PubMed Health⌕ Search

Biomedical subjects

W Neupert

Publications and source records attributed to W Neupert.

At least 163 records · Page 9Linked to original sources
PubMed1994

Mitochondrial molecular chaperones: their role in protein translocation.

After synthesis in the cytosol, most mitochondrial proteins must traverse mitochondrial membranes to reach their functional location. During this process, proteins become unfolded and then refold to attain their native conformation after crossing the lipid bilayers. Mitochondrial molecular chaperones play an essential mechanistic role at various steps of this process. They facilitate presequence translocation, unfolding of the cytosol-localized domains of precursor proteins, movement across the mitochondrial membranes and, finally, folding of newly imported proteins within the matrix.

Biological Transport↗
PubMed1994

A crucial role of the mitochondrial protein import receptor MOM19 for the biogenesis of mitochondria.

The novel genetic method of "sheltered RIP" (repeat induced point mutation) was used to generate a Neurospora crassa mutant in which MOM19, a component of the protein import machinery of the mitochondrial outer membrane, can be depleted. Deficiency in MOM19 resulted in a severe growth defect, but the cells remained viable. The number of mitochondrial profiles was not grossly changed, but mutant mitochondria were highly deficient in cristae membranes, cytochromes, and protein synthesis activity. Protein import into isolated mutant mitochondria was decreased by factors of 6 to 30 for most proteins from all suborganellar compartments. Proteins like the ADP/ATP carrier, MOM19, and cytochrome c, whose import into wild-type mitochondria occurs independently of MOM19 became imported normally showing that the reduced import activities are solely caused by a lack of MOM19. Depletion of MOM19 reveals a close functional relationship between MOM19 and MOM22, since loss of MOM19 led to decreased levels of MOM22 and reduced protein import through MOM22. Furthermore, MOM72 does not function as a general backup receptor for MOM19 suggesting that these two proteins have distinct precursor specificities. These findings demonstrate that the import receptor MOM19 fulfills an important role in the biogenesis of mitochondria and that it is essential for the formation of mitochondria competent in respiration and phosphorylation.

Cytochromes↗
PubMed1994

Mitochondrial heat shock protein 70, a molecular chaperone for proteins encoded by mitochondrial DNA.

Mitochondrial heat shock protein 70 (mt-Hsp70) has been shown to play an important role in facilitating import into, as well as folding and assembly of nuclear-encoded proteins in the mitochondrial matrix. Here, we describe a role for mt-Hsp70 in chaperoning proteins encoded by mitochondrial DNA and synthesized within mitochondria. The availability of mt-Hsp70 function influences the pattern of proteins synthesized in mitochondria of yeast both in vivo and in vitro. In particular, we show that mt-Hsp70 acts in maintaining the var1 protein, the only mitochondrially encoded subunit of mitochondrial ribosomes, in an assembly competent state, especially under heat stress conditions. Furthermore, mt-Hsp70 helps to facilitate assembly of mitochondrially encoded subunits of the ATP synthase complex. By interacting with the ATP-ase 9 oligomer, mt-Hsp70 promotes assembly of ATP-ase 6, and thereby protects the latter protein from proteolytic degradation. Thus mt-Hsp70 by acting as a chaperone for proteins encoded by the mitochondrial DNA, has a critical role in the assembly of supra-molecular complexes.

DNA, Mitochondrial↗
PubMed1994

Inactivation of the Neurospora crassa gene encoding the mitochondrial protein import receptor MOM19 by the technique of "sheltered RIP".

We have used a technique referred to as "sheltered RIP" (repeat induced point mutation) to create mutants of the mom-19 gene of Neurospora crassa, which encodes an import receptor for nuclear encoded mitochondrial precursor proteins. Sheltered RIP permits the isolation of a mutant gene in one nucleus, even if that gene is essential for the survival of the organism, by sheltering the nucleus carrying the mutant gene in a heterokaryon with an unaffected nucleus. Furthermore, the nucleus harboring the RIPed gene contains a selectable marker so that it is possible to shift nuclear ratios in the heterokaryons to a state in which the nucleus containing the RIPed gene predominates in cultures grown under selective conditions. This results in a condition where the target gene product should be present at very suboptimal levels and allows the study of the mutant phenotype. One allele of mom-19 generated by this method contains 44 transitions resulting in 18 amino acid substitutions. When the heterokaryon containing this allele was grown under conditions favoring the RIPed nucleus, no MOM19 protein was detectable in the mitochondria of the strain. Homokaryotic strains containing the RIPed allele exhibit a complex and extremely slow growth phenotype suggesting that the product of the mom-19 gene is important in N. crassa.

Amino Acid Sequence↗
PubMed1993

A matrix ATP requirement for presequence translocation across the inner membrane of mitochondria.

