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PubMed2003

The inner membrane protein Mdm33 controls mitochondrial morphology in yeast.

Mitochondrial distribution and morphology depend on MDM33, a Saccharomyces cerevisiae gene encoding a novel protein of the mitochondrial inner membrane. Cells lacking Mdm33 contain ring-shaped, mostly interconnected mitochondria, which are able to form large hollow spheres. On the ultrastructural level, these aberrant organelles display extremely elongated stretches of outer and inner membranes enclosing a very narrow matrix space. Dilated parts of Delta mdm33 mitochondria contain well-developed cristae. Overexpression of Mdm33 leads to growth arrest, aggregation of mitochondria, and generation of aberrant inner membrane structures, including septa, inner membrane fragments, and loss of inner membrane cristae. The MDM33 gene is required for the formation of net-like mitochondria in mutants lacking components of the outer membrane fission machinery, and mitochondrial fusion is required for the formation of extended ring-like mitochondria in cells lacking the MDM33 gene. The Mdm33 protein assembles into an oligomeric complex in the inner membrane where it performs homotypic protein-protein interactions. Our results indicate that Mdm33 plays a distinct role in the mitochondrial inner membrane to control mitochondrial morphology. We propose that Mdm33 is involved in fission of the mitochondrial inner membrane.

Amino Acid Sequence↗

PubMed2006

Mitochondrial preprotein translocases as dynamic molecular machines.

Proteomic studies have demonstrated that yeast mitochondria contain roughly 1000 different proteins. Only eight of these proteins are encoded by the mitochondrial genome and are synthesized on mitochondrial ribosomes. The remaining 99% of mitochondrial precursors are encoded within the nuclear genome and after their synthesis on cytosolic ribosomes must be imported into the organelle. Targeting of these proteins to mitochondria and their import into one of the four mitochondrial subcompartments--outer membrane, intermembrane space (IMS), inner membrane and matrix--requires various membrane-embedded protein translocases, as well as numerous chaperones and cochaperones in the aqueous compartments. During the last years, several novel protein components involved in the import and assembly of mitochondrial proteins have been identified. The picture that emerges from these exciting new findings is that of highly dynamic import machineries, rather than of regulated, but static protein complexes. In this review, we will give an overview on the recent progress in our understanding of mitochondrial protein import. We will focus on the presequence translocase of the inner mitochondrial membrane, the TIM23 complex and the presequence translocase-associated motor, the PAM complex. These two molecular machineries mediate the multistep import of preproteins with cleavable N-terminal signal sequences into the matrix or inner membrane of mitochondria.

Fungal Proteins↗

PubMed2006

The alpha-subunit of Leishmania F1 ATP synthase hydrolyzes ATP in presence of tRNA.

Import of tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania requires the tRNA-dependent hydrolysis of ATP leading to the generation of membrane potential through the pumping of protons. Subunit RIC1 of the inner membrane RNA import complex is a bi-functional protein that is identical to the alpha-subunit of F1F0 ATP synthase and specifically binds to a subset (Type I) of importable tRNAs. We show that recombinant, purified RIC1 is a Type I tRNA-dependent ATP hydrolase. The activity was insensitive to oligomycin, sensitive to mutations within the import signal of the tRNA, and required the cooperative interaction between the ATP-binding and C-terminal domains of RIC1. The ATPase activity of the intact complex was inhibited by anti-RIC1 antibody, while knockdown of RIC1 in Leishmania tropica resulted in deficiency of the tRNA-dependent ATPase activity of the mitochondrial inner membrane. Moreover, RIC1 knockdown extracts failed to generate a membrane potential across reconstituted proteoliposomes, as shown by a rhodamine 123 uptake assay, but activity was restored by adding back purified RIC1. These observations identify RIC1 as a novel form of the F1 ATP synthase alpha-subunit that acts as the major energy transducer for tRNA import.

Adenosine Triphosphate↗

PubMed1996

Regulated protein degradation in mitochondria.

