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Akio Ito - One of the best experts on this subject based on the ideXlab platform.
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Glutamate Residues Required for Substrate Binding and Cleavage Activity in Mitochondrial Processing Peptidase*
2008Co-Authors: Sakae Kitada, Tadashi Ogishima, Kunitoshi Shimokata, Katsuhiko Kojima, Akio ItoAbstract:Mitochondrial Processing Peptidase, a metalloendoPeptidase consisting of �- and �-subunits, specifically recognizes a large variety of Mitochondrial precursor proteins and cleaves off N-terminal extension peptides. The enzyme requires the basic amino acid residues in the extension peptides for effective and specific cleavage. To elucidate the mechanism involved in the molecular recognition of substrate by the enzyme, several glutamates around the active site of the rat �-subunit, which has a putative metal-binding motif, H 56 XXEH 60, were mutated to alanines or aspartates, and effects on kinetic parameters, metal binding, and substrate binding of the enzyme were analyzed. None of mutant proteins analyzed was impaired in dimer formation with the �-subunit. Mutation of glutamates at positions 79
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spatial orientation of Mitochondrial Processing Peptidase and a preprotein revealed by fluorescence resonance energy transfer
Journal of Biochemistry, 2007Co-Authors: Tomonori G Nishino, Akio Ito, Tadashi Ogishima, Katsuhiko Kojima, Ken Kitano, Sakae KitadaAbstract:Mitochondrial Processing Peptidase (MPP), which is composed of heterodimeric alpha-MPP and beta-MPP subunits. It specifically recognizes Mitochondrial preproteins and removes their basic N-terminal signal prepeptides. In order to elucidate the spatial orientation of the preproteins toward MPP, which has been missed by crystal structures of a yeast MPP including a synthetic prepeptide in its acidic proteolytic chamber, we analysed the fluorescence resonance energy transfer (FRET) between EGFP fused to a yeast aconitase presequence (preEGFP) and regiospecific 7-dietylamino-3-(4'-maleimidyl phenyl)-4-methyl coumarin (CPM)-labelled yeast MPPs. FRET efficiencies of 65 and 55% were observed between the EGFP chromophore and CPM-Ser(84) and -Lys(156) of beta-MPP, respectively, leading to calculated distances between the molecules of 48 and 50 A, respectively. Considering the FRET results and the structural validity based on the crystal structure of the MPP-presequence complex, a plausible model of preEGFP associated with MPP was constructed in silico. The modelled structure indicated that amino acid residues on the C-terminal side of the cleavage site in the preprotein were orientated tail out from the large cavity of MPP and interacted with the glycine-rich loop of alpha-MPP. Thus, MPP orientates preproteins at the specific cleft between the catalytic domain and the flexible glycine-rich loop which seems to pinch the extended polypeptide.
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a protein from a parasitic microorganism rickettsia prowazekii can cleave the signal sequences of proteins targeting mitochondria
Journal of Bacteriology, 2007Co-Authors: Sakae Kitada, Tsuneo Uchiyama, Tomoyuki Funatsu, Yumiko Kitada, Tadashi Ogishima, Akio ItoAbstract:The obligate intracellular parasitic bacteria rickettsiae are more closely related to mitochondria than any other microbes investigated to date. A rickettsial putative Peptidase (RPP) was found to resemble the α and β subunits of Mitochondrial Processing Peptidase (MPP), which cleaves the transport signal sequences of Mitochondrial preproteins. RPP showed completely conserved zinc-binding and catalytic residues compared with β-MPP but barely contained any of the glycine-rich loop region characteristic of α-MPP. When the biochemical activity of RPP purified from a recombinant source was analyzed, RPP specifically hydrolyzed basic peptides and presequence peptides with frequent cleavage at their MPP-Processing sites. Moreover, RPP appeared to activate yeast β-MPP so that it processed preproteins with shorter presequences. Thus, RPP behaves as a bifunctional protein that could act as a basic peptide Peptidase and a somewhat regulatory protein for other protein activities in rickettsiae. These are the first biological and enzymological studies to report that a protein from a parasitic microorganism can cleave the signal sequences of proteins targeted to mitochondria.
