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G C Shore

Publications and source records attributed to G C Shore.

At least 73 records · Page 4Linked to original sources
PubMed1987

A signal sequence domain essential for processing, but not import, of mitochondrial pre-ornithine carbamyl transferase.

Studies using deletion mutagenesis indicate that a processing recognition site lies proximal to the normal cleavage position between gln32 and ser33 of pre-ornithine carbamyl transferase (pOCT). pOCT cDNA was manipulated to delete codons specifying amino acids 22-30 of the signal sequence. The mutant precursor, designated pOCT delta 22-30, was imported to the matrix compartment by purified mitochondria, but remained largely unprocessed; the low level of processing that was observed did not involve the normal cleavage site. Several manipulations performed downstream of the normal pOCT processing site (deletion, substitution, and hybrid protein constructions) affected neither import nor correct processing. Our data suggest that domains specifying import and processing site recognition may be functionally segregated within the signal peptide; that processing is not requisite for import of pOCT; and that a proximal region, not involving the normal signal peptide cleavage site, is required for processing site recognition.

Amino Acid Sequence↗
PubMed1986

Structural analysis and amphiphilic properties of a chemically synthesized mitochondrial signal peptide.

Anionic phospholipids induce a marked conformational change in a synthetic peptide corresponding to residues 1-27 of pre-ornithine carbamyltransferase. The peptide designated, pO-(1-27)-peptide amide, becomes more alpha-helical in the presence of cardiolipin or dimyristoylphosphatidylglycerol but not in the presence of dimyristoylphosphatidylcholine. The greater helix-promoting action of anionic versus zwitterionic lipids is predicted by helix-coil transition theory. This statistical mechanical theory also predicts that a shorter peptide, N-acetyl-pO-(16-27)-peptide amide, has less helix-forming tendency, even in the presence of sodium dodecyl sulfate, despite the fact that it has a comparable number of positive charges. The N-acetyl-pO-(16-27)-peptide amide has no helical structure in buffer with or without dimyristoylphosphatidylglycerol but it has a small (5%) helical content in methanol. Thus, the ability of anionic lipids to promote helix formation requires more than the presence of cationic groups on the peptide. The angular dependence of the hydrophobic moment of the putative helical segment of pO-(1-27)-peptide amide demonstrates that any helical structure which is formed would have some amphiphilic character. The pO-(1-27)-peptide amide disrupts large lipid aggregates to form discoid micelles about 30 to 50 nm in diameter. The ability to lyse membranes into disc-shaped micelles is characteristic of peptides containing an amphiphathic helix. In the case of the mitochondrial signal peptide, this membrane-lytic behavior may contribute to the translocation of the protein into the organelle.

Cardiolipins↗
PubMed1986

Rat liver and intestinal mucosa differ in the developmental pattern and hormonal regulation of carbamoyl-phosphate synthetase I and ornithine carbamoyl transferase gene expression.

cDNA probes were employed to measure levels of carbamoyl-phosphate synthetase I (CPS) and ornithine carbamoyltransferase (OCT) mRNAs in fetal and neonatal livers and intestines. In the fetal liver, significant levels of OCT mRNA were present at 15-days gestation while CPS mRNA could not be detected until day 17 of fetal development. Apart from a small decline just after birth, amounts of both mRNAs increased steadily to reach adult levels in postnatal life. In contrast to the situation in liver, CPS and OCT mRNA levels in the fetal intestine rose rapidly to peak at day 21 of gestation and then declined steadily in the first seven days after birth. Using the methyl-sensitive restriction isoschizomeric pair, MspI/HpaII, the 5' ends of both the CPS and OCT genes were shown to undergo demethylation during development. In the case of the OCT gene, however, the hypomethylation characteristic of the adult liver and intestinal mucosa was not observed in the 15-day-old fetal liver, where significant levels of gene expression had already been established. Levels of CPS and OCT mRNA in livers of adults responded to glucagon in normal animals (1.5-fold and 2.2-fold increases, respectively) and to dexamethasone in experimentally induced diabetic animals (3-fold increase in CPS mRNA with no change in OCT mRNA). These treatments were all without effect on the levels of CPS and OCT mRNA in intestinal mucosa.

