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The I326T mutation in the TRNT1 gene encoding human tRNA nucleotidyltransferase (tRNA-NT) is linked to a relatively mild form of SIFD. Previous work indicated that the I326T variant was unable to incorporate AMP into tRNAs in vitro, however, expression of the mutant allele from a strong heterologous promoter supported in vivo CCA addition to both cytosolic and mitochondrial tRNAs in a yeast strain lacking tRNA-NT. To address this discrepancy, we determined the biochemical and biophysical characteristics of the I326T variant enzyme and the related variant, I326A. Our in vitro analysis revealed that the I326T substitution decreases the thermal stability of the enzyme and causes a ten-fold reduction in enzyme activity. We propose that the structural changes in the I326T variant that lead to these altered parameters result from a rearrangement of helices within the body domain of the protein which can be probed by the inability of the monomeric enzyme to form a covalent dimer in vitro mediated by C373. In addition, we confirm that the effects of the I326T or I326A substitutions are relatively mild in vivo by demonstrating that the mutant alleles support both mitochondrial and cytosolic CCA-addition in yeast.

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Mutations in the human TRNT1 gene encoding tRNA nucleotidyltransferase (tRNA-NT), an essential enzyme responsible for addition of the CCA (cytidine-cytidine-adenosine) sequence to the 3'-termini of tRNAs, have been linked to disease phenotypes including congenital sideroblastic anemia with B-cell immunodeficiency, periodic fevers and developmental delay (SIFD) or retinitis pigmentosa with erythrocyte microcytosis. The effects of these disease-linked mutations on the structure and function of tRNA-NT have not been explored. Here we use biochemical and biophysical approaches to study how five SIFD-linked amino acid substitutions (T154I, M158V, L166S, R190I and I223T), residing in the N-terminal head and neck domains of the enzyme, affect the structure and activity of human tRNA-NT in vitro. Our data suggest that the SIFD phenotype is linked to poor stability of the T154I and L166S variant proteins, and to a combination of reduced stability and altered catalytic efficiency in the M158 V, R190I and I223T variants.

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In addition to two genes (ENO1 and ENO2) known to code for enolase (EC4.2.1.11), the Saccharomyces cerevisiae genome contains three enolase-related regions (ERR1, ERR2 and ERR3) which could potentially encode proteins with enolase function. Here, we show that products of these genes (Err2p and Err3p) have secondary and quaternary structures similar to those of yeast enolase (Eno1p). In addition, Err2p and Err3p can convert 2-phosphoglycerate to phosphoenolpyruvate, with kinetic parameters similar to those of Eno1p, suggesting that these proteins could function as enolases in vivo. To address this possibility, we overexpressed the ERR2 and ERR3 genes individually in a double-null yeast strain lacking ENO1 and ENO2, and showed that either ERR2 or ERR3 could complement the growth defect in this strain when cells are grown in medium with glucose as the carbon source. Taken together, these data suggest that the ERR genes in Saccharomyces cerevisiae encode a protein that could function in glycolysis as enolase. The presence of these enolase-related regions in Saccharomyces cerevisiae and their absence in other related yeasts suggests that these genes may play some unique role in Saccharomyces cerevisiae. Further experiments will be required to determine whether these functions are related to glycolysis or other cellular processes.

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Green fluorescent protein (GFP) is used extensively as a reporter protein to monitor cellular processes, including intracellular protein trafficking and secretion. In general, this approach depends on GFP acting as a passive reporter protein. However, it was recently noted that GFP oligomerizes in the secretory pathway of endocrine cells. To characterize this oligomerization and its potential role in GFP transport, cytosolic and secretory forms of enhanced GFP (EGFP) were expressed in GH4C1 and AtT-20 endocrine cells. Biochemical analysis showed that cytosolic EGFP existed as a 27 kDa monomer, whereas secretory forms of EGFP formed disulphide-linked oligomers. EGFP contains two cysteine residues (Cys(49) and Cys(71)), which could play a role in this oligomerization. Site-directed mutagenesis of Cys(49) and Cys(71) showed that both cysteine residues were involved in disulphide interactions. Substitution of either cysteine residue resulted in a reduction or loss of oligomers, although dimers of the secretory form of EGFP remained. Mutation of these residues did not adversely affect the fluorescence of EGFP. EGFP oligomers were stored in secretory granules and secreted by the regulated secretory pathway in endocrine AtT-20 cells. Similarly, the dimeric mutant forms of EGFP were still secreted via the regulated secretory pathway, indicating that the higher-order oligomers were not necessary for sorting in AtT-20 cells. These results suggest that the oligomerization of EGFP must be considered when the protein is used as a reporter molecule in the secretory pathway.

