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Biomedical subjects

W Neupert

Publications and source records attributed to W Neupert.

At least 181 records · Page 10Linked to original sources
PubMed1992

Mapping of the protein import machinery in the mitochondrial outer membrane by crosslinking of translocation intermediates.

Mitochondria contain a complex machinery for the import of nuclear-encoded proteins. Receptor proteins exposed on the outer membrane surface are required for the specific binding of precursor proteins to mitochondria, either by binding of cytosolic signal recognition factors or by direct recognition of the precursor polypeptides. Subsequently, the precursors are inserted into the outer membrane at the general insertion site GIP (general insertion protein). Here we report the analysis of receptors and GIP by crosslinking of translocation intermediates and by coimmunoprecipitation. Surface-accumulated precursors were crosslinked to the receptors MOM19 and MOM72, suggesting a direct interaction of preproteins with surface receptors. We identified three novel mitochondrial outer membrane proteins, MOM7, MOM8, and MOM30 that, together with the previously identified MOM38, seem to form the GIP site and are present in the mitochondrial receptor complex.

Animals↗
PubMed1992

Protein translocation across mitochondrial membranes.

Protein translocation across biological membranes is of fundamental importance for the biogenesis of organelles and in protein secretion. We will give an overview of the recent achievements in the understanding of protein translocation across mitochondrial membranes. In particular we will focus on recently identified components of the mitochondrial import apparatus.

Biological Transport↗
PubMed1992

Import of cytochrome c heme lyase into mitochondria: a novel pathway into the intermembrane space.

Cytochrome c heme lyase (CCHL) catalyses the covalent attachment of the heme group to apocytochrome c during its import into mitochondria. The enzyme is membrane-associated and is located within the intermembrane space. The precursor of CCHL synthesized in vitro was efficiently translocated into isolated mitochondria from Neurospora crassa. The imported CCHL, like the native protein, was correctly localized to the intermembrane space, where it was membrane-bound. As with the majority of mitochondrial precursor proteins, CCHL uses the MOM19-GIP receptor complex in the outer membrane for import. In contrast to proteins taking the general import route, CCHL was imported independently of both ATP-hydrolysis and an electrochemical potential as external energy sources. CCHL which lacks a cleavable signal sequence apparently does not traverse the inner membrane to reach the intermembrane space; rather, it translocates through the outer membrane only. Thus, CCHL represents an example of a novel, 'non-conservative' import pathway into the intermembrane space, thereby also showing that the import apparatus in the outer membrane acts separately from the import machinery in the inner membrane.

Adenosine Triphosphate↗
PubMed1991

Targeting of the master receptor MOM19 to mitochondria.

The targeting of proteins to mitochondria involves the recognition of the precursor proteins by receptors on the mitochondrial surface followed by insertion of the precursors into the outer membrane at the general insertion site GIP. Most mitochondrial proteins analyzed so far use a mitochondrial outer membrane protein of 19 kilodaltons (MOM19) as an import receptor. The gene encoding MOM19 has now been isolated. The deduced amino acid sequence predicts that MOM19 is anchored in the outer membrane by an NH2-terminal hydrophobic sequence, while the rest of the protein forms a hydrophilic domain exposed to the cytosol. MOM19 was targeted to the mitochondria via a pathway that is independent of protease-accessible surface receptors and controlled by direct assembly of the MOM19 precursor with GIP.

Amino Acid Sequence↗
PubMed1991

Biogenesis of the mitochondrial phosphate carrier.

The mitochondrial phosphate carrier (PiC) is a member of the family of inner-membrane carrier proteins which are generally synthesized without a cleavable presequence. Surprisingly, the cDNA sequences of bovine and rat PiC suggested the existence of an amino-terminal extension sequence in the precursor of PiC. By expressing PiC in vitro, we found that PiC is indeed synthesized as a larger precursor. This precursor was imported and proteolytically processed by mitochondria, whereby the correct amino-terminus of the mature protein was generated. Import of PiC showed the characteristics of mitochondrial protein uptake, such as dependence on ATP and a membrane potential and involvement of contact sites between mitochondrial outer and inner membranes. The precursor imported in vitro was correctly assembled into the functional form, demonstrating that the authentic import and assembly pathway of PiC was reconstituted when starting with the presequence-carrying precursor. These results are discussed in connection with the recently postulated role of PiC as an import receptor located in the outer membrane.

Amino Acid Sequence↗
PubMed1991

Mitochondrial import receptors for precursor proteins.

The specific targeting of precursor proteins synthesized in the cytosol to various cell organelles is a central aspect of intracellular protein traffic. Several hundred different proteins are imported from the cytosol into the mitochondria. Recent studies have identified the mitochondrial outer membrane proteins MOM19, MOM72, MOM38 (approximately ISP42) and p32 which have a role in initial steps of protein import. The first three components are present in a multi-subunit complex that catalyses recognition and membrane insertion of precursor proteins.

Biological Transport, Active↗
PubMed1991

Protein folding causes an arrest of preprotein translocation into mitochondria in vivo.

With vital yeast cells, a hybrid protein consisting of the amino-terminal third of the precursor to cytochrome b2 and of the entire dihydrofolate reductase was arrested on the import pathway into mitochondria. Accumulation of the protein in the mitochondrial membranes was achieved by inducing a stable tertiary structure of the dihydrofolate reductase domain. Thereby, three salient features of mitochondrial protein uptake in vivo were demonstrated: its posttranslational character; the requirement for unfolding of precursors; and import through translocation contact sites. The permanent occupation of translocation sites by the fusion protein inhibited the import of other precursors; it did, however, not lead to leakage of mitochondrial ions, implying the existence of a channel that is sealed around the membrane spanning polypeptide segment.