Animals↗
PubMed1986

Import and processing of hybrid proteins by mammalian mitochondria in vitro.

Normal and hybrid proteins were synthesized following transcription of pSP64 recombinant plasmids with SP6 polymerase and translation of resultant mRNAs in a rabbit reticulocyte lysate. The precursor to the rat liver mitochondrial matrix enzyme, ornithine carbamyltransferase (Mr = approximately 40,000), was efficiently imported by rat heart mitochondria in vitro and processed to mature protein. Import of the precursor to a second matrix enzyme, carbamyl-phosphate synthetase I (Mr = approximately 165,000), could not be demonstrated. However, a 33-kDa hybrid protein, bearing the precarbamyl-phosphate synthetase I signal sequence (38 amino acids) and 55 amino acids from the amino terminus of the mature enzyme, followed by the carboxyl-terminal 209 amino acids of ornithine carbamyltransferase, was imported and processed under identical conditions. The topogenic function of the preornithine carbamyltransferase signal sequence (32 amino acids) was confirmed by constructing a hybrid protein bearing the signal sequence and five amino acids of mature enzyme, followed by 250 amino acids of the cytosolic enzyme of Escherichia coli, asparagine synthetase; the hybrid was transported to the matrix compartment of heart mitochondria where processing, albeit incorrect, took place. A hybrid asparagine synthetase bearing the pre-ornithine carbamyltransferase signal sequence plus 28 amino acids of mature ornithine carbamyltransferase, however, was imported and processed with apparent fidelity.

Amino Acid Sequence↗
PubMed1986

Expression in Escherichia coli of functional precursor to the rat liver mitochondrial enzyme, ornithine carbamyl transferase. Precursor import and processing in vitro.

cDNA encoding the full-length cytosolic precursor of rat liver ornithine carbamyl transferase, a mitochondrial matrix enzyme, was inserted into pKK223-3 and expressed under the control of the tac promoter. Following transformation of E. coli strain JM105 and induction by isopropylthiogalactoside, a polypeptide was synthesized which reacted with antibody against ornithine carbamyl transferase and co-migrated in SDS-polyacrylamide gels with authentic enzyme precursor (pOCT, Mr = 40,000 kDa); it constituted approximately 0.1% of total E. coli protein. pOCT was synthesized in vitro by coupled transcription-translation of the recombinant plasmid in an E. coli S30 extract; upon subsequent addition of purified rat liver or heart mitochondria, the precursor was imported and processed to mature form. Synthesis of pOCT in a bacterial system, therefore, results in production of a functional precursor polypeptide which does not require additional cytosolic factors from eukaryotic cells to support its uptake and processing by mitochondria in vitro.

Animals↗
PubMed1985

A synthetic signal peptide blocks import of precursor proteins destined for the mitochondrial inner membrane or matrix.

A peptide corresponding to amino acids 1-27 of preornithine carbamyltransferase (pOCT) has been chemically synthesized. When added to energized mitochondria in vitro, 20 microM of the peptide, designated pO(1-27), resulted in a collapse of the electrochemical potential across the mitochondrial inner membrane. This effect on transmembrane potential was not observed, however, when pO(1-27) was added to energized mitochondria under conditions that support in vitro import of precursor proteins (i.e. in the presence of reticulocyte lysate). The latter finding, therefore, made possible an examination of the ability of pO(1-27) to block import of homologous and heterologous proteins into the organelle. At 5-10 microM, pO(1-27) prevented import of pOCT in vitro; inhibition was overcome by increasing the concentration of pOCT. In contrast, pO(16-27), a peptide corresponding to amino acids 16-27 of pOCT and exhibiting a charge:mass ratio similar to pO(1-27) had no such inhibitory effect. pO(1-27) blocked import of other unrelated precursor proteins destined either for the mitochondrial matrix (pre-malate dehydrogenase and a hybrid protein containing the signal sequence of pre-carbamyl phosphate synthetase) or for the mitochondrial inner membrane (pre-thermogenin).

Amino Acid Sequence↗
PubMed1985

Expression of nuclear genes encoding the urea cycle enzymes, carbamoyl-phosphate synthetase I and ornithine carbamoyl transferase, in rat liver and intestinal mucosa.