Various adenosine triphosphate (ATP)-dependent proteases were identified within mitochondria which mediate selective mitochondrial protein degradation and fulfill crucial functions in mitochondrial biogenesis. The matrix-localized PIM1 protease, a homologue of the Escherichia coli Lon protease, is required for respiration and maintenance of mitochondrial genome integrity. Degradation of non-native polypeptides by PIM1 protease depends on the chaperone activity of the mitochondrial Hsp70 system, posing intriguing questions about the relation between the proteolytic system and the folding machinery in mitochondria. The mitochondrial inner membrane harbors two ATP-dependent metallopeptidases, the m- and the i-AAA protease, which expose their catalytic sites to opposite membrane surfaces and cooperate in the degradation of inner membrane proteins. In addition to its proteolytic activity, the m-AAA protease has chaperone-like activity during the assembly of respiratory and ATP-synthase complexes. It constitutes a quality control system in the inner membrane for membrane-embedded protein complexes.

Adenosine Triphosphate↗

PubMed1997

Two modes of ligand binding in maltose-binding protein of Escherichia coli. Electron paramagnetic resonance study of ligand-induced global conformational changes by site-directed spin labeling.

Binding of ligands to the maltose-binding protein (MBP) of Escherichia coli often causes a global conformational change involving the closure of its two lobes. We have introduced a cysteine residue onto each of these lobes by site-directed mutagenesis and modified these residues with spin labels. Using EPR spectroscopy, we examined the changes, caused by the ligand binding, in distance between the two spin labels, hence between the two lobes. The binding of both maltose and maltotetraose induced a considerable closure of the N- and C-terminal lobes of MBP. Little closure occurred upon the binding of maltotetraitol or beta-cyclodextrin. Previous study by fluorescence and UV differential absorbance spectroscopy (Hall, J. A., Gehring, K., and Nikaido, H. (1997) J. Biol. Chem. 272, 17605-17609) showed that maltose and a large portion of maltotetraose bound to MBP via one mode (R mode or "end-on" mode), which is physiologically active and leads to the subsequent transport of the ligands across the cytoplasmic membrane. In contrast, maltotetraitol and beta-cyclodextrin bound to MBP via a different mode (B mode or "middle" mode), which is physiologically inactive. The present work suggests that the B mode is nonproductive because ligands binding in this manner prevent the closure of the two domains of MBP, and, as a result, the resulting ligand-MBP complex is incapable of interacting properly with the inner membrane-associated transporter complex.

ATP-Binding Cassette Transporters↗

PubMed1999

Tim9, a new component of the TIM22.54 translocase in mitochondria.

We have identified Tim9, a new component of the TIM22.54 import machinery, which mediates transport of proteins into the inner membrane of mitochondria. Tim9, an essential protein of Saccharomyces cerevisiae, shares sequence similarity with Tim10 and Tim12. Tim9 is located in the mitochondrial intermembrane space and is organized into two distinct hetero-oligomeric assemblies with Tim10 and Tim12. One complex contains Tim9 and Tim10. The other complex contains Tim9, Tim10 and Tim12 and is tightly associated with Tim22 in the inner membrane. The TIM9.10 complex is more abundant than the TIM9.10.12 complex and mediates partial translocation of mitochondrial carriers proteins across the outer membrane. The TIM9.10.12 complex assists further translocation into the inner membrane in association with TIM22.54.

Amino Acid Sequence↗

PubMed2002

Tim50 is a subunit of the TIM23 complex that links protein translocation across the outer and inner mitochondrial membranes.