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Endocrine, neuroendocrine and exocrine cells store regulated secretory proteins in secretory granules, while constitutive and constitutive-like secretory proteins are secreted directly without storage. Sorting of secretory proteins takes place in the trans-Golgi network (sorting for entry) or immature secretory granules (sorting by retention). The relative contribution of these sorting steps and the sorting signals and mechanisms involved in each step has been the subject of intense studies and debate in recent years. New evidence now suggests that: (1) two proteins with structurally similar sorting signals can use different sorting mechanisms; (2) one protein with multiple sorting signals can be sorted differently in different cell types; and (3) one cell type can recognize different sorting signals and use different sorting mechanisms. The latter finding suggests that sorting must be a regulated event. While the current image of sorting is complex, recent findings are pointing to common features that form a mosaic of related sorting mechanisms.

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AIMS/HYPOTHESIS: Sorting of proinsulin to the regulated secretory pathway of pancreatic beta cells and retention of insulin in dense-core granules of this pathway is remarkably efficient. To monitor the specificity of these events, the secretion of two exogenous secretory proteins not known to carry information for sorting or retention in the regulated pathway was investigated in INS-1 cells. METHODS: SEGFP, a fusion protein consisting of a signal peptide N-terminal to EGFP (mutant green fluorescent protein with enhanced fluorescence) and secreted alkaline phosphatase (SEAP) were expressed in INS-1 cells by transfection and by infection with recombinant adenovirus, respectively. Secretion of SEGFP was monitored by quantitative western blotting and that of SEAP by its activity. RESULTS: Secreted alkaline phosphatase showed high basal secretion (6.6% total) but only modest (3.6-fold) stimulation of secretion by secretagogues, in keeping with secretion largely through the constitutive pathway. By contrast SEGFP had a secretory pattern similar to insulin, with low basal secretion (0.8% total) and 16-fold stimulation by secretagogues. Granular localization of SEGFP was confirmed by high resolution electron microscopy immunocytochemistry. Pulse-chase experiments indicated retention of SEGFP in granules at least 24 h after synthesis. The secretory SEGFP, but not cytosolic EGFP, formed disulphide-linked oligomers. This could be implicated in its regulated secretion. CONCLUSION/INTERPRETATION: These data indicate that in INS-1 cells SEGFP, but not SEAP, is unexpectedly handled as a regulated secretory protein and stored along with insulin in granules. This raises questions about the specificity and mechanism of the sorting of proteins to granules in INS-1 cells or their retention therein or both.

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A gene (KlCCA1) encoding ATP(CTP):tRNA specific tRNA nucleotidyltransferase (EC 2.7.7.25) was isolated from Kluyveromyces lactis by complementation of the Saccharomyces cerevisiae cca1-1 mutation. Sequencing of a 2665 bp EcoRI-SpeI restriction fragment revealed an open reading frame potentially encoding a protein of 489 amino acids with 57% sequence similarity to its S. cerevisiae homologue. Southern hybridization revealed a single copy of KlCCA1 in the K. lactis genome. KlCCA1 was able to complement both the mitochondrial and cytosolic defects in the cca1-1 mutant, suggesting that, as in S. cerevisiae, the K. lactis gene encodes a sorting isozyme that is targeted to mitochondria and the nucleus and/or cytosol. An altered KlCCA1 gene encoding a tRNA nucleotidyltransferase that lacked its first 35 amino acids was able to complement the nuclear/cytosolic but not the mitochondrial defect in the S. cerevisiae cca1-1 mutant, suggesting that the 35 amino-terminal amino acids are necessary for targeting to mitochondria but are not required for enzyme activity. Our results suggest that the mechanisms for production and distribution of mitochondrial and nuclear/cytosolic tRNA nucleotidyltransferase in K. lactis differ from those seen in S. cerevisiae.