RNA dot-blot, quantitative electron microscope immunocytochemistry, and electrophoretic immunoblotting techniques were employed to investigate the expression of carbamoyl-phosphate synthetase I (CPS) and ornithine carbamoyl transferase (OCT) genes in rat liver and intestinal mucosa. Comparing only those cell types in the two tissues which express these enzymes, we show that the concentration of CPS and OCT in hepatocyte mitochondria is 2.3-times and 1.2-times greater, respectively, than in intestinal epithelial cell mitochondria. As a percentage of total tissue protein, however, liver homogenates contain 10-20 times more CPS and 5-10 times more OCT than is found in intestinal mucosa. These relatively large differences in enzyme protein levels between the two tissues are not reflected by differences in their mRNA levels. As a percentage of total translational activity in vitro (based on incorporation of [35S]methionine), total liver mRNA directed synthesis of about twice as much precursor CPS (pCPS) and precursor OCT (pOCT) than did equivalent amounts of mRNA from intestinal mucosa. The ratio of pCPS and pOCT mRNA levels between the two tissues (2:1, liver:intestinal mucosa) was confirmed by dot-blot and Northern hybridizations employing specific cDNA probes. The sizes of the respective mRNAs were the same for the two tissues: about 6000 residues for pCPS mRNA and about 1700 residues for pOCT mRNA.

Animals↗
PubMed1985

The precursor to ornithine carbamyl transferase is transported to mitochondria as a 5S complex containing an import factor.

The precursor to ornithine carbamyl transferase (Mr = 40,000) was synthesized in a rabbit reticulocyte lysate system and purified by immunoaffinity chromatography. Import of purified precursor by isolated mitochondria depended upon the presence of import factor(s) in fresh reticulocyte lysate. Velocity sedimentation analyses indicated that import factor binds to precursor to form a 5S complex (approximately 90 kDa); in this form, precursor was efficiently imported by isolated mitochondria. The ability of the 5S complex to deliver precursor into mitochondria was not affected by pretreatment with high concentrations of RNase. Import factor did not bind to mitochondria in the absence of precursor; upon binding of precursor to mitochondria in the presence of import factor, subsequent transmembrane uptake of precursor did not require the continued presence of additional lysate components.

Animals↗
PubMed1985

Expression of carbamoyl-phosphate synthetase I mRNA in Reuber hepatoma H-35 cells. Regulation by glucocorticoid and insulin.

Reuber hepatoma H-35 cells actively synthesize the urea cycle enzyme, carbamoyl-phosphate synthetase I. Treatment of H-35 cells with dexamethasone (0.14 microM), however, enhanced synthesis of the enzyme (as measured by incorporation of [35S]methionine) by 4-5-fold. Insulin (0.18 microM) completely inhibited dexamethasone-dependent stimulation of enzyme synthesis. In vitro translation and cDNA hybridization assays were employed to measure effects of dexamethasone plus or minus insulin on levels of mRNA encoding the biosynthetic precursor of carbamoyl-phosphate synthetase I (pCPS) in Reuber H-35 cells. Both measurements yielded similar results: dexamethasone increased pCPS mRNA levels by 4-5-fold and insulin suppressed this response, but only by 50%. Specific cDNA hybridization assays also demonstrated that Reuber H-35 cells, even after hormone treatments, contain only very low levels of albumin mRNA, and no detectable ornithine carbamoyl-transferase mRNA.

Albumins↗
PubMed1985

In vitro synthesis and assembly of a 68 kDa outer mitochondrial membrane protein from rat liver.

Outer mitochondrial membrane was purified from rat liver. Its constituent proteins were analyzed by SDS-polyacrylamide gel electrophoresis and by electrophoretic immunoblotting employing antibodies raised against total outer mitochondrial membrane. Anti-outer mitochondrial membrane antiserum reacted with only one polypeptide (15 kDa) in rough microsomes, whereas no immunological cross-reactivity was observed with other mitochondrial compartments (intermembrane space, inner membrane, or matrix) or with lysosomes or total cytosol. The antiserum was employed to characterize precursors of outer mitochondrial membrane proteins synthesized in vitro in a rabbit reticulocyte cell-free system. One product (a 68 kDa polypeptide designated OMM-68) bound efficiently to mitochondria in vitro but did not interact with either dog pancreas or rat liver microsomes, either co-translationally or post-translationally. OMM-68 was synthesized exclusively by the membrane-free class of polyribosomes. Attachment of precursor OMM-68 to mitochondria was not accompanied by processing of the polypeptide to a different size.

