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N Pfanner

Publications and source records attributed to N Pfanner.

At least 91 records · Page 5Linked to original sources
PubMed1995

Cyclophilin 20 is involved in mitochondrial protein folding in cooperation with molecular chaperones Hsp70 and Hsp60.

We studied the role of mitochondrial cyclophilin 20 (CyP20), a peptidyl-prolyl cis-trans isomerase, in preprotein translocation across the mitochondrial membranes and protein folding inside the organelle. The inhibitory drug cyclosporin A did not impair membrane translocation of preproteins, but it delayed the folding of an imported protein in wild-type mitochondria. Similarly, Neurospora crassa mitochondria lacking CyP20 efficiently imported preproteins into the matrix, but folding of an imported protein was significantly delayed, indicating that CyP20 is involved in protein folding in the matrix. The slow folding in the mutant mitochondria was not inhibited by cyclosporin A. Folding intermediates of precursor molecules reversibly accumulated at the molecular chaperones Hsp70 and Hsp60 in the matrix. We conclude that CyP20 is a component of the mitochondrial protein folding machinery and that it cooperates with Hsp70 and Hsp60. It is speculated that peptidyl-prolyl cis-trans isomerases in other cellular compartments may similarly promote protein folding in cooperation with chaperone proteins.

Amino Acid Isomerases↗
PubMed1995

Posttranslational mitochondrial protein import in a homologous yeast in vitro system.

Posttranslational import of preproteins into mitochondria has been reported to be inefficient in a homologous yeast in vitro system, suggesting a requirement for coupling of protein synthesis and import. We have characterized a homologous yeast in vitro system which allows posttranslational mitochondrial import of preproteins. The efficiency is comparable to that of the heterologous system with rabbit reticulocyte lysate and isolated yeast mitochondria. Import in the homologous system depends on mitochondrial surface receptors, a membrane potential and the matrix heat shock protein Hsp70. Import is not blocked by the protein synthesis inhibitor cycloheximide, but is impaired by induction of stable folding in preproteins. Our studies demonstrate a posttranslational translocation mechanism in the homologous system, strongly supporting the validity of conclusions drawn from the widely used heterologous import system.

Animals↗
PubMed1994

Identification of the essential yeast protein MIM17, an integral mitochondrial inner membrane protein involved in protein import.

We analyzed four Saccharomyces cerevisiae mutants defective in mitochondrial protein import and found that they are complemented by a novel gene encoding a 17 kDa protein. The protein is integrally located in the mitochondrial inner membrane and is termed MIM17. It shows significant homology to MIM23/Mas6p, a previously identified mitochondrial inner membrane protein required for the import of preproteins. Like MIM23, the precursor of MIM17 is synthesized without a presequence. A deletion of MIM17 is lethal. MIM17 thus joins the small group of mitochondrial proteins that are essential for the viability of yeast. We propose that MIM17 is an essential component of the preprotein import machinery of the mitochondrial inner membrane.

Amino Acid Sequence↗
PubMed1994

The polytopic mitochondrial inner membrane proteins MIM17 and MIM23 operate at the same preprotein import site.

Three proteins of the mitochondrial inner membrane are known that are essential for the viability of yeast and seem to be involved in import of preproteins; the integral membrane proteins MIM17 and MIM23 and the peripheral membrane protein MIM44, MIM17 and MIM23 are homologous to each other in their hydrophobic domain, expose their termini to the intermembrane space, and span the inner membrane up to four times, each. A preprotein in transit across the mitochondrial membrane is specifically cross-linked to MIM17, MIM23, MIM44, and matrix hsp70. We conclude that MIM17 and MIM23 are integral parts of a preprotein translocation channel and cooperate with MIM44 and hsp70 at the same protein import site.

Amino Acid Sequence↗
PubMed1994

Preproteins of chloroplast envelope inner membrane contain targeting information for receptor-dependent import into fungal mitochondria.

