PubMed Health⌕ Search

Biomedical subjects

W Neupert

Publications and source records attributed to W Neupert.

At least 19 recordsLinked to original sources
PubMed2001

Role of the deafness dystonia peptide 1 (DDP1) in import of human Tim23 into the inner membrane of mitochondria.

Tim8 and Tim13 of yeast belong to a family of evolutionary conserved zinc finger proteins that are organized in hetero-oligomeric complexes in the mitochondrial intermembrane space. Mutations in DDP1 (deafness dystonia peptide 1), the human homolog of Tim8, are associated with the Mohr-Tranebjaerg syndrome, a progressive neurodegenerative disorder. We show that DDP1 acts with human Tim13 in a complex in the intermembrane space. The DDP1.hTim13 complex is in direct contact with translocation intermediates of human Tim23 in mammalian mitochondria. The human DDP1.hTim13 complex complements the function of the TIM8.13 complex in yeast and facilitates import of yeast and human Tim23. Thus, the pathomechanism underlying the Mohr-Tranebjaerg syndrome may involve an impaired biogenesis of the human TIM23 complex causing severe pleiotropic mitochondrial dysfunction.

Animals↗
PubMed2001

Tom40, the pore-forming component of the protein-conducting TOM channel in the outer membrane of mitochondria.

Tom40 is the main component of the preprotein translocase of the outer membrane of mitochondria (TOM complex). We have isolated Tom40 of Neurospora crassa by removing the receptor Tom22 and the small Tom components Tom6 and Tom7 from the purified TOM core complex. Tom40 is organized in a high molecular mass complex of approximately 350 kD. It forms a high conductance channel. Mitochondrial presequence peptides interact specifically with Tom40 reconstituted into planar lipid membranes and decrease the ion flow through the pores in a voltage-dependent manner. The secondary structure of Tom40 comprises approximately 31% beta-sheet, 22% alpha-helix, and 47% remaining structure as determined by circular dichroism measurements and Fourier transform infrared spectroscopy. Electron microscopy of purified Tom40 revealed particles primarily with one center of stain accumulation. They presumably represent an open pore with a diameter of approximately 2.5 nm, similar to the pores found in the TOM complex. Thus, Tom40 is the core element of the TOM translocase; it forms the protein-conducting channel in an oligomeric assembly.

Fungal Proteins↗
PubMed2001

Mba1, a novel component of the mitochondrial protein export machinery of the yeast Saccharomyces cerevisiae.

The biogenesis of mitochondria requires the integration of many proteins into the inner membrane from the matrix side. The inner membrane protein Oxa1 plays an important role in this process. We identified Mba1 as a second mitochondrial component that is required for efficient protein insertion. Like Oxa1, Mba1 specifically interacts both with mitochondrial translation products and with conservatively sorted, nuclear-encoded proteins during their integration into the inner membrane. Oxa1 and Mba1 overlap in function and substrate specificity, but both can act independently of each other. We conclude that Mba1 is part of the mitochondrial protein export machinery and represents the first component of a novel Oxa1-independent insertion pathway into the mitochondrial inner membrane.

Cell Division↗
PubMed2001

Modular structure of the TIM23 preprotein translocase of mitochondria.

The TIM23 complex mediates import into mitochondria of nuclear encoded preproteins with a matrix-targeting signal. It is composed of the integral membrane proteins Tim17 and Tim23 and the peripheral membrane protein Tim44, which recruits mitochondrial Hsp70 to the sites of protein import. We have analyzed the functions of these constituents using a combined genetic and biochemical approach. Depletion of either Tim17 or Tim23 led to loss of import competence of mitochondria and to a reduction in the number of preprotein-conducting channels. Upon depletion of Tim44, mitochondria also lost their ability to import proteins but maintained normal numbers of import channels. In the absence of Tim44 precursor protein was specifically recognized. The presequence was translocated in a Delta psi-dependent manner across the inner membrane and cleaved by matrix-processing peptidase. However, the preprotein did not move further into the matrix but rather underwent retrograde sliding out of the TIM23 complex. Thus, the TIM23 complex is composed of functionally independent modules. Tim17 and Tim23 are necessary for initiating translocation, whereas Tim44 and mitochondrial Hsp70 are indispensable for complete transport of preproteins and for unfolding of folded domains of preproteins.

Carrier Proteins↗
PubMed2001

The mitochondrial proteins Ssq1 and Jac1 are required for the assembly of iron sulfur clusters in mitochondria.

Mitochondria of the yeast Saccharomyces cerevisiae contain three different Hsp70 chaperones, Ssc1, Ecm10 and Ssq1. Ssc1 is an essential protein that mediates the import of nuclear-encoded proteins into the organelle and their subsequent folding. The nucleotide state of Ssc1 is thereby regulated by the nucleotide exchange factor Mge1. Here, we show that Mge1 interacts with Ssq1 in an ATP-dependent manner, suggesting that Mge1 also regulates Ssq1 function. In contrast to Ssc1, Ssq1 does not associate with the Tim44 subunit of the protein translocating complex, indicating a different function of both chaperones. Mutants in Ssq1 were reported to have low levels of iron sulfur (FeS) cluster-containing enzymes. Employing an assay that allowed us to monitor the conversion of the apoform of mitochondrial ferredoxin into its FeS-containing holoform, Ssq1 was demonstrated to be required for the FeS cluster assembly in mitochondria. The mitochondrial DnaJ homolog Jac1 is crucial for this process, whereas Mdj1 function is dispensable. Furthermore, the presence of frataxin is necessary for FeS cluster assembly into ferredoxin suggesting a role for frataxin at the level of the formation of holo-ferredoxin.

