Inhibitors of mitochondrial protein synthesis

Although a few subunits of mitochondrial membrane proteins are coded by mitochondrial DNA and synthesized in the mitochondrial matrix, most membrane proteins including the adenine nucleotide carrier are coded by nuclear genes and synthesized on cytoplasmic ribosomes [80,81], Chloramphenicol, an inhibitor of mitochondrial protein synthesis, does not inhibit incorporation of radioactive leucine into the carrier in growing Neurospora crassa, but cycloheximide, an inhibitor of cytoplasmic protein synthesis, does inhibit leucine incorporation [78]. Also, a yeast nuclear respiratory mutant has been shown to cause a defect in adenine nucleotide transport [81], and the nuclear gene responsible for coding the carrier in yeast is currently being cloned for further studies [82]. 

We have performed extensive studies on the sensitivity of mouse and rat embryos to inhibitors of mitochondrial protein synthesis during the late stages of organogenesis. 

The evidence indicates that a high rate of mitochondrial protein synthesis takes place in early organogenesis in rodent embryos. A high sensitivity of these developing embryos to inhibitors of mitochondrial protein synthesis exists. This suggests that from implantation to placenta-... 

Proliferation Inhibitor of DNA polymerase-gamma (e.g., nucleoside reverse transcriptase inhibitors) inhibition of mitochondrial protein synthesis (e.g., oxazolidinone antibiotics) mitochondrial DNA mutation (e.g.. oxidative injury by ethanol)... 

Because of the diflBculties of separating the hydrophobic proteins of the inner membrane by gel electrophoresis, studies were more successful in which the product of mitochondrial protein synthesis was charae-terized using specific inhibitors. As a result the activity of a few mitochondrial enzymes seems to be linked to the function of the mitochondrial genome, or at least to the function of mitoribosomes. 

An essential part of the rationale presented above under (a) consists of the identification of altered products of mitochondrial protein synthesis as a result of the mutation. Although this is not a sufficient criterion for mitochondrial specification (since an altered protein might arise as a result of a mutational alteration in a component of the mitochondrial protein-synthesizing machinery, i.e., one of the mt r- or tRNAs, (as in poky Neu-rospora), it is a necessary one. We have therefore devoted considerable effort to demonstrating the capability of the mitochondria in the mutant to perform some form of protein synthesis. We did this by showing that they were capable of incorporating (1) labeled formate into formylmethionyl-puromycin as a measure of mitochondrial polypeptide-chain initiations (see also next section) (2) labeled leucine into mitochondrial membrane proteins in a reaction that is insensitive to cycloheximide (CHX), but sensitive to chloramphenicol (CAP) and (3) that continued exposure of cells to the latter led to their conversion to petite phenocopies, s5 of characteristic aspects of their phenotype, such as the presence of cytochrome b, and that this change was reversed on removal of the inhibitor (see also Table I). 

The existence of mitochondrial protein synthesis in S. pombe is indicated by the specific inhibition of growth on glycerol—but not on glucose— by mitochondrial protein-synthesis inhibitors. Antimycin-sensitive respiration and the absorption peaks of cytochromes aa and disappear in cells grown at pH 6.5 in glucose media supplemented with either 10 Mg ethidium bromide, 20 Mg of acriflavin, or 2 mg/ml chloramphenicol. ... 

Cytosolic ATP dynamics of HeLa cell during apoptosis was measured using a luminometer (Fig.l). Total amount of ATP increased within 20 to 120 min and decreased gradually within 10 h via three types of apoptotic induction (STS, FCCP and CHX). As CHX is an inhibitor of protein synthesis, ATP elevation after apoptotic induction is not due to elevation of luciferase synthesis. On the other hand, ATP elevation is suppressed by glucose-free medium and 2-deoxy-D-glucose. It leads to a hypothesis that glycolysis pathway contributes to an elevation of cytosolic ATP.5 This hypothesis is supported by the result using FCCP, an inhibitor of mitochondrial ATP production. 

The mitochondrial ribosomes of mammals are small (about 555) and insensitive to erythromycin and lincomycin, although these antibiotics penetrate freely into the mitochondria. These ribosomes are sensitive to chloramphenicol which, fortunately, does not normally penetrate into mammalian mitochondria. Chloroplasts have ribosomes that are sensitive to most of the protein synthesis inhibitors that injure bacteria (Kiintzel and Noll, 1967). 

The use of inhibitors has helped characterize the mitochondrial protein synthesis reaction in two ways. On the one hand, information on the mechanism may be gained with some inhibitors. And on the other hand, some inhibitors (especially antibiotics) are useful in distinguishing mitochondrial protein synthesis from that in the microsomal fraction. These inhibitors may be divided into three different groups (1) Those which interfere with both mitochondrial and microsomal protein synthesis (2) compounds which selectively inhibit microsomal protein synthesis and (3) inhibitors which interfere only with mitochondrial protein synthesis. 

Incorporation of labeled amino acids by other components of the incubation medium can be excluded. Bacterial contamination of the chloroplast preparation is minimized by using sterile glassware and media. If the preparations are solubilized in 2% Triton X-100 at the end of the incubation, less than 0.1% of the radioactivity incorporated into protein is present in the pellet spun down at 10,000 for 10 min. This indicates that incorporation is not due to bacteria, nuclei, or whole leaf cells. The large stimulation of incorporation by light and the sensitivity to inhibitors of photophosphorylation argue for chloroplast, rather than mitochondrial, protein synthesis. If stored for 1 h at O C, the activity of the crude chloroplast suspensions decreases by about 50%. Therefore, we have not attempted to purify the chloroplasts from other cellular components present in the suspension. 

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