Induction of rat liver metallothionein mRNA and its distribution between free and membrane-bound polyribosomes.

Total, membrane-bound and free polyribosomes were purified from livers of Zn2+-treated and control rats. Polyadenylated RNA was separated from the polyribosomal RNA extracts by oligo(dT)--cellulose chromatography and translated in a wheat-germ cell-free translation system. Newly synthesized 35S-labelled metallothionein was isolated from the other [35S]methionine-labelled translation products by activated-thiol--Sepharose 4B chromatography. The purity of the 35S-labelled metallothionein product was substantiated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Zinc administration resulted in an elevation of metallothionein mRNA activity to 11% of the total polyribosomal mRNA activity. The vast majority of biologically active metallothionein mRNA was localized in the free polyribosomal pool, at least 94% and 97% in control and zinc-treated rats respectively. The increase in the percentage of polyribosomal mRNA coding for metallothionein after zinc administration was 3-fold, whether measured directly in total polyribosomal mRNA or as a combination derived from membrane-bound and free polyribosomal mRNA. These data indicate that the induction of metallothionein mRNA by zinc involves only free polyribosomes and suggest that the function of metallothionein is limited to intracellular processes.     

Role of membrane-bound and free polyribosomes in the synthesis of cytochrome c in rat liver

The functional distinction of membrane-bound and free polyribosomes for the synthesis of exportable and non-exportable proteins respectively is not so strict as was initially thought, and it was therefore decided to investigate their relative contribution to the elaboration of an internal protein integrated into a cell structure. Cytochrome c was chosen as an example of a soluble mitochondrial protein, and the incorporation of [(14)C]leucine and delta-amino[(14)C]laevulinate into the molecule was studied by using different ribosomal preparations from regenerating rat liver. A new procedure was devised for the purification of cytochrome c, based on ion-exchange chromatography combined with sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. In spite of cytochrome c being a non-exportable protein, the membrane-bound polyribosomes were at least as active as the free ribosomes in the synthesis in vitro of the apoprotein and the haem moiety. The detergent-treated ribosomes could also effect the synthesis of cytochrome c, although at a lower rate. Since in liver more than two-thirds of the ribosomes are bound to the endoplasmic-reticulum membranes, it is considered that in vivo they are responsible for the synthesis of most of the cytochrome c content of the cell. This suggests that in secretory tissues the endoplasmic reticulum plays a predominant role in mitochondrial biogenesis, although free ribosomes may participate in the partial turnover of some parts of the organelle. The hypothesis on the functional specialization of the different kinds of ribosomes was therefore modified to account for their parallel intervention in the synthesis of proteins associated with membranous structures.     

Evidence that free polysomes are not the precursors of membrane-bound polysomes in rat liver

We tested, in rat liver, the postulate that free polysomes were precursors of membrane-bound polysomes. Three methods were used to isolate free and membrane-bound ribosomes from either post-nuclear or post-mitochondrial supernatants of rat liver. Isolation and quantitation of 28 S and 18 S rRNA allowed determination of the 40 S and 60 S subunit composition of free and membrane-bound ribosomal populations, while pulse labeling of 28 S and 18 S rRNA with [6-¹⁴C]orotic acid and inorganic [³²P]phosphate allowed assessment of relative rates of subunit renewal. Throughout the extra-nuclear compartment, 40 S and 60 S subunits were present in essentially equal numbers, but, free ribosomes contained a stoichiometric excess of 40 S subunits, while membrane-bound ribosomes contained a complementary excess of 60 S subunits. Experiments with labeled precursors showed that throughout the extra-nuclear compartment, 40 S and 60 S subunits accumulated isotopes at essentially equal rates, however, free ribosomes accumulated isotopes faster than membrane-bound ribosomes. Among free ribosomes or polysomes, 40 S subunits accumulated isotopes faster than 60 S subunits, but, this relationship was not seen among membrane-bound ribosomes. Here, 40 S subunits accumulated isotope more slowly than 60 S subunits. This distribution of labeled precursors does not support the postulate that free polysomes are precursors of membrane-bound polysomes, but, these data suggest that membrane-bound polysomes could be precursors of free polysomes.     

