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W Neupert

Publications and source records attributed to W Neupert.

At least 37 records · Page 2Linked to original sources
PubMed2001

Structural requirements of Tom40 for assembly into preexisting TOM complexes of mitochondria.

Tom40 is the major subunit of the translocase of the outer mitochondrial membrane (the TOM complex). To study the assembly pathway of Tom40, we have followed the integration of the protein into the TOM complex in vitro and in vivo using wild-type and altered versions of the Neurospora crassa Tom40 protein. Upon import into isolated mitochondria, Tom40 precursor proteins lacking the first 20 or the first 40 amino acid residues were assembled as the wild-type protein. In contrast, a Tom40 precursor lacking residues 41 to 60, which contains a highly conserved region of the protein, was arrested at an intermediate stage of assembly. We constructed mutant versions of Tom40 affecting this region and transformed the genes into a sheltered heterokaryon containing a tom40 null nucleus. Homokaryotic strains expressing the mutant Tom40 proteins had growth rate defects and were deficient in their ability to form conidia. Analysis of the TOM complex in these strains by blue native gel electrophoresis revealed alterations in electrophoretic mobility and a tendency to lose Tom40 subunits from the complex. Thus, both in vitro and in vivo studies implicate residues 41 to 60 as containing a sequence required for proper assembly/stability of Tom40 into the TOM complex. Finally, we found that TOM complexes in the mitochondrial outer membrane were capable of exchanging subunits in vitro. A model is proposed for the integration of Tom40 subunits into the TOM complex.

Amino Acid Sequence↗
PubMed2000

Ecm10, a novel hsp70 homolog in the mitochondrial matrix of the yeast Saccharomyces cerevisiae.

Members of the heat shock protein 70 (Hsp70) family are found in most of the compartments of eukaryotic cells where they play essential roles in protein metabolism. In yeast mitochondria, two Hsp70 proteins are known: Ssc1 and Ssq1. We identified Ecm10 as a third Hsp70 protein in the mitochondrial matrix. Ecm10 shares 82% amino acid identity with Ssc1 and 54% with Ssq1. Overexpression of Ecm10 mitigates protein import defects in ssc1 mutants suggesting that Ecm10 can play a role in protein translocation. Like Ssc1, Ecm10 interacts with the nucleotide exchange factor Mge1 in an ATP-dependent manner. Deletion of ecm10 leads to synthetic growth defects with ssc1 mutations at low temperature. Our data suggest an overlapping function of Ecm10 and Ssc1.

Amino Acid Sequence↗
PubMed2000

The role of the TIM8-13 complex in the import of Tim23 into mitochondria.

Tim8 and Tim13 are non-essential, conserved proteins of the mitochondrial intermembrane space, which are organized in a hetero-oligomeric complex. They are structurally related to Tim9 and Tim10, essential components of the import machinery for mitochondrial carrier proteins. Here we show that the TIM8-13 complex interacts with translocation intermediates of Tim23, which are partially translocated across the outer membrane but not with fully imported or assembled Tim23. The TIM8-13 complex binds to the N-terminal or intermediate domain of Tim23. It traps the incoming precursor in the intermembrane space thereby preventing retrograde translocation. The TIM8-13 complex is strictly required for import of Tim23 under conditions when a low membrane potential exists in the mitochondria. The human homologue of Tim8 is encoded by the DDP1 (deafness/dystonia peptide 1) gene, which is associated with the Mohr-Tranebjaerg syndrome (MTS), a progressive neurodegenerative disorder leading to deafness. It is demonstrated that import of human Tim23 is dependent on a high membrane potential. A mechanism to explain the pathology of MTS is discussed.

Animals↗
PubMed2000

Recognition of preproteins by the isolated TOM complex of mitochondria.