Adenosine Triphosphate↗
PubMed2001

Oxa1p acts as a general membrane insertion machinery for proteins encoded by mitochondrial DNA.

Oxa1p is a member of the conserved Oxa1/YidC/Alb3 protein family involved in the membrane insertion of proteins. Oxa1p has been shown previously to directly facilitate the export of the N-terminal domains of membrane proteins across the inner membrane to the intermembrane space of mitochondria. Here we report on a general role of Oxa1p in the membrane insertion of proteins. (i) The function of Oxa1p is not limited to the insertion of membrane proteins that undergo N-terminal tail export; rather, it also extends to the insertion of other polytopic proteins such as the mitochondrially encoded Cox1p and Cox3p proteins. These are proteins whose N-termini are retained in the mitochondrial matrix. (ii) Oxa1p interacts directly with these substrates prior to completion of their synthesis. (iii) The interaction of Oxa1p with its substrates is particularly strong when nascent polypeptide chains are inserted into the inner membrane, suggesting a direct function of Oxa1p in co-translational insertion from the matrix. Taken together, we conclude that the Oxa1 complex represents a general membrane protein insertion machinery in the inner membrane of mitochondria.

Carrier Proteins↗
PubMed2001

Assembly of Tom6 and Tom7 into the TOM core complex of Neurospora crassa.

Translocation of preproteins across the mitochondrial outer membrane is mediated by the translocase of the outer mitochondrial membrane (TOM) complex. We report the molecular identification of Tom6 and Tom7, two small subunits of the TOM core complex in the fungus Neurospora crassa. Cross-linking experiments showed that both proteins were found to be in direct contact with the major component of the pore, Tom40. In addition, Tom6 was observed to interact with Tom22 in a manner that depends on the presence of preproteins in transit. Precursors of both proteins are able to insert into the outer membrane in vitro and are assembled into authentic TOM complexes. The insertion pathway of these proteins shares a common binding site with the general import pathway as the assembly of both Tom6 and Tom7 was competed by a matrix-destined precursor protein. This assembly was dependent on the integrity of receptor components of the TOM machinery and is highly specific as in vitro-synthesized yeast Tom6 was not assembled into N. crassa TOM complex. The targeting and assembly information within the Tom6 sequence was found to be located in the transmembrane segment and a flanking segment toward the N-terminal, cytosolic side. A hybrid protein composed of the C-terminal domain of yeast Tom6 and the cytosolic domain of N. crassa Tom6 was targeted to the mitochondria but was not taken up into TOM complexes. Thus, both segments are required for assembly into the TOM complex. A model for the topogenesis of the small Tom subunits is discussed.

Amino Acid Sequence↗
PubMed2001

Connection of the mitochondrial outer and inner membranes by Fzo1 is critical for organellar fusion.

Mitochondrial membrane fusion is a process essential for the maintenance of the structural integrity of the organelle. Since mitochondria are bounded by a double membrane, they face the challenge of fusing four membranes in a coordinated manner. We provide evidence that this is achieved by coupling of the mitochondrial outer and inner membranes by the mitochondrial fusion machinery. Fzo1, the first known mediator of mitochondrial fusion, spans the outer membrane twice, exposing a short loop to the intermembrane space. The presence of the intermembrane space segment is required for the localization of Fzo1 in sites of tight contact between the mitochondrial outer and inner membranes. Mutations in the intermembrane space domain of yeast Fzo1 relieve the association with the inner membrane. This results in a loss of function of the protein in vivo. We propose that the mitochondrial fusion machinery forms membrane contact sites that mediate mitochondrial fusion. A fusion machinery that is in contact with both mitochondrial membranes appears to be functionally important for coordinated fusion of four mitochondrial membranes.

Cell Polarity↗
PubMed2001

Biogenesis of porin of the outer mitochondrial membrane involves an import pathway via receptors and the general import pore of the TOM complex.

Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial. We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin. Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria. A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore. Using an assay established to monitor the assembly of in vitro-imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane [TOM] core complex). The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40. Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway. We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore.

Amino Acid Sequence↗
PubMed2001

Antineutrophil cytoplasmic antibodies induce human monocytes to produce oxygen radicals in vitro.