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Recognition and Processing of a nuclear-encoded polyprotein precursor by Mitochondrial Processing Peptidase
Biochemical Journal, 2005Co-Authors: Tsutomu Oshima, Akio Ito, Tadashi Ogishima, Eiki Yamasaki, Koh Ichi Kadowaki, Sakae KitadaAbstract:The nuclear-encoded protein RPS14 (ribosomal protein S14) of rice mitochondria is synthesized in the cytosol as a polyprotein consisting of a large N-terminal domain comprising preSDHB (succinate dehydrogenase B precursor) and the C-terminal RPS14. After the preSDHB–RPS14 polyprotein is transported into the Mitochondrial matrix, the protein is processed into three peptides: the N-terminal prepeptide, the SDHB domain and the C-terminal mature RPS14. Here we report that the general MPP (Mitochondrial Processing Peptidase) plays an essential role in Processing of the polyprotein. Purified yeast MPP cleaved both the N-terminal presequence and the connector region between SDHB and RPS14. Moreover, the connector region was processed more rapidly than the presequence. When the site of cleavage between SDHB and RPS14 was determined, it was located in an MPP Processing motif that has also been shown to be present in the N-terminal presequence. Mutational analyses around the cleavage site in the connector region suggested that MPP interacts with multiple sites in the region, possibly in a similar manner to the interaction with the N-terminal presequence. In addition, MPP preferentially recognized the unfolded structure of preSDHB–RPS14. In mitochondria, MPP may recognize the stretched polyprotein during passage of the precursor through the translocational apparatus in the inner membrane, and cleave the connecting region between the SDHB and RPS14 domains even before Processing of the presequence.
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determination of the cleavage site of the presequence by Mitochondrial Processing Peptidase on the substrate binding scaffold and the multiple subsites inside a molecular cavity
Journal of Biological Chemistry, 2003Co-Authors: Sakae Kitada, Katsuhiko Kojima, Eiki Yamasaki, Akio ItoAbstract:Abstract Mitochondrial Processing Peptidase (MPP) recognizes a large variety of basic presequences of Mitochondrial preproteins and cleaves the single site, often including arginine, at the −2 position (P2). To elucidate the recognition and specific Processing of the preproteins by MPP, we mutated to alanines at acidic residues conserved in a large internal cavity formed by the MPP subunits, α-MPP and β-MPP, and analyzed the Processing efficiencies for various preproteins. We report here that alanine mutations at a subsite in rat β-MPP interacting with the P2 arginine cause a shift in the Processing site to the C-terminal side of the preprotein. Because of reduced interactions with the P2 arginine, the mutated enzymes recognize not only the N-terminal authentic cleavage site with P2 arginine but also the potential C-terminal cleavage site without a P2arginine. In fact, it competitively cleaves the two sites of the preprotein. Moreover, the acidified site of α-MPP, which binds to the distal basic site in the long presequence, recognized the authentic P2 arginine as the distal site in compensation for ionic interaction at the proximal site in the mutant MPP. Thus, MPP seems to scan the presequence from β- to α-MPP on the substrate binding scaffold inside the MPP cavity and finds the distal and P2arginines on the multiple subsites on both MPP subunits. A possible mechanism for substrate recognition and cleavage is discussed here based on the notable character of a subsite-deficient mutant of MPP in which the substrate specificity is altered.
Sakae Kitada - One of the best experts on this subject based on the ideXlab platform.