The mitochondrial presequence initiates protein translocation across the inner membrane of mitochondria in a delta psi-dependent step. We have investigated the role of matrix ATP in this process. When matrix ATP was reduced to interfere with the function of mitochondrial heat shock protein 70, presequence translocation across the inner membrane was strongly inhibited. This was accompanied by the accumulation of an import intermediate that was unprocessed and accessible to protease added to the intact mitochondria. Both delta psi and matrix ATP were required for further translocation of this intermediate into the matrix. When ATP levels are insufficient to support protein import, it appears that the presequence becomes translocated across the inner membrane, but delta psi does not maintain it in the matrix. Presequence translocation across the inner membrane is thus a reversible reaction, and a step dependent on matrix ATP is required to make it unidirectional. Based on these observations, a model on the role of delta psi, mthsp70, and matrix ATP in presequence translocation across the inner membrane is presented.

Adenosine Triphosphate↗
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

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↗
PubMed1993

Functional independence of the protein translocation machineries in mitochondrial outer and inner membranes: passage of preproteins through the intermembrane space.

The protein translocation machineries of the outer and inner mitochondrial membranes usually act in concert during translocation of matrix and inner membrane proteins. We considered whether the two machineries can function independently of each other in a sequential reaction. Fusion proteins (pF-CCHL) were constructed which contained dual targeting information, one for the intermembrane space present in cytochrome c heme lyase (CCHL) and the other for the matrix space contained in the signal sequence of the precursor of F1-ATPase beta-subunit (pF1 beta). In the absence of a membrane potential, delta psi, the fusion proteins moved into the intermembrane space using the CCHL pathway. In contrast, in the presence of delta psi they followed the pF1 beta pathway and eventually were translocated into the matrix. The fusion protein pF51-CCHL containing 51 amino acids of pF1 beta, once transported into the intermembrane space in the absence of a membrane potential, could be further chased into the matrix upon re-establishing delta psi. The sequential and independent movement of the fusion protein across the two membranes demonstrates that the translocation machineries act as distinct entities. Our results support a model in which the two translocation machineries can function independently of each other, but generally interact in a dynamic fashion to achieve simultaneous translocation across both membranes. In addition, the results provide information about the targeting sequences within CCHL. The protein does not contain a signal for retention in the intermembrane space; rather, it lacks matrix targeting information, and therefore is unable to undergo delta psi-dependent interaction with the protein translocation apparatus in the inner membrane.

Biological Transport↗
PubMed1993

Targeting of cytochrome b2 into the mitochondrial intermembrane space: specific recognition of the sorting signal.

UNLABELLED: Cytochrome b2 contains 2-fold targeting information: an amino-terminal signal for targeting to the mitochondrial matrix, followed by a second cleavable sorting signal that functions in directing the precursor into the mitochondrial intermembrane space. The role of the second sorting sequence was analyzed by replacing one, two or all of the three positively charged amino acid residues which are present at the amino-terminal side of the hydrophobic core by uncharged residues or an acidic residue. With a number of these mutant precursor proteins, processing to the mature form was reduced or completely abolished and at the same time targeting to the matrix space occurred. The accumulation in the matrix depended on a high level of intramitochondrial ATP. At low levels of matrix ATP, the mutant proteins were sorted into the intermembrane space like the wild-type precursors. THE RESULTS: (i) suggest the existence of one or more matrix components that specifically recognize the second sorting signal and thereby trigger the translocation into the intermembrane space; (ii) indicate that the mutant signals have reduced ability to interact with the recognition component(s) and then embark on the default pathway into the matrix by interacting with mitochondrial hsp70 in conjunction with matrix ATP; (iii) strongly argue against a mechanism by which the hydrophobic segment of the sorting sequence stops translocation in the hydrophobic phase of the inner membrane.

Adenosine Triphosphate↗
PubMed1993

Translocation and insertion of precursor proteins into isolated outer membranes of mitochondria.

Nuclear-encoded proteins destined for mitochondria must cross the outer or both outer and inner membranes to reach their final sub-mitochondrial locations. While the inner membrane can translocate preproteins by itself, it is not known whether the outer membrane also contains an endogenous protein translocation activity which can function independently of the inner membrane. To selectively study the protein transport into and across the outer membrane of Neurospora crassa mitochondria, outer membrane vesicles were isolated which were sealed, in a right-side-out orientation, and virtually free of inner membranes. The vesicles were functional in the insertion and assembly of various outer membrane proteins such as porin, MOM19, and MOM22. Like with intact mitochondria, import into isolated outer membranes was dependent on protease-sensitive surface receptors and led to correct folding and membrane integration. The vesicles were also capable of importing a peripheral component of the inner membrane, cytochrome c heme lyase (CCHL), in a receptor-dependent fashion. Thus, the protein translocation machinery of the outer mitochondrial membrane can function as an independent entity which recognizes, inserts, and translocates mitochondrial preproteins of the outer membrane and the intermembrane space. In contrast, proteins which have to be translocated into or across the inner membrane were only specifically bound to the vesicles, but not imported. This suggests that transport of such proteins involves the participation of components of the intermembrane space and/or the inner membrane, and that in these cases the outer membrane translocation machinery has to act in concert with that of the inner membrane.

