No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Processing peptidase of Neurospora mitochondria. Two-step cleavage of imported ATPase subunit 9
Abstract
Subunit 9 (dicyclohexylcarbodiimide binding protein, 'proteolipid') of the mitochondrial F1F0-ATPase is a nuclearly coded protein in Neurospora crassa. It is synthesized on free cytoplasmic ribosomes as a larger precursor with an NH2-terminal peptide extension. The peptide extension is cleaved off after transport of the protein into the mitochondria. A processing activity referred to as processing peptidase that cleaves the precursor to subunit 9 and other mitochondrial proteins is described and characterized using a cell-free system. Precursor synthesized in vitro was incubated with extracts of mitochondria. Processing peptidase required Mn2+ for its activity. Localization studies suggested that it is a soluble component of the mitochondrial matrix. The precursor was cleaved in two sequential steps via an intermediate-sized polypeptide. The intermediate form in the processing of subunit 9 was also seen in vivo and upon import of the precursor into isolated mitochondria in vitro. The two cleavage sites in the precursor molecule were determined. The data indicate that: the correct NH2-terminus of the mature protein was generated, the NH2-terminal amino acid of the intermediate-sized polypeptide is isoleucine in position -31. The cleavage sites show similarity of primary structure. It is concluded that processing peptidase removes the peptide extension from the precursor to subunit 9 (and probably other precursors) after translocation of these polypeptides (or the NH2-terminal part of these polypeptides) into the matrix space of mitochondria.
... Precursor proteins are synthesized in the cytosol and appear to contain targeting signals which direct them to mitochondria (15,18,19,20,42). In many cases these signals are NH2-terminal sequences which are cleaved off upon entry into the mitochondrial matrix by a processing peptidase (3,8,24,25,38), though noncleavable signals also exist (1,45). Proteinaceous structures are involved in the initial binding step of the precursors to the outer mitochondrial membrane (17,30,46,47). ...
... Subfractionation with Digitonin. Treatment of mitochondria with digitonin was performed as described (38) with the following modifications. Samples of mimchondrial suspensions (30 gl at 10 mg protein/ml in SEM buffer containing 1 mM PMSF) were incubated with the equivalent volume of digitonin solutions (0-1.6 % wt/vol in SEM buffer containing 1 mM PMSF) for 5 rain at 0°C. ...
... In experiments where mitochondria were fractionated into outer membrane and mitoplasts by shearing forces, 300 o.g mitochondrial protein were incubated in 1.5 ml of reticulocyte lysate to generate translocational intermediate as described below. The mitochondria were then reisolated by centrifugation at 14,000 g for 7 min and resuspended together with 10 nag of unlabeled mitochondria in 4 ml swelling buffer (5 mM KPi, pH 7.2, 1 mM PMSF, 2 nag cytosolic protease inhibitor fraction from N. crassa (38)). After 15 min at 0°C, the mitochondria were ruptured by five strokes in a homogenizer with a tight-fitting teflon pestle. ...
Import of proteins into the mitochondrial matrix requires translocation across two membranes. Translocational intermediates of mitochondrial proteins, which span the outer and inner membrane simultaneously and thus suggest that translocation occurs in one step, have recently been described (Schleyer, M., and W. Neupert, 1985, Cell, 43:339-350). In this study we present evidence that distinct membrane areas are involved in the translocation process. Mitochondria that had lost most of their outer membrane by digitonin treatment (mitoplasts) still had the ability to import proteins. Import depended on proteinaceous structures of the residual outer membrane and on a factor that is located between the outer and inner membranes and that could be extracted with detergent plus salt. Translocational intermediates, which had been preformed before fractionation, remained with the mitoplasts under conditions where most of the outer membrane was subsequently removed. Submitochondrial vesicles were isolated in which translocational intermediates were enriched. Immunocytochemical studies also suggested that the translocational intermediates are located in areas where outer and inner membranes are in close proximity. We conclude that the membrane-potential-dependent import of precursor proteins involves translocation contact sites where the two membranes are closely apposed and are linked in a stable manner.
... In contrast, for cells expressing the control HA-Ub (without an MTS), conjugates signals were detected in both mitochondrial and cytosolic fractions, and no conjugates signals were detected at all in cells harboring the empty plasmid (no HA-Ub) ( Figure 3B). The Su9 MTS (preSu9) is a well-established mitochondrial matrix targeting signal [19][20][21], and therefore, preSu9-HA-Ub was employed for the rest of this study. Taken together, these observations indicate the occurrence of protein ubiquitination in mitochondria. ...
... Taken together, these observations indicate the occurrence of protein ubiquitination in mitochondria. The Su9 MTS (preSu9) is a well-established mitochondrial matrix targeting signal [19][20][21], and therefore, preSu9-HA-Ub was employed for the rest of this study. Taken together, these observations indicate the occurrence of protein ubiquitination in mitochondria. ...
Ubiquitination is a critical type of post-translational modification in eukaryotic cells. It is involved in regulating nearly all cellular processes in the cytosol and nucleus. Mitochondria, known as the metabolism heart of the cell, are organelles that evolved from bacteria. Using the subcellular compartment-dependent α-complementation, we detect multiple components of ubiquitination machinery as being eclipsed distributed to yeast mitochondria. Ubiquitin conjugates and mono-ubiquitin can be detected in lysates of isolated mitochondria from cells expressing HA-Ub and treated with trypsin. By expressing MTS (mitochondrial targeting sequence) targeted HA-tagged ubiquitin, we demonstrate that certain ubiquitination events specifically occur in yeast mitochondria and are independent of proteasome activity. Importantly, we show that the E2 Rad6 affects the pattern of protein ubiquitination in mitochondria and provides an in vivo assay for its activity in the matrix of the organelle. This study shows that ubiquitination occurs in the mitochondrial matrix by eclipsed targeted components of the ubiquitin machinery, providing a new perspective on mitochondrial and ubiquitination research.
... Most mitochondrial protein precursors carry positively charged N-terminal presequences that are cleaved in a single step by MPP during or upon translocation to the mitochondrial matrix (16). Two-step processing by MPP is a rare variation that was first described for the N. crassa ATPase subunit 9 (17) and, more recently, yeast and human frataxin (Ref. 12 and this study). ...
... The targeting signals of these three proteins are 68, 51, and 55 residues long, 1.5-2 times the average mitochondrial presequence (20 -40 residues) (26,27), which may explain why they are removed in more than one cleavage. It has been proposed that a longer targeting signal is required by the ATPase subunit 9 to compensate for the hydrophobicity of the mature protein, which could otherwise affect solubility and import competence (17). By analogy, longer targeting signals may be required by yeast and human frataxin to neutralize the acidity of the mature protein, which may interfere with the initial steps of mitochondrial import (16). ...
We showed previously that maturation of the human frataxin precursor (p-fxn) involves two cleavages by the mitochondrial processing peptidase (MPP). This observation was not confirmed by another group, however, who reported only one cleavage. Here, we demonstrate conclusively that MPP cleaves p-fxn in two sequential steps, yielding a 18,826-Da intermediate (i-fxn) and a 17,255-Da mature (m-fxn) form, the latter corresponding to endogenous frataxin in human tissues. The two cleavages occur between residues 41–42 and 55–56, and both match the MPP consensus sequence RX ↓ (X/S). Recombinant rat and yeast MPP catalyze the pài step 4 and 40 times faster, respectively, than the i à m step. In isolated rat mitochondria, p-fxn undergoes a sequence of cleavages, p à i à m à d1 à d2, with d1 and d2 representing two C-terminal fragments of m-fxn produced by an unknown protease. The iàm step is limiting, and the overall rate of p à i à m does not exceed the rate of mà d1 à d2, such that the levels of m-fxn do not change during incubations as long as 3 h. Inhibition of the iàm step by a disease-causing frataxin mutation (W173G) leads to nonspecific degradation of i-fxn. Thus, the second of the two processing steps catalyzed by MPP limits the levels of mature frataxin within mitochondria.
... As is the case with the authentic N. crassa preprotein, the Su9-DHFR presequence is cleaved twice upon import, after residues 35 and 66. This two step removal of the presequence is catalyzed by the matrix-localized processing peptidase (Schmidt et al., 1984;Hawlitschek et al., 1988). Mitochondria were isolated from wild-type, ssd-2 and sscl-3 yeast cells that were grown at the permissive temperature of 25°C. ...
... Figure 9. Co-immunoprecipitation of precursor and intermediate forms of Su9-DHFR with mt-hspT0. (A) Denatured Su9-DHFR was imported into ssd-2 mitochondria (in the presence of ATP) and immunoprecipitations were performed as described in the legend of Fig. 8, except that the mitochondrial processing peptidase was partially inactivated by performing the import in the presence of 1 mM o-phenanthroline and 5 mM EDTA (Schmidt et al., 1984;Hawlitschek et al., 1988). (B) Su9-DHFR accumulated in the absence of a membrane potential is not co-immunoprecipitated with mt-hsp70. ...
The role of mitochondrial 70-kD heat shock protein (mt-hsp70) in protein translocation across both the outer and inner mitochondrial membranes was studied using two temperature-sensitive yeast mutants. The degree of polypeptide translocation into the matrix of mutant mitochondria was analyzed using a matrix-targeted preprotein that was cleaved twice by the processing peptidase. A short amino-terminal segment of the preprotein (40-60 amino acids) was driven into the matrix by the membrane potential, independent of hsp70 function, allowing a single cleavage of the presequence. Artificial unfolding of the preprotein allowed complete translocation into the matrix in the case where mutant mt-hsp70 had detectable binding activity. However, in the mutant mitochondria in which binding to mt-hsp70 could not be detected the mature part of the preprotein was only translocated to the intermembrane space. We propose that mt-hsp70 fulfills a dual role in membrane translocation of preproteins. (a) Mt-hsp70 facilitates unfolding of the polypeptide chain for translocation across the mitochondrial membranes. (b) Binding of mt-hsp70 to the polypeptide chain is essential for driving the completion of transport of a matrix-targeted preprotein across the inner membrane. This second role is independent of the folding state of the preprotein, thus identifying mt-hsp70 as a genuine component of the inner membrane translocation machinery. Furthermore we determined the sites of the mutations and show that both a functional ATPase domain and ATP are needed for mt-hsp70 to bind to the polypeptide chain and drive its translocation into the matrix.
