Mechanisms of protein import into mitochondria.
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Biological Transport↗
Biomedical subjects
W Neupert
Publications and source records attributed to W Neupert.
Topogenesis of inner membrane proteins of mitochondria.
Proteins of the inner mitochondrial membrane display a diverse range of topological arrangements. These proteins, encoded either by the nuclear or mitochondrial genomes, contain sequence determinants, so-called topogenic signals, which direct newly synthesized proteins to their final orientation in the membrane. The recent analysis of sorting pathways of a number of proteins highlights that these topogenic signals might act in different ways.
Genome↗
Uniform nomenclature for the protein transport machinery of the mitochondrial membranes.
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Biological Transport↗
Topogenesis of cytochrome oxidase subunit II. Mechanisms of protein export from the mitochondrial matrix.
Cytochrome c oxidase subunit II (COXII) in yeast mitochondria is synthesized as a precursor (preCOXII) and is sorted across the inner membrane, whereby both N and C termini become exposed to the intermembrane space. We describe here how this process can be experimentally dissected into a number of distinct stages. Our results demonstrate that the translation of COXII is not obligatorily coupled to translocation. Insertion into the inner membrane and export of the N- and C-terminal domains require an energized inner membrane. The export of COXII is independent of both maturation by the Imp1p protease and assembly into the cytochrome c oxidase complex. When linked to a mitochondrial matrix-targeting sequence, the N-terminal portion of preCOXII (fused to mouse dihydrofolate reductase) can be imported into the mitochondrial matrix. Following accumulation in the matrix, this chimeric protein can become exported across the inner membrane, delivering the N terminus into the intermembrane space where it undergoes processing by the Imp1p protease. This export process displays a number of similarities to bacterial protein export and supports the view that the principles of sorting are conserved from prokaryotes to eukaryotic organelles.
Animals↗
Assembly of the preprotein receptor MOM72/MAS70 into the protein import complex of the outer membrane of mitochondria.
Membrane integration and assembly of MOM72 from Neurospora crassa and its yeast homolog MAS70 was studied with isolated mitochondria. After synthesis in vitro, the precursors of MOM72/MAS70 are tightly folded and expose only their N-terminal amino acid residues comprising the targeting and the membrane anchor domain. Insertion of the protein into the mitochondrial outer membrane (MOM) occurs in a time- and temperature-dependent manner and is stimulated by ATP. MOM72/MAS70 is then assembled into the outer membrane MOM complex. Whereas membrane insertion occurred independently of the presence of protease-sensitive surface components, the assembly reaction depended on such components. In the MOM complex MOM72 and MAS70 were found in the neighborhood of different components in yeast and N. crassa mitochondria. MOM72 was found in association with MOM22 in N. crassa mitochondria, whereas MAS70 was in proximity to a 37-kDa component in yeast outer mitochondrial membrane. The interaction with the 37-kDa protein is important for integration of MAS70 into the MOM complex. Thus, the 37-kDa protein plays an important role in the biogenesis of MAS70.
Adenosine Triphosphate↗
Biogenesis of mitochondrial heme lyases in yeast. Import and folding in the intermembrane space.
Heme lyases are components of the mitochondrial intermembrane space facilitating the covalent attachment of heme to the apoforms of c-type cytochromes. The precursors of heme lyases are synthesized in the cytosol without the typical N-terminal mitochondrial targeting signal. Here, we have analyzed the mode of import and folding of the two heme lyases of the yeast Saccharomyces cerevisiae, namely of cytochrome c heme lyase and of cytochrome c1 heme lyase. For transport into mitochondria, both proteins use the general protein import machinery of the outer membrane. Import occurred independently of a membrane potential, delta psi, across the inner membrane and ATP in the matrix space, suggesting that the inner membrane is not required for transport along this direct sorting pathway. The presence of a large folded domain in heme lyases was utilized to study their folding in the intermembrane space. Formation of this domain occurred at the same rate as import, indicating that heme lyases fold either during or immediately after their transfer across the membrane. Folding was not affected by depletion of ATP and delta psi or by inhibitors of peptidylprolyl cis-trans isomerases, i.e. it does not involve homologs of known folding factors (like Hsp60 and Hsp70). The energy derived from folding cannot be regarded as a major driving force for import, since the folded domain could be imported into mitochondria with the same efficiency as the intact protein. We conclude that protein folding in the intermembrane space obeys principles different from those established for other subcellular compartments.
