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    Mitochondrial protein import

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    Mitochondrial protein import

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    The transport of nuclear-encoded proteins from the cytosol into mitochondria is mediated by targeting (signal) sequences present on precursor forms. Most precursors of the mitochondrial matrix possess amino-terminal signals which characteristically contain hydroxylated and basic amino acids and lack acidic residues. With a minority of precursor proteins, internal sequence motifs can direct proteins to the mitochondria (Pfanner, N., Hoeben, P., Tropschug, M. and Neupert, W. (1987) J. Biol. Chem. 262, 14851–14854). The presence of a mitochondrial targeting sequence alone, however, is not sufficient for specific targeting to the organelle and further to the various subcompartments. There is the need for components which recognise the targeting sequences and others which keep the precursor protein in a translocation-competent form. Beyond the recognition step, components are required which mediate translocation across the mitochondrial membranes. Mitochondria possess two translocation machineries, one in the outer membrane and one in the inner membrane. The matrix space harbors a number of factors which participate in the import of proteins, in their unfolding and folding. Energy is required at several steps of these processes

    Mitochondrial protein import

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    The precursor of F0-ATPase subunit 9 was bound to mitochondria in the absence of a mitochondrial membrane potential (delta psi). Binding was mediated by a protease-sensitive component on the mitochondrial surface. When delta psi was reestablished, bound precursor was directly imported without prior release from the mitochondrial membranes. A chimaeric protein consisting of the complete subunit 9 precursor fused to cytosolic dihydrofolate reductase (DHFR) was also specifically bound to mitochondria in the absence of delta psi. Two other fusion proteins, consisting either of the entire presequence of subunit 9 and DHFR or of part of the presequence and DHFR, were imported in the presence of delta psi. In the absence of delta psi, however, specific binding to mitochondria did not take place. We suggest that the hydrophobic mature part of subunit 9 is involved in the delta psi-independent binding of the subunit 9 precursor to receptor sites on the mitochondrial surface

    Mitochondrial protein import

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    Proteolytic degradation of receptor sites on the mitochondrial surface strongly reduces the efficiency of mitochondrial protein import. The remaining residual import still involves basic mechanisms of protein import, including: insertion of precursors into the outer membrane, requirement for ATP and a membrane potential, and translocation through contact sites between both membranes. The import of a chloroplast protein into isolated mitochondria which occurs with a low rate is not inhibited by a protease-pretreatment of mitochondria, indicating that this precursor only follows the bypass pathway. The low efficiency of bypass import suggests that this unspecific import does not disturb the uniqueness of mitochondrial protein composition. We conclude that mitochondrial protein import involves a series of steps in which receptor sites appear to be responsible for the specificity of protein uptake

    Mitochondrial Protein Import

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    The role of nucleoside triphosphates (NTPs) in mitochondrial protein import was investigated with the precursors of N. crassa ADP/ATP carrier, F1-ATPase subunit β, F0-ATPase subunit 9, and fusion proteins between subunit 9 and mouse dihydrofolate reductase. NTPs were necessary for the initial interaction of precursors with the mitochondria and for the completion of translocation of precursors from the mitochondrial surface into the mitochondria. Higher levels of NTPs were required for the latter reactions as compared with the early stages of import. Import of precursors having identical presequences but different mature protein parts required different levels of NTPs. The sensitivity of precursors in reticulocyte lysate to proteases was decreased by removal of NTPs and increased by their readdition. We suggest that the hydrolysis of NTPs is involved in modulating the folding state of precursors in the cytosol, thereby conferring import competence

    Mitochondrial protein import

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    Mitochondrial protein import

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    Transport of nuclear-encoded precursor proteins into mitochondria includes proteolytic cleavage of aminoterminal targeting sequences in the mitochondrial matrix. We have isolated the processing activity from Neurospora crassa. The final preparation (enriched ca. 10,000-fold over cell extracts) consists of two proteins, the matrix processing peptidase (MPP, 57 kd) and a processing enhancing protein (PEP, 52 kd). The two components were isolated as monomers. PEP is about 15-fold more abundant in mitochondria than MPP. It is partly associated with the inner membrane, while MPP is soluble in the matrix. MPP alone has a low processing activity whereas PEP alone has no apparent activity. Upon recombining both, full processing activity is restored. Our data indicate that MPP contains the catalytic site and that PEP has an enhancing function. The mitochondrial processing enzyme appears to represent a new type of “signal peptidase,” different from the bacterial leader peptidase and the signal peptidase of the endoplasmic reticulum

    Mitochondrial protein import

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    Mitochondrial protein import

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    The precursors of the mitochondrial proteins ADP/ATP carrier (AAC) and F1-ATPase subunit β (F1β) were accumulated at the stages of binding to receptor sites on the mitochondrial outer membrane, or in contact sites between outer and inner membranes. Specific antibodies raised against the mature proteins were added to the isolated mitochondria and efficiently bound to these translocation intermediates. Further movement of the precursors to consecutive steps along their import pathway was thereby inhibited. Controls showed that precursor proteins which were inserted into or translocated across the outer membrane were not recognized by the antibodies unless the mitochondrial membranes were disrupted. We conclude that the trapped translocation intermediates have antigenic sites exposed to the outside of the outer membrane

    Efficient High-Dimensional Importance Sampling

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    The paper describes a simple, generic and yet highly accurate Efficient Importance Sampling (EIS) Monte Carlo (MC) procedure for the evaluation of high-dimensional numerical integrals. EIS is based upon a sequence of auxiliary weighted regressions which actually are linear under appropriate conditions. It can be used to evaluate likelihood functions and byproducts thereof, such as ML estimators, for models which depend upon unobservable variables. A dynamic stochastic volatility model and a logit panel data model with unobserved heterogeneity (random effects) in both dimensions are used to provide illustrations of EIS high numerical accuracy, even under small number of MC draws. MC simulations are used to characterize the finite sample numerical and statistical properties of EIS-based ML estimators.
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