Animals↗
PubMed1985

Involvement of Golgi apparatus and a restructured nuclear envelope during biogenesis and transport of herpes simplex virus glycoproteins.

Following infection of BHK-21 cells with Herpes simplex virus type 1 (HSV-1), progeny nucleocapsids in the nucleus acquire a glycoprotein-rich envelope by budding through host-cell nuclear membranes. To investigate the nature of the glycoprotein products assembled in the virion at the nuclear envelope, infected cells were pulse-labeled with [3H]-mannose, an oligosaccharidal core sugar, or [3H]-fucose, a terminal sugar. After various chase periods, the incorporation of these sugars was monitored by electron microscope radioautography. The results show that HSV glycoproteins accumulate very rapidly in nuclear membranes, where they exist only as core-glycosylated precursors, i.e., containing [3H]-mannose but not [3H]-fucose. [3H]-fucose grains are seen mainly over Golgi membranes and over virions located in the Golgi and in other cytoplasmic vesicular structures. Our data support a model where addition of terminal sugars (e.g., fucose) to HSV-1 glycoprotein precursors can occur at the surface of newly enveloped viral particles as the virions themselves egress from the cell via the Golgi apparatus.

Animals↗
PubMed1984

Regulation and expression of carbamyl phosphate synthetase I mRNA in developing rat liver and Morris hepatoma 5123D.

A cDNA clone complementary to mRNA encoding the precursor (Mr = 165,000) to the rat liver mitochondrial matrix enzyme carbamyl phosphate synthetase I (Mr = 160,000) was employed to compare relative amounts of the messenger in adult and fetal liver and in Morris hepatoma 5123D and 3924A cells. Northern blot analysis gave a size estimate for the messenger of 6,500-6,700 nucleotides. Carbamyl phosphate synthetase mRNA levels in 15-day-old fetal liver were less than 10% of adult levels; 5123D cells expressed the messenger at levels about 2-fold higher than normal adult liver, but the messenger was undetectable in 3924A cells. Albumin mRNA was also expressed in the former but not in the latter. Maintaining rats for 5 days on a diet containing 60% casein augmented the relative amount of carbamyl phosphate synthetase mRNA by about 2-fold, while a protein-free diet resulted in reduced levels of the mRNA (about 50% compared to animals on a normal diet). Finally, the pattern of hybridization of carbamyl phosphate synthetase cDNA to HindIII-digested genomic DNA showed no differences between normal liver and its corresponding hepatoma; however, a HindIII site polymorphism was observed between Buffalo and ACI rats.

Animals↗
PubMed1983

Effects of glucagon on biosynthesis of the mitochondrial enzyme, carbamoyl-phosphate synthase I, in primary hepatocytes and Morris hepatoma 5123D.

When freshly-dispersed rat hepatocytes are maintained in primary monolayer cultures, they quickly lose their capacity to synthesize the urea cycle enzyme, carbamoyl-phosphate synthase. The ability to synthesize many other proteins, e.g., serum proteins including albumin, is retained. After an initial recovery period following cell isolation (24-48 h), glucagon is able to restore the ability of cultured hepatocytes to make carbamoyl-phosphate synthase. mRNA encoding the enzyme is about 4-times higher in hepatocytes maintained for 48 h in the presence of glucagon compared to hepatocytes without the hormone, as judged by in vitro translational assays. The level of carbamoyl-phosphate synthase activity expressed in transformed hepatocytes is unique to each hepatoma. Here we show that Morris hepatoma 5123D has retained such expression, and actively synthesizes the enzyme when 5123D cells are placed in monolayer cultures. Unlike normal hepatocytes, however, synthesis continues uninterrupted at a high level whether or not glucagon is present. 5123D has higher levels of translatable carbamoyl-phosphate synthase mRNA than normal liver.

Animals↗
PubMed1983

Membrane and cytosolic components affecting transport of the precursor for ornithine carbamyltransferase into mitochondria.

