Biomedical subjects

W Neupert

Publications and source records attributed to W Neupert.

At least 91 records · Page 5Linked to original sources

PubMed1997

Autocatalytic processing of the ATP-dependent PIM1 protease: crucial function of a pro-region for sorting to mitochondria.

The biogenesis of the ATP-dependent PIM1 protease of mitochondria was studied by mutational analysis. The ATPase and proteolytic activities of PIM1 were shown to be essential for mitochondrial function. A proteolytically inactive mutant form of PIM1 protease accumulated as a pro-form in mitochondria, revealing a two-step processing of PIM1: the matrix targeting signal is removed by the mitochondrial processing peptidase and then a pro-region of 61 amino acids is cleaved off in an autocatalytic reaction. This latter process depended on the ATP-dependent assembly of PIM1 protease subunits and can occur by an intermolecular and, most probably, also an intramolecular pathway. The respiratory competence of cells harboring mutant PIM1 protease lacking the pro-region was strongly impaired. Subcellular fractionation revealed a cytosolic localization of mutant PIM1 protease. This demonstrates the requirement for the propeptide for efficient sorting of PIM1 protease to mitochondria.

ATP-Dependent Proteases↗

PubMed1997

Oxa1p mediates the export of the N- and C-termini of pCoxII from the mitochondrial matrix to the intermembrane space.

Oxa1p is a mitochondrial protein reported to be involved in the assembly of the cytochrome oxidase complex. In the absence of a functional Oxa1p, subunit II of the cytochrome oxidase accumulates as its precursor form (pCoxII). Using mitochondria isolated from a yeast strain bearing a temperature sensitive mutation in the Oxa1p, pet ts1402, we have analyzed the function of the Oxa1p protein. We demonstrate that the accumulation of pCoxII in the pet ts1402 mitochondria does not reflect a compromised Imp1p activity in this mutant. Furthermore, measurement of the membrane potential has shown it to be sufficient to support the export of CoxII from the matrix. Rather, we found that newly synthesized pCoxII accumulates in the matrix of the pet ts1402 mitochondria, because export across the inner membrane is inhibited in the pet ts1402 mitochondria. In conclusion, Oxa1p mediates the export of the N- and C-termini of the mitochondrially encoded subunit II of cytochrome oxidase from the matrix to the intermembrane space.

Biological Transport↗

PubMed1997

Functional cooperation and stoichiometry of protein translocases of the outer and inner membranes of mitochondria.

The qualitative relationship between preprotein translocases in the mitochondrial outer and inner membranes was determined by both a functional analysis and a determination of characteristic components of the translocases. Translocation contact sites of isolated mitochondria were saturated with intermediates of a matrix-targeted precursor of the beta-subunit of the F1-ATPase (pF1beta), and import of preproteins into the different mitochondrial subcompartments was monitored. A strong inhibition (75-95%) was observed for preproteins with an N-terminal matrix targeting signal, indicating that a significant portion of the contact sites was blocked by accumulated F1beta. Insertion of preproteins into the outer membrane and import into the intermembrane space of preproteins without matrix targeting signals was inhibited by about 45%, indicating that functional outer membrane translocases were available despite saturation of contact sites. Similarly, import of members of the mitochondrial carrier family into the inner membrane was only partly inhibited (40-50%), demonstrating that functional Tim22 translocases were available to cooperate with the Tom machinery in the import of carrier proteins. The stoichiometry of Tom40, Tim23, and Tim22 in mitochondria was determined to be 5:1:0.22. We conclude that translocases of the outer membrane are present in excess over translocases of the inner membrane.

Biological Transport↗

PubMed1997

Mdj2p, a novel DnaJ homolog in the mitochondrial inner membrane of the yeast Saccharomyces cerevisiae.

Members of the heat shock protein 70 (Hsp70) family mediate import, folding, assembly and degradation of proteins in mitochondria. The function of Hsp70 proteins is dependent on their interaction with cofactors, including members of the DnaJ protein family. The mitochondrial DnaJ homolog, Mdj1p, has been shown to cooperate with the major mitochondrial Hsp70, mt-Hsp70. We describe the identification of a second mitochondrial DnaJ homolog, Mdj2p, in the yeast Saccharomyces cerevisiae. The protein possesses an N-terminal transmembrane domain that anchors it in the mitochondrial inner membrane. The C-terminal J-domain shares 30% amino acid identity with the J-domain of Escherichia coli DnaJ and is exposed to the mitochondrial matrix. Mdj2p carries a putative internal mitochondrial targeting signal and is imported into mitochondria in a membrane potential-dependent manner. Deletion of the MDJ2 gene did not result in a detectable growth defect. Double mutants of mdj1 and mdj2 showed severe growth defects at elevated temperature, indicating a distinct overlap of the functions of Mdj1p and Mdj2p.