A multisubunit complex in the mitochondrial outer membrane, the TOM complex, mediates targeting and membrane translocation of nuclear-encoded preproteins. We have isolated the TOM holo complex, containing the preprotein receptor components Tom70 and Tom20, and the TOM core complex, which lacks these receptors. The interaction of recombinant mitochondrial preproteins with both types of soluble TOM complex was analyzed. Preproteins bound efficiently in a specific manner to the isolated complexes in the absence of chaperones and lipids in a bilayer structure. Using fluorescence correlation spectroscopy, a dissociation constant in the nanomolar range was determined. The affinity was lower when the preprotein was stabilized in its folded conformation. Following the initial binding, the presequence was transferred into the translocation pore in a step that required unfolding of the mature part of the preprotein. This translocation step was also mediated by protease-treated TOM holo complex, which contains almost exclusively Tom40. Thus, the TOM core complex, consisting of Tom40, Tom22, Tom6 and Tom7, is a molecular machine that can recognize and partially translocate mitochondrial precursor proteins.

Binding Sites↗
PubMed2000

What fuels polypeptide translocation? An energetical view on mitochondrial protein sorting.

Protein sorting into mitochondria is achieved by the concerted action of at least four translocation complexes. Vectorial transport of polypeptide chains by these complexes requires different driving forces. In particular, Deltapsi, matrix adenosine triphosphate and the free energy of the binding to other protein components are used in series to achieve sorting of proteins to the various mitochondrial subcompartments. The processes providing the translocation energy are presented in this review and their impact for protein sorting into and within mitochondria is discussed.

Adenosine Triphosphatases↗
PubMed2000

The cytochrome bc1 and cytochrome c oxidase complexes associate to form a single supracomplex in yeast mitochondria.

The mitochondrial electron transport chain complexes are large multisubunit complexes embedded in the inner membrane. We report here that in the yeast Saccharomyces cerevisiae, the cytochrome bc(1) and cytochrome c oxidase complexes co-exist as a larger complex of approximately 1000 kDa in the mitochondrial membrane. Following solubilization with a mild detergent, the cytochrome bc(1)-cytochrome c oxidase complex remains stable. It was analyzed using the techniques of gel filtration and blue native-polyacrylamide gel electrophoresis. Direct physical association of subunits of the cytochrome bc(1) complex with those of the cytochrome c oxidase complex was verified by co-immunoprecipitation analysis. Our data indicate that the cytochrome bc(1) complex is exclusively in association with the cytochrome c oxidase complex in yeast mitochondria. We term this complex the cytochrome bc(1)-cytochrome c oxidase supracomplex.

Electron Transport↗
PubMed2000

Tim23 links the inner and outer mitochondrial membranes.

Tim23, a key component of the mitochondrial preprotein translocase, is anchored in the inner membrane by its C-terminal domain and exposes an intermediate domain in the intermembrane space that functions as a presequence receptor. We show that the N-terminal domain of Tim23 is exposed on the surface of the outer membrane. The two-membrane-spanning topology of Tim23 is a novel characteristic in membrane biology. By the simultaneous integration into two membranes, Tim23 forms contacts between the outer and inner mitochondrial membranes. Tethering the inner membrane translocase to the outer membrane facilitates the transfer of precursor proteins from the TOM complex to the TIM23 complex and increases the efficiency of protein import.

Animals↗
PubMed2000

The chaperonin-related protein Tcm62p ensures mitochondrial gene expression under heat stress.

Tcm62p, distantly related to chaperonins, is required for the assembly of succinate dehydrogenase in mitochondria of Saccharomyces cerevisiae and was proposed to exert chaperone activity. We demonstrate here crucial functions of Tcm62p under heat stress. It ensures mitochondrial gene expression at elevated temperatures and prevents heat-aggregation of the ribosomal subunit Var1p. Similar to chaperonins, Tcm62p forms a high molecular mass protein complex of approximately 850 kDa in the mitochondrial matrix space. These results suggest a more general chaperone function of Tcm62p in mitochondria.

Cell Respiration↗
PubMed2000

Identification of Cox20p, a novel protein involved in the maturation and assembly of cytochrome oxidase subunit 2.