Adenosine Triphosphate↗
MOM22 is a receptor for mitochondrial targeting sequences and cooperates with MOM19.
Recognition of targeting signals is a crucial step in protein sorting within the cell. So far, only a few components capable of deciphering targeting signals have been identified, and insights into the chemical nature of the interaction between the signals and their receptors are scarce. Using highly purified mitochondrial outer membrane vesicles, we demonstrate that MOM22 and MOM19, components of the protein import complex of the outer membrane, bind preproteins at the mitochondrial surface in a reversible fashion. Interaction specifically and directly occurs with the N-terminal presequence and is abolished after inactivation of either MOM22 or MOM19. Binding is salt sensitive, suggesting that recognition involves electrostatic forces between the positive charges of the presequence and the acidic cytosolic domain of MOM22. MOM19 and MOM22 can be cross-linked with high efficiency. We propose that the two proteins form a complex which functions as the presequence receptor at the mitochondrial surface and facilitates the movement of preproteins into the translocation pore.
Animals↗
Hsp78, a Clp homologue within mitochondria, can substitute for chaperone functions of mt-hsp70.
Hsp78 is a Clp homologue within mitochondria of Saccharomyces cerevisiae. Deletion of HSP78 does not cause any detectable changes in wild type cells, but results in a petite phenotype in the ssc1-3 mutant strain carrying a temperature-sensitive allele of mt-hsp70. When overexpressed in the ssc1-3 mutant strain, hsp78 suppresses the defect in mitochondrial protein import under permissive conditions in vitro and interacts directly with newly imported polypeptide chains. As a molecular chaperone, hsp78 prevents the aggregation of misfolded proteins in the matrix of mitochondria under conditions of impaired mt-hsp70 function. However, unlike misfolded proteins associated with mt-hsp70, hsp78-bound polypeptides are not efficiently degraded by the ATP-dependent PIM1 protease. Thus, hsp78 can partially substitute for mt-hsp70 functions in the assembly of mitochondria and may be part of a salvage pathway if mt-hsp70 is limiting.
Amino Acid Sequence↗
Conservative sorting of F0-ATPase subunit 9: export from matrix requires delta pH across inner membrane and matrix ATP.
In an attempt to understand the mechanisms of sorting of mitochondrial inner membrane proteins, we have analyzed the import of subunit 9 of the mitochondrial F1F0-ATPase (Su9) from Neurospora crassa, an integral inner membrane protein. A chimeric protein was used consisting of the presequence and the first transmembrane domain of Su9 fused to mouse dihydrofolate reductase (preSu9(1-112)-DHFR). This protein attains the correct topology across the inner membrane (Nout-Cin) following import. The transmembrane domain becomes first completely imported into the matrix, where after processing of the presequence, it mediates membrane insertion and export of the N-terminal tail. Import and export steps can be experimentally dissected into two distinct events. Translocation of the N-terminal hydrophilic tail out of the matrix was blocked when the presequence was not processed, indicating an important role of the sequences and charges flanking the hydrophobic domain. Furthermore, export was supported by a delta pH and required matrix ATP hydrolysis. Thus the hydrophobic transmembrane domain operates as a membrane insertion signal and not as a stop-transfer signal. Our findings suggest that several aspects of this sorting process have been conserved from their prokaryotic ancestors.
Adenosine Triphosphate↗
The role of the GrpE homologue, Mge1p, in mediating protein import and protein folding in mitochondria.
Mge1p, a mitochondrial GrpE homologue, has recently been identified in the yeast Saccharomyces cerevisiae and a role for this protein in precursor import has been reported. To dissect the molecular mechanism of Mge1p function, conditional mge1 mutants were constructed. Cells harbouring mutant mge1 accumulated precursor proteins at restrictive temperature. Both kinetics and efficiency of import were reduced in mitochondria isolated from strains possessing mutant mge1. Binding of mitochondrial-Hsp70 (mt-Hsp70) to incoming precursor proteins was abolished at restrictive temperature. Nucleotide-dependent dissociation of mt-Hsp70 from the import component MIM44 was reduced in mitochondria from mutant mge1 strains. Furthermore, at restrictive temperature an increase of incompletely folded, newly imported protein and enhanced protein aggregation was observed in mitochondria isolated from the mutant strains. We conclude that Mge1p exerts an essential function in import and folding of proteins by controlling the nucleotide-dependent binding of mt-Hsp70 to substrate proteins and the association of mt-Hsp70 with MIM44.
