Activation of a Matrix Processing Peptidase from the Crystalline Cytochrome bc1 Complex of Bovine Heart Mitochondria

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Mature core I and core II proteins of the bovine heart mitochondrial cytochrome bc 1 complex were individually overexpressed in Escherichia coli as soluble proteins using the expression vector pET-I and pET-II, respectively. Purified recombinant core I and core II alone show no mitochondrial processing peptidase (MPP) activity. When these two proteins are mixed together, MPP activity is observed. Maximum activity is obtained when the molar ratio of these two core proteins reaches 1. This indicates that only the two core subunits of thebc 1 complex are needed for MPP activity. The properties of reconstituted MPP are similar to those of Triton X-100-activated MPP in the bovine bc 1 complex. When Rieske iron-sulfur protein precursor is used as substrate for reconstituted MPP, the processing activity stops when the amount of product formation (subunit IX) equals the amount of reconstituted MPP used in the system. Addition of Triton X-100 to the product-inhibited reaction mixture restores MPP activity, indicating that Triton X-100 dissociates bound subunit IX from the active site of reconstituted MPP. The aromatic group, rather than the hydroxyl group, at Tyr 57 of core I is essential for reconstitutive activity.Most nuclear-encoded mitochondrial proteins are synthesized on cytoplasmic ribosomes as larger precursors with presequences for targeting into mitochondria (1). These presequences are proteolytically removed during or after import of the precursors into mitochondria. Three types of processing peptidases are involved in removal of the presequence from precursors: mitochondrial processing peptidase (MPP) 1 (2), mitochondrial intermediate peptidase (3), and inner membrane protease I (4, 5).MPP cleaves all or part of the presequence as the initial processing step. Many proteins are mature after a one-step cleavage by MPP. Mitochondrial intermediate peptidase catalyzes a second-step cleavage in the two-step processing of some precursor proteins. The inner membrane protease I cleaves intermediate forms of proteins routed to the intermembrane space. The last two peptidases act sequentially after cleavage of the matrix targeting sequences by MPP. Thus, MPP plays an important role in the proteolytic processing of precursor proteins in the mitochondria.MPP is located in the matrix of fungal and mammalian mitochondria and in the inner membrane of plant mitochondria (2). Matrix-localized MPP has been studied extensively and purified to homogeneity from Neurospora crassa (6), Saccharomyces cerevisiae (7), and rat liver (8, 9). Purified, matrix-localized MPP contains two nonidentical subunits, ␣-MPP and ␤-MPP, each with molecular mass of around 50 kDa. The cDNAs encoding ␣-and ␤-MPP from these three sources have been cloned, sequenced (6, 7, 9 -13), and overexpressed in Escherichia coli cells (13-15). MPP is classified in the pitrilysin family (16) of zinc metalloproteases because of the presence of an inverted zinc binding motif, HXXEH 76 H, in the ␤-MPP (17). Processing activity requires both subunits because recombinant...

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confidence: 99%

Reconstitution of Mitochondrial Processing Peptidase from the Core Proteins (Subunits I and II) of Bovine Heart Mitochondrial Cytochrome bc1 Complex

Deng

Shenoy

Tso

et al. 2001

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“…The matrix region of Complex III may also be important for stabilization of the complex itself as bc 1 complexes without core proteins are generally less stable and have lower activity (15). If the interaction between the matrix enzymes and the bc 1 complex exists, this region would be a good choice for the contact.It also should be noted that core I and core II subunits of the bovine complex have mitochondrial processing peptidase-like activity (16,17). The presequence peptide of ISP is cleaved by core I and core II.…”

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confidence: 99%

“…It also should be noted that core I and core II subunits of the bovine complex have mitochondrial processing peptidase-like activity (16,17). The presequence peptide of ISP is cleaved by core I and core II.…”

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confidence: 99%

Cross-talk between Mitochondrial Malate Dehydrogenase and the Cytochrome bc1 Complex

