Reguladtion of Citrate Efflux from Mitochondria Oleaginou and Non‐Oleaginous Yeasts by Adenine Nucleotides

Abstract

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 curuata 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 nonoleaginous).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-oxoglutarate 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 mitochondrial NADC-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 nonoleaginous yeast.These results indicate that the supply ofcitrate (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.Work in our laboratory has been concerned with the biochemistry of various oleaginous yeasts and moulds which can accumulate large quantities of lipid [l, 21. When a nutrient, usually nitrogen, becomes depleted from the growth medium, protein and nucleic acid synthesis ceases but the excess carbon continues to be metabolised to lipid [3-51. Under such conditions, non-oleaginous yeasts still do not accumulate lipid. We have so far explained microbial oleaginicity [6] by showing that under conditions of nitrogen limitation, citrate accumulates in the mitochondria due primarily to a decrease in the intracellular AMP concentration which leads to a decline in the activity of the AMP-requiring, NAD +-dependent isocitrate dehydrogenase within the mitochondria. This citrate is then transported across the mitochondrial membrane in exchange for L-malate [7] and is cleaved in the cytosol by ATP:citrate lyase to yield acetyl-CoA (and oxaloacetate) from which fatty acids are synthesized. The presence of ATP :citrate lyase is confined to oleaginous yeasts and moulds K91...

Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621

“…In contrast to results reported by Evans et al [9] aad Wills and Melham [71 the e~yme pyruvate carboxylase was found to be localized i~ the cytosol in bodl S. cerevisiae and C. utilis. It c,~u~d be argued that this would result from leakage of the enzyme from the mitochondrial matrix.…”

“…However, in contrast to the report by Evans et al [9], also in C. utilis the enzyme was exclusively recovered in the soluble fraction (Table I). The activity of the enzyme was approximately the same in both organisms.…”

“…on quantitative aspects of pyruvate metabolism. Of special interest was the localization of pyruvate carboxylase, in view of reported differences between S. cerevisiae and C. utilis [6][7][8][9]. Moreover, the regulation of f.he enzyme pyruvate decarboxylase by inorganic phosphate and other possible effectors was studied, since Boiteux and Hess [10] reported inhibition of this enzyme by phosphate.…”

Regulation of Citrate Efflux from Mitochondria of Oleaginous and Non‐Oleaginous Yeass by Long‐Chain Fatty Acyl‐CoA Esters

“…Efflux of citrate could be controlled by (a) limiting the amount of intramitochondrial citrate made available for binding to the citrate translocase in the inner mitochondrial membrane, (b) by the availability of cytosolic L-malate as counter-anion for the operation of the citrate-malate exchange and (c) by directly affecting the efficiency of the citrate carrier from promoting exchange. The importance of points (a) and (b) with regard to oleaginicity in yeasts has been discussed previously [5,6]. The direct regulation of the mitochondrial carrier by long-chain fatty-acyl-CoA esters forms the subject of this report.…”

“…The following yeasts were used in this study: Methods used for growth of yeasts, lipid extraction and preparation of mitochondria were as reported earlier [5]. Citrate was assayed enzymatically [36] in supernatants and mitochondrial extracts.…”