Biomedical subjects

W Neupert

Publications and source records attributed to W Neupert.

At least 73 records · Page 4Linked to original sources

PubMed1998

Mcx1p, a ClpX homologue in mitochondria of Saccharomyces cerevisiae.

Members of the Hsp100/Clp-family of molecular chaperones form regulatory subunits of ATP-dependent Clp proteases and fulfill crucial roles for cellular thermotolerance. We have identified a Clp-like protein in Saccharomyces cerevisiae, Mcx1p, which shares approximately 30% sequence identity with ClpX-proteins in bacteria, plants and nematodes. Mcx1p localizes to the matrix space of mitochondria and is peripherally associated with the inner membrane. A homologue of E. coli ClpP protease was not identified when screening the yeast genome. We therefore propose that Mcx1p represents a novel molecular chaperone of mitochondria with non-proteolytic function.

ATPases Associated with Diverse Cellular Activitie↗

PubMed1998

Import into mitochondria, folding and retrograde movement of fumarase in yeast.

A single translation product of the FUM1 gene encoding fumarase is distributed between the cytosol and mitochondria of Saccharomyces cerevisiae. All fumarase translation products are targeted and processed in mitochondria before distribution. Here we show that targeting of fumarase is coupled to translation and initially involves insertion of the protein across the mitochondrial membranes and processing by the matrix protease. Rapid folding of fumarase may determine its requirement for coupling of its translocation with translation and unique route of distribution. The amino termini of most fumarase molecules are translocated across the mitochondrial membranes and processed. Unlike the in vivo situation where these molecules are released into the cytosol, in vitro they remain externally attached to the mitochondria, thereby positioned for release from the organelle. Our model suggests that fumarase displays a unique mechanism of targeting and distribution, which occurs cotranslationally and involves folding and retrograde movement of the processed protein back through the translocation pore.

Biological Transport↗

PubMed1998

The formation of respiratory chain complexes in mitochondria is under the proteolytic control of the m-AAA protease.

Yta10p (Afg3p) and Yta12p (Rcal1p), members of the conserved AAA family of ATPases, are subunits of the mitochondrial m-AAA protease, an inner membrane ATP-dependent metallopeptidase. Deletion of YTA10 or YTA12 impairs degradation of non-assembled inner membrane proteins and assembly of respiratory chain complexes. Mutations of the proteolytic sites in either YTA10 or YTA12 have been shown to inhibit proteolysis of membrane-integrated polypeptides but not the respiratory competence of the cells, suggesting additional activities of Yta10p and Yta12p. Here we demonstrate essential proteolytic functions of the m-AAA protease in the biogenesis of the respiratory chain. Cells harbouring proteolytically inactive forms of both Yta10p and Yta12p are respiratory deficient and exhibit a pleiotropic phenotype similar to Deltayta10 and Deltayta12 cells. They show deficiencies in expression of the intron-containing mitochondrial genes COX1 and COB. Splicing of COX1 and COB transcripts is impaired in mitochondria lacking m-AAA protease, whilst transcription and translation can proceed in the absence of Yta10p or Yta12p. The function of the m-AAA protease appears to be confined to introns encoding mRNA maturases. Our results reveal an overlapping substrate specificity of the subunits of the m-AAA protease and explain the impaired assembly of respiratory chain complexes by defects in expression of intron-containing genes in mitochondria lacking m-AAA protease.

Adenosine Triphosphatases↗

PubMed1998

Fzo1p is a mitochondrial outer membrane protein essential for the biogenesis of functional mitochondria in Saccharomyces cerevisiae.

Fzo1p is a novel component required for the biogenesis of functional mitochondria in the yeast Saccharomyces cerevisiae. The protein is homologous to Drosophila Fzo, the first known protein mediator of mitochondrial fusion. Deletion of the FZO1 gene results in a petite phenotype, loss of mitochondrial DNA, and a fragmented mitochondrial morphology. Fzo1p is an integral protein of the mitochondrial outer membrane exposing its major part to the cytosol. It is imported into the outer membrane in a receptor-dependent manner. Fzo1p is part of a larger protein complex of 800 kDa, and presumably is the first identified component of the yeast mitochondrial fusion machinery.