... The Genbank Accession Nos are as follows: HsSHMmit, U23143; HsSHMcyt, L11931; ScSHMmit, L22528; ScSHMcyt, L22529; CaSHMmit, AF009965; CaSHMcyt, AF009966. VHRRGL present in the S. cerevisiae mitochondrial SHMT (McNeil et al., 1994) and conforms approximately to the cleavage site consensus: hydrophobic-polar-lys-arg-small-bulky hydrophobic residues derived by Schmidt et al. (1984) for some mitochondrial proteins. Like the human mitochondrial SHMT, the N-terminal extension of the C. albicans Shm1 protein is rich in arg, leu and ser residues which, in the case of the human enzyme, forms an amphipathic-helix that favours mitochondrial import (Schmidt et al., 1984). ...
... VHRRGL present in the S. cerevisiae mitochondrial SHMT (McNeil et al., 1994) and conforms approximately to the cleavage site consensus: hydrophobic-polar-lys-arg-small-bulky hydrophobic residues derived by Schmidt et al. (1984) for some mitochondrial proteins. Like the human mitochondrial SHMT, the N-terminal extension of the C. albicans Shm1 protein is rich in arg, leu and ser residues which, in the case of the human enzyme, forms an amphipathic-helix that favours mitochondrial import (Schmidt et al., 1984). The C. albicans Shm1 protein is also more similar in sequence to the S. cerevisiae mitochondrial isoform (68% identity) than the cytosolic SHMT (59% identity). ...
Genes encoding the mitochondrial (SHM1) and cytosolic (SHM2) serine hydroxymethyltransferases, and the L-threonine aldolase gene (GLY1) from Candida albicans were cloned and sequenced. All three genes are involved in glycine metabolism. The C. albicans Shm1 protein is 82% identical to that from Saccharomyces cerevisiae and 56% identical to that from Homo sapiens. The corresponding identities for the Shm2 proteins are 68% and 53%. The Gly1 protein shares significant identity with the S. cerevisiaeL-threonine aldolase (55%) and also with threonine aldolases from Aeromonas jandiae (36%) and Escherichia coli (36%). Genetic ablation experiments show that GLY1 is a non-essential gene in C. albicans and that L-threonine aldolase plays a lesser role in glycine metabolism than it does in S. cerevisiae. GenBank Accession Nos of the C. albicansSHM1 and SHM2 are AF009965 and AF009966, respectively. Accession No. for C. albicansGLY1 is AF009967. Copyright © 2000 John Wiley & Sons, Ltd.
... In contrast, for cells expressing the control HA-Ubi (without an MTS), conjugate signals were detected in both mitochondrial and cytosolic fractions, and no conjugate signals were detected, at all, in cells harbouring the empty plasmid (no HA-Ubi) (Fig 3B). The Su9 MTS (preSu9) is a well-established mitochondrial matrix targeting signal [18][19][20] and therefore, preSu9-HA-Ubi was employed for the rest of this study. Taken together these observations indicate the occurrence of protein ubiquitination in mitochondria. ...
Ubiquitination is a critical type of post translational modification in eukaryotic cells. It is involved in regulating nearly all cellular processes in the cytosol and nucleus. Mitochondria, known as the metabolism heart of the cell, are organelles that evolved from bacteria. Using the subcellular compartment-dependent α-complementation, we detect multiple components of ubiquitination machinery as being eclipsed distributed to yeast mitochondria. Subsequently, the results with respect to MTS (mitochondrial targeting sequence) targeted HA-tagged ubiquitin demonstrate that certain ubiquitination events specifically occur in yeast mitochondria and are independent of proteasome activity in the cytosol/nucleus. Importantly, we show that the E2 Rad6 affects the pattern of protein ubiquitination in mitochondria and provides an in vivo assay for its activity in the matrix of the organelle. This study shows that ubiquitination occurs in the mitochondrial matrix by eclipsed targeted components of the ubiquitin machinery, providing a new perspective of mitochondrial and ubiquitination research.
... Indeed, the mitochondrial FTSH4 protease precursor is cleaved by MPP at two different sites (Kmiec et al., 2012). The two-step processing carried out by MPP has been previously reported for yeast and human frataxin (Branda et al., 1999;Cavadini et al., 2000) and ATP synthase subunit 9 in Neurospora crassa (Schmidt et al., 1984). Using the precursor of mitochondrial tandem protein Arg5,6 as a model substrate from S. cerevisiae mitochondria, Friedl et al. (2020) have demonstrated that MPP not only removes the N-terminal targeting sequence from this precursor tandem protein but is also required for specific internal processing generating two functional enzymes, Arg5 and Arg6. ...
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.
... The import of preCOX4-mCherry is strictly dependent on the presence of an active membrane potential, and import is, therefore, proportional to the membrane potential, causing the preCOX4-mCherry to relocate to the cytoplasm upon weakening of the membrane potential (19). The preSU9 is a well-established strong mitochondrial presequence that is less sensitive to the membrane potential than other presequences and can drive import even at low membrane potential (19)(20)(21)(22). The import strength of the preSU9 presequence has been demonstrated in experiments using addition of increasing concentrations of the uncoupler cyanide m-chlorophenyl hydrazone (CCCP) in order to gradually decrease the membrane potential (19,22). ...
Saccharomyces cerevisiae constitutes a popular eukaryal model for research on mitochondrial physiology. Being Crabtree-positive, this yeast has evolved the ability to ferment glucose to ethanol and respire ethanol once glucose is consumed. Its transition phase from fermentative to respiratory metabolism, known as the diauxic shift, is reflected by dramatic rearrangements of mitochondrial function and structure. To date, the metabolic adaptations that occur during the diauxic shift have not been fully characterized at the organelle level. In this study, the absolute proteome of mitochondria was quantified alongside precise parametrization of biophysical properties associated with the mitochondrial network using state-of-the-art optical-imaging techniques. This allowed the determination of absolute protein abundances at a subcellular level. By tracking the transformation of mitochondrial mass and volume, alongside changes in the absolute mitochondrial proteome allocation, we could quantify how mitochondria balance their dual role as a biosynthetic hub as well as a center for cellular respiration. Furthermore, our findings suggest that in the transition from a fermentative to a respiratory metabolism, the diauxic shift represents the stage where major structural and functional reorganizations in mitochondrial metabolism occur. This metabolic transition, initiated at the mitochondria level, is then extended to the rest of the yeast cell.
... Downloaded from https://academic.oup.com/jxb/advance-article-abstract/doi/10.1093/jxb/erz365/5570685 by Univ Western Australia user on 17 September 2019 has also been observed, where precursor proteins contain two MPP recognition motifs (Schmidt et al., 1984;Branda et al., 1999;Kmiec et al., 2012). The presence of a -3R motif has stirred up some controversy due to the fact that the residue that interacts with MPP is -2R as determined by structural studies (Taylor et al., 2001). ...
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.
... The negatively charged pocket of the MPP catalytic site is capable of binding the positively charged arginine residue at the P2 or P3 from the precursor cleavage site [21]. Its catalytic action occurs in a single processing step at a conserved Arginine residue (−2R) position although a two-step processing might be observed, primarily when the presequence contains two MPP recognition motifs [119][120][121]. Presequences can also be subsequently cleaved by distinct peptidases such as intermediate cleavage peptidase 55 kDa (Icp55) [122], Oct1 [123], and the inner membrane peptidase (IMP) [124,125]. ...
... Once in the mitochondrial matrix, the pre-protein has its N-terminal targeting signal cleaved off by the mitochondrial processing peptidase (MPP) [11][12][13]. MPP is a heterodimer formed by the alpha-MPP recognition subunit and the beta-MPP subunit with a catalytic activity [14][15][16]. A small group of mitochondrial proteins requires a second cleavage after MPP processing, a step that is catalyzed by either the inner membrane peptidase (IMP) [17], the intermediate cleaving peptidase 55 (Icp55) [18] or the mitochondrial intermediate peptidase (MIP) [19]. ...
Upon their translocation into the mitochondrial matrix, the N-terminal pre-sequence of nuclear-encoded proteins undergoes cleavage by mitochondrial processing peptidases. Some proteins require more than a single processing step, which involves several peptidases. Down-regulation of the putative Trypanosoma brucei mitochondrial intermediate peptidase (MIP) homolog by RNAi renders the cells unable to grow after 48 hours of induction. Ablation of MIP results in the accumulation of the precursor of the trypanosomatid-specific trCOIV protein, the largest nuclear-encoded subunit of the cytochrome c oxidase complex in this flagellate. However, the trCOIV precursor of the same size accumulates also in trypanosomes in which either alpha or beta subunits of the mitochondrial processing peptidase (MPP) have been depleted. Using a chimeric protein that consists of the N-terminal sequence of a putative subunit of respiratory complex I fused to a yellow fluorescent protein, we assessed the accumulation of the precursor protein in trypanosomes, in which RNAi was induced against the alpha or beta subunits of MPP or MIP. The observed accumulation of precursors indicates MIP depletion affects the activity of the cannonical MPP, or at least one of its subunits.
... As one of the prevailing models in metabolism research, we established the system in budding yeast allowing us a thorough examination of its functionality by testing different growth conditions, genetic mutations, and chemical treatments. For robust labelling of mitochondria independent of their MMP in yeast, we fused GFP to the fungal mitochondrial localisation signal of the F 0 -ATPase subunit 9 (preSU9) of Neurospora crassa, an established MMP independent localisation signal (Merz and Westermann, 2009;Schmidt et al., 1984;Westermann and Neupert, 2000). To generate the second marker, an mCherry protein was fused to the N-terminal localisation sequence of cytochrome C oxidase 4 ((COX4), preCOX4, [1:28]), which is imported into mitochondria proportional to the MMP (Bevis and Glick, 2002;Garipler et al., 2014;Veatch et al., 2009). ...