Based on the results of site-specific photocrosslinking of translocation intermediates, we have identified Tim50, a component of the yeast TIM23 import machinery, which mediates translocation of presequence-containing proteins across the mitochondrial inner membrane. Tim50 is anchored to the inner mitochondrial membrane, exposing the C-terminal domain to the intermembrane space. Tim50 interacts with the N-terminal intermembrane space domain of Tim23. Functional defects of Tim50 either by depletion of the protein or addition of anti-Tim50 antibodies block the protein translocation across the inner membrane. A translocation intermediate accumulated at the TOM complex is crosslinked to Tim50. We suggest that Tim50, in cooperation with Tim23, facilitates transfer of the translocating protein from the TOM complex to the TIM23 complex

Carrier Proteins↗

PubMed1994

Biochemical evidence for the orientation of cytochrome b in the yeast mitochondrial membrane in the eight-helix model.

The topographical localization of the N-terminus of cytochrome b in the inner mitochondrial membrane was determined by mild proteolysis of the yeast mitochondrial cytochrome bc1 complex and identification of the proteolytic fragments derived from subunits of the complex with an established orientation in the inner membrane. The cytochrome bc1 complex was incorporated into proteoliposomes which were separated by cytochrome c affinity chromatography into two populations in either the mitochondrial or the submitochondrial orientation. Core protein I which protrudes from the matrix side of the inner membrane was digested by proteinase K only in proteoliposomes with the submitochondrial orientation and not in those with the mitochondrial orientation. By contrast, cytochrome c1 with protrudes from the cytoplasmic side of the inner membrane was digested by proteinase K only in proteoliposomes with the mitochondrial orientation and not in those with the submitochondrial orientation. Cytochrome b was digested by SV8 protease only in proteoliposomes with the mitochondrial orientation to yield two aggregating fragments of 25.6 and 24.5 kDa. These peptides were isolated by preparative gel chromatography and sequenced to establish that the cleavage of cytochrome b by SV8 protease occurred at glutamate residues 59 and 66. These residues are localized in the extramembranous loop between the two hydrophobic membrane-spanning helices A and B and thus face the cytoplasmic side of the inner mitochondrial membrane. These results indicate that the N-terminus of yeast cytochrome b protrudes from the matrix side of the inner membrane consistent with the eight-helix model for the orientation of cytochrome b in the membrane.

Amino Acid Sequence↗

PubMed1999

Different import pathways through the mitochondrial intermembrane space for inner membrane proteins.

Earlier work on the protein import system of yeast mitochondria has identified two soluble 70 kDa protein complexes in the intermembrane space. One complex contains the essential proteins Tim9p and Tim10p and mediates transport of cytosolically-made metabolite carrier proteins from the outer to the inner membrane. The other complex contains the non-essential proteins Tim8p and Tim13p as well as loosely associated Tim9p; its function was unclear, but it interacted structurally or functionally with the Tim9p-Tim10p complex. We now show that the two 70 kDa complexes each mediate the import of a different subset of integral inner membrane proteins and that they can transfer these proteins to one of three different membrane insertion sites: the TIM22 complex, the TIM23 complex or an as yet uncharacterized insertion site. Yeast mitochondria thus use multiple pathways for escorting hydrophobic inner membrane proteins across the aqueous intermembrane space.

Biological Transport↗

PubMed1999

Biogenesis of Tim proteins of the mitochondrial carrier import pathway: differential targeting mechanisms and crossing over with the main import pathway.

Two major routes of preprotein targeting into mitochondria are known. Preproteins carrying amino-terminal signals mainly use Tom20, the general import pore (GIP) complex and the Tim23-Tim17 complex. Preproteins with internal signals such as inner membrane carriers use Tom70, the GIP complex, and the special Tim pathway, involving small Tims of the intermembrane space and Tim22-Tim54 of the inner membrane. Little is known about the biogenesis and assembly of the Tim proteins of this carrier pathway. We report that import of the preprotein of Tim22 requires Tom20, although it uses the carrier Tim route. In contrast, the preprotein of Tim54 mainly uses Tom70, yet it follows the Tim23-Tim17 pathway. The positively charged amino-terminal region of Tim54 is required for membrane translocation but not for targeting to Tom70. In addition, we identify two novel homologues of the small Tim proteins and show that targeting of the small Tims follows a third new route where surface receptors are dispensable, yet Tom5 of the GIP complex is crucial. We conclude that the biogenesis of Tim proteins of the carrier pathway cannot be described by either one of the two major import routes, but involves new types of import pathways composed of various features of the hitherto known routes, including crossing over at the level of the GIP.