Fungal Proteins↗

PubMed1998

ATP-dependent proteolysis in mitochondria. m-AAA protease and PIM1 protease exert overlapping substrate specificities and cooperate with the mtHsp70 system.

To analyze protein degradation in mitochondria and the role of molecular chaperone proteins in this process, bovine apocytochrome P450scc was employed as a model protein. When imported into isolated yeast mitochondria, P450scc was mislocalized to the matrix and rapidly degraded. This proteolytic breakdown was mediated by the ATP-dependent PIM1 protease, a Lon-like protease in the mitochondrial matrix, in cooperation with the mtHsp70 system. In addition, a derivative of P450scc was studied to which a heterologous transmembrane region was fused at the amino terminus. This protein became anchored to the inner membrane upon import and was degraded by the membrane-embedded, ATP-dependent m-AAA protease. Again, degradation depended on the mtHsp70 system; it was inhibited at non-permissive temperature in mitochondria carrying temperature-sensitive mutant forms of Ssc1p, Mdj1p, or Mge1p. These results demonstrate overlapping substrate specificities of PIM1 and the m-AAA protease, and they assign a central role to the mtHsp70 system during the degradation of misfolded polypeptides by both proteases.

ATP-Dependent Proteases↗

PubMed1998

The preprotein translocation channel of the outer membrane of mitochondria.

The preprotein translocase of the outer membrane of mitochondria (TOM complex) facilitates the recognition, insertion, and translocation of nuclear-encoded mitochondrial preproteins. We have purified the TOM complex from Neurospora crassa and analyzed its composition and functional properties. The TOM complex contains a cation-selective high-conductance channel. Upon reconstitution into liposomes, it mediates integration of proteins into and translocation across the lipid bilayer. TOM complex particles have a diameter of about 138 A, as revealed by electron microscopy and image analysis; they contain two or three centers of stain-filled openings, which we interpret as pores with an apparent diameter of about 20 A. We conclude that the structure reported here represents the protein-conducting channel of the mitochondrial outer membrane.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1998

The ATP-dependent PIM1 protease is required for the expression of intron-containing genes in mitochondria.

The ATP-dependent PIM1 protease, a Lon-like protease localized in the mitochondrial matrix, is required for mitochondrial genome integrity in yeast. Cells lacking PIM1 accumulate lesions in the mitochondrial DNA (mtDNA) and therefore lose respiratory competence. The identification of a multicopy suppressor, which stabilizes mtDNA in the absence of PIM1, enabled us to characterize novel functions of PIM1 protease during mitochondrial biogenesis. The synthesis of mitochondrially encoded cytochrome c oxidase subunit I (CoxI) and cytochrome b (Cob) is impaired in pim1 mutants containing mtDNA. PIM1-mediated proteolysis is required for the translation of mature COXI mRNA. Moreover, deficiencies in the splicing of COXI and COB transcripts, which appear to be restricted to introns encoding mRNA maturases, were observed in cells lacking the PIM1 gene. Transcripts of COXI and COB genes harboring multiple introns are degraded in the absence of PIM1. These results establish multiple, essential functions of the ATP-dependent PIM1 protease during mitochondrial gene expression.

ATP-Dependent Proteases↗

PubMed1998

An import signal in the cytosolic domain of the Neurospora mitochondrial outer membrane protein TOM22.