... As one of the prevailing models in metabolism research, we established the system in budding yeast allowing us a thorough examination of its functionality by testing different growth conditions, genetic mutations, and chemical treatments. For robust labelling of mitochondria independent of their MMP in yeast, we fused GFP to the fungal mitochondrial localisation signal of the F0-ATPase subunit 9 (preSU9) of Neurospora crassa, an established MMP independent localisation signal (Merz and Westermann, 2009;Schmidt et al., 1984;. We genetically coupled this marker to a mCherry protein fused to the N-terminal localisation sequence of cytochrome C oxidase 4 ((COX4), preCOX4, [1:28]), which is imported into mitochondria proportional to the MMP (Bevis and Glick, 2002;Garipler et al., 2014;Veatch et al., 2009). ...
Mitochondria assemble into flexible networks. Here we present a simple method for the simultaneous quantification of mitochondrial membrane potential and network morphology that is based on computational co-localisation analysis of differentially imported fluorescent marker proteins. Established in, but not restricted to, Saccharomyces cerevisiae, MitoLoc reproducibly measures changes in membrane potential induced by the uncoupling agent CCCP, by oxidative stress, in respiratory deficient cells, and in ∆fzo1, ∆ref2, and ∆dnm1 mutants that possess fission and fusion defects. In combination with super-resolution images, MitoLoc uses 3D reconstruction to calculate six geometrical classifiers which differentiate network morphologies in ∆fzo1, ∆ref2, and ∆dnm1 mutants, under oxidative stress and in cells lacking mtDNA, even when the network is fragmented to a similar extent. We find that mitochondrial fission and a decline in membrane potential do regularly, but not necessarily, co-occur. MitoLoc hence simplifies the measurement of mitochondrial membrane potential in parallel to detect morphological changes in mitochondrial networks. Marker plasmid open-source software as well as the mathematical procedures are made openly available.
... Partially cleaved forms of yeast cytochrome b2 (29, 32) (a soluble enzyme of the intermembrane space), cytochrome c~ of yeast (12,29) and Neurospora (36), Fe/S protein of ubiquinol-cytochrome c reductase (14) (proteins located in the inner membrane and exposed to the intermembrane space), and ATPase subunit 9 (37) (inner membrane protein) have been demonstrated in vitro and in vivo. These precursors are first cleaved within the leader to an intermediate form by a matrix-located protease, then cleaved again by a protease located either in the matrix (37) or in the intermembrane space (29,32). The twostep processing which has been described for the precursors of yeast cytochrome b2 and the Fe/S protein of Neurospora has been shown to be important in determining the subrnitochondrial localization of these enzymes. ...
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.
... Transfer into and across the inner membrane requires the electrical potential (A"P) across the inner membrane . The presequences are proteolytically cleaved by the processing peptidase of the mitochondrial matrix (B6hni et al., 1980Conboy et al., 1982;McAda and Douglas, 1982;Miura et al., 1982;Schmidt et al., 1984;Hawlitschek et al., 1988). Several precursors destined for the intermembrane space or the outside of the inner membrane are retranslocated from the matrix back across the inner membrane (Hartl et al., 1986(Hartl et al., , 1987. ...
The precursor of porin, a mitochondrial outer membrane protein, competes for the import of precursors destined for the three other mitochondrial compartments, including the Fe/S protein of the bc1-complex (intermembrane space), the ADP/ATP carrier (inner membrane), subunit 9 of the F0-ATPase (inner membrane), and subunit beta of the F1-ATPase (matrix). Competition occurs at the level of a common site at which precursors are inserted into the outer membrane. Protease-sensitive binding sites, which act before the common insertion site, appear to be responsible for the specificity and selectivity of mitochondrial protein uptake. We suggest that distinct receptor proteins on the mitochondrial surface specifically recognize precursor proteins and transfer them to a general insertion protein component (GIP) in the outer membrane. Beyond GIP, the import pathways diverge, either to the outer membrane or to translocation contact-sites, and then subsequently to the other mitochondrial compartments.
... The supernatants and pellets were separated and kept on ice until enzyme activities were determined. Adenylate kinase (AK) activity was measured indirectly by following the NADH decrease at 340 nm (37°C, 3 min) [22], whereas monoamine oxidase (MAO) activity was measured by following the decrease of A360, caused by the oxidation of kynuramine, for 10 min at 37°C [23]. Furthermore, fumarase (FUM) activity was assayed by monitoring the increase in A250 caused by the conversion of L-malate into fumarate [24]. ...
The effects of the glycoalkaloids α-solanine, α-chaconine and α-tomatine on different cell types were studied in order to investigate the membrane action of these compounds. Hemolysis of erythrocytes was compared to 6-carboxyfluorescein leakage from both ghosts and erythrocyte lipid vesicles, whereas leakage of enzymes from mitochondria and the apical and baso-lateral side of Caco-2 cells was determined. Furthermore, the effects of glycoalkaloids on the gap-junctional communication between Caco-2 cells was studied. From these experiments, it was found that glycoalkaloids specifically induced membrane disruptive effects of cholesterol containing membranes as was previously reported in model membrane studies. In addition, α-chaconine was found to selectively decrease gap-junctional intercellular communication. Furthermore, the glycoalkaloids were more potent in permeabilizing the outer membrane of mitochondria compared to digitonin at the low concentrations used.
... The media used in the above assays were adjusted to 0.2% Triton X-100 to obtain maximal enzyme activities in muscle homogenates and mitochondrial fractions. Total ATPase activities in suspensions of intact mitochondria were measured as described previously [46,49]. Care was taken to avoid detergent contamination and no Triton X-100 was added. ...
... Whether the absence of i-MDH in yeast represents differences in processing of species specific p-MDH or simply reflects the difficulty in detection of yeast i-MDH is not clear. Although the leader peptides of two matrix localised and three membrane-bound proteins processed in two steps share a common three-amino acid motif (Hendrick et al., 1989) this motif is not found in the twice cleaved subunit 9 of Neurosporu crmm F,/F,-ATPase (Schmidt et al., 1984;Hawlitschek et al., 1988) consistent with the apparent overall lack of sequence specificity of the mitochondrial matrix proteases. These observations stress that the degree of two-step processing in mitochondria is unknown and that the need to establish simple procedures for the detection of these processing events are needed. ...
Mitochondrial preornithine transcarbamylase (p-OTC) and premalate dehydrogenase (p-MDH) are the only two matrix-located preproteins so far identified for which the proteolytic processing in vitro requires the formation of genuine processinw intermediates, i-OTC and i-MDH, respectively. To establish the processing of other preproteins during import with respect to the two-step processing of p-PTC and p-MDH, the chelators EDTA and 1,10-phenanthroline were used to study the import and processing of rat prechaperonin 60 (p-cpn60) and p-OTC by mitochondria from four cpn60-containing organs. We found no evidence for a secondary processing step in the maturation of p-cpn60, but a clear requirement for two-step processing of p-OTC, even in three organs which do not contain ornithine transcarbamylase. The metal-ion requirement of the p-OTC processing activities in the organelle is consistent with the proposition that the mitochondrial processing protease (MPP) and mitochondrial intermediate peptidase (MIP) activities defined in vitro [Kalousek, F., Hendrick, J. P. & Rosenberg, L. E. (1988) Proc. Natl Acad. Sci. USA 85, 7536–7540] are responsible for precursor processing in vivo. The authenticity of two-step processing in vivo was, furthermore, established by demonstrating that i-OTC accumulates to high levels in Spodoptora frugiperda insect cells supplemented with MnCl2. The inability of the insest cells to process p-OTC fully is not a characteristic of cells grown in culture since cultured rat hepatoma cells process p-OTC fully processed m-OTC. Finally, we find that the import and processing of p-cpn60 and p-OTC is inhibited in an identical fashion by presequence–bovine-serum-qlbumin conjugates. The differenses in proteolytic maturation between p-cpn60 and p-OTC are therefore not likely to result from different import pathways as the two precursors compete for common components of the import apparatus.
... In yeast, the mitochondrial processing peptidase (MPP) is responsible for processing of the majority of mitochondrial precursors, with a fraction of them undergoing a second step of processing, carried out by either metalloproteases (Oct1/MIP, Icp55) or serine proteases (Imp, Pcp1) (reviewed in Teixeira and Glaser, 2012). Unusual two-step processing in which both processing steps are carried out by MPP has been reported for yeast and human frataxin (Branda et al., 1999) and for Neurospora crassa mitochondrial ATP synthase subunit 9 (Schmidt et al., 1984). ...
Targeting of the majority of proteins to mitochondria requires an N-terminal extension designated presequence. Upon import through the mitochondrial inner membrane the presequence is cleaved off in a reaction termed processing. Most of our knowledge regarding mitochondrial processing comes from analyses performed in Saccharomyces cerevisiae. In yeast as much as 75% of mitochondrially targeted proteins undergo processing. Mitochondrial protein processing in plants is believed to be similarly common, but its understanding is only fragmentary.Here we analyzed processing of the precursor of an ATP-dependent metalloprotease, AtFtsH4, in plant mitochondria. As most mitochondrial proteins, the AtFtsH4 precursor is synthesized in the cytosol and then imported to mitochondria. Upon the import, the presequence of AtFtsH4 is cleaved off in two independent steps. We show that both processing steps are carried out in the matrix by the mitochondrial processing peptidase (MPP). This is the first report on two-step MPP-dependent processing in plant mitochondria. These results broaden our knowledge about mechanism of action of plant MPP and mitochondrial precursors maturation in plants.
Introducing nitrogen fixation (nif ) genes into eukaryotic genomes and targeting Nif components to mitochondria or chloroplasts is a promising strategy for engineering nitrogen-fixing plants. A prerequisite for achieving nitrogen fixation in crops is stable and stoichiometric expression of each component in organelles. Previously, we designed a polyprotein-based nitrogenase system depending on Tobacco Etch Virus protease (TEVp) to release functional Nif components from five polyproteins. Although this system satisfies the demand for specific expression ratios of Nif components in Escherichia coli, we encountered issues with TEVp cleavage of polyproteins targeted to yeast mitochondria. To overcome this obstacle, a version of the Nif polyprotein system was constructed by replacing TEVp cleavage sites with minimal peptide sequences, identified by knowledge-based engineering, that are susceptible to cleavage by the endogenous mitochondrial-processing peptidase. This replacement not only further reduces the number of genes required, but also prevents potential precleavage of polyproteins outside the target organelle. This version of the polyprotein-based nitrogenase system achieved levels of nitrogenase activity in E. coli, comparable to those observed with the TEVp-based polyprotein nitrogenase system. When applied to yeast mitochondria, stable and balanced expression of Nif components was realized. This strategy has potential advantages, not only for transferring nitrogen fixation to eukaryotic cells, but also for the engineering of other metabolic pathways that require mitochondrial compartmentalization.