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Calcium-induced aggregation has been proposed to play a role in the sorting and storage of secretory proteins in secretory granules of endocrine cells. The regulation of this process is not known. Hexahistidine epitope tags were used to create aggregation chaperones that enhance the calcium-induced aggregation of secretory granule proteins in vitro. Indeed, 100% recovery of the aggregating target protein was achieved without any modification of the target protein. The aggregation chaperone is not trapped in the aggregates. Co-expression of His(6)-tagged secreted alkaline phosphatase and the regulated secretory protein chromogranin A resulted in an increased chromogranin storage in secretory granules, and stimulated secretion of chromogranin A increased 50%. However, secretion of secreted alkaline phosphatase was not affected by the hexahistidine epitope tag. Thus, calcium-induced aggregation is not a passive process; rather, aggregation and sorting of secretory proteins can be regulated by aggregation chaperones in the secretory pathway of endocrine cells.

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Chromogranins are a family of regulated secretory proteins that are stored in secretory granules in endocrine and neuroendocrine cells and released in response to extracellular stimulation (regulated secretion). A conserved N-terminal disulfide bond is necessary for sorting of chromogranins in neuroendocrine PC12 cells. Surprisingly, this disulfide bond is not necessary for sorting of chromogranins in endocrine GH4C1 cells. To investigate the sorting mechanism in GH4C1 cells, we made several mutant forms removing highly conserved N- and C-terminal regions of bovine chromogranin A. Removing the conserved N-terminal disulfide bond and the conserved C-terminal dimerization and tetramerization domain did not affect the sorting of chromogranin A to the regulated secretory pathway. In contrast, removing the C-terminal 90 amino acids of chromogranin A caused rerouting to the constitutive secretory pathway and impaired aggregation properties as compared with wild-type chromogranin A. Since this mutant was sorted to the regulated secretory pathway in PC12 cells, these results demonstrate that chromogranins contain independent N- and C-terminal sorting domains that function in a cell type-specific manner. Moreover, this is the first evidence that low pH/calcium-induced aggregation is necessary for sorting of a chromogranin to the regulated secretory pathway of endocrine cells.

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The Candida glabrata ADE2 gene encoding aminoimidazole ribonucleotide (AIR) carboxylase (EC 4.1.1.21) was isolated by complementation of the ade2-1 mutation in Saccharomyces cerevisiae. The predicted amino acid (aa) sequence is 75% identical to that of S. cerevisiae. Integrative transformation was used to produce a C. glabrata strain bearing a deletion of ADE2 coding sequences. A high-copy-number shuttle vector bearing the ADE2 gene was constructed and contains a fragment of S. cerevisiae mitochondrial (mt) DNA that confers the ability to replicate autonomously in C. glabrata.

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ATP (CTP):tRNA nucleotidyltransferase (EC 2.7.7.25) was purified to apparent homogeneity from a crude extract of Lupinus albus seeds. Purification was accomplished using a multistep protocol including ammonium sulfate fractionation and chromatography on anion-exchange, hydroxylapatite and affinity columns. The lupin enzyme exhibited a pH optimum and salt and ion requirements that were similar to those of tRNA nucleotidyltransferases from other sources. Oligonucleotides, based on partial amino acid sequence of the purified protein, were used to isolate the corresponding cDNA. The cDNA potentially encodes a protein of 560 amino acids with a predicted molecular mass of 64 164 Da in good agreement with the apparent molecular mass of the pure protein determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The size and predicted amino acid sequence of the lupin enzyme are more similar to the enzyme from yeast than from Escherichia coli with some blocks of amino acid sequence conserved among all three enzymes. Functionality of the lupin cDNA was shown by complementation of a temperature-sensitive mutation in the yeast tRNA nucleotidyltransferase gene. While the lupin cDNA compensated for the nucleocytoplasmic defect in the yeast mutant it did not enable the mutant strain to grow at the non-permissive temperature on a non-fermentable carbon source.

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The TRM1 gene of Saccharomyces cerevisiae codes for a tRNA modification enzyme, N2,N2-dimethylguanosine-specific tRNA methyltransferase (m2(2)Gtase), shared by mitochondria and nuclei. Immunofluorescent staining at the nuclear periphery demonstrates that m2(2)Gtase localizes at or near the nuclear membrane. In determining sequences necessary for targeting the enzyme to nuclei and mitochondria, we found that information required to deliver the enzyme to the nucleus is not sufficient for its correct subnuclear localization. We also determined that mislocalizing the enzyme from the nucleus to the cytoplasm does not destroy its biological function. This change in location was caused by altering a sequence similar to other known nuclear targeting signals (KKSKKKRC), suggesting that shared enzymes are likely to use the same import pathway as proteins that localize only to the nucleus. As with other well-characterized mitochondrial proteins, the mitochondrial import of the shared methyltransferase depends on amino-terminal amino acids, and removal of the first 48 amino acids prevents its import into mitochondria. While this truncated protein is still imported into nuclei, the immunofluorescent staining is uniform throughout rather than at the nuclear periphery, a staining pattern identical to that described for a fusion protein consisting of the first 213 amino acids of m2(2)Gtase in frame with beta-galactosidase. As both of these proteins together contain the entire m2(2)Gtase coding region, the information necessary for association with the nuclear periphery must be more complex than the short linear sequence necessary for nuclear localization.

