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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have designed a fusion gene encoding a chimeric mitochondrial precursor protein (avidin fusion protein) that consists of the mitochondrial presequence followed by mouse dihydrofolate reductase, a spacer segment, and streptavidin. The avidin fusion protein synthesized in vitro formed a tetramer at the avidin moiety on incubation with biotin during or after the translation reaction. The avidin fusion protein purified from the Escherichia coli overexpresser cells also formed the tetramer on dilution from 6M urea into buffer containing biotin. In in vitro import experiments with isolated yeast mitochondria, the tetramer of the avidin fusion protein became stuck across both mitochondrial membranes, with its N-terminal dihydrofolate reductase moiety in the matrix and its C-terminal avidin moiety exposed on the mitochondrial surface. Accumulation of the translocation intermediate of the fusion protein inhibited the import of a mitochondrial precursor protein, and allowed us to estimate the number of mitochondrial import sites.
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PMID:Avidin fusion protein as a tool to generate a stable translocation intermediate spanning the mitochondrial membranes. 857 89

The cauliflower mosaic virus (CaMV) inclusion body protein (pVI) is able to specifically interact with the viral capsid precursor protein (pIV). By using the yeast two-hybrid system and a blot assay, the pIV region required for the recognition of pVI was mapped to the lysine-rich domain. This region of only 48 amino acids when fused to dihydrofolate reductase (DHFR) mediated pVI and DNA binding in vitro. Competition experiments confirmed that pVI and DNA bind to the same region of pIV. Since pVI is absent from the mature virus, models are discussed in which pVI plays an accessory role in CaMV assembly, in addition to its function in transactivating translation.
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PMID:Interaction between cauliflower mosaic virus inclusion body protein and capsid protein: implications for viral assembly. 859 99

In eukaryotic evolution, the earliest branch of organisms to have mitochondria are the trypanosomatids. Their mitochondrial biogenesis not only includes import of most proteins, but also, unlike in other organisms, import of the whole set of tRNAs. In order to investigate these processes, we devised novel procedures for the isolation of mitochondria from two trypanosomatid species: Trypanosoma brucei and Leishmania tarentolae. Isotonic cell lysis followed by equilibrium density centrifugation in Nycodenz gradients yielded mitochondrial fractions exhibiting a membrane potential. Furthermore, we have used these fractions to reconstitute import of mitochondrial matrix proteins in vitro. Energy-dependent uptake of an artificial precursor protein, containing a trypanosomal presequence attached to mouse dihydrofolate reductase and of yeast mitochondrial alcohol dehydrogenase could be demonstrated. The presequences of both proteins were processed in T. brucei whereas only the trypanosomal one was cleaved in L. tarentolae. Trypsin pretreatment abolished the ability of the mitochondria to import proteins, indicating the involvement of proteinaceous components at the surface of mitochondria.
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PMID:In vitro import of proteins into mitochondria of Trypanosoma brucei and Leishmania tarentolae. 883 75

The mitochondrial phenotype within cardiac muscle cells is dramatically altered by thyroid hormone. We report here that this can be accounted for, in part, by modifications in the rate of mitochondrial protein import. The import of matrix-localized precursor proteins malate dehydrogenase (MDH) and ornithine carbamoyltransferase was augmented, whereas the insertion of the outer membrane protein Bcl-2 was unaffected by thyroid hormone treatment. Coincident with increases in the import of these matrix-localized precursors were thyroid hormone-induced elevations in the outer membrane receptor Tom20 and the matrix heat-shock protein mthsp70. The phospholipid cardiolipin was not involved in mediating the thyroid hormone-induced increase in import, as judged from adriamycin inhibition studies. When the import reaction was supplemented with rat heart cytosol, we found that 1) MDH import was stimulated, but Bcl-2 import was inhibited and 2) thyroid hormone did not influence the effect of the cytosol on import rates. Thus distinct requirements exist for the mitochondrial import of precursor proteins, destined for different organellar compartments. Although import of these matrix-localized proteins was augmented by thyroid hormone treatment, the proteolysis of matrix proteins was unaffected as indicated by the degradation of cytob2(167)RIC-dihydrofolate reductase, a chimeric protein missorted to the matrix. Thus our data indicate that at least some thyroid hormone-induced modifications of the mitochondrial phenotype occur due to the compartment-specific upregulation of precursor protein import rates, likely mediated via changes in the expression of protein import machinery components.
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PMID:Thyroid hormone modifies mitochondrial phenotype by increasing protein import without altering degradation. 984 12

We investigated the effect of L and D enantiomers of a 25-residue peptide derived from the N-terminal region of the presequence of Nicotiana plumbaginifolia F1beta subunit of the ATP synthase, pF1beta(1, 25), on import into spinach leaf mitochondria. Three in vitro synthesized precursor proteins using different import pathways were used. Import of the precursor proteins of F1beta subunit of the ATP synthase, pre-F1beta, and the alternative oxidase, pre-AOX, required addition of external ATP. whereas the chimeric precursor containing the N-terminal 84 amino acids of the cytochrome b2 precursor protein linked to dihydrofolate reductase, pre-b2(1, 84)-DHFR was not dependent on ATP. Import of pre-F1beta, and pre-AOX was inhibited already at 1 microM and 3 microM concentration of the L and D enantiomers, whereas inhibition of import of pre-b2(1, 84)-DHFR, occurred at concentrations >10 microM of both enantiomers. Binding efficiency of the precursor proteins was not affected by addition of the L and D enantiomers. There was no correlation between inhibition of import of pre-F1beta and pre-AOX and dissipation of membrane potential measured as a decrease of Rhodamine 123 fluorescence quenching. The inhibitory effect of the L and D presequence enantiomers on import of pre-F1beta and pre-AOX was concluded to occur within the outer membrane translocase machinery beyond the initial precursor receptor interaction. Furthermore, the fact that the D enantiomer had the same effect as the natural peptide showed that interaction of the presequence with the import machinery was not dependent on chiral properties of the presequence.
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PMID:L and D presequence peptides derived from the precursor of F1beta subunit of the ATP synthase inhibit mitochondrial protein import by interaction with import machinery. 1178 42


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