Biological Transport↗
PubMed1992

Two related genes encoding extremely hydrophobic proteins suppress a lethal mutation in the yeast mitochondrial processing enhancing protein.

The processing enhancing protein of mitochondria (PEP) is an essential component that has been shown to participate in proteolytic removal of NH2-terminal signal peptides from precursor proteins imported into the mitochondrial matrix. Using a yeast strain bearing a PEP mutation that renders it temperature-sensitive, an approach of genetic suppression was taken in order to identify additional components that could be involved with protein import: high copy plasmids comprising a yeast genomic library were tested for ability to suppress the 37 degrees C growth defect. Two plasmids were isolated, pSMF1 and pSMF2, which suppressed the growth defect nearly as well as the cloned PEP gene itself. Sequence analysis of the rescuing genes predicted extremely hydrophobic proteins with sizes of 63 and 60 kDa, respectively. Remarkably, the predicted SMF1 and SMF2 products are 49% identical to each other overall. To test the requirement for SMF1 and SMF2, the chromosomal genes were disrupted. Individual disruption was without effect, but cells in which both genes were disrupted grew poorly. When mitochondria were prepared from the double disruption strain grown in a nonfermentable carbon source, they were morphologically normal but defective for translocation of radiolabeled precursor proteins. SMF1 protein was provisionally localized to the mitochondrial membranes using epitope tagging. We suggest that SMF1 and SMF2 are mitochondrial membrane proteins that influence PEP-dependent protein import, possibly at the step of protein translocation.

Amino Acid Sequence↗
PubMed1992

Transport of proteins into the various subcompartments of mitochondria.

The import of proteins into mitochondria is an intricate process comprised of multiple steps. The first step involves the sorting of cytosolically synthesized precursor proteins to the mitochondrial surface. There precursor proteins are recognized by specific receptors which deliver them to the general import site present in the outer membrane. The second stage of import involves a series of complex intraorganelle sorting events which results in the delivery of the proteins to one of the four possible submitochondrial destinations, namely the outer and inner membranes, the matrix and intermembrane space. Here in this review, we discuss the current knowledge on these intramitochondrial sorting events. We especially focus on targeting of proteins to the intermembrane space. Sorting to the intermembrane space represents a particularly interesting situation, as at least three separate targeting pathways to this subcompartment are known to exist.

Biological Transport↗
PubMed1992

Identification of the mitochondrial receptor complex in Saccharomyces cerevisiae.

Mitochondrial protein import involves the recognition of preproteins by receptors and their subsequent translocation across the outer membrane. In Neurospora crassa, the two import receptors, MOM19 and MOM72, were found in a complex with the general insertion protein, GIP (formed by MOM7, MOM8, MOM30 and MOM38) and MOM22. We isolated a complex out of S. cerevisiae mitochondria consisting of MOM38/ISP42, the receptor MOM72, and five new yeast proteins, the putative equivalents of N. crassa MOM7, MOM8, MOM19, MOM22 and MOM30. A receptor complex isolated out of yeast cells transformed with N. crassa MOM19 contained the N. crassa master receptor in addition to the yeast proteins. This demonstrates that the yeast complex is functional, and provides strong evidence that we also have identified the yeast MOM19.

Biological Transport↗
PubMed1992

Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space.

Cytochrome b2 reaches the intermembrane space of mitochondria by transport into the matrix followed by export across the inner membrane. While in the matrix, the protein interacts with hsp60, which arrests its folding prior to export. The bacterial-type export sequence in pre-cytochrome b2 functions by inhibiting the ATP-dependent release of the protein from hsp60. Release for export apparently requires, in addition to ATP, the interaction of the signal sequence with a component of the export machinery in the inner membrane. Export can occur before import is complete provided that a critical length of the polypeptide chain has been translocated into the matrix. Thus, hsp60 combines two activities: catalysis of folding of proteins destined for the matrix, and maintaining proteins in an unfolded state to facilitate their channeling between the machineries for import and export across the inner membrane. Anti-folding signals such as the hydrophobic export sequence in cytochrome b2 may act as switches between these two activities.

Base Sequence↗