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Glutamate Residues Required for Substrate Binding and Cleavage Activity in Mitochondrial Processing Peptidase*
2008Co-Authors: Sakae Kitada, Tadashi Ogishima, Kunitoshi Shimokata, Katsuhiko Kojima, Akio ItoAbstract:Mitochondrial Processing Peptidase, a metalloendoPeptidase consisting of �- and �-subunits, specifically recognizes a large variety of Mitochondrial precursor proteins and cleaves off N-terminal extension peptides. The enzyme requires the basic amino acid residues in the extension peptides for effective and specific cleavage. To elucidate the mechanism involved in the molecular recognition of substrate by the enzyme, several glutamates around the active site of the rat �-subunit, which has a putative metal-binding motif, H 56 XXEH 60, were mutated to alanines or aspartates, and effects on kinetic parameters, metal binding, and substrate binding of the enzyme were analyzed. None of mutant proteins analyzed was impaired in dimer formation with the �-subunit. Mutation of glutamates at positions 79
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spatial orientation of Mitochondrial Processing Peptidase and a preprotein revealed by fluorescence resonance energy transfer
Journal of Biochemistry, 2007Co-Authors: Tomonori G Nishino, Akio Ito, Tadashi Ogishima, Katsuhiko Kojima, Ken Kitano, Sakae KitadaAbstract:Mitochondrial Processing Peptidase (MPP), which is composed of heterodimeric alpha-MPP and beta-MPP subunits. It specifically recognizes Mitochondrial preproteins and removes their basic N-terminal signal prepeptides. In order to elucidate the spatial orientation of the preproteins toward MPP, which has been missed by crystal structures of a yeast MPP including a synthetic prepeptide in its acidic proteolytic chamber, we analysed the fluorescence resonance energy transfer (FRET) between EGFP fused to a yeast aconitase presequence (preEGFP) and regiospecific 7-dietylamino-3-(4'-maleimidyl phenyl)-4-methyl coumarin (CPM)-labelled yeast MPPs. FRET efficiencies of 65 and 55% were observed between the EGFP chromophore and CPM-Ser(84) and -Lys(156) of beta-MPP, respectively, leading to calculated distances between the molecules of 48 and 50 A, respectively. Considering the FRET results and the structural validity based on the crystal structure of the MPP-presequence complex, a plausible model of preEGFP associated with MPP was constructed in silico. The modelled structure indicated that amino acid residues on the C-terminal side of the cleavage site in the preprotein were orientated tail out from the large cavity of MPP and interacted with the glycine-rich loop of alpha-MPP. Thus, MPP orientates preproteins at the specific cleft between the catalytic domain and the flexible glycine-rich loop which seems to pinch the extended polypeptide.
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a protein from a parasitic microorganism rickettsia prowazekii can cleave the signal sequences of proteins targeting mitochondria
Journal of Bacteriology, 2007Co-Authors: Sakae Kitada, Tsuneo Uchiyama, Tomoyuki Funatsu, Yumiko Kitada, Tadashi Ogishima, Akio ItoAbstract:The obligate intracellular parasitic bacteria rickettsiae are more closely related to mitochondria than any other microbes investigated to date. A rickettsial putative Peptidase (RPP) was found to resemble the α and β subunits of Mitochondrial Processing Peptidase (MPP), which cleaves the transport signal sequences of Mitochondrial preproteins. RPP showed completely conserved zinc-binding and catalytic residues compared with β-MPP but barely contained any of the glycine-rich loop region characteristic of α-MPP. When the biochemical activity of RPP purified from a recombinant source was analyzed, RPP specifically hydrolyzed basic peptides and presequence peptides with frequent cleavage at their MPP-Processing sites. Moreover, RPP appeared to activate yeast β-MPP so that it processed preproteins with shorter presequences. Thus, RPP behaves as a bifunctional protein that could act as a basic peptide Peptidase and a somewhat regulatory protein for other protein activities in rickettsiae. These are the first biological and enzymological studies to report that a protein from a parasitic microorganism can cleave the signal sequences of proteins targeted to mitochondria.
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Recognition and Processing of a nuclear-encoded polyprotein precursor by Mitochondrial Processing Peptidase
Biochemical Journal, 2005Co-Authors: Tsutomu Oshima, Akio Ito, Tadashi Ogishima, Eiki Yamasaki, Koh Ichi Kadowaki, Sakae KitadaAbstract:The nuclear-encoded protein RPS14 (ribosomal protein S14) of rice mitochondria is synthesized in the cytosol as a polyprotein consisting of a large N-terminal domain comprising preSDHB (succinate dehydrogenase B precursor) and the C-terminal RPS14. After the preSDHB–RPS14 polyprotein is transported into the Mitochondrial matrix, the protein is processed into three peptides: the N-terminal prepeptide, the SDHB domain and the C-terminal mature RPS14. Here we report that the general MPP (Mitochondrial Processing Peptidase) plays an essential role in Processing of the polyprotein. Purified yeast MPP cleaved both the N-terminal presequence and the connector region between SDHB and RPS14. Moreover, the connector region was processed more rapidly than the presequence. When the site of cleavage between SDHB and RPS14 was determined, it was located in an MPP Processing motif that has also been shown to be present in the N-terminal presequence. Mutational analyses around the cleavage site in the connector region suggested that MPP interacts with multiple sites in the region, possibly in a similar manner to the interaction with the N-terminal presequence. In addition, MPP preferentially recognized the unfolded structure of preSDHB–RPS14. In mitochondria, MPP may recognize the stretched polyprotein during passage of the precursor through the translocational apparatus in the inner membrane, and cleave the connecting region between the SDHB and RPS14 domains even before Processing of the presequence.
