Gene/Protein Disease Symptom Drug Enzyme Compound
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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)

A technique is described for the isolation and purification of intact, respiratory-competent mitochondria from Schizosaccharomyces pombe. The purified mitochondria are capable of oxidizing NADH and succinate as respiratory substrates, indicating the presence of succinate dehydrogenase and an NADH dehydrogenase located on the outer surface of the inner membrane. Mitochondria display good respiratory control with an ADP/O ratio of < 2. Respiratory activity is linearly dependent upon the redox poise of the quinone pool, suggesting the presence of an unbranched respiratory pathway to molecular oxygen. Immunogold labelling using antisera raised against mitochondrial HSP70 proteins (SSP1, SSC1 and PHSP1) from three different species, namely S. pombe, Saccharomyces cerevisiae and the plant Pisum sativum respectively, has been used to investigate the presence and ultrastructure of the mitochondria isolated by this procedure. The immunocytochemistry was carried out using cells containing wild-type levels of SSP1 protein and cells over-expressing the protein. These results also demonstrate the capacity of mitochondria to import increased levels of protein in vivo. In vitro import experiments using COXIV-DHFR indicate that purified S. pombe mitochondria can efficiently import this precursor, and that protein translocation is dependent upon an oxidizable substrate and a membrane potential.
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PMID:Schizosaccharomyces pombe mitochondria: morphological, respiratory and protein import characteristics. 148 70

We show that a synthetic peptide corresponding to the N-terminal 22 residues of the cytochrome c oxidase subunit IV presequence blocked import of pre-subunit IV into yeast mitochondria. The 22-residue peptide pL4-(1-22) did not alter the electrical potential across the mitochondrial inner membrane (the delta psi). Inhibition of import was reversible and could be overcome by the addition of increased amounts of precursor. Two other peptides, pL4-(1-16) and pL4-(1-23), which correspond to, respectively, the N-terminal 16 and 23 residues of the same presequence, also blocked import of pre-subunit IV. However, pL4-(1-16) was a much weaker inhibitor of import, while the inhibitory effect of pL4-(1-23) was due to its ability to completely collapse the delta psi. pL4-(1-22) seems to be a general inhibitor of mitochondrial import, in that it also blocked uptake of several other proteins. These included the precursors of the yeast proteins cytochrome c oxidase subunit Va, the F1-ATPase beta subunit, mitochondrial malate dehydrogenase, and the ATP/ADP carrier. In addition, uptake of two non-yeast precursor proteins (human ornithine transcarbamylase and a cytochrome oxidase subunit IV-dihydrofolate reductase fusion), was also blocked by the peptide. Subsequent studies revealed that pL4-(1-22) did not block the initial recognition or binding of proteins to mitochondria. Rather, our results suggest that the peptide acts at a subsequent translocation step which is common to the import pathways of many different precursor proteins.
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PMID:A synthetic presequence reversibly inhibits protein import into yeast mitochondria. 216 Apr 69

Fusions between the TRM1 gene of Saccharomyces cerevisiae and COXIV or DHFR were made to examine the mitochondrial targeting signals of N2,N2-dimethylguanosine-specific tRNA methyltransferase [tRNA (m2(2)G)dimethyltransferase]. This enzyme is responsible for the modification of both mitochondrial and cytoplasmic tRNAs. We have previously shown that two forms of the enzyme are translated from two in-frame ATGs in this gene, that they differ by a 16-amino-acid amino-terminal extension, and that both the long and short forms are imported into mitochondria. Results of studies to test the ability of various TRM1 sequences to serve as surrogate mitochondrial targeting signals for passenger protein import in vitro and in vivo showed that the most efficient signal derived from tRNA (m2(2)G)dimethyltransferase included a combination of sequences from both the amino-terminal extension and the amino terminus of the shorter form of the enzyme. The amino-terminal extension itself did not serve as an independent mitochondrial targeting signal, whereas the amino terminus of the shorter form of tRNA (m2(2)G)dimethyltransferase did function in this regard, albeit inefficiently. We analyzed the first 48 amino acids of tRNA (m2(2)G)dimethyltransferase for elements of primary and secondary structure shared with other known mitochondrial targeting signals. The results lead us to propose that the most efficient signal spans the area around the second ATG of TRM1 and is consistent with the idea that there is a mitochondrial targeting signal present at the amino terminus of the shorter form of the enzyme and that the amino-terminal extension augments this signal by extending it to form a larger, more efficient mitochondrial targeting signal.
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PMID:Amino-terminal extension generated from an upstream AUG codon increases the efficiency of mitochondrial import of yeast N2,N2-dimethylguanosine-specific tRNA methyltransferases. 265