We have identified Cox20p, a 23.8-kDa protein of the mitochondrial inner membrane that is involved in the biogenesis of the yeast cytochrome oxidase complex. Cytochrome oxidase subunit 2 (Cox2p) accumulates as a precursor in cox20 mutants, suggesting a defect in biogenesis of this mitochondrially encoded protein. The inability of cox20 mutants to process the subunit 2 precursor (pCox2p) is not due to impaired export of the protein across the inner membrane or to an inactive Imp1p/Imp2p peptidase. Rather, Cox20p specifically binds the newly synthesized pCox2p, a step required to present the exported pCox2p as a substrate to the Imp1p peptidase. All of the endogenous pCox2p accumulated in an Deltaimp1 mutant, and a small fraction of Cox2p in wild type yeast, is detected in a complex with Cox20p. Following maturation Cox2p remained associated with Cox20p, prior to assembling into the cytochrome oxidase complex. We propose that Cox20p acts as a membrane-bound chaperone necessary for cleavage of pCox2p and for interaction of the mature protein with other subunits of cytochrome oxidase in a later step of the assembly process.

Amino Acid Sequence↗
PubMed2000

MITOP, the mitochondrial proteome database: 2000 update.

MITOP (http://www.mips.biochem.mpg.de/proj/medgen/mitop/) is a comprehensive database for genetic and functional information on both nuclear- and mitochondrial-encoded proteins and their genes. The five species files--Saccharomyces cerevisiae, Mus musculus, Caenorhabditis elegans, Neurospora crassa and Homo sapiens--include annotated data derived from a variety of online resources and the literature. A wide spectrum of search facilities is given in the overlapping sections 'Gene catalogues', 'Protein catalogues', 'Homologies', 'Pathways and metabolism' and 'Human disease catalogue' including extensive references and hyperlinks to other databases. Central features are the results of various homology searches, which should facilitate the investigations into interspecies relationships. Precomputed FASTA searches using all the MITOP yeast protein entries and a list of the best human EST hits with graphical cluster alignments related to the yeast reference sequence are presented. The orthologue tables with cross-listings to all the protein entries for each species in MITOP have been expanded by adding the genomes of Rickettsia prowazeckii and Escherichia coli. To find new mitochondrial proteins the complete yeast genome has been analyzed using the MITOPROT program which identifies mitochondrial targeting sequences. The 'Human disease catalogue' contains tables with a total of 110 human diseases related to mitochondrial protein abnormalities, sorted by clinical criteria and age of onset. MITOP should contribute to the systematic genetic characterization of the mitochondrial proteome in relation to human disease.

Animals↗
PubMed2000

Mitochondria-targeted green fluorescent proteins: convenient tools for the study of organelle biogenesis in Saccharomyces cerevisiae.

We describe the construction and characterization of a novel set of plasmids for expression of mitochondria-targeted green fluorescent protein (GFP) in Saccharomyces cerevisiae. The vectors include constructs with strong regulatable and constitutive promoters, four different auxotrophic markers for yeast transformation, and a green (S65T) and a blue-shifted (P4-3) variant of GFP. Mitochondria are brightly fluorescent in living yeast cells grown on different carbon sources and at different temperatures, with virtually no background staining. Specific staining of mitochondria is also shown for a respiratory-deficient mutant with abnormal mitochondrial morphology. The plasmids facilitate convenient analysis of mutants defective in mitochondrial morphology or inheritance and, at the same time, are suitable vectors for easy construction of different kinds of GFP fusion proteins to study various aspects of organelle biogenesis in living yeast cells.

Cloning, Molecular↗
PubMed2000

[Management of comminuted and open fractures of the mandible and fractures in atrophic mandibles with titanium mesh].

Miniplate osteosynthesis is a standard method for the surgical treatment of mandible fractures today. Apart from easy handling, in the majority of cases it also ensures adequate fracture stability. The treatment of fractures of very atrophic mandibles as well as mandibular defects and comminuted fractures with miniplates and rigid plates is often difficult or only possible to an inadequate extent. Titanium mesh has proved successful for the treatment of such fractures. Compared with conventional miniplates this mesh is outstanding for its considerably higher stability due to its given geometry. The titanium mesh can be adapted and screwed individually in any situation, in contrast to miniplates and rigid plates. As a result, complications such as fracture gap infections and pseudoarthrosis can be avoided to a great extent.

Adult↗
PubMed2000

Protein translocation into mitochondria: the role of TIM complexes.

