exceptions to this general import pathway are known. Precursors of outer membrane proteins are transported directly into the outer membrane in a receptor-dependent manner. The precursor of cytochrome c is directly translocated across the outer membrane and thereby reaches the intermembrane space. In addition to the general sequence of events which occurs during mitochondrial protein import, current research focuses on the molecules themselves that are involved in these processes.
Representative samples of mitochondrial and chloroplast targeting peptides have been analyzed in terms of amino acid composition, positional amino acid preferences and amphiphilic character. No highly conserved 'homology blocks' are found in either class of topogenic sequence. Mitochondrial-matrix-targeting peptides are composed of two domains with different amphiphilic properties. Arginine is frequently found either at position -10 or -2 relative to the cleavage site, suggesting that some targeting peptides may be cleaved twice in succession by two different matrix proteases. In stroma-targeting chloroplast transit peptides three distinct regions are evident: an uncharged amino-terminal domain, a central domain lacking acidic residues and a carboxy-terminal domain with the potential to form an amphiphilic beta-strand. Targeting peptides that route proteins to the mitochondrial intermembrane space or the lumen of chloroplast thylakoids have a mosaic design with an amino-terminal matrix- or stroma-targeting part attached to a carboxy-terminal extension that shares many characteristics with secretory signal peptides.
We have compiled sequences of precursor proteins for 50 mitochondrial proteins for which the mature amino terminus has been determined by amino acid sequence analysis. Included in this set are 8 precursors that have leader peptides that are cleaved in two places by mitochondrial matrix proteases. When these eight leader peptides are aligned and compared, a highly conserved three-amino acid motif is identified as being common to this class of leader peptides. This motif includes an arginine at position -10, a hydrophobic residue at position -8, and serine, threonine, or glycine at position -5 relative to the mature amino terminus. The initial cleavage of these peptides by matrix processing protease occurs within the motif, between residues at -9 and -8, such that arginine at position -10 is at position -2 relative to the cleaved bond. The rest of the motif is within the octapeptide removed by subsequent cleavage catalyzed by intermediate-specific protease. An additional 14 leader peptides in this collection (all of those that contain an arginine at -10) conform to this motif. Assuming that these 14 precursors are matured in two steps, we compared the internal cleavage sites at position -8 with the ends of the other 30 leader peptides in the collection. We find that 74% of matrix processing protease cleavage sites follow an arginine at position -2 relative to cleavage.
A processing protease has been purified from the matrix fraction of rat liver mitochondria. The purified protease contained two protein subunits of 55 kd (P-55) and 52 kd (P-52) as determined by SDS-PAGE. The processing protease was estimated to be 105 kd in gel filtration, indicating that the two protein subunits form a heterodimeric complex. At high ionic conditions, the two subunits dissociated. The purified processing protease cleaved several mitochondrial protein precursors destined to different mitochondrial compartments, including adrenodoxin, malate dehydrogenase, P-450(SCC) and P-450(11 beta), but the processing efficiencies were different each other. The endoprotease nature of the processing protease was confirmed with the purified enzyme using adrenodoxin precursor as the substrate; both the mature form and the extension peptide were detected after the processing. The processing activity of the protease was inhibited by metal chelators, and reactivated by Mn2+, indicating that the protease is a metalloprotease.
The primary sequence motif HExxH has been found in many zinc-dependent endopeptidases. We show that a larger signature comprising this sequence is common to most of the known zinc-dependent endopeptidases, and that the presence of the signature can be indicative of membership in the family. A search of the protein sequence databases for entries containing the signature retrieved several unexpected potential zinc endopeptidases.
Transport of nuclear-encoded precursor proteins into mitochondria includes proteolytic cleavage of amino-terminal targeting sequences in the mitochondrial matrix. We have isolated the processing activity from Neurospora crassa. The final preparation (enriched ca. 10,000-fold over cell extracts) consists of two proteins, the matrix processing peptidase (MPP, 57 kd) and a processing enhancing protein (PEP, 52 kd). The two components were isolated as monomers. PEP is about 15-fold more abundant in mitochondria than MPP. It is partly associated with the inner membrane, while MPP is soluble in the matrix. MPP alone has a low processing activity whereas PEP alone has no apparent activity. Upon recombining both, full processing activity is restored. Our data indicate that MPP contains the catalytic site and that PEP has an enhancing function. The mitochondrial processing enzyme appears to represent a new type of "signal peptidase," different from the bacterial leader peptidase and the signal peptidase of the endoplasmic reticulum.
The Fe/S protein of complex III is encoded by a nuclear gene, synthesized in the cytoplasm as a precursor with a 32 residue amino-terminal extension, and transported to the outer surface of the inner mitochondrial membrane. Our data suggest the following transport pathway. First, the precursor is translocated via translocation contact sites into the matrix. There, cleavage to an intermediate containing an eight residue extension occurs. The intermediate is then redirected across the inner membrane, processed to the mature subunit, and assembled into complex III. We suggest that the folding and membrane-translocation pathway in the endosymbiotic ancestor of mitochondria has been conserved during evolution of eukaryotic cells; transfer of the gene for Fe/S protein to the nucleus has led to addition of the presequence, which routes the precursor back to its "ancestral" assembly pathway.