Yeast cytochrome c oxidase subunit IV (an imported mitochondrial protein) is made as a larger precursor with a transient pre-sequence of 25 amino acids. If this pre-sequence is fused to the amino terminus of mouse dihydrofolate reductase (a cytosolic protein) the resulting fusion protein is imported into the matrix space, and cleaved to a smaller size, by isolated yeast mitochondria. We have now fused progressively shorter amino-terminal segments of the subunit IV pre-sequence to dihydrofolate reductase and tested each fusion protein for import into the matrix space and cleavage by the matrix-located processing protease. The first 12 amino acids of the subunit IV pre-sequence were sufficient to direct dihydrofolate reductase into the mitochondrial matrix, both in vitro and in vivo. However, import of the corresponding fusion protein into the matrix was no longer accompanied by proteolytic processing. Fusion proteins containing fewer than nine amino-terminal residues from the subunit IV pre-piece were not imported into isolated mitochondria. The information for transporting attached mouse dihydrofolate reductase into mitochondria is thus contained within the first 12 amino acids of the subunit IV pre-sequence.
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PMID:The first twelve amino acids (less than half of the pre-sequence) of an imported mitochondrial protein can direct mouse cytosolic dihydrofolate reductase into the yeast mitochondrial matrix. 299 81

Sequences that are capable of restoring mitochondrial targeting to a truncated yeast cytochrome c oxidase subunit IV presequence are encoded within the genome of Escherichia coli and within the gene for a higher eukaryotic cytosolic protein, mouse dihydrofolate reductase. These sequences, which resemble authentic presequences in their overall amino acid composition and degree of hydrophobicity, are rather frequent; greater than 2.7% of clones generated from E. coli DNA and greater than 5% of clones from the dihydrofolate reductase gene were functional in our screening system. These results suggest that, during evolution, mitochondrial precursor proteins could arise as a result of DNA rearrangements that place potential mitochondrial presequences at the amino terminus of existing open reading frames. Primitive eukaryotic cells may have used this mechanism to target proteins to their endosymbiotic protomitochondria.
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PMID:Sequences from a prokaryotic genome or the mouse dihydrofolate reductase gene can restore the import of a truncated precursor protein into yeast mitochondria. 303 34

The cleavable prepiece of the precursor to yeast cytochrome c oxidase subunit IV (an imported mitochondrial protein) was attached to the amino-terminus of mouse dihydrofolate reductase (a cytosolic protein) by gene fusion. The resulting fusion protein was imported into the matrix of isolated, energized yeast mitochondria and cleaved to a polypeptide whose size was similar to that of authentic dihydrofolate reductase.
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PMID:The cleavable prepiece of an imported mitochondrial protein is sufficient to direct cytosolic dihydrofolate reductase into the mitochondrial matrix. 609 70

The interaction of several basic peptides with yeast mitochondria has been analyzed. The peptides were selected for their ability to block a cationic channel of the outer membrane, the peptide-sensitive channel. These peptides possess common characteristics, such as a net positive charge superior to 2 and the capacity to form amphiphilic structures. They can be divided into two classes as follows: peptides of class I derived from mitochondrial signal peptides, such as the presequence of cytochrome c oxidase subunit IV, e.g. pCyt OX IV (1-12) Y; and peptides of class II unrelated to the mitochondria, such as dynorphin B (1-13). Class I peptides inhibited the translocation of a chimeric protein, cytochrome b2-DHFR, into the mitochondrial matrix, whereas peptides of class II failed to inhibit this import. Experiments with iodinated pCyt OX IV (1-12) Y and dynorphin B (1-13) showed, however, that both types of peptides were imported into yeast mitochondria in vitro and subsequently degraded. At 30 degrees C, two import mechanisms could be distinguished; the mitochondrial presequences (class I) were translocated into the matrix in a temperature- and potential-sensitive manner, probably along the general import pathway, while class II dynorphin B (1-13) was imported into the intermembrane space by a process that was neither temperature- nor potential-sensitive. At 0 degree C, both peptides were imported in a class II manner. The class II characteristics suggested the existence of a direct pathway into the intermembrane space, which may be associated with the peptide-sensitive channel. This hypothesis is substantiated by the competition for the import into the mitochondria between peptides of the two classes. The import of pCyt OX (1-12) Y was inhibited at 30 degrees C only by peptides of class I, IV whereas, at 0 degree C, this import was also inhibited by peptides of class II. Import of peptides of the latter class was inhibited by peptides of the two classes both at 0 degree C and 30 degrees C.
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PMID:Basic peptides can be imported into yeast mitochondria by two distinct targeting pathways. Involvement of the peptide-sensitive channel of the outer membrane. 751 2