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determination of the cleavage site of the presequence by Mitochondrial Processing Peptidase on the substrate binding scaffold and the multiple subsites inside a molecular cavity
Journal of Biological Chemistry, 2003Co-Authors: Sakae Kitada, Katsuhiko Kojima, Eiki Yamasaki, Akio ItoAbstract:Abstract Mitochondrial Processing Peptidase (MPP) recognizes a large variety of basic presequences of Mitochondrial preproteins and cleaves the single site, often including arginine, at the −2 position (P2). To elucidate the recognition and specific Processing of the preproteins by MPP, we mutated to alanines at acidic residues conserved in a large internal cavity formed by the MPP subunits, α-MPP and β-MPP, and analyzed the Processing efficiencies for various preproteins. We report here that alanine mutations at a subsite in rat β-MPP interacting with the P2 arginine cause a shift in the Processing site to the C-terminal side of the preprotein. Because of reduced interactions with the P2 arginine, the mutated enzymes recognize not only the N-terminal authentic cleavage site with P2 arginine but also the potential C-terminal cleavage site without a P2arginine. In fact, it competitively cleaves the two sites of the preprotein. Moreover, the acidified site of α-MPP, which binds to the distal basic site in the long presequence, recognized the authentic P2 arginine as the distal site in compensation for ionic interaction at the proximal site in the mutant MPP. Thus, MPP seems to scan the presequence from β- to α-MPP on the substrate binding scaffold inside the MPP cavity and finds the distal and P2arginines on the multiple subsites on both MPP subunits. A possible mechanism for substrate recognition and cleavage is discussed here based on the notable character of a subsite-deficient mutant of MPP in which the substrate specificity is altered.
Elzbieta Glaser - One of the best experts on this subject based on the ideXlab platform.
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mutagenesis and computer modelling approach to study determinants for recognition of signal peptides by the Mitochondrial Processing Peptidase
Plant Journal, 2001Co-Authors: Xiaoping Zhang, James Whelan, Sara Sjoling, Marcel Tanudji, Laszlo Somogyi, David Andreu, L Goran E Eriksson, Astrid Graslund, Elzbieta GlaserAbstract:Determinants for the recognition of a Mitochondrial presequence by the Mitochondrial Processing Peptidase (MPP) have been investigated using mutagenesis and bioinformatics approaches. All plant mit ...
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studies on the topology of the protein import channel in relation to the plant Mitochondrial Processing Peptidase integrated into the cytochrome bc1 complex
Plant Journal, 2000Co-Authors: Patrick Dessi, Charlotta Rudhe, Elzbieta GlaserAbstract:The Mitochondrial Processing Peptidase (MPP) specifically cleaves N-terminal targeting signals from hundreds of nuclear-encoded, matrix-targeted precursor proteins. In contrast to yeast and mammals, the plant MPP is an integral component of the respiratory cytochrome bc1 complex. The topology of the protein import channel in relation to MPP/bc1 in plants was studied using chimeric precursors containing truncated cytochrome b2 (cyt b2) proteins of 55-167 residues in length, fused to dihydrofolate reductase (DHFR). The DHFR domain could be tightly folded by methotrexate (MTX), generating translocation intermediates trapped in the import channel with only the cyt b2 pre-sequence/mature domain protruding into the matrix. Spinach and soybean mitochondria imported and processed unfolded precursors. MTX-folded intermediates were not processed in spinach but the longest (1-167) MTX-folded cyt b2-DHFR construct was processed in soybean, while yeast mitochondria successfully processed even shorter MTX-folded constructs. The MTX-folded precursors were cleaved with high efficiency by purified spinach MPP/bc1 complex. We interpret these results as indicating that the protein import channel is located distantly from the MPP/bc1 complex in plants, and that there is no link between protein translocation and protein Processing.