Bacterial Proteins↗
Sorting of cytochrome b2 to the intermembrane space of mitochondria. Kinetic analysis of intermediates demonstrates passage through the matrix.
Precytochrome b2 is targeted to the mitochondrial intermembrane space by a dual targeting sequence comprising 80 amino acids. A kinetic analysis of intramitochondrial sorting was performed. The intermediate-size form accumulated transiently in the matrix. When import was performed in the presence of metal chelators to prevent the first processing by the matrix processing peptidase, > 40% of the imported precursor was localized in the matrix. A deletion of 13 amino acids in the intermembrane space sorting sequence caused partial inhibition of the first processing, and a transient accumulation of the precursor form in the matrix was also observed. The decrease in this matrix-localized precursor form paralleled an increase in the mature-size form in the intermembrane space. A point mutation in the mitochondrial targeting sequence (N-terminal to the sorting sequence) resulted in missorting to the matrix space. Furthermore, a chimeric protein consisting of the initial 85 residues of cytochrome b2 fused to dihydrofolate reductase was partially targeted to the matrix at 15 degrees C, but not at 25 degrees C. Together, the results presented here indicate that cytochrome b2 passes through the matrix on its sorting pathway to the intermembrane space.
Amino Acid Sequence↗
The MIM complex mediates preprotein translocation across the mitochondrial inner membrane and couples it to the mt-Hsp70/ATP driving system.
We have identified a complex in mitochondria that functions as a part of the preprotein import machinery of the inner membrane (MIM complex). Two known components, MIM23 and MIM17, and two novel components, MIM33 and MIM14, were found as constituents of this complex. In the presence of a translocating chain, the outer membrane import machinery (MOM complex) and the MIM complex form translocation contact sites. On the matrix side, the MIM complex is associated with the mt-Hsp70-MIM44 system. We propose a structure of the import machinery in which the MIM complex constitutes a proteinaceous channel that accepts preproteins from the MOM complex, facilitates their reversible transmembrane movement, and mediates unidirectional transport by linkage to the ATP-dependent mt-Hsp70-MIM44 system.
Adenosine Triphosphate↗
Translocation of apocytochrome c across the outer membrane of mitochondria.
Apocytochrome c follows a unique pathway into mitochondria. Import does not require the general protein translocation machinery, protease-sensitive components of the outer membrane, or a membrane potential across the inner membrane. We investigated the membrane binding and translocation steps of the import reaction using purified outer membrane vesicles (OMV) from Neurospora crassa mitochondria. OMV specifically bound, but did not import apocytochrome c. If, however, specific antibodies were enclosed inside OMV, apocytochrome c was accumulated in soluble form in the lumen. Import was reversible, since apocytochrome c became accessible to external protease after release from the antibodies. Thus, OMV are competent of translocating apocytochrome c into their lumen, but lack a binding partner which traps the apoprotein. In intact mitochondria, cytochrome c heme lyase (CCHL), a peripheral protein of the inner membrane, serves such a function by stably associating with apocytochrome c in a complex which is detectable by co-immunoprecipitation. We suggest a model for the import mechanism of apocytochrome c in which the apoprotein specifically associates with and reversibly passes across the outer membrane. Translocation is rendered unidirectional by stable association with CCHL which serves as a "trans side receptor." Finally, heme is attached by CCHL and the holoprotein folds into its native structure.
Apoproteins↗
A novel intermediate on the import pathway of cytochrome b2 into mitochondria: evidence for conservative sorting.