MOD5, a gene responsible for the modification of A37 to isopentenyl A37 of both cytosolic and mitochondrial tRNAs, encodes two isozymes. Initiation of translation at the first AUG of the MOD5 open reading frame generates delta 2-isopentenyl pyrophosphate:tRNA isopentanyl transferase I (IPPT-I), which is located predominantly, but not exclusively, in the mitochondria. Initiation of translation at a second AUG generates IPPT-II, which modifies cytoplasmic tRNA. IPPT-II is unable to target to mitochondria. The N-terminal sequence present in IPPT-I and absent in IPPT-II is therefore necessary for mitochondrial targeting. In these studies, we fused MOD5 sequences encoding N-terminal regions to genes encoding passenger proteins, pseudomature COXIV and dihydrofolate reductase, and studied the ability of these chimeric proteins to be imported into mitochondria both in vivo and in vitro. We found that the sequences necessary for mitochondrial import, amino acids 1 to 11, are not sufficient for efficient mitochondrial targeting and that at least some of the amino acids shared by IPPT-I and IPPT-II comprise part of the mitochondrial targeting information. We used indirect immunofluorescence and cell fractionation to locate the MOD5 isozymes in yeast. IPPT-I was found in two subcellular compartments: mitochondria and the cytosol. We also found that IPPT-II had two subcellular locations: nuclei and the cytosol. The nuclear location of this protein is surprising because the A37-->isopentenyl A37 modification had been predicted to occur in the cytoplasm. MOD5 is one of the first genes reported to encode isozymes found in three subcellular compartments.
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PMID:Subcellular locations of MOD5 proteins: mapping of sequences sufficient for targeting to mitochondria and demonstration that mitochondrial and nuclear isoforms commingle in the cytosol. 813 35

We have utilized a homologous cell-free mitochondrial protein import system derived from the yeast Saccharomyces cerevisiae, in addition to performing a series of in vivo experiments in yeast, to investigate the coupling between cytosolic protein synthesis and protein transport into mitochondria. We found that the import of bulk mitochondrial proteins was inhibited in both the homologous in vitro reaction and in vivo upon arrest of cytosolic protein synthesis with the addition of cycloheximide. Tight coupling of synthesis and import was also demonstrated in vivo for the beta subunit of the mitochondrial F1-ATPase. We also investigated the effect of the antifolate methotrexate on the import of a fusion protein consisting of the mitochondrial targeting signal of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase (the COXIV-DHFR fusion protein). Methotrexate has previously been shown to inhibit posttranslational import of COXIV-DHFR by preventing the DHFR moiety from unfolding. However, we found that antifolate addition had no inhibitory effect on the import of COXIV-DHFR in vivo, suggesting that its import into mitochondria in yeast cells occurs cotranslationally. Further, when we treated yeast with the proton ionophore carbonyl cyanide m-chlorophenylhydrazone to collapse the mitochondrial membrane potential and induce the accumulation of extramitochondrial precursor pools, we found that the ability to be imported by a strictly posttranslational mechanism upon reestablishing the membrane potential varied from one precursor to another, suggesting that cotranslational import may be mandatory for the import of some proteins in vivo. In summary, our findings are entirely consistent with the notion that import of proteins into yeast mitochondria occurs cotranslationally under normal conditions in vivo.
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PMID:Coupling of cytosolic protein synthesis and mitochondrial protein import in yeast. Evidence for cotranslational import in vivo. 838 May 82