Mitochondria are double-membraned organelles found in eukaryotic cells. The integrity of mitochondrial structure and function determines cell destiny. Mitochondria are also the “energy factories of cells.” The production of energy is accompanied by reactive oxygen species (ROS) generation. Generally, the production and consumption of ROS maintains a balance in cells. Ozone is a highly oxidizing, harmful substance in ground-level atmosphere. Ozone inhalation causes oxidative injury owing to the generation of ROS, resulting in mitochondrial oxidative stress overload. Oxidative damage to the mitochondria induces a vicious cycle of ROS production which might destroy mitochondrial DNA and mitochondrial structure and function in cells. ROS can alter the phosphorylation of various signaling molecules, triggering a series of downstream signaling pathway reactions. These include inflammatory responses, pyroptosis, autophagy, and apoptosis. Changes involving these molecular mechanisms may be related to the occurrence of disease. According to numerous epidemiological investigations, ozone exposure induces respiratory, cardiovascular, and nervous system diseases in humans. In addition, these systems require large quantities of energy. Hence, the mitochondrial damage caused by ozone may act as a bridge between human diseases. However, the specific molecular mechanisms involved require further investigation. This review discusses our understanding of the structure and function of mitochondria the mechanisms underlying ozone-induced mitochondrial damage.
The cyt-20-1 mutant of Neurospora crassa is a temperature-sensitive, cytochrome b- and aa3-deficient strain that is severely deficient in both mitochondrial and cytosolic protein synthesis (R.A. Collins, H. Bertrand, R.J. LaPolla, and A.M. Lambowitz, Mol. Gen. Genet. 177:73-84, 1979). We cloned the cyt-20+ gene by complementation of the cyt-20-1 mutation and found that it contains a 1,093-amino-acid open reading frame (ORF) that encodes both the cytosolic and mitochondrial valyl-tRNA synthetases (vaIRSs). A second mutation, un-3, which is allelic with cyt-20-1, also results in temperature-sensitive growth, but not in gross deficiencies in cytochromes b and aa3 or protein synthesis. The un-3 mutant had also been reported to have pleiotropic defects in cellular transport process, resulting in resistance to amino acid analogs (M.S. Kappy and R.L. Metzenberg, J. Bacteriol. 94:1629-1637, 1967), but this resistance phenotype is separable from the temperature sensitivity in crosses and may result from a mutation in a different gene. The 1,093-amino-acid ORF encoding vaIRSs is the site of missense mutations resulting in temperature sensitivity in both cyt-20-1 and un-3 and is required for the transformation of both mutants. The opposite strand of the cyt-20 gene encodes an overlapping ORF of 532 amino acids, which may also be functional but is not required for transformation of either mutant. The cyt-20-1 mutation in the vaIRS ORF results in severe deficiencies of both mitochondrial and cytosolic vaIRS activities, whereas the un-3 mutation does not appear to result in a deficiency of these activities or of mitochondrial or cytosolic protein synthesis sufficient to account for its temperature-sensitive growth. The phenotype of the un-3 mutant raises the possibility that the vaIRS ORF has a second function in addition to protein synthesis.
The biogenesis of cytochrome c1 involves a number of steps including: synthesis as a precursor with a bipartite signal sequence, transfer across the outer and inner mitochondrial membranes, removal of the first part of the presequence in the matrix, reexport to the outer surface of the inner membrane, covalent addition of heme, and removal of the remainder of the presequence. In this report we have focused on the steps of heme addition, catalyzed by cytochrome c1 heme lyase, and of proteolytic processing during cytochrome c1 import into mitochondria. Following translocation from the matrix side to the intermembrane-space side of the inner membrane, apocytochrome c1 forms a complex with cytochrome c1 heme lyase, and then holocytochrome c1 formation occurs. Holocytochrome c1 formation can also be observed in detergent-solubilized preparations of mitochondria, but only after apocytochrome c1 has first interacted with cytochrome c1 heme lyase to produce this complex. Heme linkage takes place on the intermembrane-space side of the inner mitochondrial membrane and is dependent on NADH plus a cytosolic cofactor that can be replaced by flavin nucleotides. NADH and FMN appear to be necessary for reduction of heme prior to its linkage to apocytochrome c1. The second proteolytic processing of cytochrome c1 does not take place unless the covalent linkage of heme to apocytochrome c1 precedes it. On the other hand, the cytochrome c1 heme lyase reaction itself does not require that processing of the cytochrome c1 precursor to intermediate size cytochrome c1 takes place first. In conclusion, cytochrome c1 heme lyase catalyzes an essential step in the import pathway of cytochrome c1, but it is not involved in the transmembrane movement of the precursor polypeptide. This is in contrast to the case for cytochrome c in which heme addition is coupled to its transport directly across the outer membrane into the intermembrane space.
The precursor protein of pig mitochondrial aspartate aminotransferase (pre-mAspAT) contains a 29-residue presequence (Joh, T., Nomiyama, H., Maeda, S., Shimada, K., and Morino, Y. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1–5). Pre-mAspAT produced in an in vitro transcription and translation system was avidly imported into pig and rat liver mitochondria to be processed to the mature form of the enzyme. The pre-mAspAT was also processed to the mature form upon incubation with mitochondrial extracts. We synthesized precursor proteins with alterations within the presequence and compared quantitatively the effects of these mutations on the rates of both import and processing. Single and multiple substitutions of four basic residues with neutral amino acids at positions 5, 8, 18, and 28 showed that each residue contributes differentially to import and processing. Substitutions of His⁵ and Arg⁸ with glycines abolished the import activity but did not appreciably affect the rate of processing. Substitution of Arg²⁸ with leucine at the position adjacent to the cleavage site seriously impaired the processing without appreciably affecting the rate of import. Analysis of deletions revealed that the amino-terminal region from position 2 to 8 was essential for both the import and processing. Thus the positive charges in the amino-terminal region are critical for import while the amino-terminal peptide segment and the cleavage site region appear to be requisite for recognition by a processing protease.
Mitochondrial Targeting Sequences (MTSs) are responsible for trafficking nuclear-encoded proteins into mitochondria. Once entering the mitochondria, the MTS is recognized and cleaved off. Some MTSs are long and undergo two-step processing, as in the case of the human frataxin (FXN) protein (80aa), implicated in Friedreich's ataxia (FA). Therefore, we chose the FXN protein to examine whether nuclear-encoded mitochondrial proteins can efficiently be targeted via a heterologous MTS (hMTS) and deliver a functional protein into mitochondria.
The biogenesis of mitochondrial proteins requires two genetic systems. Less than 5% of mitochondrial proteins are encoded by mitochondrial genes and are synthesized within the organelle. The vast majority of mitochondrial proteins are encoded by nuclear genes and are synthesized as precursor proteins on cytosolic polysomes (Douglas et al., 1986; Rosenberg et al, 1987; Attardi and Schatz, 1988; Pfanner et al., 1988a; Hartl et al., 1989; Pfanner and Neupert, 1989; Lonsdale and Grienenberger, 1992) (Fig. 1). Therefore, several hundred different proteins have to be imported into mitochondria. The transport of precursor proteins into mitochondria comprises a complex series of steps, including targeting, membrane translocation, and assembly of the proteins.
A new strategy for the controlled genetic manipulation of mitochondrial gene products is described, focusing on subunits of the mitochondrial ATP synthase (mtATPase) complex of Saccharomyces cerevisiae. Subunit 8 is an F0-sector subunit 48 amino acids long and is normally a product of the mitochondrial aap1 gene. An imported version of subunit 8 has been produced using an artificial nuclear gene. Import was mediated in yeast cells by use of an expression vector which directed the production of a precursor protein consisting of an N-terminal cleavable transit peptide, derived from the nuclearly encoded mtATPase subunit 9 from Neurospora crassa, fused to subunit 8. The imported version of subunit 8 assembles with the mitochondrially encoded subunits 6 and 9 in yeast to produce a functional F0-sector. Parallel experiments are in progress with a version of yeast subunit 9 (76 amino acids long) specified by an artificial nuclear gene. These studies open the way to a directed mutagenesis approach to the analysis of mitochondrially encoded membrane-associated proteins of mitochondrial ATP synthase.
Several thousand different polypeptides are synthesized within an eukaryotic cell. In general, protein synthesis takes place in the cytosol, but many proteins have their functional location in subcellular compartments that are separated from the cytosol by at least one membrane. Certain proteins, for example, those residing in the lumen of chloroplast thylakoids, have to be translocated across as many as three bilayers to reach their destination. Others, for example, proteins exerting transport functions, become integrated into membranes, often in arrangements spanning the bilayer many times. How are proteins targeted to the correct membrane compartment? What are the signals involved, and how can polypeptides, i.e. macromolecules containing many hydrophilic groups, penetrate lipid bilayers at all? By describing the main principles of protein translocation across biological membranes I shall try to answer some of these basic questions in molecular cell biology.
The majority of mitochondrial proteins are encoded by nuclear genes and are imported into the organelle after being synthesized on cytoplasmic ribosomes. During the last years our knowledge of the different stages of this import process of proteins into mitochondria has increased greatly.
A 2.3-kb BamHI-KpnI fragment was isolated from a partial genomic library and shown by nucleotide sequence analysis to contain the entire coding region of the gene encoding the beta subunit of the Blastocladiella mitochondrial processing peptidase (beta-MPP). The predicted beta-MPP protein has 465 amino acids and a calculated molecular mass of 50.8 kDa, S1 nuclease protection assays revealed an intron, 209 bp in size, interrupting the coding region between the putative signal sequence and the mature protein. Northern blot analysis showed that beta-MPP mRNA levels decrease significantly during B. emersonii sporulation, reaching basal levels in the zoospore stage, The amount of beta-MPP protein, determined in Western blots, unlike its mRNA, does not vary significantly throughout the fungal life cycle.