The imported precursors of the mammalian matrix enzymes malate dehydrogenase [(S)-malate:NAD+ oxidoreductase, EC 1.1.1.37] and ornithine transcarbamylase (carbamoyl-phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) are cleaved to their mature subunits in two steps, each catalyzed by matrix-localized processing proteases. The number and properties of these proteases are the subjects of this report. We have identified and characterized two distinct protease activities in a crude matrix fraction from rat liver: processing protease I, which cleaves these precursors to the corresponding intermediate form; and processing protease II, which cleaves the intermediate forms to mature subunits. Protease I is insensitive to chelation by EDTA and to inactivation with N-ethylmaleimide; protease II is inhibited by 5 mM EDTA and is inactivated by treatment with N-ethylmaleimide. We have prepared from mitochondrial matrix an 800-fold-enriched protease I fraction free of protease II activity by using the following steps: ion exchange, hydroxyapatite, molecular sieving, and hydrophobic chromatography. Using similar procedures, we also have prepared an approximately 2000-fold-enriched protease II fraction, which has a trace amount of contaminating protease I. This enriched protease II fraction has little or no cleavage activity toward mitochondrial precursors but rapidly and efficiently converts intermediate forms to mature size. Finally, we show that protease I alone is sufficient to cleave the precursor of a third nuclear-encoded mitochondrial protein subunit--the beta subunit of propionyl-CoA carboxylase [propanoyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.3]--to its mature size.
The degradation of the prolipoprotein signal peptide in vitro by membranes, cytoplasmic fraction, and two purified major signal peptide peptidases from Escherichia coli was followed by reverse-phase liquid chromatography (RPLC). The cytoplasmic fraction hydrolyzed the signal peptide completely into amino acids. In contrast, many peptide fragments accumulated as final products during the cleavage by a membrane fraction. Most of the peptides were similar to the peptides formed during the cleavage of the signal peptide by the purified membrane-bound signal peptide peptidase, protease IV. Peptide fragments generated during the cleavage of the signal peptide by protease IV and a cytoplasmic enzyme, oligopeptidase A, were identified from their amino acid compositions, their retention times during RPLC, and knowledge of the amino acid sequence of the signal peptide. Both enzymes were endopeptidases, as neither dipeptides nor free amino acids were formed during the cleavage reactions. Protease IV cleaved the signal peptide predominantly in the hydrophobic segment (residues 7 to 14). Protease IV required substrates with hydrophobic amino acids at the primary and the adjacent substrate-binding sites, with a minimum of three amino acids on either side of the scissile bond. Oligopeptidase A cleaved peptides (minimally five residues) that had either alanine or glycine at the P'1 (primary binding site) or at the P1 (preceding P'1) site of the substrate. These results support the hypothesis that protease IV is the major signal peptide peptidase in membranes that initiates the degradation of the signal peptide by making endoproteolytic cuts; oligopeptidase A and other cytoplasmic enzymes further degrade the partially degraded portions of the signal peptide that may be diffused or transported back into the cytoplasm from the membranes.
- F.-U Hartl
- B Schmidt
- E Wachter
- H Weiss
- W Neupert
Hartl, F.-U., Schmidt, B., Wachter, E., Weiss, H. & Neupert, W.
(1986) Cell 47, 939-951.
- G Von Heijne
- J Steppuhn
- R G Hermann
von Heijne, G., Steppuhn, J. & Hermann, R. G. (1989) Eur. J.
Biochem. 180, 535-545.
- E S Sztul
- J P Hendrick
- J P Kraus
- D Wall
- F Kalousek
- L E Rosenberg
Sztul, E. S., Hendrick, J. P., Kraus, J. P., Wall, D., Kalousek, F.
& Rosenberg, L. E. (1987) J. Cell Biol. 105, 2631-2639.
- G Hawlitschek
- B Schneider
- B Schmidt
- M Tropschug
- F.-U Hard
- W Neupert
Hawlitschek, G., Schneider, B., Schmidt, B., Tropschug, M., Hard,
F.-U. & Neupert, W. (1988) Cell 53, 785-806.
- A Pierotti
- K.-W Dong
- M J Glucksman
- M Orlowski
- J L Roberts
Pierotti, A., Dong, K.-W., Glucksman, M. J., Orlowski, M. &
Roberts, J. L. (1990) Biochemistry 29, 10323-10329.
- M R Yang
- R E Jensen
- M P Yaffe
- W Opplinger
- G Schatz
Yang, M. R., Jensen, R. E., Yaffe, M. P., Opplinger, W. & Schatz,
G. (1988) EMBO J. 7, 3857-3862.
- S G Oliver
Oliver, S. G., et al. (1992) Nature (London) 357, 38-46.
- R E Jensen
- M P Yaffe
Jensen, R. E. & Yaffe, M. P. (1988) EMBO J. 7, 3863-3871.
- C Witte
- R E Jensen
- M P Jaffe
- G Schatz
Witte, C., Jensen, R. E., Jaffe, M. P. & Schatz, G. (1988) EMBOJ.
7, 1439-1447.
- F Kalousek
- G Isaya
- L E Rosenberg
Kalousek, F., Isaya, G. & Rosenberg, L. E. (1992) EMBO J., in
press.