Wang

2010

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The interactions between the mitochondrial cytochrome bc 1 complex and matrix-soluble proteins were studied by a precipitation pulldown technique. Purified, detergent-dispersed bc 1 complex was incubated with mitochondrial matrix proteins followed by dialysis in the absence of detergent. The interacting protein(s) was co-precipitated with bc 1 complex upon centrifugation. One of the matrix proteins pulled down by bc 1 complex was identified as mitochondrial malate dehydrogenase (MDH) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and confirmed by Western blotting with anti-MDH antibody. Using a cross-linking technique, subunits I, II (core I and II), and V of the bc 1 complex were identified as the interacting sites for MDH. Incubating purified MDH with the detergent dispersed bc 1 complex results in an increase of the activities of both the bc 1 complex and MDH. The effect of the bc 1 complex on the activities of MDH is unidirectional (oxaloacetate 3 malate). These results suggest that the novel cross-talk between citric acid cycle enzymes and electron transfer chain complexes might play a regulatory role in mitochondrial bioenergetics.The mitochondrial cytochrome bc 1 complex catalyzes electron transfer from ubiquinol to cytochrome c with the concomitant translocation of protons across the membrane to generate a proton gradient and membrane potential for ATP synthesis (1). Three-dimensional structures of mitochondrial bc 1 complexes from various sources have become available in recent years (2-11). All mitochondrial cytochrome bc 1 complexes contain three essential subunits that house four redox prosthetic groups and seven to eight non-redox prosthetic groups containing supernumerary subunits. The three essential subunits are: the cytochrome b subunit, housing two b-type hemes (b H and b L ); the cytochrome c 1 subunit, housing one c-type heme (c 1 ); the Rieske iron-sulfur protein (ISP), 2 housing a high potential [2Fe-2S] cluster.Although the structural and functional studies of the mitochondrial bc 1 complex have been intensive, few studies of the interactions between the bc 1 complex and the soluble matrix enzymes have been available. Complex II (succinate-ubiquinone oxidoreductase) is the only enzyme that participates in both the citric acid cycle and the electron transport chain (12). Complex II is composed of a succinate dehydrogenase, which is a citric acid cycle enzyme, and a membrane-anchoring protein fraction, which houses ubiquinone. Complex II catalyzes the oxidation of succinate to fumarate and the reduction of ubiquinone to ubiquinol. Other enzymes of citric acid cycle are known to couple to the electron transfer chain through their product NADH at complex I (13). Structurally the mitochondrial bc 1 complex can be divided into three regions: matrix, transmembrane, and the intermembrane space regions. The matrix region contains subunits I (core I), II (core II), VI, IX, and the N-terminal part of subunits V (ISP) and VII (2, 4). Although this region contains the m...

“…Subunit 9 of the bovine cytochrome bc 1 complex corresponds to the presequence of the bovine Rieske iron-sulfur protein and is the only known example of a presequence that is retained in the mitochondria and integrated as a subunit of an oligomeric membrane-bound protein complex (31). The precursor of the bovine Rieske iron-sulfur protein is targeted and assembled into the bc 1 complex before the presequence is cleaved off, presumably by the core proteins in the bc 1 complex (31)(32)(33).…”

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confidence: 99%

Isolation and Identification of a Novel Mitochondrial Metalloprotease (PreP) That Degrades Targeting Presequences in Plants

Ståhl¹,

Moberg²,

Ytterberg³

et al. 2002

116

Most of the nuclear encoded mitochondrial precursor proteins contain an N-terminal extension called the presequence that carries targeting information and that is cleaved off after import into mitochondria. The presequences are amphiphilic, positively charged, membrane-interacting peptides with a propensity to form ␣-helices. Here we have investigated the proteolysis of the presequences that have been cleaved off inside mitochondria. A presequence derived from the overexpressed F 1 ␤ subunit of the ATP synthase and specific synthetic fluorescent peptides (Pep Tag Protease assay) have been shown to undergo rapid degradation catalyzed by a matrix located protease. We have developed a three-step chromatographic procedure including affinity and anion exchange chromatography for isolation of the protease from potato tuber mitochondria. Twodimensional gel electrophoresis of the isolated proteolytically active fraction followed by electrospray ionization-mass spectrometry/mass spectrometry and data base searches allowed identification of the presequence peptide-degrading protease in Arabidopsis thaliana data base as a novel mitochondrial metalloendoprotease with a molecular mass of 105 kDa. The identified metalloprotease contains an inverted zinc-binding motif and belongs to the pitrilysin family.