Amino Acid Sequence↗

PubMed1997

Mitochondrial protein import. Tom40 plays a major role in targeting and translocation of preproteins by forming a specific binding site for the presequence.

During preprotein transport across the mitochondrial outer membrane, the N-terminal presequence initially binds to a surface-exposed site, termed cis site, of the protein translocation complex of this membrane (the TOM complex). The presequence then moves into the translocation pore and becomes exposed at the intermembrane space side. Membrane passage is driven by specific interaction of the presequence with the trans site. We have used chemical cross-linking to identify components in the vicinity of the translocating presequence. Preproteins bound to the surface-exposed cis site can be cross-linked via their N-terminal presequence to Tom20 and Tom22, demonstrating their direct association with this part of the preprotein. In addition, the presequence establishes an early contact to Tom40, a membrane-embedded protein of the TOM complex. Upon further entry of the preprotein into the translocation pore, the presequence loses its contact with Tom20/Tom22, but remains in firm association with Tom40. Our study suggests that Tom40 plays an important function in guiding the presequence of a preprotein across the mitochondrial outer membrane. We propose that Tom40 forms a major part of the trans presequence binding site.

Animals↗

PubMed1997

Yeast mitochondrial F1F0-ATPase: the novel subunit e is identical to Tim11.

We report here the identification of the novel subunit of the mitochondrial F1F0-ATPase from Saccharomyces cerevisiae, ATPase subunit e. Yeast ATPase subunit e displays significant similarities in both amino acid sequence, properties (hydropathy and predicted coiled-coil structure) and orientation in the inner membrane, with previously identified mammalian ATPase subunit e proteins. Estimation of its native molecular mass and ability to be co-immunoprecipitated with a subunit of the F1-ATPase, demonstrate that subunit e is a subunit of the F1F0-ATPase. Stable expression of subunit e requires the presence of the mitochondrially encoded subunits of the F0-ATPase. Subunit e had been previously identified as Tim11 and was proposed to be involved in the process of sorting of proteins to the mitochondrial inner membrane.

Amino Acid Sequence↗

PubMed1997

N-terminal hydrophobic sorting signals of preproteins confer mitochondrial hsp70 independence for import into mitochondria.

The requirement of mitochondrial hsp70 (mt-hsp70) for the import of a series of preproteins containing hydrophobic sorting signals into isolated yeast mitochondria was investigated. Here we demonstrate that the presence of such a sorting signal in proximity to the N-terminal matrix-targeting sequence of a preprotein can secure a translocating polypeptide chain in the import channel in a manner that does not require mt-hsp70 activity. Trapping the translocating chain in this fashion leads to efficient processing by the mitochondrial processing peptidase and to complete translocation across the outer mitochondrial membrane into the intermembrane space. These mt-hsp70-independent effects appear to be exerted at the level of the inner membrane through an interaction of the hydrophobic core of the sorting signal with component(s) of the translocase of the inner membrane. Hydrophobic sorting signals of inner membrane proteins inserted into the membrane from the matrix, as well as those of intermembrane space proteins, are capable of causing this mt-hsp70-independent stabilization, demonstrating that this phenomenon is not unique to those preproteins normally sorted to the intermembrane space.

Adenosine Triphosphate↗

PubMed1997

Insertion into the mitochondrial inner membrane of a polytopic protein, the nuclear-encoded Oxa1p.