The dominant paradigm of current biological thought supposes that all the characteristics of living organisms are caused ultimately by the properties of the proteins that they contain. The extraordinary diversity of living organisms is mirrored by the plethora of different proteins that exist and by the complexity of the interactions that these proteins undergo. The large size and polymeric nature of protein molecules are responsible for the abundance of the reactions and structures that they create and control. This chapter distils some basic principles of protein targeting from the current literature and discusses selected examples that illustrate these principles. Most research on protein targeting has employed systems derived from animals and microorganisms, but what little has been carried out with plant cells suggests that the same principles apply to them also. Indeed, one of the most striking features of the understanding of protein targeting is the universality of its mechanisms, even between prokaryotes and eukaryotes. Finally, the chapter describes the problems and possibilities presented by this field of research.
Mitochondria are the only organelles in animal and fungal cells with their own genome and the machinery for its expression. Despite this fact, the majority of mitochondrial proteins are coded by the nuclear genome and synthesized on cytoplasmic ribosomes (1). These proteins must be delivered to their functional location in outer membrane, inner membrane, intermembrane space, or matrix (Figure 8.1). Therefore, central problems in mitochondrial biogenesis are: 1) How are nuclear-coded mitochondrial proteins guided to their correct location in the mitochondrion? 2) How is the insertion and/or transfer of these proteins into or across the mitochondrial membrane(s) accomplished? 3) How do imported proteins acquire their final functional characteristics?
N-Acetylglutamate kinase (AGK) and N-acetyl-gamma-glutamyl-phosphate reductase (AGPR) function as two separate mitochondrial enzymes, but are encoded by a single nuclear gene in several fungi. The Neurospora crassa arg-6 gene encoding these enzymes has been cloned and sequenced, and the enzymes responsible for processing the polyprotein precursor have been identified. The 871-amino acid precursor contains a normal N-terminal mitochondrial targeting sequence, an internal connecting region (approximately 200 amino acids) upstream of the distal reductase domain, and coding regions with N-terminal amino acid sequences identical with those of purified N-acetylglutamate kinase and N-acetyl-gamma-glutamyl-phosphate reductase. Sequence comparisons of the coding regions indicate high levels of conservation between prokaryotic and fungal proteins. Regions suggesting ancestral relationships to N-acetylglutamate synthase and aspartate beta-semialdehyde dehydrogenase have been identified. Both the N-terminal targeting sequence and the connecting region contain consensus sites for cleavage by the mitochondrial processing peptidase and processing enhancing protein. In vitro processing assays with intact mitochondria, solubilized mitochondria, and purified enzymes have shown that the mitochondrial processing peptidase and processing enhancing protein cleave not only the N-terminal mitochondrial targeting sequence but also process the polyprotein precursor into the two mature enzymes.
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 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.
The biogenesis and functionality of mitochondria and chloroplasts depend on the constant turnover of their proteins. The majority of mitochondrial and chloroplastic proteins are imported as precursors via their N-terminal targeting peptides. After import, the targeting peptides are cleaved off and degraded. Recent work has elucidated a pathway involved in the degradation of targeting peptides in mitochondria and chloroplasts, with two proteolytic components: the presequence protease (PreP) and the organellar oligopeptidase (OOP). PreP and OOP are specialized in degrading peptides of different lengths, with the substrate restriction being dictated by the structure of their proteolytic cavities. The importance of the intraorganellar peptide degradation is highlighted by the fact that elimination of both oligopeptidases affects growth and development of Arabidopsis thaliana.
The identification of quite a number of protein components involved in mitochondrial protein import has contributed significantly to understand the mechanism of this complex process. Mitochondrial preprotein import is a multi-step process, which is facilitated by the sequential and coordinated action of two separate import machineries located in the outer and inner mitochondrial membrane (Tom and Tim complex). The Tom machinery translocates the charged presequences of matrix targeted preproteins across the outer membrane but not the entire protein. Preproteins destined for the outer membrane and the intermembrane space can be imported and inserted by the Tom machinery independently of the inner membrane. The driving forces for these reactions are unknown; the free energies of membrane insertion and membrane association and folding could represent such forces. From the trans-site, the matrix targeting sequence is passed on to the inner membrane complex (Tim). The components on the inner membrane, which receive the presequence and probably initiate the transfer into the matrix remain to be identified.
The view that the inner membrane possesses a structurally independent protein import system has been substantiated by the recent identification of three mitochondrial inner membrane proteins, which are essential components of the transport machinery. This chapter focuses on the properties of these translocase of the mitochondrial inner membrane (Tim)-proteins. The identification of the Tim-proteins now opens a broad area for characterization of the import apparatus. This will include the topology of the Tims, their mode of interaction with preproteins, and the specificity of the transport machinery. Of particular importance will be the analysis of the interaction of the Tims with each other and with mt-Hsp70 and partner proteins, as well as the identification of putative further components required for inner membrane transport. The process of mitochondrial protein uptake appears to be unidirectional and irreversible, that is, an imported protein is unable to move back into the cytosol. The conformational changes accompanying the import, folding, and assembly of proteins are by themselves usually not sufficient to drive an unidirectional transport. This is particularly obvious in the case ofprotein translocation across the mitochondrial inner membrane. The supply of two external energy sources, ATP and the membrane potential AY across the inner membrane, is needed to drive protein import. The chapter discusses that with some preproteins short segments may move back across the inner membrane when the function of the matrix Hsp70 is strongly impaired—for example, by ATP-depletion of the matrix, supporting the view of an energy-driven unidirectional transport.
Mitochondrial protein quality control incorporates anelaborate network of chaperones and proteases that survey the organelle for misfolded or unfolded proteins and toxic aggregates.Repair of misfolded or aggregated protein and proteolytic removal of irreversibly damaged proteins arecarried out by the mitochondrial protein quality control system. Initial maturation and folding of the nuclear or mitochondrial-encoded mitochondrial proteins aremediated by processing peptidases and chaperones that interact with the protein translocation machinery. Mitochondrial proteins are subjected to cumulative oxidative damage. Thus, impairment ofquality control processes maycause mitochondrial dysfunction. Ageing has been associated with a marked decline in the effectiveness of mitochondrial protein quality control. Here, we present an overview of the chaperones and proteases involved in the initial folding and maturation of new, incoming precursor molecules, and the subsequent repair and removal of oxidized aggregated proteins. In addition, we highlight the link between mitochondrial protein quality control mechanisms andthe ageing process. This article is part of a Special Issue entitled: Protein Modifications.
Subunit 8 is a small integral membrane protein of the proton-translocating F0 sector of the mitochondrial ATP synthase complex. We here review our current understanding of the structure, expression and membrane integration of this protein, which is naturally encoded by the mitochondrial aapl gene in bakers’ yeast Saccharomyces cerevisiae. Genetic, biochemical and immunological analyses of yeast mutants deficient in subunit 8 production have begun to reveal the role of subunit 8 in the assembly and function of the mitochondrial ATPase complex. A recent major advance has been the recoding of the gene encoding subunit 8 to achieve its relocation to the nucleus such that nuclearly encoded subunit 8 can be demonstrated to functionally assemble into the mitochondrial ATPase complex. Further, the expression of subunit 8 in vitro, in the form of a chimaeric precursor bearing an N-terminal cleavable presequence, has permitted study of the import of the protein into isolated mitochondria and its assembly into the enzyme complex. The powerful combination of in vivo and in vitro approaches has now led to the systematic manipulation of subunit 8 using site-directed mutagenesis in order to gain further insight into its structure and function.
Mitochondrial processing peptidase, a metalloendopeptidase consisting of α- and β-subunits, specifically recognizes a large
variety of mitochondrial precursor proteins and cleaves off amino-terminal extension peptides. The α-subunit has a characteristic
glycine-rich segment in the middle portion. To elucidate the role of the region in processing functions of the enzyme, deletion
or site-directed mutations were introduced, and effects on kinetic parameters and substrate binding of the enzyme were analyzed.
Deletion of three residues of the region, Phe289 to Ala291, led to a dramatic reduction in processing activity to practically zero. Mutation of Phe289, Lys296, and Met298 to alanine resulted in a decrease in the activity, but these mutations had no apparent effect on interactions between the
two subunits, indicating that reduction in processing activity is not due to structural disruption at the interface interacting
with the β-subunit. Although the mutant enzymes, Phe289Ala, Lys296Ala, and Met298Ala, had an approximate 10-fold less affinity
for substrate peptides than did that of the wild type, the deletion mutant, Δ289–291, showed an extremely low affinity. Thus,
shortening of the glycine-rich stretch led to a dramatic reduction of interaction between the enzyme and substrate peptides
and cleavage reaction, whereas mutation of each amino acid in this region seemed to affect primarily the cleavage reaction.
Depending on the organism, mitochondria consist approximately of 500-1,400 different proteins. By far most of these proteins are encoded by nuclear genes and synthesized on cytosolic ribosomes. Targeting signals direct these proteins into mitochondria and there to their respective subcompartment: the outer membrane, the intermembrane space (IMS), the inner membrane, and the matrix. Membrane-embedded translocation complexes allow the translocation of proteins across and, in the case of membrane proteins, the insertion into mitochondrial membranes. A small number of proteins are encoded by the mitochondrial genome: Most mitochondrial translation products represent hydrophobic proteins of the inner membrane which-together with many nuclear-encoded proteins-form the respiratory chain complexes. This chapter gives an overview on the mitochondrial protein translocases and the mechanisms by which they drive the transport and assembly of mitochondrial proteins.
