Mitochondrial translocation and processing of the precursor to the α-subunit of rat liver succinyl-CoA synthetase

1990 ◽  
Vol 68 (1) ◽  
pp. 292-299 ◽  
Author(s):  
Ramanath Majumdar ◽  
William A. Bridger
Keyword(s):  
Rat Liver ◽  
Signal Sequence ◽  
Proteolytic Processing ◽  
In Vitro Translation ◽  
Rat Liver Mitochondria ◽  
Translation System ◽  
Cdna Insert ◽  
Α Subunit ◽  
Mitochondrial Translocation ◽  
Succinyl Coa Synthetase

Succinyl-CoA synthetase functions in the mitochondrial matrix as an αβ-dimer. Its constitutive subunits are thus expected to be encoded in the nucleus and synthesized in the cytoplasm as precursors containing signal sequences for mitochondrial translocation. We have previously reported the isolation and sequence of a rat liver cDNA clone (λSCS19) that apparently encodes the cytoplasmic precursor to the α-subunit. Here we report the preparation of mRNA transcripts of this cDNA insert and their in vitro translation to produce labeled protein that can be translocated across the membranes of subsequently added rat liver mitochondria. Translocation is accompanied by proteolytic processing to convert the 34.5-kilodalton precursor to the 32-kilodalton mature form of the subunit. The N-terminal sequence of the mature α-subunit from the GTP-specific isozyme has been determined by sequential Edman degradation and compared with the amino acid sequence deduced from the cDNA. This confirms that the cloned sequence encodes the GTP-specific α-subunit, and establishes that the point of cleavage is between histidyl and glycyl residues and that the signal sequence consists of 27 residues. The signal sequence shares characteristics of other mitochondrial targeting sequences that have been elucidated (largely of yeast mitochondrial precursors), including the potential to form an amphiphilic helix. Import is dependent upon the presence of ATP and is inhibited by compounds that diminish mitochondrial membrane potential. Translocation of the precursor is effective for precursor produced by the reticulocyte translation system, but is not seen for the product that is translated by a wheat germ extract, indicating that the latter may lack a factor or component that is necessary for the targeting and import process.Key words: succinyl-CoA synthetase, mitochondria, protein translocation, signal sequence.

scholarly journals Role of polyamines in the transport in vitro of the precursor of ornithine transcarbamylase

1991 ◽  
Vol 279 (3) ◽  
pp. 815-820 ◽  
Author(s):  
C González-Bosch ◽  
M J Marcote ◽  
J Hernández-Yago
Keyword(s):  
Rat Liver ◽  
Signal Sequence ◽  
Chimeric Protein ◽  
Liver Mitochondria ◽  
Ornithine Transcarbamylase ◽  
Rat Liver Mitochondria ◽  
Surface Transport ◽  
Binding To Mitochondria

Polyamines induce the transport in vitro of the rat liver precursor of ornithine transcarbamylase (pOTC) into isolated rat liver mitochondria. The accumulation of this precursor at the level of binding to the mitochondrial surface has allowed us to establish that polyamines are involved in the interaction of the precursor with the mitochondrial surface. Transport of a chimeric protein having the signal sequence of pOTC fused to a fragment of the cytosolic protein human arginosuccinate lyase was also induced by polyamines. The sensitivity of the pOTC synthesized in vitro and of the chimeric protein to proteinases decreases in the presence of polyamines. This result suggests that polyamines may play a role in modulating the folding of precursors to favour their binding to mitochondria.

scholarly journals Import of rat liver mitochondrial transhydrogenase

1988 ◽  
Vol 252 (3) ◽  
pp. 833-836 ◽  
Author(s):  
L N Y Wu ◽  
I M Lubin ◽  
R R Fisher
Keyword(s):  
Rat Liver ◽  
Proteinase Inhibitors ◽  
Rat Hepatocytes ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Translation System ◽  
Isolated Rat Hepatocytes ◽  
Matrix Fraction ◽  
Translation Mixture ◽  
Isolated Rat

The biosynthesis of pyridine dinucleotide transhydrogenase has been studied in isolated rat hepatocytes and in a rabbit reticulocyte-lysate translation system supplemented with either intact isolated rat liver mitochondria or the soluble matrix fraction from isolated mitochondria. In intact hepatocytes, the transhydrogenase precursor was short-lived in the cytosol and was efficiently imported into the membranous fraction. When the cell-free translation mixture was incubated with intact mitochondria, the transhydrogenase precursor was processed to the mature form, to an extent that depended on the amount of added mitochondria. Incubation of the translation mixture with the soluble mitochondria matrix fraction converted the precursor to a mature-sized protein with 75% efficiency, this being blocked by various proteinase inhibitors such as EDTA, 1,10-phenanthroline and leupeptin.

scholarly journals Rat liver β-glucuronidase. cDNA cloning, sequence comparisons and expression of a chimeric protein in COS cells

1988 ◽  
Vol 250 (2) ◽  
pp. 547-555 ◽  
Author(s):  
P P Powell ◽  
J W Kyle ◽  
R D Miller ◽  
J Pantano ◽  
J H Grubb ◽  
...  
Keyword(s):  
Amino Acid ◽  
Liver Enzyme ◽  
Rat Liver ◽  
Signal Sequence ◽  
Proteolytic Processing ◽  
Amino Acid Difference ◽  
Amino Acid Residues ◽  
Sequence Comparisons ◽  
Chimeric Clone ◽  
Cos Cells

