Newly processed ornithine transcarbamylase subunits are assembled to trimers in rat liver mitochondria

We have characterized further the biogenesis in vitro of ornithine transcarbamylase, a homotrimeric mitochondrial matrix enzyme synthesized in the cytoplasm as a larger precursor. When cell-free translation mixtures containing the ornithine transcarbamylase precursor (40 kDa) were chromatographed on Bio-Gel P-200 columns, all of the precursor eluted as aggregates or complexes with molecular weights greater than 200 kDa. None of the precursor bound to a ligand affinity column containing delta-N-(phosphonoacetyl)-L-ornithine (delta-PALO), a transition-state analog and competitive inhibitor of carbamyl phosphate binding, which recognizes native ornithine transcarbamylase. In contrast, a significant portion of the labeled mature-sized subunits, formed when intact mitochondria processed the precursor, bound specifically to the delta-PALO column, were eluted by carbamyl phosphate, and chromatographed on a Bio-Gel P-300 column with a mobility identical to that of native, trimeric ornithine transcarbamylase. No such binding to delta-PALO was observed for the mature-sized monomer or dimer, or for the intermediate-sized ornithine transcarbamylase polypeptide. Moreover, processing by a mitochondrial matrix fraction failed to yield trimeric enzyme, despite producing ample amounts of mature-sized monomer. We conclude that delta-PALO recognizes only trimeric ornithine transcarbamylase composed of mature-sized subunits and that such trimers can be assembled in vitro by intact mitochondria following translocation and proteolytic processing.     

One amino acid change in rat SMR1 polypeptide induces a 1 kDa difference in its apparent molecular mass determined by electrophoretic analysis

SMR1 is a male-specific, 19 kDa, in vitro translation product of Wistar rat submaxillary glands, which may be the precursor of a small hormone resembling the TRH. In Sprague-Dawley and Fischer rats, instead of SMR1, a male-specific 18 kDa polypeptide may be found. We have cloned the cDNA encoding the 18 kDa polypeptide. We show that the 19 and the 18 kDa polypeptides have the same sequence except for one amino and change.     

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.     

Cytochrome c1 of bakers' yeast. II. Synthesis on cytoplasmic robosomes and influence of oxygen and heme on accumulation of the apoprotein.

In the preceding paper (Ross, E., and Schatz, G. (1976) J. Biol. Chem. 251, 1991-1996) yeast cytochrome c1 was characterized as a 31,000 dalton polypeptide with a covalently bound heme group. In order to determine the site of translation of this heme-carrying polypeptide, yeast cells were labeled with [H]leu(be under the following conditions: (a) in the absence of inhibitors, (b) in the presence of acriflavin (an inhibitor of mitochondrial translation), or (c) in the presence of cycloheximide (an inhibitor of cytoplasmic translation). The incorporation of radioactivity into the hemeprotein was measured by immunoprecipitating it from mitochondrial extracts and analyzing it by dodecyl sulfate-polyacrylamide gel electrophoresis. Label was incorporated into the cytochrome c1 apoprotein only in the presence of acriflavin or in the absence of inhibitor, but not in the presence of cycloheximide. Cytochrome c1 is thus a cytoplasmic translation product. This conclusion was further supported by the demonstration that a cytolasmic petite mutant lacking mitochondrial protein synthesis still contained holocytochrome c1 that was indistinguishable from cytochrome c1 of wild type yeast with respect to molecular weight, absorption spectru, the presence of a covalently bound heme group, and antigenic properties. Cytochrome c1 in the mitochondria of the cytoplasmic petite mutant is firmly bound to the membrane, and its concentration approaches that typical of wild type mitochondria. However, its lability to proteolysis appeared to be increased. A mitochondrial translation product may thus be necessary for the correct conformation or orientation of cytochrome c1 in the mitochondrial inner membrane. Accumulation of cytochrome c1 protein in mitochondria is dependent on the abailability of heme. This was shown with a delta-aminolevulinic acid synthetase-deficient yeast mutant which lacks heme and any light-absorbing peaks attributable to cytochromes. Mitochondria from mutant cells grown without added delta-aminolevulinic acid contained at least 20 times less protein immunoprecipitable by cytochrome c1-antisera than mitochondria from cells grown in the presence of the heme precursor. Similarly, the respiration-deficient promitochondria of anaerobically grown wild type cells are almost completely devoid of material cross-reacting with cytochrome c1-antisera. A 105,000 X g supernatant of aerobically grown wild type cells contains a 29,000 dalton polypeptide that is precipitated by cytochrome c1-antiserum but not by nonimmune serum. This polypeptide is also present in high speed supernatants from the heme-deficient mutant or from anaerobically gorwn wild type cells. The possible identity of this polypeptide with soluble apocytochrome c1 is being investigated.     

Studies on the import and processing of the alternative oxidase precursor by isolated soybean mitochondria

Import of the synthetic precursor of the alternative oxidase from soybean was shown to be dependent on a membrane potential and ATP. The membrane potential in soybean mitochondria may be formed either by respiration through the cytochrome pathway, or through the alternative oxidase pathway with NAD⁺-linked substrates. Import of the alternative oxidase precursor in the presence of succinate as respiratory substrate was inhibited by KCN. Import in the presence of malate was insensitive to KCN and SHAM added separately, but was inhibited by KCN and SHAM added together (inhibitors of the cytochrome and alternative oxidases respectively). Import of the alternative oxidase was accompanied by processing of the precursor to a single 32 kDa product in both cotyledon and root mitochondria. This product had a different mobility than the two alternative oxidase bands detected by immunological means (34 and 36 kDa), suggesting that the enzyme had been modified in situ. When the cDNA clone of the alternative oxidase was modified by a single mutation (–2 Arg changed to –2 Gly), the processing of the precursor was inhibited.     

Expression and processing of a recombinant human macrophage colony-stimulating factor in mouse cells.

A human macrophage colony-stimulating factor encoded by a 4-kilobase cDNA was expressed with bovine papillomavirus vectors in mouse cells. Pulse-chase analyses revealed that the 62-kilodalton (kDa) translation product was glycosylated, cleaved, and efficiently secreted within 1 h of synthesis. The secreted product contained both N-linked and O-linked oligosaccharide chains. Macrophage colony-stimulating factor was present extracellularly as an 80-kDa homodimer and as a multimeric species of greater than 200 kDa that may be associated with other glycoproteins.