TOM22 is an integral component of the preprotein translocase of the mitochondrial outer membrane (TOM complex). The protein is anchored to the lipid bilayer by a central trans-membrane segment, thereby exposing the amino-terminal domain to the cytosol and the carboxyl-terminal portion to the intermembrane space. Here, we describe the sequence requirements for the targeting and correct insertion of Neurospora TOM22 into the outer membrane. The orientation of the protein is not influenced by the charges flanking its trans-membrane segment, in contrast to observations regarding proteins of other membranes. In vitro import studies utilizing TOM22 preproteins harboring deletions or mutations in the cytosolic domain revealed that the combination of the trans-membrane segment and intermembrane space domain of TOM22 is not sufficient to direct import into the outer membrane. In contrast, a short segment of the cytosolic domain was found to be essential for the import and assembly of TOM22. This sequence, a novel internal import signal for the outer membrane, carries a net positive charge. A mutant TOM22 in which the charge of the import signal was altered to -1 was imported less efficiently than the wild-type protein. Our data indicate that TOM22 contains physically separate import and membrane anchor sequences.

Biological Transport↗

PubMed1998

cis and trans sites of the TOM complex of mitochondria in unfolding and initial translocation of preproteins.

Translocation of preproteins across the mitochondrial outer membrane is mediated by the TOM complex. Our previous studies led to the concept of two preprotein binding sites acting in series, the surface-exposed cis site and the trans site exposed to the intermembrane space. We report here that preproteins are bound to the cis site in a labile fashion even at low ionic strength, whereas intermediates arrested at the trans site remained firmly bound at higher salt concentration. The stability of the trans site intermediate results from interactions of both the presequence and unfolded parts of the mature part of the preprotein with the TOM complex. Binding to the trans site proceeded at rates comparable with those of unfolding of the mature domain and appeared to be kinetically limited by the unfolding reaction. Efficient binding to the trans site and unfolding were observed with both outer membrane vesicles and intact mitochondria whose membrane potential, DeltaPsi, was dissipated. Upon re-establishing DeltaPsi, trans site-bound preprotein resumed translocation into the matrix. The rates of unfolding and binding to the trans site were the same as those for translocation into intact energized mitochondria. We conclude that preprotein unfolding in intact mitochondria can take place without the involvement of the translocation machinery of the inner membrane and, in particular, the matrix Hsp70 chaperone. Further, preprotein unfolding at the outer membrane can be a rate-limiting step for formation of the trans site intermediate and for the entire translocation reaction.

Animals↗

PubMed1998

Sorting of D-lactate dehydrogenase to the inner membrane of mitochondria. Analysis of topogenic signal and energetic requirements.

D-Lactate dehydrogenase (D-LD) is located in the inner membrane of mitochondria. It spans the membrane once in an Nin-Cout orientation with the bulk of the protein residing as a folded domain in the intermembrane space. D-LD is synthesized as a precursor with an N-terminal cleavable presequence and is imported into the mitochondria in a Deltapsi-dependent, but mt-Hsp70-independent manner. Upon import in vitro D-LD folds in the intermembrane space to attain a conformation indistinguishable from endogenous D-LD. Sorting of D-LD to the inner membrane is directed by a composite topogenic signal consisting of the hydrophobic transmembrane segment and a cluster of charged amino acids C-terminal to it. We propose a model for the mode of operation of the sorting signal of D-LD. This model also accounts for the driving force of translocation across the outer membrane, in the apparent absence of mt-Hsp70-dependent assisted import and involves the folding of the D-LD in the intermembrane space.

Cell Membrane↗

PubMed1998

Biogenesis of Tim23 and Tim17, integral components of the TIM machinery for matrix-targeted preproteins.

We analysed the import pathway of Tim23 and of Tim17, components of the mitochondrial import machinery for matrix-targeted preproteins. Tim23 contains two independent import signals. One is located within the first 62 amino acid residues of the hydrophilic domain that, in the assembled protein, is exposed to the intermembrane space. This signal mediates translocation of Tim23 across the outer membrane independently of the membrane potential, DeltaPsi. A second import signal is located in the C-terminal membrane-integrated portion of Tim23. It mediates translocation across the outer membrane and insertion into the inner membrane in a strictly DeltaPsi-dependent fashion. Structurally, Tim17 is related to Tim23 but lacks a hydrophilic domain. It contains an import signal in the C-terminal half and its import requires DeltaPsi. The DeltaPsi-dependent import signals of Tim23 and Tim17 are located at corresponding sites in these two homologous proteins. They exhibit features reminiscent of the positively charged N-terminal presequences of matrix-targeted precursors. Import of Tim23 and its insertion into the inner membrane requires Tim22 but not functional Tim23. Thus, biogenesis of the Tim23.17 complex depends on the Tim22 complex, which is the translocase identified as mediating the import of carrier proteins.