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integration of the Mitochondrial Processing Peptidase into the cytochrome bc1 complex in plants
Journal of Bioenergetics and Biomembranes, 1999Co-Authors: Elzbieta Glaser, Patrick DessiAbstract:The plant Mitochondrial cytochrome bc 1 complex, like nonplant Mitochondrial complexes,consists of cytochromes b and c 1, the Rieske iron–sulfur protein, two Core proteins, and fivelow-molecular mass subunits. However, in contrast to nonplant sources, the two Core proteinsare identical to subunits of the general Mitochondrial Processing Peptidase (MPP). The MPPis a fascinating enzyme that catalyzes the specific cleavage of the diverse presequence peptidesfrom hundreds of the nuclear-encoded Mitochondrial precursor proteins that are synthesizedin the cytosol and imported into the mitochondrion. Integration of the MPP into the bc 1complex renders the bc 1 complex in plants bifunctional, being involved both in electrontransport and in protein Processing. Despite the integration of MPP into the bc 1 complex,electron transfer as well as translocation of the precursor through the import channel areindependent of the protein-Processing activity. Recognition of the Processing site by MPPoccurs via the recognition of higher-order structural elements in combination with charge andcleavage-site properties. Elucidation of the three-dimensional (3-D) structure of the mammaliancytochrome bc 1 complex is highly useful for understanding of the mechanism of action of MPP.
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determinants for Processing by the Mitochondrial Processing Peptidase mpp integrated into the cytochrome bc1 complex in plants
1998Co-Authors: Elzbieta Glaser, Sara SjolingAbstract:Determinants for Processing by the Mitochondrial Processing Peptidase (MPP) integrated into the cytochrome bc1 complex in plants
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Mitochondrial Processing Peptidase integrated into the bc1 complex of the respiratory chain in spinach
1997Co-Authors: Sara Sjoling, Elzbieta GlaserAbstract:Mitochondrial Processing Peptidase integrated into the bc1 complex of the respiratory chain in Spinach.
Frantisek Kalousek - One of the best experts on this subject based on the ideXlab platform.
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substrate binding changes conformation of the α but not the β subunit of Mitochondrial Processing Peptidase
Archives of Biochemistry and Biophysics, 2001Co-Authors: Oleksandr Gakh, Jiri Adamec, Jaroslav Spizek, Tomas Obsil, Evzen Amler, Jiri Janata, Frantisek KalousekAbstract:Abstract Lifetime analysis of tryptophan fluorescence of the Mitochondrial Processing Peptidase (MPP) from Saccharomyces cerevisiae clearly proved that substrate binding evoked a conformational change of the α-subunit while presence of substrate influenced neither the lifetime components nor the average lifetime of the tryptophan excited state of the β-MPP subunit. Interestingly, lifetime analysis of tryptophan fluorescence decay of the α-MPP subunit revealed about 11% of steady-state fractional intensity due to the long-lived lifetime component, indicating that at least one tryptophan residue is partly buried at the hydrophobic microenvironment. Computer modeling, however, predicted none of three tryptophans, which the α-subunit contains, as deeply buried in the protein matrix. We conclude this as a consequence of a possible dimeric (oligomeric) structure.
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complementation between Mitochondrial Processing Peptidase mpp subunits from different species
Archives of Biochemistry and Biophysics, 1999Co-Authors: Jiri Adamec, Jaroslav Spizek, Olexandre Gakh, Frantisek KalousekAbstract:Abstract Mitochondrial Processing Peptidase (MPP), a dimer of nonidentical subunits, is the primary Peptidase responsible for the removal of leader peptides from nuclearly encoded Mitochondrial proteins. Alignments of the α and β subunits of MPP (α- and β-MPP) from different species show strong protein sequence similarity in certain regions, including a highly negatively charged region as well as a domain containing a putative metal ion binding site. In this report, we describe experiments in which we combine the subunits of MPP from yeast, rat, and Neurospora crassa, both in vivo and in vitro and mesure the resultant Processing activity. For in vivo complementation, we used the temperature sensitive mif1 and mif2 yeast mutants, which lack MPP activity at the nonpermissive temperature (37°C). We found that the defective α-MPP of mif2 cannot be substituted for by the α-MPP from rat or Neurospora. On the other hand, the β-MPP from rat and Neurospora can fully substitute for the defective β-MPP in the mif1 mutant. These results were confirmed in in vitro experiments in which individually expressed subunits were combined. Only combinations of the α-MPP from yeast with the β-MPP from rat or Neurospora produced active MPP.