A cDNA for rat liver beta-glucuronidase was isolated, its sequence determined and its expression after transfection into COS cells studied. The deduced amino acid sequence of the rat liver clone showed 77% homology with that from the cDNA for human placental beta-glucuronidase and 47% homology with that deduced from the cDNA for Escherichia coli beta-glucuronidase. Several differences were found between the cDNA from rat liver and that previously reported from rat preputial gland. Only one change leads to an amino acid difference in the mature enzyme. A chimeric clone was constructed by using a fragment encoding the first 18 amino acid residues of the signal sequence from the human placental cDNA clone and a fragment from the rat clone encoding four amino acid residues of the signal sequence, all 626 amino acid residues of the mature rat enzyme, and all of the 3′ untranslated region. After transfection into COS cells the chimeric clone expressed beta-glucuronidase activity that was specifically immunoprecipitated by antibody to rat beta-glucuronidase. The Mr value of 76,000 of the expressed gene product was characteristic of the glycosylated rat enzyme. It was proteolytically processed in COS cells to Mr 75,000 6 h after metabolic labelling. At least 50% of the expressed enzyme was secreted at 60 h post-transfection, but the secreted enzyme did not undergo proteolytic processing. These results provide evidence that the partial cDNA isolated from a rat liver library contains the complete coding sequence for the mature rat liver enzyme and that the chimeric signal sequence allows normal biosynthesis and processing of the transfected rat liver enzyme in COS cells.

scholarly journals Rubella Virus Nonstructural Protein Protease Domains Involved in trans- and cis-Cleavage Activities

2000 ◽  
Vol 74 (12) ◽  
pp. 5412-5423 ◽  
Author(s):  
Yuying Liang ◽  
Jiansheng Yao ◽  
Shirley Gillam
Keyword(s):  
Rubella Virus ◽  
Nonstructural Protein ◽  
Proteolytic Processing ◽  
Open Reading Frames ◽  
In Vitro Translation ◽  
Translation System ◽  
Nonstructural Proteins ◽  
Cleavage Activity ◽  
Domain Of Unknown Function

ABSTRACT Rubella virus (RV) genomic RNA contains two large open reading frames (ORFs): a 5′-proximal ORF encoding nonstructural proteins (NSPs) that function primarily in viral RNA replication and a 3′-proximal ORF encoding the viral structural proteins. Proteolytic processing of the RV NSP ORF translation product p200 is essential for viral replication. Processing of p200 to two mature products (p150 and p90) in the order NH2-p150-p90-COOH is carried out by an RV-encoded protease residing in the C-terminal region of p150. The RV nonstructural protease (NS-pro) belongs to a viral papain-like protease family that cleaves the polyprotein both in trans and incis. A conserved X domain of unknown function was found from previous sequence analysis to be associated with NS-pro. To define the domains responsible for cis- andtrans-cleavage activities and the function of the X domain in terms of protease activity, an in vitro translation system was employed. We demonstrated that the NSP region from residue 920 to 1296 is necessary for trans-cleavage activity. The domain from residue 920 to 1020 is not required for cis-cleavage activity. The X domain located between residues 834 and 940, outside the regions responsible for both cis- andtrans-cleavage activities of NS-pro, was found to be important for NS-pro trans-cleavage activity but not forcis-cleavage activity. Analysis of sequence homology and secondary structure of the RV NS-pro catalytic region reveals a folding structure similar to that of papain.

Effects of 5-5'-Diphenyl-2-thiohydantoin (DPTH) and Thyroxine on the Ultrastructure and Chemical Composition of Rat Liver

1971 ◽  
Vol 29 ◽  
pp. 570-571
Author(s):  
E. A. Elfont ◽  
R. B. Tobin ◽  
D. G. Colton ◽  
M. A. Mehlman
Keyword(s):  
Chemical Composition ◽  
Rat Liver ◽  
Future Study ◽  
Mode Of Action ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Effective Inhibitor ◽  
Biochemical Effects ◽  
Glycerophosphate Dehydrogenase ◽  
Stimulation Of

Summary5,-5'-diphenyl-2-thiohydantoin (DPTH) is an effective inhibitor of thyroxine (T4) stimulation of α-glycerophosphate dehydrogenase in rat liver mitochondria. Because this finding indicated a possible tool for future study of the mode of action of thyroxine, the ultrastructural and biochemical effects of DPTH and/or thyroxine on rat liver mere investigated.Rats were fed either standard or DPTH (0.06%) diet for 30 days before T4 (250 ug/kg/day) was injected. Injection of T4 occurred daily for 10 days prior to sacrifice. After removal of the liver and kidneys, part of the tissue was frozen at -50°C for later biocheailcal analyses, while the rest was prefixed in buffered 3.5X glutaraldehyde (390 mOs) and post-fixed in buffered 1Z OsO4 (376 mOs). Tissues were embedded in Araldlte 502 and the sections examined in a Zeiss EM 9S.Hepatocytes from hyperthyroid rats (Fig. 2) demonstrated enlarged and more numerous mitochondria than those of controls (Fig. 1). Glycogen was almost totally absent from the cytoplasm of the T4-treated rats.

Energy and Antioxidant Status of Rat Liver Mitochondria during Hypoxia- Reoxygenation of Different Duration

2016 ◽  
Vol 7 (4) ◽  
pp. 349-361
Author(s):  
Olga A. Gonchar ◽  
Valentina I. Nosar ◽  
Larisa. V. Bratus ◽  
I. N. Tymchenko ◽  
N. N. Steshenko ◽  
...  
Keyword(s):  
Rat Liver ◽  
Antioxidant Status ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Hypoxia Reoxygenation
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