Amino Acid Sequence↗

PubMed1998

Mam33p, an oligomeric, acidic protein in the mitochondrial matrix of Saccharomyces cerevisiae is related to the human complement receptor gC1q-R.

Mam33p (mitochondrial acidic matrix protein) is a soluble protein, located in mitochondria of Saccharomyces cerevisiae. It is synthesized as a precursor with an N-terminal mitochondrial targeting sequence that is processed on import. Mam33p assembles to a homo-oligomeric complex in the mitochondrial matrix. It can bind to the sorting signal of cytochrome b2 that directs this protein into the intermembrane space. Mam33p is encoded by an 801 bp open reading frame. Gene disruption did not result in a significant growth defect. Mam33p exhibits sequence similarity to gC1q-R, a human protein that has been implicated in the binding of complement factor C1q and kininogen.

Amino Acid Sequence↗

PubMed1998

Oxa1p, an essential component of the N-tail protein export machinery in mitochondria.

A number of nuclear encoded inner membrane proteins of mitochondria span the membrane in such a manner that their N termini are located in the intermembrane space. Many of these proteins attain this membrane orientation by undergoing an export step from the matrix across the inner membrane. This export process, which resembles bacterial N-tail export from energetic and topogenic signal requirements, is facilitated by Oxa1p, a protein that has homologues throughout prokaryotes and eukaryotes. Oxa1p, as we have previously shown, is required to export the N and C termini of the mitochondrially encoded pCoxII to the intermembrane space. We demonstrate here that imported nuclear encoded proteins physically interact with Oxa1p and depend on Oxa1p for efficient export of their N termini to the intermembrane space. Furthermore, Oxa1p interacts with nascent polypeptide chains synthesized in mitochondria, including the fully synthesized pCoxII and CoxIII species. Thus, Oxa1p represents a component of a general export machinery of the mitochondrial inner membrane.

Cross-Linking Reagents↗

PubMed1998

Carrier protein import into mitochondria mediated by the intermembrane proteins Tim10/Mrs11 and Tim12/Mrs5.

Import of nuclear-encoded precursor proteins into mitochondria and their subsequent sorting into mitochondrial subcompartments is mediated by translocase enzymes in the mitochondrial outer and inner membranes. Precursor proteins carrying amino-terminal targeting signals are translocated into the matrix by the integral inner membrane proteins Tim23 and Tim17 in cooperation with Tim44 and mitochondrial Hsp70. We describe here the discovery of a new pathway for the transport of members of the mitochondrial carrier family and other inner membrane proteins that contain internal targeting signals. Two related proteins in the intermembrane space, Tim10/Mrs11 and Tim12/Mrs5, interact sequentially with these precursors and facilitate their translocation across the outer membrane, irrespective of the membrane potential. Tim10 and Tim12 are found in a complex with Tim22, which takes over the precursor and mediates its membrane-potential-dependent insertion into the inner membrane. This interaction of Tim10 and Tim12 with the precursors depends on the presence of divalent metal ions. Both proteins contain a zinc-finger-like motif with four cysteines and bind equimolar amounts of zinc ions.

Amino Acid Sequence↗

PubMed1998

Two distinct and independent mitochondrial targeting signals function in the sorting of an inner membrane protein, cytochrome c1.