Amino Acid Sequence↗

PubMed2000

Coupling of mitochondrial translation with the formation of respiratory complexes in yeast mitochondria.

In contrast to most other eukaryotic organisms, yeast can survive without respiration. This ability has been exploited to investigate nuclear genes required for expression of mitochondrial DNA. Availability of complete Saccharomyces cerevisiae genomic sequence has provided additional help in detailed molecular analysis. Seven of the eight major products encoded by mitochondrial DNA are hydrophobic subunits of respiratory complexes in the inner membrane. Localization of the translation process in the same cellular compartment ensures synthesis of mitochondrially encoded proteins near sites of their assembly into multimeric respiratory complexes. Association of mitochondrial ribosomes with the membrane is mediated by mRNA-specific translational activators, that are involved in the recognition of initiation codon. The newly synthesized mitochondrial proteins are transferred to membrane by a specific export system. This review discusses the role of membrane-localized factors responsible for quality control and turnover of mitochondrially synthesized subunits as well as for assembly of respiratory complexes.

Electron Transport↗

PubMed1992

Partial Purification and Characterization of Complex I, NADH:Ubiquinone Reductase, from the Inner Membrane of Beetroot Mitochondria.

A NADH dehydrogenase was isolated from an inner membrane-enriched fraction of beetroot mitochondria (Beta vulgaris L.) by solubilization with sodium deoxycholate and purified using gel filtration and affinity chromatography. The NADH dehydrogenase preparation contained a minor ATPase contamination. Beetroot mitochondria were chosen as the isolation material for purifying the enzymes responsible for oxidizing matrix NADH due to the absence of the externally facing NADH dehydrogenase in the variety we have used. The purified NADH dehydrogenase complex catalyzed the reduction of various electron acceptors with NADH as the electron donor, was not sensitive to rotenone inhibition, and had a slow NADPH-ubiquinone 5 reductase activity. The isolated complex contained 14 major polypeptides. It was concluded that the dehydrogenase represented a form of the plant mitochondrial complex I and not the internally facing rotenone-insensitive NADH dehydrogenase found in plant mitochondria because of its complex structure, its cross-reactivity with antisera raised against bovine heart mitochondrial complex I, and the similarity of its kinetics and inhibitor responses to rotenone-sensitive NADH oxidation by beetroot submitochondrial particles.

Journal Article↗

PubMed1987

[Effect of a number of biologically active substances on the activity of membrane-bound Na+,K+-ATPase from the bovine brain].

Cellular Na+, K+-transport ATPase appears to be more resistant to the influence of DNA intercalators and other agents than ATP-synthetase complex of inner mitochondrial membrane. Neither synthetic intercalators, nor antibiotics olivomycin and actinomycin D, some uncouplers of oxidative phosphorylation, alkaloids and inorganic bivalent cations selectively (up to 10(-4) M) inhibit Na+, K+-ATPase activity. Besides ouabain, alkaloid sanguinarine and cation Ag+ (the latter in the absence of anion Cl-) cause a significant decrease of Na+, K+-ATPase activity. Ag+ is most potent as inhibitor, while sanguinarine is weaker than ouabain. Inhibition of Na+, K+-ATPase by sanguinarine may result presumably from modification of SH-groups of the enzyme.

Alkaloids↗

PubMed1977

[Characterization of the membrane attached to the folded chromosome isolated from Escherichia coli].