Oxa1p, a nuclear-encoded protein of the mitochondrial inner membrane with five predicted transmembrane (TM) segments is synthesized as a precursor (pOxa1p) with an N-terminal presequence. It becomes imported in a process requiring the membrane potential, matrix ATP, mt-Hsp70 and the mitochondrial processing peptidase (MPP). After processing, the negatively charged N-terminus of Oxa1p (approximately 90 amino acid residues) is translocated back across the inner membrane into the intermembrane space and thereby attains its native N(out)-C(in) orientation. This export event is dependent on the membrane potential. Chimeric preproteins containing N-terminal stretches of increasing lengths of Oxa1p fused on mouse dehydrofolate reductase (DHFR) were imported into isolated mitochondria. In each case, their DHFR moieties crossed the inner membrane into the matrix. Thus Oxa1p apparently does not contain a stop transfer signal. Instead the TM segments are inserted into the membrane from the matrix side in a pairwise fashion. The sorting pathway of pOxa1p is suggested to combine the pathways of general import into the matrix with a bacterial-type export process. We postulate that at least two different sorting pathways exist in mitochondria for polytopic inner membrane proteins, the evolutionarily novel pathway for members of the ADP/ATP carrier family and a conserved Oxa1p-type pathway.

Adenosine Triphosphate↗

PubMed1997

Peripheral noxious stimulation releases spinal PGE2 during the first phase in the formalin assay of the rat.

Injection of formalin (5%; 50 microl) into the dorsal surface of the hind paw of rats evoked a characteristic biphasic flinching behaviour of the injured paw accompanied by a significant increase in the interstitial prostaglandin E2 (PGE2) concentration of the dorsal lumbar spinal cord. Interestingly, the increase in PGE2 concentration was only observed during the first phase of the formalin behavioural response (during the 0-10 and 10-20 min microdialysis-sample). Saline paw injection did not have a significant effect on behaviour or on PGE2 concentration. These data suggest that spinal release of PGE2 is involved in nociceptive processing in the formalin-induced hyperalgesia model of the rat during the first but not second phase.

Animals↗

PubMed1997

Effects of ibuprofen enantiomers and its coenzyme A thioesters on human prostaglandin endoperoxide synthases.

1. Ibuprofen enantiomers and their respective coenzyme A thioesters were tested in human platelets and blood monocytes to determine their selectivity and potency as inhibitors of cyclo-oxygenase activity of prostaglandin endoperoxide synthase-1 (PGHS-1) and PGHS-2. 2. Human blood from volunteers was drawn and allowed to clot at 37 degrees C for 1 h in the presence of increasing concentrations of the test compounds (R-ibuprofen, S-ibuprofen, R-ibuprofenoyl-CoA, S-ibuprofenoyl-CoA, NS-398). Immunoreactive (ir) thromboxane B2 (TXB2) concentrations in serum were determined by a specific EIA assay as an index of the cyclo-oxygenase activity of platelet PGHS-1. 3. Heparin-treated blood from the same donors was incubated at 37 degrees C for 24 h with the same concentrations of the test compounds in the presence of lipopolysaccharide (LPS, 10 microg ml[-1]). The contribution of PGHS-1 was suppressed by pretreatment of the volunteers with aspirin (500 mg; 48 h before venepuncture). As a measure of LPS induced PGHS-2 activity immunoreactive prostaglandin E2 (irPGE2) plasma concentrations were determined by a specific EIA assay. 4. S-ibuprofen inhibited the activity of PGHS-1 (IC50 2.1 microM) and PGHS-2 (IC50 1.6 microM) equally. R-ibuprofen inhibited PGHS-1 (IC50 34.9) less potently than S-ibuprofen and showed no inhibition of PGHS-2 up to 250 microM. By contrast R-ibuprofenoyl-CoA thioester inhibited PGE2 production from LPS-stimulated monocytes almost two orders of magnitude more potently than the generation of TXB2 (IC50 5.6 vs 219 microM). 5. Western blotting of PGHS-2 after LPS induction of blood monocytes showed a concentration-dependent inhibition of PGHS-2 protein expression by ibuprofenoyl-CoA thioesters. 6. These data confirm that S-ibuprofen represents the active entity in the racemate with respect to cyclo-oxygenase activity. More importantly the data suggest a contribution of the R-enantiomer to therapeutic effects not only by chiral inversion to S-ibuprofen but also via inhibition of induction of PGHS-2 mediated by R-ibuprofenoyl-CoA thioester. 7. The data may explain why racemic ibuprofen is ranked as one of the safest non-steroidal anti-inflammatory drugs (NSAIDs) so far determined in epidemiological studies.

Blood Platelets↗

PubMed1997

Disposition and effects of flurbiprofen enantiomers in human serum and blister fluid.