We have analyzed how translocation intermediates of imported mitochondrial precursor proteins, which span contact sites, interact with the mitochondrial membranes. F1-ATPase subunit β(F1β) was trapped at contact sites by importing it into Neurospora mitochondria in the presence of low levels of nucleoside triphosphates. This F1β translocation intermediate could be extracted from the membranes by treatment with protein denaturants such as alkaline pH or urea. By performing import at low temperatures, the ADP/ATP carrier was accumulated in contact sites of Neurospora mitochondria and cytochrome b2 in contact sites of yeast mitochondria. These translocation intermediates were also extractable from the membranes at alkaline pH. Thus, translocation of precursor proteins across mitochondrial membranes seems to occur through an environment which is accessible to aqueous perturbants. We propose that proteinaceous structures are essential components of a translocation apparatus present in contact sites.
We have determined the nucleotide sequence of the Aspergillus nidulans nuclear gene oliC31, which encodes subunit 9 of mitochondrial ATP synthase. The open reading frame contains no introns and specifies a predicted protein of 143 amino acids comprising a pre-sequence of 62 residues and a mature protein of 81 residues. The amino acid homology with the equivalent Neurospora crassa protein is 50% for the pre-sequence and 80% for the mature protein. A comparison with this and other imported mitochondrial proteins has revealed conserved regions which may be important for transport or subsequent processing. Multiple transcription initiation and polyadenylation sites have been identified. The promoter region of the oliC31 gene is characterised by long pyrimidine-rich tracts preceding the transcription initiation sites.
The protein-processing system of spinach leaf mitochondria was investigated using different mitochondrial fractions and in vitro transcribed and translated mitochondrial precursor proteins, the F1β subunit of the F0F1-ATP synthases of Nicotiana plumbaginifolia and Neurospora crassa and the Neurospora Rieske FeS protein. Processing resulted in cleavage of the precursor proteins to the mature size products. The processing activity of spinach leaf mitochondria was found to be located in the mitochondrial membrane fraction. The activity could not be dissociated from the membrane by treatment of the membranes with 100 mM KCl, 4 M urea or at pH 11. The membrane-bound processing activity could not be stimulated by addition of the mitochondrial matrix fraction. These results show that the spinach leaf mitochondrial processing proteinase that catalyzes cleavage of the mitochondrial F1β and Rieske FeS precursors is a membrane-associated protein. These results are in contrast to the results described for yeast, Neurospora and rat liver, where the corresponding processing proteinase was found to be a matrix enzyme. The membrane-bound processing activity of spinach leaf mitochondria was stimulated by the divalent cations Mn2+, Zn2+ and Co2+ and inhibited by the metal chelators orthophenanthroline and EDTA, indicating that the processing proteinase is a metalloproteinase. Western blots of spinach leaf and potato tuber mitochondria incubated with antibodies against Neurospora processing enhancing protein (PEP) showed strong cross-reactivity with a protein of 63 kDa in spinach mitochondria and a protein of 59 kDa in potato mitochondria. Antibodies against Neurospora mitochondrial processing peptidase (MPP) did not cross-react with any spinach or potato mitochondrial proteins.
The rutamycin-sensitive ATPase complex of yeast mitocondria consists of the ATPase, F1, of an easily extractable protein (OSCP) which is concerned with the binding of F1 to the membrane, and of another membrane factor which contains at least four distinct subunit proteins. When glucose-repressed yeast are incubated in a low glucose medium containing cycloheximide and radioactive leucine, label is incorporated into a fraction which forms a precipitable complex with antiserum to the rutamycin-sensitive ATPase. Analysis of the antibody precipitate by gel electrophoresis has revealed that at least four distinct proteins are labeled. The labeled products comigrate with known subunits of the rutamycin-sensitive ATPase and comprise those protein components of the ATPase which are most firmly associated with the membrane. These results indicate that with the exception of F1 and OSCP, which are synthesized in the cytoplasm, the remaining subunits of the ATPase are made by the mitochondrion.
The precursor proteins to the subunits of ubiquinol:cytochrome c reductase (cytochrome bc1 complex) of Neurospora crassa were synthesized in a reticulocyte lysate. These precursors were immunoprecipitated with antibodies prepared against the individual subunits and compared to the mature subunits immunoprecipitated or isolated from mitochondria. Most subunits were synthesized as precursors with larger apparent molecular weights (subunits I, 51,500 versus 50,000; subunit II, 47,500 versus 45,000; subunit IV (cytochrome c1), 38,000 versus 31,000; subunit V (Fe-S protein), 28,000 versus 25,000; subunit VII, 12,000 versus 11,500; subunit VIII, 11,600 versus 11,200). Subunit VI (14,000) was synthesized with the same apparent molecular weight. The post-translational transfer of subunits I, IV, V, and VII was studied in an in vitro system employing reticulocyte lysate and isolated mitochondria. The transfer and proteolytic processing of these precursors was found to be dependent on the mitochondrial membrane potential. In the transfer of cytochrome c1, the proteolytic processing appears to take place in two separate steps via an intermediate both in vivo and in vitro. In vivo, the intermediate form accumulated when cells were kept at 8 degrees C and was chased into mature cytochrome c1 at 25 degrees C. Both processing steps were energy-dependent.
A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
Subunit 9 of mitochondrial ATPase (Su9) is synthesized in reticulocyte lysates programmed with Neurospora poly A-RNA, and in a Neurospora cell free system as a precursor with a higher apparent molecular weight than the mature protein (Mr 16,400 vs. 10,500). The RNA which directs the synthesis of Su9 precursor is associated with free polysomes. The precursor occurs as a high molecular weight aggregate in the postribosomal supernatant of reticulocyte lysates. Transfer in vitro of the precursor into isolated mitochondria is demonstrated. This process includes the correct proteolytic cleavage of the precursor to the mature form. After transfer, the protein acquires the following properties of the assembled subunit: it is resistant to added protease, it is soluble in chloroform/methanol, and it can be immunoprecipitated with antibodies to F1-ATPase. The precursor to Su9 is also detected in intact cells after pulse labeling. Processing in vivo takes place posttranslationally. It is inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). A hypothetical mechanism is discussed for the intracellular transfer of Su9. It entails synthesis on free polysomes, release of the precursor into the cytosol, recognition by a receptor on the mitochondrial surface, and transfer into the inner mitochondrial membrane, which is accompanied by proteolytic cleavage and which depends on an electrical potential across the inner mitochondrial membrane.
Isolated yeast mitochondria were able to take up Neurospora ATPase subunit 9 in vitro although the homologous yeast protein is synthesized within the mitochondria and inserted into the membrane from the matrix side (Tzagoloff, A., and Meagher, P. (1972) J. Biol. Chem. 247, 594-603). The transfer of the protein was dependent on an energized mitochondrial inner membrane. It was accompanied by proteolytic processing of the precursor to the mature protein with the correct NH2 terminus as determined by Edman degradation of the transferred protein. The possibility is discussed that there are common features in the uptake machinery neither specific for one species nor specific for individual precursor proteins in the same species.
Many nuclear-coded mitochondrial proteins are synthesized as larger precursor polypeptides that are proteolytically processed during import into the mitochondrion. This processing appears to be catalyzed by a soluble, metal-dependent protease localized in the mitochondrial matrix. In this report we employ an in vitro system to investigate the role of processing in protein import. Intact Neurospora crassa mitochondria were incubated with radiolabeled precursors in the presence of the chelator o-phenanthroline. Under these conditions, the processing of the precursors of the beta-subunit of F1-ATPase (F1 beta) and subunit 9 of the F0F1-ATPase was strongly inhibited. Protease-mapping studies indicated that import of the precursor proteins into the mitochondria continued in the absence of processing. Upon readdition of divalent metal to the treated mitochondria, the imported precursors were quantitatively converted to their mature forms. This processing of imported precursors occurred in the absence of a mitochondrial membrane potential and was extremely rapid even at 0 degrees C. This suggests that all or part of the polypeptide chain of the imported precursors had been translocated into the matrix location of the processing enzyme. Localization experiments suggested that the precursor to F1 beta is peripherally associated with the mitochondrial membrane while the precursor to subunit 9 appeared to be tightly bound to the membrane. We conclude that proteolytic processing is not necessary for the translocation of precursor proteins across mitochondrial membranes, but rather occurs subsequent to this event. On the basis of these and other results, a hypothetical pathway for the import of F1 beta and subunit 9 is proposed.
The precursor proteins to the subunits of ubiquinol:cytochrome c reductase (cytochrome bc1 complex) of Neurospora crassa were synthesized in a reticulocyte lysate. These precursors were immunoprecipitated with antibodies prepared against the individual subunits and compared to the mature subunits immunoprecipitated or isolated from mitochondria. Most subunits were synthesized as precursors with larger apparent molecular weights (subunits I, 51,500 versus 50,000; subunit II, 47,500 versus 45,000; subunit IV (cytochrome c1), 38,000 versus 31,000; subunit V (Fe-S protein), 28,000 versus 25,000; subunit VII, 12,000 versus 11,500; subunit VIII, 11,600 versus 11,200). Subunit VI (14,000) was synthesized with the same apparent molecular weight. The post-translational transfer of subunits I, IV, V, and VII was studied in an in vitro system employing reticulocyte lysate and isolated mitochondria. The transfer and proteolytic processing of these precursors was found to be dependent on the mitochondrial membrane potential. In the transfer of cytochrome c1, the proteolytic processing appears to take place in two separate steps via an intermediate both in vivo and in vitro. In vivo, the intermediate form accumulated when cells were kept at 8 degrees C and was chased into mature cytochrome c1 at 25 degrees C. Both processing steps were energy-dependent.