A higher molecular weight precursor (Mr = 39,000) to the liver mitochondrial matrix enzyme, ornithine carbamyltransferase (Mr = 36,000), is imported and processed by heart mitochondria in vitro in a manner similar to liver mitochondria. In both systems, however, an additional 37-kDa ornithine carbamyltransferase polypeptide appears, but this arises from nonspecific events and, therefore, does not represent a bona fide intermediate in the overall processing sequence. Our experiments demonstrate that the outer mitochondrial membrane of mitochondria contains a protease-sensitive (5 micrograms of trypsin or chymotrypsin/ml, 15 min at 2 degrees C), salt-resistant (1.0 M KCl) protein which is required to maintain import functions. In addition, functional post-translational import requires a component of the reticulocyte lysate (i.e. cytosol) that is used for initially synthesizing precursor enzyme. The component is retained by Sephadex G-25. Import of Sephadex G-25-excluded precursor is restored by fresh reticulocyte lysate but not by a combination of other additives, including Mg2+, K+, ATP, ADP, Pi, succinate, and total translation mixture (minus lysate).

Animals↗
PubMed1983

Localization of the coding region for a 35000 Dalton polypeptide on the genome of herpes simplex virus type 2.

The cloned BglII N fragment of herpes simplex virus type 2 (HSV-2) DNA has been shown to code for a 35K polypeptide. Subfragments made by cleavage with XhoI, BamHI, SstII and XorII were then cloned and used with RNA extracted from HSV-2-infected cells for mRNA selection and in vitro protein synthesis. We found that the major translation product of such hybrid-selected mRNA has a molecular weight of 35 000. By further mapping, DNA coding sequences for this mRNA were located within the region of BglII N, at approximately 0.585 to 0.596 genome map units. DNA sequences complementary to mRNA encoding a 56K polypeptide were located between 0.607 and 0.612 map units.

Chromosome Mapping↗
PubMed1982

Intracellular transport of the transmembrane glycoprotein G of vesicular stomatitis virus through the Golgi apparatus as visualized by electron microscope radioautography.

The intracellular migration of G protein in vesicular stomatitis virus-infected cells was visualized by light and electron microscope radioautography after a 2-min pulse with [3H]mannose followed by nonradioactive chase for various intervals. The radioactivity initially (at 5-10 min) appeared predominantly in the endoplasmic reticulum, and the [3H]mannose-labeled G protein produced was sensitive to endoglycosidase H. Silver grains were subsequently (at 30-40 min) observed over the Golgi apparatus, and the [3H]mannose-labeled G protein became resistant to endoglycosidase H digestion. Our data directly demonstrate the intracellular transport of a plasmalemma-destined transmembrane glycoprotein through the Golgi apparatus.

Animals↗
PubMed1982

Immunocytochemical localization of mitochondrial proteins in the rat hepatocyte.

Two mitochondrial proteins, carbamyl phosphate synthetase (CPS) and a structural membrane protein, OMM-35, were specifically localized in the hepatocyte mitochondrial matrix and inner and outer membranes, respectively, using the protein A-gold technique. Three embedding media, Epon, glycol methacrylate (GMA), and Lowicryl K4M, were tested for their ability to provide good ultrastructural preservation of mitochondrial membranes, while at the same time retaining protein antigenicity in embedded liver. Epon embedding proved to be relatively unsuitable, since mitochondrial membranes were poorly preserved. GMA and Lowicryl however gave excellent ultrastructural preservation and retained protein antigenicity sufficiently well to enable the localization of the structural membrane protein. Both qualitative and quantitative immunocytochemical demonstration of CPS have ascertained its localization to the rat hepatocyte mitochondrial matrix. The enzyme was undetectable, however, in the mitochondria of liver endothelial cells. OMM-35 was specifically located in the mitochondrial membranes and the quantitative evaluation confirms the biochemical data that OMM-35 is clearly enriched in the outer mitochondrial membrane. OMM-35 was detected in mitochondria of both hepatocytes and endothelial cells. The labeling of a relatively minor structural membrane protein such as OMM-35 gives and indication of the high sensitivity of the protein A-gold immunocytochemical technique.

Animals↗