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Citrate efflux from mitochondria of l0 different yeasts was investigated. 1. Isolated mitochondria of all the yeasts show the possession of a carrier system for dicarboxylic and tricarboxylic anions, phosphate and pyruvate. 2. The tricarboxylate carrier is also specific for L-malate in all 10 yeasts. 3. In citrate-loaded mitochondria, citrate efflux and L-malate uptake are directly proportional to the concentration of extramitochondrial L-malate until the translocator becomes saturated (> 8 mM L-malate in Candida curvata D). 4. Rates of citrate efflux in the presence of L-malate are linear for a period of 2 min and are approximately 2.5-times greater in oleaginous than in non-oleaginous yeasts. 5. The malate-citrate translocator has a significantly lower Km for L-malate in oleaginous yeasts (≈ 4.0 mM) than in non-oleaginous yeasts (≈ 8.4 mM). 6. Both pyruvate and phosphate appear to stimulate the malate-citrate exchange in mitochondria of C. curvata D. 7. Oleaginous yeast mitochondria contain 3–4-times higher intramitochondrial citrate levels than non-oleaginous yeast. In all these yeasts the enzymes for synthesis and metabolism of citrate were exclusively mitochondrial, the only exception being the possession of a cytosolic ATP : citrate lyase by oleaginous yeasts. 8. In metabolically active mitochondria, once the malate-citrate carrier is saturated with L-malate, efflux of citrate is directly proportional to the concentration of extramitochondrial pyruvate (as acetyl donor). 9. The rates of efflux of newly synthesised citrate from metabolically active mitochondria, in the presence of saturating L-malate and pyruvate, are 6–8-times greater in oleaginous yeasts and the citric-acid-producing yeast. 10. The inhibition of aconitase by fluorocitrate produced a ninefold increase in citrate efflux from non-oleaginous yeast mitochondria but only a slight increase in oleaginous yeasts. 11. It is concluded that in fully functional yeast mitochondria, citrate efflux is limited by the amount of intramitochondrial citrate made available to the citrate translocator for exchange. [ABSTRACT FROM AUTHOR]

The optimum conditions for transcription in vitro of yeast mtDNA into biologically relevant RNA by Escherichia coli RNA polymerase holoenzyme and yeast mitochondrial RNA polymerase was found to critically depend on salt concentration. RNA was transcribed (at 0.25 M KCl concentration) from high-molecular-weight mtDNA which was then translated in an E. coli (S-30) cell-free protein synthesising system. Efficient translation of mitochondrial RNA was achieved using conditions which had also been determined to be optimal in other systems. Identification of the polypeptides produced in the translation system was made using antiserum raised against mitochondrial membranes. Electrophoresis of the completely dissociated antigen-antibody complexes using dodecylsulphate-polyacrylamide gels revealed that the system in vitro produced polypeptides of similar molecular weight to those synthesised in vivo by cycloheximide-inhibited whole cells. [ABSTRACT FROM AUTHOR]

1. Citrate efflux from a wide range of yeast mitochondria was inhibited by long-chain fatty-acyl-CoA esters. 2. Fatty-acyl-CoA esters with chain lengths of between 14 and 18 carbons were the most potent inhibitors of citrate efflux which was unaffected by the fatty acids themselves. 3. 50% inhibition of citrate transport was observed using palmitoyl-CoA and oleoyl-CoA at approx. 4--5 μM when the tricarboxylate carrier was saturated with L-malate as counter-anion. 4. The inhibition with palmitoyl-CoA and oleoyl-CoA was competitive with L-malate. 5. The possibility that the fatty-acyl-CoA esters were exerting their effect by acting as detergent was eliminated because of the low concentrations used and appropriate comparisons being made with non-specific detergents. Although detergents inhibited citrate efflux they also released citrate by causing membrane damage. 6. The effect of fat ty-acyl-CeA esters on citrate efflux could be decreased by using higher mitochondrial protein levels and by adding bovine serum albumin. 7. The possibility is discussed that this inhibition represents a genuine feedback inhibition which could regulate the amount of lipid being synthesized by an oleaginous yeast. [ABSTRACT FROM AUTHOR]

The regulation of mitochondrial citrate metabolism has been investigated in oleaginous and non-oleaginous yeasts to ascertain its importance in controlling the rate of citrate efflux from mitochondria. The following observations were made: 1. Citrate efflux from mitochondria of the oleaginous yeast candida curvata D, in the presence of L-malate and pyruvate, was stimulated by adding ATP and reduced by AMP. In the non-oleaginous yeast, Candida utilis 359 there was very little stimulation of citrate efflux by ATP but it was reduced by AMP. These effects appeared to be generalized as similar results were obtained in an examination of eight further yeasts (seven oleaginous and one non- oleaginous). 2. The effects of ATP and AMP were not observed in mitochondria whose metabolism had been inhibited by antimycin A and rotenone indicating the direct regulation of the citrate translocase was not involved. 3. In C. curvata D, ATP increased the total mitochondrial citrate content and reduced that of 2-oxog1utarate whereas AMP had the reverse effect, in C. utilis 359, AMP had a similar effect but that of ATP was much smaller 4. To explain these observations the mitochondria! NAD 4-dependent isocitrate dehydrogenase was studied in a number of yeasts. The enzyme from oleaginous yeasts had a requirement for AMP for activity and was inhibited by ATP. in non-oleaginous yeasts the enzyme was active in the absence of AMP and increased in activity as the isocitrate concentration increased, 5. The enzyme in C. curvata D was constantly more sensitive to Increasing energy charge than that of the non- olcaginous yeast. These results indicate that the supply of citrate (and hence acetyl-CoA) to the cytosol is controlled by the activity of the intramitochondrial NAD+ -dependent isocitrate dehydrogenase which in turn is regulated by adenine nucleotides. The sensitivity of this enzyme to the ATP/AMP ratio during lipogenesis is therefore an important control in the accumulation of lipid by yeasts. [ABSTRACT FROM AUTHOR]

A mutant of Saccharomyces cerevisiae has been selected which has temperature-sensitive mitochondrial protein synthesis. This has been demonstrated by incorporation into cycloheximide-inhibited whole cells, isolated mitochondria and hybrid cell-free systems. The temperature sensitivity is associated with the mitochondrial ribosomes and the mutation is recessive and nuclear. [ABSTRACT FROM AUTHOR]