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Yeast and human frataxin are processed to mature form in two sequential steps by the Mitochondrial Processing Peptidase.
The Journal of biological chemistry, 1999Co-Authors: Steven S. Branda, Patrizia Cavadini, Jiri Adamec, Frantisek Kalousek, Franco Taroni, Grazia IsayaAbstract:Frataxin is a nuclear-encoded Mitochondrial protein which is deficient in Friedreich's ataxia, a hereditary neurodegenerative disease. Yeast mutants lacking the yeast frataxin homologue (Yfh1p) show iron accumulation in mitochondria and increased sensitivity to oxidative stress, suggesting that frataxin plays a critical role in Mitochondrial iron homeostasis and free radical toxicity. Both Yfh1p and frataxin are synthesized as larger precursor molecules that, upon import into mitochondria, are subject to two proteolytic cleavages, yielding an intermediate and a mature size form. A recent study found that recombinant rat Mitochondrial Processing Peptidase (MPP) cleaves the mouse frataxin precursor to the intermediate but not the mature form (Koutnikova, H., Campuzano, V., and Koenig, M. (1998) Hum. Mol. Gen. 7, 1485–1489), suggesting that a different Peptidase might be required for production of mature size frataxin. However, in the present study we show that MPP is solely responsible for maturation of yeast and human frataxin. MPP first cleaves the precursor to intermediate form and subsequently converts the intermediate to mature size protein. In this way, MPP could influence frataxin function and indirectly affect Mitochondrial iron homeostasis.
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mutational analysis of both subunits from rat Mitochondrial Processing Peptidase
Archives of Biochemistry and Biophysics, 1996Co-Authors: Hans Martin Striebel, Jiri Adamec, Petr Rysavy, Jaroslav Spizek, Frantisek KalousekAbstract:Abstract Rat liver Mitochondrial Processing Peptidase (MPP) is the primary Peptidase that cleaves leader peptides from nuclearly encoded Mitochondrial proteins following their transport from the cytosol to the Mitochondrial matrix. This enzyme consists of two nonidentical subunits that have overall similarity to each other and share certain amino acid motifs. These include the putative metal-ion binding HFLEH motif in the β-subunit and the HFLEK motif of the α-subunit, as well as a possibly helical amino acid stretch bearing a high concentration of negatively charged residues about 70 amino acids downstream of these motifs in both subunits. In order to achieve a better understanding of the role of certain amino acids in rat MPP, we performed site-directed mutagenesis on both of its subunits. Our results show that whereas both histidines and the glutamate of the HFLEH motif in the β-subunit are crucial for MPP function, this holds true only for the glutamate in the related HFLEK motif in the α-subunit. In addition, functionally important negatively charged residues in the region 70 amino acids downstream occur only in the β-subunit and not in the α-subunit. This indicates a functional asymmetry between the subunits, with the β-subunit containing a majority of residues participating in the active center.