Proteins of the mitochondrial inner membrane display a wide variety of orientations, many spanning the membrane more than once. Some of these proteins are synthesized with NH2-terminal cleavable targeting sequences (presequences) whereas others are targeted to mitochondria via internal signals. Here we report that two distinct mitochondrial targeting signals can be present in precursors of inner membrane proteins, an NH2-terminal one and a second, internal one. Using cytochrome c1 as a model protein, we demonstrate that these two mitochondrial targeting signals operate independently of each other. The internal targeting signal, consisting of a transmembrane segment and a stretch of positively charged amino acid residues directly following it, initially directs the translocation of the preprotein into the intermembrane space. It then inserts into the inner membrane from the intermembrane space side in a delta psi-dependent manner and thereby determines the orientation the protein attains in the inner membrane. Analysis of a number of other presequence-containing protein of the inner membrane suggest that they too contain such internal targeting signals.

Adenosine Triphosphate↗

PubMed1998

Protein translocation into mitochondria.

The mitochondrial membranes contain translocation machineries ("preprotein translocases") which facilitate transport of proteins from the cytosol into the mitochondria. The TOM complex is localized in the outer membrane. Two translocases have been characterized in the inner membrane: the Tim23-Tim17 complex and the Tim22 complex which differ in their specificities for preproteins. The TOM and TIM complexes can act in a coordinated fashion to facilitate the insertion of preproteins into the mitochondrial membranes as well as the translocation into the internal compartments, the intermembrane space and the matrix. We discuss insights into the function of components of the translocases, illustrated by examples for mechanisms of protein sorting into the different compartment of mitochondria.

Biological Transport↗

PubMed1998

Role of the negative charges in the cytosolic domain of TOM22 in the import of precursor proteins into mitochondria.

TOM22 is an essential mitochondrial outer membrane protein required for the import of precursor proteins into the organelles. The amino-terminal 84 amino acids of TOM22 extend into the cytosol and include 19 negatively and 6 positively charged residues. This region of the protein is thought to interact with positively charged presequences on mitochondrial preproteins, presumably via electrostatic interactions. We constructed a series of mutant derivatives of TOM22 in which 2 to 15 of the negatively charged residues in the cytosolic domain were changed to their corresponding amido forms. The mutant constructs were transformed into a sheltered Neurospora crassa heterokaryon bearing a tom22::hygromycin R disruption in one nucleus. All constructs restored viability to the disruption-carrying nucleus and gave rise to homokaryotic strains containing mutant tom22 alleles. Isolated mitochondria from three representative mutant strains, including the mutant carrying 15 neutralized residues (strain 861), imported precursor proteins at efficiencies comparable to those for wild-type organelles. Precursor binding studies with mitochondrial outer membrane vesicles from several of the mutant strains, including strain 861, revealed only slight differences from binding to wild-type vesicles. Deletion mutants lacking portions of the negatively charged region of TOM22 can also restore viability to the disruption-containing nucleus, but mutants lacking the entire region cannot. Taken together, these data suggest that an abundance of negative charges in the cytosolic domain of TOM22 is not essential for the binding or import of mitochondrial precursor proteins; however, other features in the domain are required.

Alleles↗

PubMed1998

Dynamics of the TOM complex of mitochondria during binding and translocation of preproteins.

Translocation of preproteins across the mitochondrial outer membrane is mediated by the TOM complex. This complex consists of receptor components for the initial contact with preproteins at the mitochondrial surface and membrane-embedded proteins which promote transport and form the translocation pore. In order to understand the interplay between the translocating preprotein and the constituents of the TOM complex, we analyzed the dynamics of the TOM complex of Neurospora crassa and Saccharomyces cerevisiae mitochondria by following the structural alterations of the essential pore component Tom40 during the translocation of preproteins. Tom40 exists in a homo-oligomeric assembly and dynamically interacts with Tom6. The Tom40 assembly is influenced by a block of negatively charged amino acid residues in the cytosolic domain of Tom22, indicating a cross-talk between preprotein receptors and the translocation pore. Preprotein binding to specific sites on either side of the outer membrane (cis and trans sites) induces distinct structural alterations of Tom40. To a large extent, these changes are mediated by interaction with the mitochondrial targeting sequence. We propose that such targeting sequence-induced adaptations are a critical feature of translocases in order to facilitate the movement of preproteins across cellular membranes.

Antibodies↗