Aminopterin↗
PubMed1991

Processing of mitochondrial precursor proteins.

The mitochondrial processing enzyme consists of two components, the mitochondrial processing peptidase (MPP) and processing enhancing protein (PEP). MPP and PEP act cooperatively in proteolytic processing of mitochondrial precursor proteins. Most of the mitochondrial precursors possess aminoterminal presequences (also called "targeting sequences" or "signal sequences"), that do not display a common motif and that show only limited similarities of the cleavage sites. The mitochondrial processing peptidase is a metal-dependent endoprotease, sensitive to sulfhydryl-modifying reagents and appears to belong to a new class of proteases. MPP and PEP, together with the core 1 and core 2 proteins of the respiratory complex III, form a new protein family.

Amino Acid Sequence↗
PubMed1990

Precursor proteins in transit through mitochondrial contact sites interact with hsp70 in the matrix.

We previously reported that hsp70 in the mitochondrial matrix (mt-hsp 70 = Ssclp) is required for import of precursor proteins destined for the matrix or intermembrane space. Here we show that mt-hsp70 is also needed for the import of mitochondrial inner membrane proteins. In particular, the precursor of ADP/ATP carrier that is known not to interact with hsp60 on its assembly pathway requires functional mt-hsp70 for import, suggesting a general role of mt-hsp70 in membrane translocation of precursors. Moreover, a precursor arrested in contact sites was specifically co-precipitated with antibodies directed against mt-hsp70. We conclude that mt-hsp70 is directly involved in the translocation of many, if not all, precursor proteins that are transported across the inner membrane.

Biological Transport↗
PubMed1990

Polypeptides traverse the mitochondrial envelope in an extended state.

Most mitochondrial proteins are synthesized as precursors in the cytosol and imported through contact sites between outer and inner mitochondrial membranes. The molecular mechanism of membrane translocation of precursor proteins is largely unclear. For this report, various hybrid proteins between portions of the precursor of cytochrome b2 and the entire dihydrofolate reductase (DHFR) were accumulated in mitochondrial contact sites. We unexpectedly found that about 50 amino acid residues of the polypeptide chain in transit were sufficient to span both membranes. This suggests a linear translocation of the polypeptide chain and presents evidence for a high degree of unfolding of polypeptides traversing the mitochondrial membranes.

Biological Transport↗
PubMed1990

Import of apocytochrome c into the mitochondrial intermembrane space along a cytochrome c1 sorting pathway.

The question of whether cytochrome c could be functionally sorted to the mitochondrial intermembrane space along a "conservative sorting" pathway was investigated using a fusion protein termed pLc1-c. pLc1-c contains 3-fold targeting information, namely, the complete bipartite presequence of the cytochrome c1 precursor joined to the amino terminus of apocytochrome c. pLc1-c could be selectively imported into the intermembrane space either directly across the outer membrane along a cytochrome c import route or along a cytochrome c1 route via the matrix. Thus, apocytochrome c could be sorted along a conservative sorting pathway; however, following reexport from the matrix, apo-Lc1-c could not be converted to its holo counterpart. Despite the apparent similarity of structure and functional location of the heme lyases and similarity of the heme binding regions in their respective apoproteins, cytochrome c heme lyase and cytochrome c1 heme lyase apparently have different and nonoverlapping substrate specificities.

Amino Acid Sequence↗
PubMed1990

Energy requirements for unfolding and membrane translocation of precursor proteins during import into mitochondria.

ATP is involved in conferring transport competence to numerous mitochondrial precursor proteins in the cytosol. Unfolded precursor proteins were found not to require ATP for import into mitochondria, suggesting a role of ATP in the unfolding of precursors. Here we report the unexpected finding that a hybrid protein containing the tightly folded passenger protein dihydrofolate reductase becomes unfolded and specifically translocated across the mitochondrial membranes independently of added ATP. Moreover, interaction of the precursor with the mitochondrial receptor components does not require ATP. The results suggest that ATP is not involved in the actual process of unfolding during membrane translocation of precursors. ATP rather appears to be necessary for preventing the formation of improper structures of precursors in the cytosol and for folding of imported polypeptides on (and release from) chaperone-like molecules in the mitochondrial matrix.

Adenosine Triphosphate↗
PubMed1990

Sorting pathways of mitochondrial inner membrane proteins.

Two distinct pathways of sorting and assembly of nuclear-encoded mitochondrial inner membrane proteins are described. In the first pathway, precursor proteins that carry amino-terminal targeting signals are initially translocated via contact sites between both mitochondrial membranes into the mitochondrial matrix. They become proteolytically processed, interact with the 60-kDa heat-shock protein hsp60 in the matrix and are retranslocated to the inner membrane. The sorting of subunit 9 of Neurospora crassa F0-ATPase has been studied as an example. F0 subunit 9 belongs to that class of nuclear-encoded mitochondrial proteins which are evolutionarily derived from a prokaryotic ancestor according to the endosymbiont hypothesis. We suggest that after import into mitochondria, these proteins follow the ancestral sorting and assembly pathways established in prokaryotes (conservative sorting). On the other hand, ADP/ATP carrier was found not to require interaction with hsp60 for import and assembly. This agrees with previous findings that the ADP/ATP carrier possesses non-amino-terminal targeting signals and uses a different import receptor to other mitochondrial precursor proteins. It is proposed that the ADP/ATP carrier represents a class of mitochondrial inner membrane proteins which do not have a prokaryotic equivalent and thus appear to follow a non-conservative sorting pathway.

Base Sequence↗