The amino-terminal transit sequences of two preproteins destined for the chloroplast inner envelope membrane show similarities to mitochondrial presequences in the prevalence of positive charges and the potential formation of an amphipathic alpha-helix. We studied if these preproteins could be imported into mitochondria and found a low, yet significant import into isolated plant mitochondria. The plant mitochondria were previously shown not to import precursors of chloroplast stromal or thylakoidal proteins. To analyze the specificity of import into mitochondria we used the established import systems of fungal mitochondria. The envelope preproteins were efficiently imported into Saccharomyces cerevisiae or Neurospora crassa mitochondria. Their import showed the characteristics of specific mitochondrial protein uptake, including a requirement for the main receptor MOM19 (mitochondrial outer membrane protein of 19 kDa) and a membrane potential across the inner membrane, and depended on the presence of the chloroplast transit sequence. We conclude that some chloroplast transit sequences contain sufficient information for specific interaction with mitochondrial import receptors (at least from fungal sources).

Ascomycota↗
PubMed1994

Specific recognition of mitochondrial preproteins by the cytosolic domain of the import receptor MOM72.

The import receptor MOM72 constitutes part of the protein translocation machinery of the outer mitochondrial membrane, the receptor-general insertion pore complex. The protein contains a membrane anchor at the NH2 terminus and a large cytosolic domain. In yeast and Neurospora crassa the cytosolic domain comprises about 570-580 amino acid residues. The cytosolic domain of yeast MOM72 was purified after expression in Escherichia coli as a homogeneous monomeric protein. It can recognize precursor proteins as demonstrated by its ability to compete for binding and import into the mitochondria and to physically interact with preproteins. A subset of preproteins including the ADP/ATP carrier and the phosphate carrier interact with very high affinity, precursors that are known to be targeted via MOM72. Thus, the cytosolic domain of MOM72 plays a critical function in the recognition of preproteins by directly binding to precursor proteins and thereby facilitating their targeting to mitochondria.

Base Sequence↗
PubMed1994

Deletion of the receptor MOM19 strongly impairs import of cleavable preproteins into Saccharomyces cerevisiae mitochondria.

The mitochondrial outer membrane proteins MOM19 and MOM72 are thought to function as import receptors for nuclear encoded preproteins. Different views exist about the importance of each receptor in the import of cleavable and noncleavable preproteins into mitochondria. Here we cloned and sequenced MOM19 from Saccharomyces cerevisiae and constructed a gene disruption mutant. Yeast cells lacking MOM19 were unable to grow on nonfermentable carbon sources and were slow in growing on a fermentable medium, while the growth of yeast cells lacking MOM72 (Mas70p) was much less impaired. delta MOM19 cells accumulated considerable amounts of mitochondrial preproteins in vivo. The import of cleavable preproteins into isolated delta MOM19 mitochondria was strongly inhibited, while import of the noncleavable ADP/ATP carrier and phosphate carrier was only slightly inhibited. The reciprocal situation was found for protein import into delta MOM72 mitochondria. In particular, import of the cleavable precursor of cytochrome c1 into delta MOM72 mitochondria was, in agreement with a previous report (Hines, V., and Schatz, G. (1993) J. Biol. Chem. 268, 449-454), found to be partially inhibited, yet a much stronger inhibition of import was seen into delta MOM19 mitochondria. The direct comparison of protein import into yeast mutants of either receptor yields a unifying hypothesis on mitochondrial preprotein targeting; both receptors have an overlapping specificity, and MOM19 plays a major role for cleavable preproteins. Interestingly, the primary sequence of MOM19 predicts the presence of a tetratricopeptide motif that was also found in MOM72, in the peroxisomal membrane protein PAS8/PAS10, and in several proteins involved in RNA synthesis or mitosis.

Amino Acid Sequence↗
PubMed1994

The protein import machinery of the mitochondrial inner membrane.

Mitochondria import most of their proteins from the cytosol. Although considerable information is available on the import machineries of the mitochondrial outer membrane and matrix, until recently little was known about the machinery of the inner membrane. Recent studies have identified three mitochondrial inner membrane proteins (MIMs) as essential components of the import machinery. MIM17 and MIM23 seem to form part of a channel, while MIM44, in cooperation with the heat-shock protein Hsp70, binds the preproteins in transit. The electrical membrane potential and ATP are needed to drive protein translocation through the MIM import machinery.

Adenosine Triphosphate↗
PubMed1994

Mitochondrial protein import: biochemical and genetic evidence for interaction of matrix hsp70 and the inner membrane protein MIM44.