Import of nuclear-encoded mitochondrial preproteins is mediated by a general translocase in the outer membrane, the TOM complex, and by two distinct translocases in the mitochondrial inner membrane, the TIM23 complex and the TIM22 complex. Both TIM complexes cooperate with the TOM complex but facilitate import of different classes of precursor proteins. Precursors with an N-terminal presequence are imported via the TIM23 complex, whereas mitochondrial carrier proteins require the TIM22 complex for insertion into the inner membrane. This review discusses recent advances in understanding the structure and function of the translocases of the inner membrane and the possible role of Tim proteins in the development of the Mohr-Tranebjaerg syndrome, a mitochondrial disorder leading to neurodegeneration.

Animals↗
PubMed2000

Membrane protein degradation by AAA proteases in mitochondria: extraction of substrates from either membrane surface.

Two AAA proteases, each with its catalytic site at the opposite membrane surface, mediate the ATP-dependent degradation of mitochondrial inner membrane proteins. We demonstrate here that a model substrate polypeptide containing hydrophilic domains at both sides of the membrane can be completely degraded by either of the AAA proteases, if solvent-exposed domains are in an unfolded state. A short protein tail protruding from the membrane surface is sufficient to allow the proteolytic attack of an AAA protease that facilitates domain unfolding at the opposite side. Our results provide a rationale for the membrane arrangement of AAA proteases in mitochondria and demonstrate that degradation of membrane proteins by AAA proteases involves an active extraction of transmembrane segments and transport of solvent-exposed domains across the membrane.

Adenosine Triphosphatases↗
PubMed2000

Protein transport into mitochondria.

Mitochondria are made up of two membrane systems that subdivide this organelle into two aqueous subcompartments: the matrix, which is enclosed by the inner membrane, and the intermembrane space, which is located between the inner and the outer membrane. Protein import into mitochondria is a complex reaction, as every protein has to be routed to its specific destination within the organelle. In the past few years, studies with mitochondria of Neurospora crassa and Saccharomyces cerevisiae have led to the identification of four distinct translocation machineries that are conserved among eukaryotes. These translocases, in a concerted fashion, mediate import and sorting of proteins into the mitochondrial subcompartments.

Biological Transport↗
PubMed2000

Treatment of complex mandibular fractures using titanium mesh.

INTRODUCTION: The treatment of complex fractures with miniplates is often difficult and unsatisfactory. Such fractures include extremely atrophic mandibles, discontinuity defects, and marked comminuted fractures. AIM: With this paper we want to call to mind once more that titanium mesh is useful for the treatment of such fractures and its use can reduce the number of complications. PATIENTS: Between January 1996 and December 1998 we treated with titanium mesh 17 patients with fractures of extremely atrophic mandibles, mandibular discontinuity defects or comminuted fractures. RESULTS: Union occurred without complication in 70% of fractures treated with titanium mesh. In 20% there were minor complications such as postoperative haematoma. In only one case did infection occur, a more severe complication. CONCLUSION: Because of its geometry and the excellent physical and biomechanical properties, titanium mesh helps to achieve better stabilization of complex mandibular fractures than conventional miniplates do. Complications such as infection and non union can largely be avoided and bony continuity of the mandible can be restored.

Adolescent↗
PubMed2000

Role of MMM1 in maintaining mitochondrial morphology in Neurospora crassa.

Mmm1p is a protein required for maintenance of mitochondrial morphology in budding yeast. It was proposed that it is required to mediate the interaction of the mitochondrial outer membrane with the actin cytoskeleton. We report the cloning and characterization of MMM1 of the filamentous fungus Neurospora crassa, an organism that uses microtubules for mitochondrial transport. Mutation of the mmm-1 gene leads to a temperature-sensitive slow growth phenotype and female sterility. Mutant cells harbor abnormal giant mitochondria at all stages of the asexual life cycle, whereas actin filament-depolymerizing drugs have no effect on mitochondrial morphology. The MMM1 protein has a single transmembrane domain near the N terminus and exposes a large C-terminal domain to the cytosol. The protein can be imported into the outer membrane in a receptor-dependent manner. Our findings suggest that MMM1 is a factor of general importance for mitochondrial morphology independent of the cytoskeletal system used for mitochondrial transport.

Amino Acid Sequence↗