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ATP (CTP):tRNA-specific tRNA nucleotidyltransferase is an enzyme required for the synthesis of functional tRNAs in eukaryotic cells. Neither the tRNA genes in the nucleus nor in organelles encode the CCA end, so it must be added post-transcriptionally. The gene that codes for the enzyme that adds the CCA end to nuclear coded tRNAs in Saccharomyces cerevisiae has been isolated (Aebi, M., Kirchner, G., Chen, J.-Y., Vijayraghavan, U., Jacobson, A., Martin, N. C., and Abelson, J. (1990) J. Biol. Chem. 265, 16216-16220). We now demonstrate that there is a mitochondrial tRNA nucleotidyltransferase activity in yeast and that it is a matrix enzyme. A comparison of purified mitochondrial enzyme with its cytoplasmic counterpart revealed no differences. These results suggest that proteins responsible for this step in the maturation of tRNAs in the nucleus and mitochondria might be identical and coded by the same nuclear gene. Accumulation of shortened mitochondrial as well as cytoplasmic tRNAs in a strain with a temperature-sensitive tRNA nucleotidyltransferase is consistent with this hypothesis. Alteration of the wild type gene such that amino-terminal truncated proteins are produced leads to a defect in mitochondrial function and a decrease in mitochondrial nucleotidyltransferase activity. This provides a direct demonstration that one gene provides this enzyme activity for the biosynthesis of tRNAs in both the nuclear/cytoplasmic and mitochondrial compartments in yeast.

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Infusion of endotoxin into domestic pigs induces an acute respiratory failure that has many similarities with the adult respiratory distress syndrome. We hypothesized that mono-hydroxyeicosatetraenoic acids (HETE) and platelet-activating factor (PAF) might be involved in this model of respiratory failure. Escherichia coli endotoxin (055-B5) was infused intravenously into anesthetized young pigs at 5 micrograms/kg the first hour, followed by 2 micrograms/kg/hour for 3 hours in the presence and absence of SRI 63-675, a specific PAF receptor antagonist. SRI 63-675 (10 mg/kg before endotoxin + 3 mg/kg/hour during endotoxemia) blocked or attenuated endotoxin-induced pulmonary hypertension, bronchoconstriction, hypoxemia, thrombocytopenia, increased permeability of the alveolar-capillary membrane, and the increases in plasma (at 3 and 4 hours) and bronchoalveolar lavage fluid concentrations of 5-, 12-, and 15-HETE. In a separate group of pigs, before treatment with SRI 63-675, ex vivo stimulation of whole blood with calcium ionophore (at -25 min) caused large increases in plasma concentrations of 5-HETE and, to a lesser extent, 12-HETE. At 4 hours, these increases were not significantly modified in blood derived from pigs treated with SRI 63-675 (10 mg/kg + 3 mg/kg/hour from 0 to 4 hours), indicating no direct inhibition of 5- or 12-lipoxygenase and suggesting that the in vivo effects were PAF receptor-mediated. We conclude that PAF contributes to the release of HETEs during endotoxemia and that this phenomenon could be important in the pathophysiology associated with endotoxin-induced lung injury in anesthetized pigs.