The import of preproteins into mitochondria involves translocation of the polypeptide chains through putative channels in the outer and inner membranes. Preprotein-binding proteins are needed to drive the unidirectional translocation of the precursor polypeptides. Two of these preprotein-binding proteins are the peripheral inner membrane protein MIM44 and the matrix heat shock protein hsp70. We report here that MIM44 is mainly exposed on the matrix side, and a fraction of mt-hsp70 is reversibly bound to the inner membrane. Mt-hsp70 binds to MIM44 in a 1:1 ratio, suggesting that mt-hsp70 is localizing to the membrane via its interaction with MIM44. Formation of the complex requires a functional ATPase domain of mt-hsp70. Addition of Mg-ATP leads to dissociation of the complex. Overexpression of mt-hsp70 rescues the protein import defect of mutants in MIM44; conversely, overexpression of MIM44 rescues protein import defects of mt-hsp70 mutants. In addition, yeast strains with conditional mutations in both MIM44 and mt-hsp70 are barely viable, showing a synthetic growth defect compared to strains carrying single mutations. We propose that MIM44 and mt-hsp70 cooperate in translocation of preproteins. By binding to MIM44, mt-hsp70 is recruited at the protein import sites of the inner membrane, and preproteins arriving at MIM44 may be directly handed over to mt-hsp70.

Adenosine Triphosphate↗
PubMed1994

Mitochondrial GrpE is present in a complex with hsp70 and preproteins in transit across membranes.

We characterized a 24-kDa protein associated with matrix hsp70 (mt-hsp70) of Neurospora crassa and Saccharomyces cerevisiae mitochondria. By using specific antibodies, the protein was identified as MGE, a mitochondrial homolog of the prokaryotic heat shock protein GrpE. MGE extracted from mitochondria was quantitatively bound to hsp70. It was efficiently released from hsp70 by the addition of Mg-ATP but not by nonhydrolyzable ATP analogs or high salt. A mutant mt-hsp70, which was impaired in release of bound precursor proteins, released MGE in an ATP-dependent manner, indicating that precursor proteins and MGE bind to different sites of hsp70. A preprotein accumulated in transit across the mitochondrial membranes was specifically coprecipitated by either antibodies directed against MGE or antibodies directed against mt-hsp70. The preprotein accumulated at the outer membrane was not coprecipitated by either antibody preparation. After being imported into the matrix, the preprotein could be coprecipitated only by antibodies against mt-hsp70. We propose that mt-hsp70 and MGE cooperate in membrane translocation of preproteins.

Ascomycota↗
PubMed1993

Targeting and translocation of the phosphate carrier/p32 to the inner membrane of yeast mitochondria.

We analyzed the submitochondrial location and biogenesis pathway of the phosphate carrier (PiC), also termed p32, of Saccharomyces cerevisiae mitochondria, PiC/p32 was found to behave as an integral membrane protein that cofractionated with the ADP/ATP carrier of the inner membrane. Import of the precursor of PiC/p32 required a membrane potential across the inner membrane, supporting its localization to the inner membrane. This makes it unlikely that the major function of PiC/p32 is that of an import receptor on the surface of the mitochondrial outer membrane. Furthermore, we found that both receptors MOM72 and MOM19 were involved in the import pathway of the precursor of PiC/p32 with MOM72 being responsible for the bulk of import. Yeast PiC/p32 is thus not only structurally homologous to the ADP/ATP carrier, but has a similar targeting mechanism and submitochondrial location, supporting its classification as a member of the inner membrane carrier family.

Antibodies, Monoclonal↗
PubMed1993

Biogenesis of the mitochondrial receptor complex. Two receptors are required for binding of MOM38 to the outer membrane surface.

Targeting of preproteins to mitochondria is mediated by the receptor complex in the outer membrane that contains two import receptors and the general insertion pore with MOM38 (38-kDa mitochondrial outer membrane protein) as major constituent. As all components of the receptor complex have to be imported from the cytosol themselves, the specificity of their targeting is fundamental for the correct assembly of mitochondria. None of the receptors is involved in its own import; the precursor of the main receptor MOM19 is even targeted without any surface receptor but directly assembles with MOM38. We report that import of the precursor of MOM38 strictly depended on surface receptors. The import followed a new highly selective mechanism in that both receptors together were needed for the specific binding of the preprotein to the outer membrane surface, which was followed by its assembly into the receptor complex. These findings suggest that targeting of the mitochondrial targeting components involves a complex system of mutual specificity control, ensuring a selective assembly of the components into preexisting import sites.