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studies on protein Processing for membrane bound spinach leaf Mitochondrial Processing Peptidase integrated into the cytochrome bc1 complex and the soluble rat liver matrix Mitochondrial Processing Peptidase
FEBS Journal, 1996Co-Authors: Sara Sjoling, Frantisek Kalousek, Elzbieta Glaser, Mary Waltner, Henry WeinerAbstract:The plant Mitochondrial Processing Peptidase (MPP) that catalyses the cleavage of the presequences from precursor proteins during or after protein import is a membrane-bound enzyme that constitutes an integral part of the bc1 complex of the respiratory chain. In contrast, MPP from mammals is soluble in the matrix space and does not form part of the respiratory chain. In the present study, we have compared the substrate specificity of the isolated spinach leaf bc1/MPP with rat liver MPP using synthetic signal peptides and different Mitochondrial precursor proteins. Inhibition studies of Processing with synthetic peptides showed a similar inhibition pattern for plant and rat MPP activity. A peptide derived from the presequence of rat liver Mitochondrial aldehyde dehydrogenase (ALDH) was a potent inhibitor of the spinach and rat MPP. Two nonprocessed signal peptides, rhodanese and linker-deleted ALDH (a form of ALDH that lacks the RGP linker connecting two helices in the presequence) had lower inhibitory effects towards each protease. The signal peptide from thiolase, another nonprocessed protein, had little inhibitory effect on MPP. Peptides derived from presequence of the plant Nicotiana plumbaginifolia F1β also showed a similar inhibitory pattern with rat MPP as with spinach MPP Processing. In-vitro synthesised precursors of plant N. plumbaginifolia F1β and rat liver ALDH were cleaved to mature form by both spinach and rat MPP. However, the efficiency of Processing was higher with the homologous precursor. Linker-deleted ALDH, rhodanese, and thiolase were not processed by the mammalian or plant MPP. However, both forms of MPP cleaved a mutated form of rhodanese that possesses a typical MPP cleavage motif, RXY S. Addition of the same cleavage motif to thiolase did not result in Processing by either MPP. These results show that similar higher-order structural elements upstream from the cleavage site are important for Processing by both the membrane-bound plant and the soluble mammalian MPP.
Tadashi Ogishima - One of the best experts on this subject based on the ideXlab platform.
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Glutamate Residues Required for Substrate Binding and Cleavage Activity in Mitochondrial Processing Peptidase*
2008Co-Authors: Sakae Kitada, Tadashi Ogishima, Kunitoshi Shimokata, Katsuhiko Kojima, Akio ItoAbstract:Mitochondrial Processing Peptidase, a metalloendoPeptidase consisting of �- and �-subunits, specifically recognizes a large variety of Mitochondrial precursor proteins and cleaves off N-terminal extension peptides. The enzyme requires the basic amino acid residues in the extension peptides for effective and specific cleavage. To elucidate the mechanism involved in the molecular recognition of substrate by the enzyme, several glutamates around the active site of the rat �-subunit, which has a putative metal-binding motif, H 56 XXEH 60, were mutated to alanines or aspartates, and effects on kinetic parameters, metal binding, and substrate binding of the enzyme were analyzed. None of mutant proteins analyzed was impaired in dimer formation with the �-subunit. Mutation of glutamates at positions 79
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spatial orientation of Mitochondrial Processing Peptidase and a preprotein revealed by fluorescence resonance energy transfer
Journal of Biochemistry, 2007Co-Authors: Tomonori G Nishino, Akio Ito, Tadashi Ogishima, Katsuhiko Kojima, Ken Kitano, Sakae KitadaAbstract:Mitochondrial Processing Peptidase (MPP), which is composed of heterodimeric alpha-MPP and beta-MPP subunits. It specifically recognizes Mitochondrial preproteins and removes their basic N-terminal signal prepeptides. In order to elucidate the spatial orientation of the preproteins toward MPP, which has been missed by crystal structures of a yeast MPP including a synthetic prepeptide in its acidic proteolytic chamber, we analysed the fluorescence resonance energy transfer (FRET) between EGFP fused to a yeast aconitase presequence (preEGFP) and regiospecific 7-dietylamino-3-(4'-maleimidyl phenyl)-4-methyl coumarin (CPM)-labelled yeast MPPs. FRET efficiencies of 65 and 55% were observed between the EGFP chromophore and CPM-Ser(84) and -Lys(156) of beta-MPP, respectively, leading to calculated distances between the molecules of 48 and 50 A, respectively. Considering the FRET results and the structural validity based on the crystal structure of the MPP-presequence complex, a plausible model of preEGFP associated with MPP was constructed in silico. The modelled structure indicated that amino acid residues on the C-terminal side of the cleavage site in the preprotein were orientated tail out from the large cavity of MPP and interacted with the glycine-rich loop of alpha-MPP. Thus, MPP orientates preproteins at the specific cleft between the catalytic domain and the flexible glycine-rich loop which seems to pinch the extended polypeptide.