The cytoplasmically made subunit V was isolated from enzymically active yeast cytochrome c oxidase and its NH2-terminal amino acid sequence was determined to be (formula; see text) In order to exclude that this NH2 terminus had been generated by proteolysis during the lengthy isolation of the subunit, subunit V was directly immunoprecipitated from yeast cells that had been pulse-labeled with [35S]methionine; radiochemical sequencing revealed methionine at position 12, in agreement with the sequence given above. When the precursor to subunit V was synthesized in vitro in the presence of either [35S]methionine, [3H]leucine, or [3H]histidine and then incubated either with isolated yeast mitochondria or the partially purified matrix protease (Böhni, P. C., Daum, G., and Schatz, G. (1983) J. Biol. Chem. 258, 4937-4943), it was converted to a polypeptide co-migrating with mature subunit V on dodecyl sulfate-polyacrylamide gels. Radiochemical sequence analysis of the processed in vitro product showed that it contained histidine, leucine, and methionine in positions 4, 6, and 12, respectively, exactly as the authentic mature protein. In contrast, the unprocessed precursor contained methionine only at position 9, but not at position 12; thus, the precursor has a NH2 terminus different from the mature polypeptide. Similarly, if the in vitro synthesized cytochrome b2 precursor is incubated with isolated mitochondria, it is converted to a polypeptide which co-migrates with mature cytochrome b2 and, like the latter, contains leucine and methionine in positions 4 and 6, respectively. These data show that isolated yeast mitochondria convert the precursors to polypeptides which have the NH2 terminus of the authentic mature polypeptides. In the case of cytochrome c oxidase subunit V, correct NH2-terminal processing was also demonstrated with the purified matrix protease.
Most mitochondrial proteins are synthesized in the cytoplasm as larger precursor polypeptides which are imported into the organelle in an energy-dependent step. The proteolytic conversion of these precursors to their mature size involves a neutral matrix-located protease which has been purified 100-fold from yeast mitochondria. It cleaves the precursors to several imported proteins of the matrix, the mitochondrial inner membrane and the intermembrane space, but is inactive against all mature mitochondrial proteins or against all nonmitochondrial proteins tested so far. As shown in the subsequent report (Cerletti, N., Böhni, P. C., and Suda, K. (1983) J. Biol. Chem. 258, 4944-4949), processing of the cytochrome c oxidase subunit V precursor with the partially purified protease yielded the correct mature NH2 terminus. Precursors to cytochrome b2 and cytochrome c1 (which are imported into the intermembrane space and the outer face of the inner membrane, respectively) are cleaved to intermediate forms which can also be detected as transient forms in vivo. The protease activity has a pH optimum of 7.5 and is inhibited by 1,10-phenanthroline, EDTA, or nucleoside triphosphates, but not by serine-protease inhibitors or by small peptide inhibitors. Its activity can be restored after chelation by excess Co2+ or Zn2+. The enzyme is coded in the nucleus and is, thus, imported into mitochondria.
The precursor form of Neurospora crassa mitochondrial ADP/ATP carrier synthesized in a cell-free protein-synthesizing system can be imported into isolated mitochondria. If the mitochondrial transmembrane potential is abolished, import does not occur but the precursor binds to the mitochondrial surface. Upon reestablishment of the membrane potential, the bound precursor is imported. This occurs without dissociation of the bound precursor from the mitochondrial surface. We conclude that the binding observed represents an interaction with receptor sites and thus is an early step in the import pathway.
A 3.1-kilobase Bgl II fragment of Saccharomyces cerevisiae carrying the nuclear gene encoding the mitochondrial polypeptide chain elongation factor (EF) Tu has been cloned on pBR327 to yield a chimeric plasmid pYYB. The identification of the gene designated as tufM was based on the cross-hybridization with the Escherichia coli tufB gene, under low stringency conditions. The complete nucleotide sequence of the yeast tufM gene was established together with its 5'- and 3'-flanking regions. The sequence contained 1,311 nucleotides coding for a protein of 437 amino acids with a calculated Mr of 47,980. The nucleotide sequence and the deduced amino acid sequence of tufM were 60% and 66% homologous, respectively, to the corresponding sequences of E. coli tufA, when aligned to obtain the maximal homology. Plasmid YRpYB was then constructed by cloning the 2.5-kilobase EcoRI fragment of pYYB carrying tufM into a yeast cloning vector YRp-7. A mRNA hybridizable with tufM was isolated from the total mRNA of S. cerevisiae D13-1A transformed with YRpYB and translated in the reticulocyte lysate. The mRNA could direct the synthesis of a protein with Mr 48,000, which was immunoprecipitated with an anti-E. coli EF-Tu antibody but not with an antibody against yeast cytoplasmic EF-1 alpha. The results indicate that the tufM gene is a nuclear gene coding for the yeast mitochondrial EF-Tu.
A post-translational processing assay of the precursor to the yeast F1-ATPase subunit has been utilized to examine a mitochondrial endoprotease which cleaves this subunit precursor to the size of a mature subunit. The endoprotease is extracted from purified mitochondria as a soluble complex of Mr = 115,000 which is composed of subunits of lower molecular weight when examined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It exhibits a pH optimum of between pH 7 and 8 and is inactive at pH 6.5 and below. The mitochondrial endoprotease is insensitive to serine esterase inhibitors, but is inhibited by EDTA and o-phenanthroline. Restoration of precursor subunit processing activity in the presence of metal chelators is strictly dependent on excess Co2+ and Mn2+ over other heavy metals examined. These and additional data indicate that this soluble metallo endoprotease is involved in the processing of other cytoplasmically synthesized precursor subunits of the ATPase complex in addition to the subunit 2 precursor. The role of this processing enzyme in the assembly of mitochondrial inner membrane complexes is discussed in light of the current model of mitochondrial biogenesis.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Subunit 9 of mitochondrial ATPase (Su9) is synthesized in reticulocyte lysates programmed with Neurospora poly A-RNA, and in a Neurospora cell free system as a precursor with a higher apparent molecular weight than the mature protein (Mr 16,400 vs. 10,500). The RNA which directs the synthesis of Su9 precursor is associated with free polysomes. The precursor occurs as a high molecular weight aggregate in the postribosomal supernatant of reticulocyte lysates. Transfer in vitro of the precursor into isolated mitochondria is demonstrated. This process includes the correct proteolytic cleavage of the precursor to the mature form. After transfer, the protein acquires the following properties of the assembled subunit: it is resistant to added protease, it is soluble in chloroform/methanol, and it can be immunoprecipitated with antibodies to F1-ATPase. The precursor to Su9 is also detected in intact cells after pulse labeling. Processing in vivo takes place posttranslationally. It is inhibited by the uncoupler carbonylcyanide m- chlorophenylhydrazone (CCCP). A hypothetical mechanism is discussed for the intracellular transfer of Su9. It entails synthesis on free polysomes, release of the precursor into the cytosol, recognition by a receptor on the mitochondrial surface, and transfer into the inner mitochondrial membrane, which is accompanied by proteolytic cleavage and which depends on an electrical potential across the inner mitochondrial membrane.
Transport of apocytochrome c across the outer mitochondrial membrane and conversion to holocytochrome c were studied in vitro. Apocytochrome c was synthesized in a cell-free homogenate from Neurospora crassa. Transfer in vitro was accomplished in a reconstituted system consisting of the postribosomal supernatant of the cell-free homogenate and of isolated and purified mitochondria from Neurospora. The reconstituted system has the following characteristics:
We conclude from these observations that apocytochrome c is transported across the outer mitochondrial membrane via receptor sites. In the presence of the heme analogue deuterohemin, binding to the receptor sites on the cytoplasmic surface of the outer mitochondrial membrane still takes place but translocation does not. The latter step is apparently coupled to the covalent linkage of the heme group. We suggest that the formation of the thioether bonds between apoprotein and heme is catalysed by an enzyme in the intermembrane space and that deuterohemin can compete with protohemin for binding to the enzyme. Finally, the data indicate that it is the heme group and not the porphyrin group which is coupled to the apoprotein.
The major protein of the outer mitochondrial membrane of Neurospora was purified. On dodecylsulfate-containing gels it displayed a single bend with an apparent molecular weight of 31000. reconstitution experiments with artifical lipid bilayers showed that this protein forms pores. Pore conductance was dependent on the voltage across the membrane. The protein inserted into the membrane in an oriented fashion, the membrane current being dependent on the sign of the voltage. Single pore conductance was 5nS, suggesting a diameter of 2nm of the open pore. This mitochondrial protein shows a number of similarities to the outer membrane porins of gram-negative bacteria.
ADP/ATP carrier protein was synthesized in heterologous cell‐free systems programmed with Neurospora poly(A)‐containing RNA and homologous cell‐free systems from Neurospora. The apparent molecular weight of the product obtained in vitro was the same as that of the authentic mitochondrial protein. The primary translation product obtained in reticulocyte lysates starts with formylmethionine when formylated initiator methionyl‐tRNA (fMet‐tRNA f Met ) was present. The product synthesized in vitro was released from the ribosomes into the postribosomal supernatant.
The evidence presented indicates that the ADP/ATP carrier is synthesized as a polypeptide with the same molecular weight as the mature monomeric protein and does not carry an additional sequence.
Rat liver mitochondria were fragmented by a combined technique of swelling, shrinking, and sonication. Fragments of inner membrane were separated by density gradient centrifugation. They differed in several respects: electronmicroscopic appearance, phospholipid and cytochrome contents, electrophoretic behaviour of proteins and enzymatic activities.
Three types of inner membrane fractions were isolated. The first type is characterized by a high activity of metal chelatase, low activities of succinate‐cytochrome c reductase and of glycerolphosphate dehydrogenase, as well as by a high phospholipid content and low contents of cytochromes aa 3 and b.
The second type displays maximal activities of glycerolphosphate dehydrogenase and metal chelatase, but contains relatively little cytochromes and has low succinate‐cytochrome c reductase activity.
The third type exhibits highest succinate‐cytochrome c reductase activity, a high metal chelatase activity and highest cytochrome contents. However, this fraction was low in both glycerolphosphate dehydrogenase activity and phospholipid content. This fraction was also richest in the following enzyme activities: cytochrome oxidase, oligomycin‐sensitive ATPase, proline oxidase, 3‐hydroxybutyrate dehydrogenase and rotenone‐sensitive NADH‐cytochrome c reductase.
Amino acid incorporation in vitro and in vivo in the presence of cycloheximide occurs predominantly into inner membrane fractions from the second type.
These data suggest that the inner membrane is composed of differently organized parts, and that polypeptides synthesized by mitochondrial ribosomes are integrated into specific parts of the inner membrane.