OBJECTIVE: Antineutrophil cytoplasmic antibodies (ANCA) are believed to play a pathogenetic role in necrotizing small-vessel vasculitis. While the involvement of neutrophils in this disease has been extensively studied in vitro, we undertook to analyze thoroughly the contribution of monocytes to tissue destruction in systemic vasculitis. METHODS: Monocytes obtained from normal human individuals were stimulated by ANCA isolated from patients with active vasculitis. The formation of oxygen radicals was measured by a fluorometric assay using 2',7'-dichlorofluorescin diacetate. RESULTS: ANCA induced monocytes to produce oxygen radicals, resulting in a mean 43% increase (range 21-84%) in oxygen radical formation compared with normal IgG. The formation of reactive oxygen species was time and concentration dependent and was also induced by ANCA F(ab')2 fragments. Normal nonspecific IgG or their corresponding F(ab')2 fragments induced no release or very little release of oxygen radicals. Preincubation of monocytes with the Fcy receptor type II-blocking monoclonal antibody IV.3 before addition of ANCA greatly reduced formation of oxygen radicals. Using ligand affinity chromatography with proteinase 3 (PR3) and myeloperoxidase (MPO), ANCA were further purified by depletion of patient IgG. The stimulation of monocytes with these pure PR3- and MPO-ANCA confirmed that cellular activation was specifically induced by ANCA. CONCLUSION: These results show that ANCA induce the formation of reactive oxygen species in human monocytes. These findings support the notion that ANCA specifically activate monocytes by several mechanisms to participate in the inflammatory process of ANCA-associated vasculitis.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed2001

Analysis of protein-protein interactions in mitochondria by coimmunoprecipitation and chemical cross-linking.

Many different techniques have been employed to analyze protein-protein interactions. Coimmunoprecipitation and chemical cross-linking have been used extensively to study mitochondrial biogenesis. Both techniques have proven to be powerful methods to investigate the sequential interactions of precursor proteins with the various components of the translocation machineries in the mitochondrial membranes. Similarly, protein-protein interactions during processes such as protein synthesis, folding, and degradation can be studied. Moreover, the composition of the oligomeric protein complexes of mitochondria, such as respiratory chain complexes or protein translocation machineries, can be determined. The general principles and protocols of these methods are described and illustrated with typical examples.

Adenosine Triphosphatases↗
PubMed2001

Import of proteins into mitochondria: a novel pathomechanism for progressive neurodegeneration.

The vast majority of mitochondrial proteins are encoded as precursors by the nuclear genome. A major aspect of mitochondrial biogenesis is therefore the transfer of nuclear-encoded, cytosplasmically synthesized precursor proteins across and into the mitochondrial membranes. During the past years the use of simple model organisms such as the yeasts S. cerevisiae and N. crassa has helped considerably to identify and unravel the structure and function of a substantial number of components involved in targeting of nuclear-encoded preproteins to mitochondria. Several pathways and a number of components were characterized that are involved in guiding mitochondrial preproteins to their specific sites of function. In particular, import of nuclear-encoded precursor proteins into and across the mitochondrial inner membrane is mediated by two distinct translocases, the TIM23 complex and the TIM22 complex. Both TIM complexes cooperate with the general preprotein translocase of the outer membrane, TOM complex. The TIM complexes differ in the their substrate specificity. While the TIM23 complex mediates import of preproteins with a positively charged matrix targeting signal, the TIM22 complex facilitates the insertion of a class of hydrophobic proteins with internal targeting signals into the inner membrane. Most recently the rapid progress of research has allowed elucidation of a new mitochondrial disease on the molecular level. This rare X-linked progressive neurodegenerative disorder, named Mohr-Tranebjaerg (MT syndrome), is caused by mutations in the DDP1 gene and includes sensorineural deafness, blindness, mental retardation and a complex movement disorder. The analysis of the novel pathomechanism is based on the homology of the affected DDP1 protein to a family of conserved yeast components acting along the TIM22 pathway. This contribution briefly summarizes the current knowledge of the pathways of protein import and proposes a mechanism to explain how defective import leads to neurodegeneration.

Animals↗
PubMed2001

Electron tomography of mitochondria after the arrest of protein import associated with Tom19 depletion.

In a mutant form of Neurospora crassa, in which sheltered RIP (repeat induced point mutation) was used to deplete Tom19, protein transport through the TOM/TIM pathway is arrested by the addition of p-fluorophenylalanine (FPA). Using intermediate-voltage electron tomography, we have generated three-dimensional reconstructions of 28 FPA-treated mitochondria at four time points (0-32 h) after the addition of FPA. We determined that the cristae surface area and volume were lost in a roughly linear manner. A decrease in mitochondrial volume was not observed until after 16 h of FPA treatment. The inner boundary membrane did not appear to shrink or contract away from the outer membrane. Interestingly, the close apposition of these membranes remained over the entire periphery, even after all of the cristae had disappeared. The different dynamics of the shrinkage of cristae membrane and inner boundary membrane has implications for compartmentalization of electron transport proteins. Two structurally distinct types of contact sites were observed, consistent with recently published work. We determined that the cristae in the untreated (control) mitochondria are all lamellar. The cristae of FPA-treated mitochondria retain the lamellar morphology as they reduce in size and do not adopt tubular shapes. Importantly, the crista junctions exhibit tubular as well as slot-like connections to the inner boundary membrane, persisting until the cristae disappear, indicating that their stability is not dependent on continuous protein import through the complex containing Tom19.

Fungal Proteins↗
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↗