Vicente J. Miralles

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

Abstract 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 ( 35 S)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.

Spermidine and spermine stimulate the transport of the precursor of ornithine carbamoyltransferase into rat liver mitochondria

We have examined the effect of low molecular weight components of the transport mixture generally used for the import of rat liver pre-ornithine carbamoyltransferase by isolated rat liver mitochondria. These studies revealed that spermidine and spermine, at physiological concentrations, stimulate the transport of the precursor of ornithine carbamoyltransferase into mitochondria. This stimulatory effect of spermidine and spermine is concentration-dependent and is completely inhibited at higher than physiological concentrations (20 mM for spermidine and 4 mM for spermine). Magnesium ions, which also have a stimulatory effect, inhibit the stimulatory effect of spermidine.

Exit of proteins and fragments thereof from mitochondria is accelerated by the import of cytosolic synthesized proteins

Most mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria. Incubation of 35S-methionine labeled mitochondria from rat hepatocytes with proteins synthesized in a cell-free system, using messenger RNA from rat liver, dramatically increased the release of mitochondrial proteins and fragments thereof into the medium. Since the synthesized proteins include cytosolic precursors of mitochondrial proteins, our results strongly suggest that import of proteins from the cytosol into mitochondria influences the half-life of proteins in these organelles. The use of this simple approach--i.e. combining the study of protein import and exit with mitochondria--to further clarify intracellular protein turnover and its regulation is suggested.

Polyamines are sufficient to drive the transport of the precursor of ornithine carbamoyltransferase into rat liver mitochondria: possible effect on mitochondrial membranes

The polyamines spermidine, spermine and putrescine, by themselves, at physiological concentrations, induce the transport of the precursor of ornithine carbamoyltransferase into isolated rat liver mitochondria. The presence of polyamines in the transport medium results in the approach of both mitochondrial membranes, suggesting a possible role of these molecules in the transport of the precursor of ornithine carbamoyltransferase into mitochondria, by the formation and/or stabilization of mitochondrial structures involved in the transport system.

Loading rat liver mitochondria with apocytochrome c provokes exit of cytochrome c

Rat liver mitochondria were loaded with cytochrome c by incubation with large amounts of [14C]apocytochrome c. After being washed they were incubated with either more apocytochrome c or cytochrome c. There was no release of labeled proteins from the mitochondria when incubated with cytochrome c. However, there was when incubated with apocytochrome c. The material released showed only one radioactive band which migrated as cytochrome c. Also no release of proteins other than cytochrome c was detected when liver mitochondria isolated from rats injected with [35S]methionine were incubated with apocytochrome c. These results suggest that the level and possibly the turnover of cytochrome c in rat liver mitochondria is regulated by the entry of apocytochrome c into mitochondria.

Apocytochrome-c competes with pre-ornithine carbamoyl transferase for transport into mitochondria

Apocytochrome c, the cytosolic precursor of cytochrome c, competes with the precursor of ornithine carbamoyltransferase (OCT) for entry into isolated rat liver mitochondria.

Transport of the precursor for rat liver glutamate dehydrogenase into mitochondria “in vitro”

Abstract Glutamate dehydrogenase of rat liver, a mitochondrial matrix enzyme, is synthesized in a larger precursor form in a rabbit reticulocyte cell-free system. When this precursor was incubated with isolated mitochondria in the absence of protein synthesis, it was processed to the mature form and rendered protease-resistant during incubation with mitochondria.

Polyamines Stimulate the “In Vitro” Transport of the Precursor of Ornithine Carbamoyltransferase into Rat Liver Mitochondria

Most mitochondrial proteins are coded by nuclear genes, synthesized outside mitochondria, and imported into these organelles in a post-translational event (1). The import process usually involves the following steps: 1) synthesis of a precursor polypeptides with an amino-terminal extension (signal peptide), on free cytoplasmic polysomes, 2) binding of the precursor to a receptor-like component on the mitochondrial surface, 3) energy-dependent translocation across both mitochondrial membranes, possibly through “contact sites” between outer and inner membranes, 4) removal of the amino-terminal extension by a chelator-sensitive protease in the mitochondrial matrix, and 5) mature proteins location into their functional sites. Exceptions to this sequence of events include apocytochrome c and all proteins of the outer membrane which are synthesized without a leading peptide and transported into mitochondria without need of a membrane potential.

In vitro transport of ornithine carbamoyltransferase precursor into rat liver mitochondria using a more homologous medium.

The precursor of ornithine carbamoyltransferase can be transported in vitro into rat liver mitochondria using the postmitochondrial supernatant from rat liver, a more homologous medium than the commonly used rabbit reticulocyte lysate. The transport of the precursor in the case of reticulocyte lysate requires a standard translation mixture. In the presence of the postmitochondrial supernatant the same is true. However, when the components of the translation mixture were added individually to the postmitochondrial supernatant, it was found that spermidine or spermine, at physiological concentrations, sufficed for the transport of the precursor of ornithine carbamoyltransferase. The activity of the postmitochondrial supernatant was inactivated by trypsin and slightly decreased by RNase treatment; it was not lost by dialysis or by heating at 100 degrees C.

An ammonium sulphate fraction from rabbit reticulocytes increases the release of proteins from rat liver mitochondria

Incubation of [35S]methionine labeled mitochondria from rat liver with rabbit reticulocyte lysate under the same conditions as those used in the import of mitochondrial protein precursors results in the release of mitochondrial proteins to the medium. Fractionation of the lysates with ammonium sulphate yields a fraction, essentially free of haemoglobin, which exhibits higher activity for the release of mitochondrial proteins than the starting lysate. The fraction has a molecular mass of > 10 kDa and is heat‐sensitive. The release is insensitive to inhibitors of reticulocyte lipoxygenase.