Construction of a cDNA clone for a nuclear-coded subunit of cytochrome c oxidase from rat liver

A cDNA library for 6S-9S poly(A)-containing RNA from rat liver was constructed in E. coli. Initial screening of the clones was carried out using single stranded 32P-labeled cDNA prepared against poly(A)-containing RNA isolated from immunoadsorbed polyribosomes enriched for the nuclear-coded subunit messenger RNAs of cytochrome c oxidase. One of the clones, pCO89, was found to hybridize with the messenger RNA for subunit VIC. The DNA sequence of the insert in pCO89 was carried out and it has got extensive homology with the C-terminal 33 amino acids of subunit VIC from beef heart cytochrome c oxidase. In addition, the insert contained 146 bp, corresponding to a portion of the 3'-non-coding region. Northern blot analysis of rat liver RNA with the nick-translated insert of pCO89 revealed that the messenger RNA for subunit VI would contain around 510 bases.     

Species-specific expression of cytochrome c oxidase subunit III mRNA

1. A cDNA probe encoding cytochrome c oxidase subunit III cloned from rat liver mitochondria was used to quantify mRNA levels in rat, mouse and rabbit tissues. This was compared to its phenotypic expression, using enzyme activity. 2. Enzyme activities were highest in mouse, intermediate in rat, and lowest in rabbit tissues. 3. Subunit III mRNA levels were easily quantified in rat, but could not be accurately measured in rabbit or mouse tissues despite high cytochrome c oxidase activities. 4. Significant subunit III sequence divergence must exist, among these species. Caution should be exercised in quantifying the expression of this mitochondrial gene.     

Synthesis of a larger precursor for the subunit IV of rat liver cytochrome c oxidase in a cell-free wheat germ system

Poly(A)+RNA from phenol-extracted rat liver polysomes was translated in a heterologous cell-free system derived from wheat germ. The RNA stimulated the incorporation of [35S]methionine into proteins 20- to 30-fold. The labeled translation products were incubated with an antiserum against cytochrome c oxidase. After binding of the antigen x immunoglobulin complex to and elution from protein A-Sepharose and sodium dodecyl sulfate (SDS)-polyacrylamide step gel electrophoresis, autoradiography was carried out. Mainly one major protein with an apparent molecular weight of 19,500 was visualized. When the unlabeled individual cytochrome c oxidase subunits IV, V, VI, or VII, isolated from preparative SDS-polyacrylamide gels, were added to the translation mixture, it was found that only subunit IV could compete with the in vitro-synthesized protein of 19.5 kilodaltons in respect to the binding to the cytochrome c oxidase antiserum. The in vitro-synthesized product was 3,000 daltons larger than the cytochrome c oxidase subunit polypeptide IV. It is concluded that the subunit IV is synthesized as a precursor. Evidence for the precursor form was obtained from translation experiments with [35S]methionine bound to a specific initiator tRNA which led to a radioactively labeled product of identical electrophoretic mobility as the 19.5 kilodalton protein. Furthermore, two dimensional tryptic fingerprints of subunit IV and its precursor show a high degree of similarity.     

Rat liver mitochondrial aspartate aminotransferase mRNA: synthesis of an higher molecular weight precursor by free polysomes

The site of synthesis of the higher molecular weight precursor of mAAT has been determined by separation of free and membrane-bound polysomes. Free and bound polysomes were isolated from rat liver and the polyA+RNA was extracted. Protein synthesis was carried out for two hours by using a reticulocyte lysate system in the presence of 35S-methionine. The samples were subjected to immunoprecipitation with specific antiserum. The precipitates were analyzed by Na-DodSO4/polyacrylamide gel electrophoresis and fluorography. The results clearly demonstrate that the enzyme is synthesized by free polysomes.     

Cell-free synthesis and processing of a large precursor of glutamate dehydrogenase of rat liver

The mitochondrial matrix protein glutamate dehydrogenase of rat liver was synthesized in a cell-free reticulocyte lysate using mRNA from free or membrane-bound polysomes from rat liver. Immunoprecipitation of the (³⁵S)methionine labeled translation mixture was performed using rabbit anti-glutamate dehydrogenase serum. Analysis after electrophoresis of the immunoprecipitate by fluorography of a dried sodium dodecyl sulfate/polyacrylamide gel showed that the glutamate dehydrogenase is synthesized ‘in vitro’ as a large precursor. A mitochondrial extract from rat liver processed the precursor synthesized “in vitro” to the mature form.