Most mitochondrial polypeptides are synthesized outside the organelle as precursors which are usually larger than the ‘mature’ polypeptides found within mitochondria. The precursors are imported into the mitochondria by a process which is independent of protein synthesis but dependent on high-energy phosphate bonds inside the mitochondria. This mechanism is basically different from that which governs the movement of secretory polypeptides across the membrane of the endoplasmic reticulum.
The F0 part of the ATP synthase complex serves as a proton channel. Recent genetical and biochemical approaches are shedding light on the structure of F0 and the role of its individual subunits.
1. Precipitating antibodies specific for apocytochrome c and holocytochrome c, respectively, were employed to study synthesis and intracellular transport of cytochrome c in Neurospora in vitro. 2. Apocytochrome c as well as holocytochrome c were found to be synthesized in a cell-free homogenate. A precursor product relationship between the two components is suggested by kinetic experiments. 3. Apocytochrome c synthesized in vitro was found in the post-ribosomal fraction and not in the mitochondrial fraction, whereas holocytochrome c synthesized in vitro was mainly detected in the mitochondrial fraction. A precursor product relationship between postribosomal apocytochrome c and mitochondrial holocytochrome c is indicated by the labelling data. In the microsomal fraction both apocytochrome c and holocytochrome c were found in low amounts. Their labeling kinetics do not subbest a precursor role of microsomal apocytochrome c or holocytochrome c. 4. Formation of holocytochrome c from apocytochrome c was observed when postribosomal supernatant containing apocytochrome c synthesized in vitro was incubated with isolated mitochondria, but not when incubated in the absence of mitochondria. The cytochrome c formed under these conditions was detected in the mitochondria. 5. Conversion of labelled apocytochrome c synthesized in vitro to holocytochrome c during incubation of a postribosomal supernatant with isolated mitochondria was inhibited when excess isolated apocytochrome c, but not when holocytochrome c was added. 6. The data presented are interpreted to show that apocytochrome c is synthesized on cytoplasmic ribosomes and released into the supernatant. It is suggested that apocytochrome c migrates to the inner mitochondrial membrane, where the heme group is covalently linked to the apoprotein. The hypothesis is put forward that the concomitant change in conformation leads to trapping of holocytochrome c in the membrane. The problems of permeability of the outer mitochondrial membrane to apocytochrome c and the site and nature of the reaction by which the heme group is linked to the apoprotein are discussed.
Sodium salicylate has been found to serve as a satisfactory fluor for enhanced detection of radioactivity in polyacrylamide gels. The compound is inexpensive, water soluble, penetrates rapidly into gels, and gives linearity and sensitivity comparable to diphenyl oxazole.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Posttranslational transfer of most precursor proteins into mitochondria is dependent on energization of the mitochondria. Experiments were carried out to determine whether the membrane potential or the intramitochondrial ATP is the immediate energy source. Transfer in vitro of precursors to the ADP/ATP carrier and to ATPase subunit 9 into isolated Neurospora mitochondria was investigated. Under conditions where the level of intramitochondrial ATP was high and the membrane potential was dissipated, import and processing of these precursor proteins did not take place. On the other hand, precursors were taken up and processed when the intramitochondrial ATP level was low, but the membrane potential was not dissipated. We conclude that a membrane potential is involved in the import of those mitochondrial precursor proteins which require energy for intracellular translocation.
Mitochondrial porin, the major protein of the outer mitochondrial membrane is synthesized by free cytoplasmic polysomes. The apparent molecular weight of the porin synthesized in homologous or heterologous cell-free systems is the same as that of the mature porin. Transfer in vitro of mitochondrial porin from the cytosolic fraction into the outer membrane of mitochondria could be demonstrated. Before membrane insertion, mitochondrial porin is highly sensitive to added proteinase; afterwards it is strongly protected. Binding of the precursor form to mitochondria occurs at 4 degrees C and appears to precede insertion into the membrane. Unlike transfer of many precursor proteins into or across the inner mitochondrial membrane, assembly of the porin is not dependent on an electrical potential across the inner membrane.
The proteolipid subunit of the mitochondrial ATP synthase from Neurospora crassa is an extremely hydrophobic protein of 81 amino acid residues, which is imported into mitochondria as a precursor of mol. wt. 15 000. The primary structure of the imported form has now been determined by isolating and analyzing cDNA clones of the preproteolipid mRNA. An initial cDNA clone was identified by hybridizing total polyadenylated RNA to pooled cDNA recombinant plasmids from an ordered clone bank and subsequent cell-free translation of hybridization-selected mRNA. Further preproteolipid clones were identified at a frequency of 0.2% by colony filter hybridization. One isolated cDNA represented the major part of the preproteolipid mRNA. The nucleotide sequence showed 243 bases corresponding to the mature proteolipid and, in addition, 178 bases coding for an amino-terminal presequence . Non-coding sequences of 48 bases at the 5' end and of 358 bases at the 3' end plus a poly(A) tail were determined. The long presequence of 66 amino acids is very polar, in contrast to the lipophilic mature proteolipid, and includes 12 basic and no acidic side chains. It is suggested that the presequence is specifically designed to solubilize the proteolipid for post-translational import into the mitochondria.
According to the signal hypothesis, a signal sequence, once having initiated export of a growing protein chain across the rough endoplasmic reticulum, is cleaved from the mature protein at a specific site. It has long been known that some part of the cleavage specificity resides in the last residue of the signal sequence, which invariably is one with a small, uncharged side-chain, but no further specific patterns of amino acids near the point of cleavage have been discovered so far. In this paper, some such patterns, based on a sample of 78 eukaryotic signal sequences, are presented and discussed, and a first attempt at formulating rules for the prediction of cleavage sites is made.
The precursor of rat liver ornithine transcarbamylase (ornithine carbamoyltransferase; carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) (pre-ornithine transcarbamylase), which was synthesized in a reticulocyte lysate cell-free system, was converted to an apparently mature form of the enzyme by isolated rat liver mitochondria. The proteolytic processing involved two steps: (i) conversion of pre-ornithine transcarbamylase (39,400 daltons) to a product of about 37,000 daltons and (ii) further conversion to the apparently mature form of the enzyme (36,00 daltons). When mitochondria were subfractionated by digitonin treatment followed by sonication of a mitoplast fraction, the proteolytic activity catalyzing the first step was recovered mainly in a matrix fraction. Some activity was found in an intermembrane space fraction. The enzyme activity in the matrix fraction has an optimal pH at about 7.5. The activity was inhibited almost completely by 2 mM leupeptin and partly by 2 mM antipain but not significantly by other microbial protease inhibitors or serine protease inhibitors. It was inhibited strongly by 2 mM EDTA, 2 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate, 2 mM p-chloromercuriphenylsulfonate, and 2 mM Hg(CH3COO)2 but not by N-ethylmaleimide or iodoacetamide. These results suggest that pre-ornithine transcarbamylase is first transported into the mitochondrial matrix and converted there to the mature form of the enzyme by a novel neutral protease(s).
Proteolytic processing of the larger precursor of the rat liver mitochondrial matrix enzyme, ornithine transcarbamylase, has been studied in a cell free system using in vitro-synthesized precursor and various submitochondrial fractions. The protease responsible for cleavage to mature-sized enzyme fractionates with matrix marker enzymes. Maximal catalytic activity of this matrix protease requires approximately 0.1 mM Zn2+, a concentration known to be in the physiological range for this divalent cation in intact mitochondria. Certain other divalent metal ions (Co2+ and Mn2+) also stimulate this protease activity, while 1,10-phenanthroline, a divalent metal ion chelator, inhibits the protease. We conclude that proteolytic cleavage by a zinc-dependent protease in the mitochondrial matrix of rat liver is a required step in the conversion of pre-ornithine transcarbamylase to the mature subunit.
- M Teintze
- M Slaughter
- H Weiss
- W Neupert
Teintze, M., Slaughter, M., Weiss, H. & Neupert, W. (1982) J.
- J Kaput
- S Goltz
- G Blobel
Kaput, J., Goltz, S. & Blobel, G. (1982) J. Biol. Chem. 257,
- G Kreil
- G Heijne
- E Schneider
- K Altendorf
Kreil, G. (1981) Annu. Rev. Biochem. 50, 317-381.
34. v. Heijne, G. (1983) Eur. J. Biochem. 133, 17-21.
35. Schneider, E. & Altendorf, K. (1984) Trends Biochem. Sci 9,
36. Nagata, S., Tsunetsugu-Yokota, Y., Naito, A. & Kaziro, Y.
15054-15058.
- C Zwizinski
- W Neupert
Zwizinski, C. & Neupert, W. (1983) J. Biol. Chem. 258,
- L Kanarek
Kanarek, L. &Hill, R. L. (1964) J. Biol. Chem. 239,4202-4206.
- A Tzagoloff
- P Meagher
Tzagoloff, A. & Meagher, P. (1972) J. Bid. Chem. 247,594-603.
(1983) Proc. Natl Acad. Sci. USA 80,6192-6196.
- M Mori
- S Miura
- M Tatibana
- P P Cohen
Mori, M., Miura, S., Tatibana, M. & Cohen, P. P. (1980) Proc.
Natl Acad. Sci. USA 77, 7044 -7048.
- B Schmidt
- B Hennig
- H Kohler
- W Neupert
Schmidt, B., Hennig, B., Kohler, H. & Neupert, W. (1983) J. Biol.
- H Freitag
- W Neupert
- R Benz
Freitag, H., Neupert, W. & Benz, R. (1982) Eur. J. Biochem. 123,
18. Harmey, M. A. & Neupert, W. (1979) FEBSLett. 108,385-389.
- U K Laemmli
Laemmli, U. K. (1970) Nature (Lond.) 227, 680-685.
- E Schneider
- K Altendorf
- S Nagata
- Y Tsunetsugu-Yokota
- A Naito
- Y Kaziro
Schneider, E. & Altendorf, K. (1984) Trends Biochem. Sci 9,
36. Nagata, S., Tsunetsugu-Yokota, Y., Naito, A. & Kaziro, Y.
15054-15058.
- A Tzagoloff
- P Meagher
Tzagoloff, A. & Meagher, P. (1972) J. Bid. Chem. 247,594-603.
(1983) Proc. Natl Acad. Sci. USA 80,6192-6196.