AIMS: To investigate the pharmacokinetics of the enantiomers of flurbiprofen and inhibition of prostanoid production in blister fluid and serum. METHODS: Eleven healthy volunteers received 75 mg R-, 75 mg S-flurbiprofen or no medication in a randomized 3-way cross-over study. Flurbiprofen concentrations were determined by h.p.l.c. TXB2 and PGE2 were determined by enzyme immunoassay and chemiluminescence immunoassay respectively. RESULTS: S-flurbiprofen produced almost complete (> 99% vs baseline) inhibition of thromboxane B2 (TXB2) in serum in all volunteers and significant inhibition of prostaglandin E2 (PGE2) generation in blister fluid, but there was a considerable inter-individual variation in the response ranging from -78 to +190% change from control PGE2 AUC. After administration of R-flurbiprofen, there was a mean maximum TXB2 inhibition of 65.2 +/- 15.0% in serum but no significant changes of PGE2 levels in blister fluid were observed. The pharmacokinetic parameters in serum and blister fluid were not significantly different between enantiomers. R- to S-inversion did not occur to a clinically relevant extent. For R-flurbiprofen, the complex rate constant of transfer into blister fluid was greater at the u.v.-exposed site (0.110 +/- 0.050) than at the control site (0.079 +/- 0.026, P < 0.05) which corresponded to a higher AUC and Cmax of R-flurbiprofen in u.v.-exposed blister as compared with control. For inhibition of TXB2 generation after administration of S-flurbiprofen, a sigmoidal log-linear concentration-response relationship was established in all subjects (EC50: 0.123 +/- 0.092 microgram ml-1). In contrast, inhibition of PGE2 production in blister showed no clear concentration-response relationship when correlated with concentrations of S-flurbiprofen in either serum or blister fluid. After administration of R-flurbiprofen, no concentration-effect relationship could be established. CONCLUSIONS: It is concluded that the blister model may have value for studying the pharmacokinetics and pharmacodynamics of antiinflammatory drugs in humans. Interestingly, inter-individual variation in the pharmacokinetics of flurbiprofen enantiomers could not account for the variability in response observed in the blister model.

Adult↗

PubMed1997

Protein import into mitochondria.

Mitochondria import many hundreds of different proteins that are encoded by nuclear genes. These proteins are targeted to the mitochondria, translocated through the mitochondrial membranes, and sorted to the different mitochondrial subcompartments. Separate translocases in the mitochondrial outer membrane (TOM complex) and in the inner membrane (TIM complex) facilitate recognition of preproteins and transport across the two membranes. Factors in the cytosol assist in targeting of preproteins. Protein components in the matrix partake in energetically driving translocation in a reaction that depends on the membrane potential and matrix-ATP. Molecular chaperones in the matrix exert multiple functions in translocation, sorting, folding, and assembly of newly imported proteins.

Adenosine Triphosphatases↗

PubMed1996

Heme binding to a conserved Cys-Pro-Val motif is crucial for the catalytic function of mitochondrial heme lyases.

Covalent attachment of heme to the apoforms of mitochondrial cytochromes c and c1 requires the activity of cytochrome c heme lyase (CCHL) and cytochrome c1 heme lyase (CC1HL), respectively. The two enzymes differ in their cytochrome specificity, but they are related in sequence, and both contain conserved Cys-Pro-Val (CPV) motifs. By using various in vitro assays we investigated whether heme can bind directly to heme lyases and whether the CPV motif may be involved in heme binding. Heme stabilized CC1HL, as a model protein, in a folded, protease-resistant conformation, stimulated the refolding of CC1HL after urea denaturation, and inhibited the import of the CC1HL precursor into mitochondria. These effects were not observed with a point mutant, CC1HLSPV, in which cysteine was replaced by serine, and with CC1HLDeltaCPV, in which the motif was deleted. These results show that heme lyases can bind heme directly, and they identify the CPV sequence as a structural element important for this interaction. The phenotype of a yeast mutant expressing CC1HLSPV is in good agreement with such a role of the CPV motif. The mutant cells accumulate the heme-free intermediate form of cytochrome c1 and display a severe deficiency in the holo form. We suggest that the CPV motif forms a crucial part of the substrate binding site for heme.

Amino Acid Sequence↗

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↗

PubMed1996

Import of carrier proteins into the mitochondrial inner membrane mediated by Tim22.