Cytochrome b2 is sorted into the intermembrane space of mitochondria by a bipartite N-terminal targeting and sorting presequence. In an attempt to define the sorting pathway we have identified an as yet unknown import intermediate. Cytochrome b2-dihydrofolate reductase (DHFR) fusion proteins were arrested in the presence of methotrexate (MTX) so that the DHFR domain was at the surface of the outer membrane while the N-terminus reached into the intermembrane space where the sorting signal was removed. This membrane-spanning, mature-sized species was efficiently chased into the mitochondria upon removal of MTX. Thus, an intermediate was generated which was exposed to the intermembrane space but was still associated with the inner membrane. This intermediate was also found upon direct import of cytochrome b2 and derived fusion proteins. These membrane-bound mature-sized cytochrome b2 species loop through the matrix and could be recovered in a complex with mt-Hsp70 and the inner membrane MIM44/ISP45, a component of the inner membrane import apparatus. This novel sorting intermediate can only be explained by a pathway in which cytochrome b2 passes through the matrix. The existence of such an intermediate is inconsistent with a pathway by which entrance of the mature part of cytochrome b2 into the matrix is stopped by the sorting sequence; however, its presence is fully consistent with the conservative sorting pathway.
HSP70 Heat-Shock Proteins↗
'Sheltered disruption' of Neurospora crassa MOM22, an essential component of the mitochondrial protein import complex.
MOM22 is a component of the protein import complex of the mitochondrial outer membrane of Neurospora crassa. Using the newly developed procedure of 'sheltered disruption', we created a heterokaryotic strain harboring two nuclei, one with a null allele of the mom-22 gene and the other with a wild-type allele. Homokaryons bearing the mom-22 disruption could not be isolated, suggesting that mom-22 is an essential gene. The mutant nucleus can be forced to predominate in the heterokaryon through the use of specific nutritional and inhibitor resistance markers. Cultivation of the heterokaryon under conditions favoring the mutant nucleus resulted in selective depletion of MOM22. MOM22-depleted cells did not grow and contained mitochondria with an altered morphology and protein composition. Protein import into isolated, MOM22-depleted mitochondria was abolished for most precursor proteins destined for all subcompartments. In contrast, precursors of MOM19, MOM22 and MOM72 became inserted normally into the outer membrane, defining a novel MOM22-independent import pathway which remained intact in mutant mitochondria. Furthermore, the specific binding of the ADP/ATP carrier to the outer membrane was unaffected, but subsequent transport across the outer membrane did not occur. Our data show that MOM22 is an essential component of Neurospora cells specifically required for the biogenesis of mitochondria.
Biological Transport↗
Mitochondrial protein import: reversible binding of the presequence at the trans side of the outer membrane drives partial translocation and unfolding.
The mechanism of translocation of matrix-targeted, cleavable preproteins across the mitochondrial outer membrane was studied using purified outer membrane vesicles. The N-terminal presequence interacts in a sequential and reversible fashion with two specific binding sites. The first one is provided by protease-sensitive receptors on the surface of the membrane (cis site); the second one is located at the inner face of the outer membrane (trans site). Binding to the trans site drives translocation of the N-terminal portion of the preprotein and, at the same time, unfolding of its mature part. We suggest that the outer membrane protein import machinery forms a translocation channel that permits reversible sliding of preproteins and prevents their lateral aggregation in the membrane. Although translocation can be initiated by the outer membrane, its completion requires coupling to the energetic system of the inner membrane. Our data assign an essential role to the presequence, not only for efficient targeting, but also for the translocation step.
Binding Sites↗
Identification and initial characterization of the cytosolic protein Ycr77p.
The nucleotide sequence of yeast chromosome III encompassing the previously the previously described open reading frames (ORFs) YCR80w, YCR77c and YCR78c (Oliver et al., 1992) has been updated. In the corrected sequence, these ORFs are replaced by two new ORFs, YCR80w (453 bp) and YCR77c (2391 bp). In addition, the orientation of Ycr79c is reversed to give ORF Ycr79w, which has an unaltered nt sequence. The predicted translation products do not exhibit significant homology to known proteins. ORF Ycr77p encodes an 88 kDa, cytosolic protein. A fraction of the protein is associated with small membranous structures in a salt-sensitive fashion. Initial characterization revealed that the protein is not essential for yeast viability, growth on non-fermentable carbon sources, mating and sporulation. The chromosome III DNA sequence that was used for the analysis has the Accession Number X59720 in the GenBank/EMBL database.
Amino Acid Sequence↗
Purification and characterization of mitochondrial processing peptidase of Neurospora crassa.
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Base Sequence↗