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We hypothesized that platelet-activating factor (PAF) and eicosanoids might be important mediators of endotoxin-induced respiratory failure in pigs. Escherichia coli endotoxin (055-B5) was infused intravenously into anesthetized 10- to 14-wk-old pigs at 5 micrograms/kg the 1st h, followed by 2 micrograms.kg-1.h-1 for 3 h in the presence and absence of SRI 63-675, a specific PAF receptor antagonist. During phase I (i.e., 0-2 h), endotoxin caused pulmonary hypertension and hypoxemia, decreased cardiac index, increased pulmonary vascular resistance, and increased plasma concentrations of thromboxane B2 (TxB2), prostaglandin (PG)F2 alpha, and 6-keto-PGF1 alpha. These phase I effects were attenuated or blocked by SRI 63-675 (10 mg/kg before endotoxin + 3 mg.kg-1.h-1 during endotoxemia). During phase II endotoxemia (i.e., 2-4 h), the PAF receptor antagonist blocked endotoxin-induced pulmonary edema and hypoxemia and increased relative permeability index of the alveolar-capillary membrane. SRI 63-675 also blocked the endotoxin-induced increases in plasma and bronchoalveolar lavage fluid concentrations of leukotriene B4 (LTB4). Ex vivo stimulation of whole blood with calcium ionophore caused large increases in plasma concentrations of TxB2 and LTB4. These increases were not significantly modified in blood derived from pigs treated with SRI 63-675, indicating no inhibition of cyclooxygenase or 5-lipoxygenase and suggesting that the in vivo effects were PAF receptor mediated. We conclude that PAF plays an important role in the release of eicosanoids during endotoxemia and in mediating, either directly or indirectly, endotoxin-induced lung injury in anesthetized pigs.

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We have identified genes encoding a "native" tRNA(Asp) (trnD-GTC) and a "chloroplast-like" tRNA(Asn) (trnN-GTT) on opposite strands and 633 bp apart within a sequenced 1640 bp RsaI restriction fragment of wheat mtDNA. The trnD gene has been found previously at a different location in wheat mtDNA (P.B.M. Joyce et al. (1988) Piant Mol. Biol. 11, 833-843); the duplicate copies of this gene are identical within the coding and immediate flanking regions (9 bp downstream and at least 68 bp upstream), after which obvious sequence similarity abruptly disappears. The trnN gene is identical to its homolog in maize ctDNA; continuation of sequence similarity beyond the coding region suggests that this gene originated as promiscuous ctDNA that is now part of the wheat mitochondrial genome. In the course of this work, we have encountered some unexpected similarities between tRNA gene regions from wheat mitochondria and other sources. Detailed analysis of these similarities leads us to suggest that trnN genes reportedly from petunia nuclear DNA (N. Bawnik et al. (1983) Nucleic Acids Res. 11, 1117-1122) and lupine mtDNA (B. Karpinska and H. Augustyniak (1988) Nucleic Acids Res. 16, 6239) are, in fact, from petunia mtDNA and lupine ctDNA, respectively, whereas a putative wheat nuclear tRNA(Ser) (trnS-TGA) gene (Z. Szwekowska-Kulinska et al. (1989) Gene 77, 163-167) is actually from wheat mtDNA. In these instances, it seems probable that the DNA samples used for cloning contained trace amounts of DNA from another sub-cellular compartment, leading to the inadvertent selection of spurious clones.

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We present the sequence of a wheat mitochondrial (mt) lysine tRNA gene (trnK-UUU). This gene more closely resembles its E. coli counterpart than it does the corresponding gene in fungal or mammalian mtDNA. Hybridization experiments with a trnK-specific probe suggest that at least two copies of this tRNALys gene are present in the wheat mitochondrial genome.

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In the course of a systematic survey of wheat mitochondrial tRNA genes, we have sequenced chloroplast-like serine (trnS-GGA), phenylalanine (trnF-GAA) and cysteine (trnC-GCA) tRNA genes and their flanking regions. These genes are remnants of 'promiscuous' chloroplast DNA that has been incorporated into wheat mtDNA in the course of its evolution. Each gene differs by one or a few nucleotides from the authentic chloroplast homolog previously characterized in wheat or other plants, and each could potentially encode a functional tRNA whose secondary structure shows no deviations from the generalized model. To determine whether these chloroplast-like tRNA genes are actually expressed, wheat mitochondrial tRNAs were resolved by a series of polyacrylamide gel electrophoreses, after being specifically end-labeled in vitro by 3'-CCA addition mediated by wheat tRNA nucleotidyltransferase. Subsequent direct RNA sequence analysis identified prominent tRNA species corresponding to the mitochondrial and not the chloroplast trnS, trnF and trnC genes. This analysis also revealed chloroplast-like elongator methionine, asparagine and tryptophan tRNAs. Our results suggest that at least some chloroplast-like tRNA genes in wheat mtDNA are transcribed, with transcripts undergoing processing, post-transcriptional modification and 3'-CCA addition, to produce mature tRNAs that may participate in mitochondrial protein synthesis.

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