Translocation of mitochondrial preproteins across the inner membrane is facilitated by the TIM machinery. Tim23 binds to matrix targeting signals and initiates membrane potential-dependent import. Tim23 and Tim17 are constituents of a translocation channel across the inner membrane. Tim44 is associated with this channel at the matrix side, and Tim44 recruits mitochondrial Hsp70 and its co-chaperone Mgel, which drive protein translocation into the matrix using ATP as an energy source. Tim22 is a new component of the import machinery of mitochondria, which shares sequence similarity with both Tim23 and Tim17. Here we report that Tim22 is required for the import of proteins of the mitochondrial ADP/ATP carrier (AAC) family into the inner membrane. Members of the yeast AAC family are synthesized without matrix targeting signals. Tim22 is in an assembly of high relative molecular mass that is distinct from the Tim23-Tim17 complex. Import of proteins of the AAC family is independent of Tim23, and import of matrix targeting signals containing preproteins is independent of Tim22.

Amino Acid Sequence↗

PubMed1996

The nucleotide exchange factor MGE exerts a key function in the ATP-dependent cycle of mt-Hsp70-Tim44 interaction driving mitochondrial protein import.

Import of preproteins into the mitochondrial matrix is driven by the ATP-dependent interaction of mt-Hsp70 with the peripheral inner membrane import protein Tim44 and the preprotein in transit. We show that Mge1p, a co-chaperone of mt-Hsp70, plays a key role in the ATP-dependent import reaction cycle in yeast. Our data suggest a cycle in which the mt-Hsp70-Tim44 complex forms with ATP: Mge1p promotes assembly of the complex in the presence of ATP. Hydrolysis of ATP by mt-Hsp70 occurs in complex with Tim44. Mge1p is then required for the dissociation of the ADP form of mt-Hsp70 from Tim44 after release of inorganic phosphate but before release of ADP. ATP hydrolysis and complex dissociation are accompanied by tight binding of mt-Hsp70 to the preprotein in transit. Subsequently, the release of mt-Hsp70 from the polypeptide chain is triggered by Mge1p which promotes release of ADP from mt-Hsp70. Rebinding of ATP to mt-Hsp70 completes the reaction cycle.

Adenosine Triphosphate↗

PubMed1996

Role of Tim23 as voltage sensor and presequence receptor in protein import into mitochondria.

Tim23, an essential component of the protein import machinery of the inner membrane of mitochondria (TIM complex), forms dimers that display a dynamic behavior. Dimer formation is promoted by the membrane potential delta psi. Binding of a matrix targeting sequence to Tim23 triggers dimer dissociation. Monomeric Tim23 is present when a preprotein chain is in transit across the TIM complex. Dimerization of Tim23 is dependent on the second half of its N-terminal hydrophilic domain, which is exposed to the intermembrane space. This segment contains a heptad leucine repeat motif with a predicted capacity for dimer formation. We propose that Tim23 exerts a key function in protein import: Tim23 dimers formed in response to delta psi act as receptors for matrix targeting sequences on the surface of the inner membrane. The ensuring dissociation of Tim23 dimer triggers opening of the TIM channel and insertion of the preprotein.

Amino Acid Sequence↗

PubMed1996

AAA proteases with catalytic sites on opposite membrane surfaces comprise a proteolytic system for the ATP-dependent degradation of inner membrane proteins in mitochondria.

The mechanism of selective protein degradation of membrane proteins in mitochondria has been studied employing a model protein that is subject to rapid proteolysis within the inner membrane. Protein degradation was mediated by two different proteases: (i) the m-AAA protease, a protease complex consisting of multiple copies of the ATP-dependent metallopeptidases Yta1Op (Afg3p) and Yta12p (Rcalp); and (ii) by Ymelp (Ytallp) that also is embedded in the inner membrane. Ymelp, highly homologous to Yta1Op and Yta12p, forms a complex of approximately 850 kDa in the inner membrane and exerts ATP-dependent metallopeptidase activity. While the m-AAA protease exposes catalytic sites to the mitochondrial matrix, Ymelp is active in the intermembrane space. The Ymelp complex was therefore termed 'i-AAA protease'. Analysis of the proteolytic fragments indicated cleavage of the model polypeptide at the inner and outer membrane surface and within the membrane-spanning domain. Thus, two AAA proteases with their catalytic sites on opposite membrane surfaces constitute a novel proteolytic system for the degradation of membrane proteins in mitochondria.

ATP-Dependent Proteases↗