Animals↗
PubMed1993

Identification of MIM23, a putative component of the protein import machinery of the mitochondrial inner membrane.

A screening for yeast mutants impaired in mitochondrial protein import led to the identification of two genes (MPII and MPI2) encoding the essential components MIM44 and MIM17 of the inner membrane import machinery. We analyzed twelve additional mutants obtained in the screening and found two further complementation groups. One group represents mutants of SSC1, the gene encoding mitochondrial hsp70, an essential matrix protein required for protein import across the inner membrane. The second complementation group represents mutants of a new gene (MP13) encoding a 23 kDa integral inner membrane protein (MIM23). MIM23 is synthesized without a presequence, and its import to the inner membrane requires a membrane potential. MIM23 contains a domain homologous to half of MIM17. We speculate that MIM23 is a new member of the protein import machinery of the mitochondrial inner membrane.

Amino Acid Sequence↗
PubMed1993

The mitochondrial receptor complex: a central role of MOM22 in mediating preprotein transfer from receptors to the general insertion pore.

The receptor complex in the mitochondrial outer membrane, which consists of at least seven different proteins, is responsible for the recognition and translocation of cytosolically synthesized preproteins. Two of its subunits, MOM19 and MOM72, function as surface receptors for preproteins. Four other subunits (MOM38, MOM30, MOM8, and MOM7) have been suggested to constitute the general insertion pore (GIP). Here we report on the structure and function of MOM22. MOM22 is anchored in the outer membrane by a single transmembrane segment. The highly negatively charged N-terminal domain is exposed to the cytosol and the C-terminal domain to the intermembrane space. MOM22 appears to be a central component of the receptor complex, required for the transfer of preproteins from the receptors to the GIP. We speculate that the negatively charged domain of MOM22 is involved in the transfer of positively charged signal sequences of preproteins.

Amino Acid Sequence↗
PubMed1993

The protein import receptor MOM19 of yeast mitochondria.

We have identified the protein import receptor MOM19 of Saccharomyces cerevisiae mitochondria. MOM19 is exposed on the outer membrane surface and present in the mitochondrial receptor complex. Antibodies raised against MOM19 strongly inhibited the import of preproteins into isolated yeast mitochondria. Fab fragments prepared from the antibodies showed the same inhibitory effect. By using mutant mitochondria, which lacked the second import receptor MOM72, we found that the import of preproteins via MOM19 did not require the presence of MOM72. We conclude that MOM19 is required for preprotein translocation across the yeast mitochondrial outer membrane and is able to function independently of the receptor MOM72.

Antibodies↗
PubMed1993

Insertion of MOM22 into the mitochondrial outer membrane strictly depends on surface receptors.

Targeting of preproteins to mitochondria and their translocation across the outer membrane are mediated by the mitochondrial receptor complex. This protein complex contains the import receptors MOM19 and MOM72 and the general insertion pore GIP. All seven components of the receptor complex are synthesized in the cytosol and thus have to be targeted to the mitochondria themselves. Here we investigated the import pathway of the precursor of MOM22 into the outer membrane. In contrast to other mitochondrial preproteins studied so far, the import of MOM22 absolutely depended on the presence of surface receptors. In fact, both receptors MOM19 and MOM72 were involved in its import pathway. The targeting of MOM22 to mitochondria is thus highly specific and controlled.

Animals↗
PubMed1993

Roles of molecular chaperones in protein targeting to mitochondria.

Molecular chaperones are essential components of the machinery facilitating import of nuclear-encoded proteins into the mitochondria. They act at several steps of the complex import pathway. Cytosolic hsp 70 appears to contribute to maintaining precursors in a translocation-competent conformation. Mitochondrial hsp 70 has a distinct role in driving translocation across outer and inner mitochondrial membranes and probably in supporting unfolding of precursors in the cytosol. Hsp 60 in the matrix is involved in facilitating folding and assembly of imported polypeptide chains.

Bacterial Proteins↗