Phospholipid analysis of the membranes associated with fast sedimenting folded chromosomes prepared by lysis of E. coli CR 34 shows that both inner and outer membranes are parts of the complex, in proportions not very different from that found in the whole bacteria. During the preparation of the folded chromosomes, the most recently synthesized molecules of phosphatidylglycerol and phosphatidylethanoamine are more sensitive to solubilisation, particularly those from the cytoplasmic membrane. Identification of a dominant fraction, the outer membrane, in some complexes, results from a preferential solubilization of the inner membrane. These results do not favor any specific association between the folded chromosome and the membranes.

Cell Fractionation↗

PubMed2003

Assembly of cytochrome c oxidase within the mitochondrion.

Cytochrome c oxidase (CcO) is an oligomeric complex localized within the mitochondrial inner membrane. Assembly of the active oxidase complex requires the coordinate assembly of subunits synthesized in both the cytoplasm and the mitochondrion. In addition, assembly is dependent on the insertion of five types of cofactors, including two hemes, three copper ions, and one Zn, Mg, and Na ion. A series of accessory proteins are critical for synthesis of the heme A cofactor and insertion of the copper ions. This Account will focus on the steps in the coordinate assembly of CcO subunits, the formation of heme A, and the delivery and insertion of copper ions.

Electron Transport Complex IV↗

PubMed1990

Localization of the alpha-oxoacid dehydrogenase multienzyme complexes within the mitochondrion.

Bovine kidney mitochondria were separated into matrix and membrane fractions by treatment with digitonin and Lubrol PX. While malate dehydrogenase was found essentially in the matrix fraction, both the pyruvate and the alpha-oxoglutarate dehydrogenase multienzyme complexes remained bound to the inner membrane fraction and became solubilized only after repeated treatments with detergents. Thus both multienzyme complexes must be associated with the inner membrane rather than located within the matrix space.

Animals↗

PubMed2006

Prohibitins interact genetically with Atp23, a novel processing peptidase and chaperone for the F1Fo-ATP synthase.

The generation of cellular energy depends on the coordinated assembly of nuclear and mitochondrial-encoded proteins into multisubunit respiratory chain complexes in the inner membrane of mitochondria. Here, we describe the identification of a conserved metallopeptidase present in the intermembrane space, termed Atp23, which exerts dual activities during the biogenesis of the F(1)F(O)-ATP synthase. On one hand, Atp23 serves as a processing peptidase and mediates the maturation of the mitochondrial-encoded F(O)-subunit Atp6 after its insertion into the inner membrane. On the other hand and independent of its proteolytic activity, Atp23 promotes the association of mature Atp6 with Atp9 oligomers. This assembly step is thus under the control of two substrate-specific chaperones, Atp10 and Atp23, which act on opposite sides of the inner membrane. Strikingly, both ATP10 and ATP23 were found to genetically interact with prohibitins, which build up large, ring-like assemblies with a proposed scaffolding function in the inner membrane. Our results therefore characterize not only a novel processing peptidase with chaperone activity in the mitochondrial intermembrane space but also link the function of prohibitins to the F(1)F(O)-ATP synthase complex.

Amino Acid Sequence↗

PubMed2005

Early encounters of a nascent membrane protein: specificity and timing of contacts inside and outside the ribosome.

An unbiased photo-cross-linking approach was used to probe the "molecular path" of a growing nascent Escherichia coli inner membrane protein (IMP) from the peptidyl transferase center to the surface of the ribosome. The nascent chain was initially in proximity to the ribosomal proteins L4 and L22 and subsequently contacted L23, which is indicative of progression through the ribosome via the main ribosomal tunnel. The signal recognition particle (SRP) started to interact with the nascent IMP and to target the ribosome-nascent chain complex to the Sec-YidC complex in the inner membrane when maximally half of the transmembrane domain (TM) was exposed from the ribosomal exit. The combined data suggest a flexible tunnel that may accommodate partially folded nascent proteins and parts of the SRP and SecY. Intraribosomal contacts of the nascent chain were not influenced by the presence of a functional TM in the ribosome.

Escherichia coli↗