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a protein from a parasitic microorganism rickettsia prowazekii can cleave the signal sequences of proteins targeting mitochondria
Journal of Bacteriology, 2007Co-Authors: Sakae Kitada, Tsuneo Uchiyama, Tomoyuki Funatsu, Yumiko Kitada, Tadashi Ogishima, Akio ItoAbstract:The obligate intracellular parasitic bacteria rickettsiae are more closely related to mitochondria than any other microbes investigated to date. A rickettsial putative Peptidase (RPP) was found to resemble the α and β subunits of Mitochondrial Processing Peptidase (MPP), which cleaves the transport signal sequences of Mitochondrial preproteins. RPP showed completely conserved zinc-binding and catalytic residues compared with β-MPP but barely contained any of the glycine-rich loop region characteristic of α-MPP. When the biochemical activity of RPP purified from a recombinant source was analyzed, RPP specifically hydrolyzed basic peptides and presequence peptides with frequent cleavage at their MPP-Processing sites. Moreover, RPP appeared to activate yeast β-MPP so that it processed preproteins with shorter presequences. Thus, RPP behaves as a bifunctional protein that could act as a basic peptide Peptidase and a somewhat regulatory protein for other protein activities in rickettsiae. These are the first biological and enzymological studies to report that a protein from a parasitic microorganism can cleave the signal sequences of proteins targeted to mitochondria.
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Recognition and Processing of a nuclear-encoded polyprotein precursor by Mitochondrial Processing Peptidase
Biochemical Journal, 2005Co-Authors: Tsutomu Oshima, Akio Ito, Tadashi Ogishima, Eiki Yamasaki, Koh Ichi Kadowaki, Sakae KitadaAbstract:The nuclear-encoded protein RPS14 (ribosomal protein S14) of rice mitochondria is synthesized in the cytosol as a polyprotein consisting of a large N-terminal domain comprising preSDHB (succinate dehydrogenase B precursor) and the C-terminal RPS14. After the preSDHB–RPS14 polyprotein is transported into the Mitochondrial matrix, the protein is processed into three peptides: the N-terminal prepeptide, the SDHB domain and the C-terminal mature RPS14. Here we report that the general MPP (Mitochondrial Processing Peptidase) plays an essential role in Processing of the polyprotein. Purified yeast MPP cleaved both the N-terminal presequence and the connector region between SDHB and RPS14. Moreover, the connector region was processed more rapidly than the presequence. When the site of cleavage between SDHB and RPS14 was determined, it was located in an MPP Processing motif that has also been shown to be present in the N-terminal presequence. Mutational analyses around the cleavage site in the connector region suggested that MPP interacts with multiple sites in the region, possibly in a similar manner to the interaction with the N-terminal presequence. In addition, MPP preferentially recognized the unfolded structure of preSDHB–RPS14. In mitochondria, MPP may recognize the stretched polyprotein during passage of the precursor through the translocational apparatus in the inner membrane, and cleave the connecting region between the SDHB and RPS14 domains even before Processing of the presequence.
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recognition of Mitochondrial protein precursor lacking arginine at position 2 by Mitochondrial Processing Peptidase Processing of bovine cytochrome p450 scc precursor
Journal of Biochemistry, 2001Co-Authors: Katsuhiko Kojima, Tadashi Ogishima, Sakae Kitada, Eiki Yamasaki, Akio ItoAbstract:Mitochondrial Processing Peptidase (MPP) specifically cleaves off the N-terminal presequence of the Mitochondrial protein precursor. Previous studies demonstrated that Arg at position -2 from the cleavage site, which is found among many precursors, plays a critical role in recognition by MPP. We analyzed the structural elements of bovine cytochrome P450 side-chain cleavage enzyme precursor [pre-P450(SCC)], which has Ala at position -2, for recognition by MPP. Replacement of Ala position -2 of pre-P450(SCC) with Arg resulted in an increase in the cleavage rate. Replacement with Gly caused a reduction in the cleavage rate and the appearance of an additional cleavage site downstream of the authentic site. A pre-P450(SCC) mutant with Met at position -2 retained cleavage efficiency equal to that of the wild type. These results indicate that -2 Ala of pre-P450(SCC) is recognized by MPP as a determinant for precise cleavage, and that the amino acid at -2 is required to have a straight methylene chain for interaction with the S 2 site. The preference for distal basic residues, a hydrophobic residue at +1, and hydroxyl residues at +2 and +3, was almost the same as those of the precursors with Arg at -2, indicating that the recognition mechanism of pre-P450(SCC) by MPP is essentially the same as that of the precursors with Arg at position -2.