Eur J Biochem. zyxwvutsrqpon 177, 207-211 (1988) I, FEBS 1988 zyxwvutsrqpo Localization of the synthesis of very-long-chain fatty acid in mitochondria from zyxwvut Saccharomyces cerevisiae Jean-Jacques BESSOULE, Rene LESS1 RE, Michel RIGOULET, Bernard GUERIN and Claude CASSAGNE Institut de Biochimie Cellulaire et Neurochimie (LP 8231), Bordeaux (Received March 17/June 16, 1988) zyxwvutsrqp - EJB 88 0157 The localization of the mitochondrial elongation activities ('elongases') from Saccharomyces cerevisiae has been investigated. It was shown, using carboxyatractyloside in the incubation mixture, that synthesis of very- long-chain Fatty acids probably occurred outside the matrix and, by fractionation experiments, that elongases are membrane-bound enzymes. The solubilization of the outer membrane by digitonin showed that three elongating activities are correlated with a marker of the outer membrane and not with an inner membrane marker. A further partial purification of the outer membrane showed that elongases are present in the outer membrane of mitochondria. The synthesis of very-long-chain fatty acids has been de- scribed in many organisms and occurs in many compartments of the cell. In procaryotes, they are synthesized by a soluble multi-enzyme complex [l - 31, while in eucaryotic cells the elongation seems to take place almost exclusively in the endomembrane system (for review see [4]). In Allium porrum epidermal cells, the existence of three different elongation activities ('elongases') has been demonstrated : an ATP-de- pendent elongase, which condenses one molecule of malonyl- CoA [5,6] with an endogenous substrate, and two other acyl- CoA elongases: a CI8-CoA elongase and a Czo-CoA elongase. The latter differ by their molecular masses (350 and 650 kDa, respectively) [7] and their intracellular location (the endoplas- mic reticulum and Golgi apparatus, respectively) [4]. In con- trast with the ATP-dependent elongase, these acyl-CoA elongases condense several molecules of malonyl-CoA with an exogenous acyl-CoA [6]. We have recently demonstrated that mitochondria from Saccharomyces cerevisiae are also able to synthesize very- long-chain Fatty acids [8] using endogenous precursors (in the presence of ATP), or exogenous acyl-CoAs. The reason for the presence of these three activities in mitochondria is far from clear, partly because of the total absence of data concern- ing their localization. Are the three activities located differently as in higher plants? Are they soluble complexes, as observed in bacteria (which would support the hypothetical procaryotic origin of mitochondria) and therefore, co-local- ized with the enzymes of the /$oxidation system, or are they localized in membranes, as in eucaryotic cells? In order to answer these questions, we investigated the localization of the activities leading to the formation of very-long-chain fatty acids in mitochondria from S. cerevisiae. In this paper, we demonstrate that the three elongation activities are localized in a unique mitochondria1 compart- C'orrespondenw to J.-J. Bcssoule, Lnstitut de Biochimie et Neuro- chirnie, 1 rue Camille Saint-Saens, F-33077 Bordeaux Ckdex, France Ahhreviution. Very-long-chain fatty acids, CH3-(CH2).-COOH with n> 18. Enzymes. ATP-dependent elongase, [acyl-carrier-protein] malo- nyltransferase (EC 2.3.1.39); C,&oA elongase and CZO-CoA elongase, zyxwvutsrq acyl-[acyl-carrier-protein] -phospholipid acyltransferases (EC 2.3.1.40). ment: the outer membrane. This elongation system is different from the mitochondrial P-oxidation system, not only from the point of view of the mechanism of reaction [8], but also in its nature (membrane-bound and not soluble). MATERIALS AND METHODS Substrates and reagents [2-'"C]Malonyl-CoA (55 Ci/mol) was obtained from Amersham International. Sucrose was purchased from Pro- labo. All other chemical products were from Sigma Chemicals. Preparation zyxwv of mitochondria Cells of the diploid wild strain S. cerevisiae (yeast foam) were grown aerobically at 28 "C in a complete medium, con- taining 1% yeast extract, 0.1% potassium phosphate, 0.12% ammonium sulfate (pH 4.9, and supplemented with 2% lac- tate as the carbon source. Cells were harvested during the logarithmic phase. Mitochondria were isolated as described previously [9]. The mitochondrial purity was tested by electron microscopy and sucrose density gradient centrifugation [lo]. Oxygen uptake The rate of oxygen uptake was measured with a Clark oxygen electrode (Gilson) at 27 "C in 3 ml buffer consisting of 10 mM Tris/maleate, 0.65 M mannitol, 1.3 mM KH2P04 and 0.3% bovine serum albumin pH 6.7. Other additions are specified in the legend of the Fig. 1. Subfractionation of mitochondria Mitochondria1 protein (3 mg, 150 pl) was diluted in 15 ml HzO and homogenized with five strokes in a Dounce homogenizer. After 5 min, the suspension was centrifuged twice at 12000 x g for 15 min. The supernatant (0.7 mg pro- tein) was then centrifuged at 65000 x g for 20 min. The pellet (0.2 mg protein) was suspended in 1 ml H20 and the super- natant (0.5 mg) centrifuged for 1 h at 105000 x g. The pellet (0.03 mg) was suspended in 0.5 ml H 2 0 . 0.45 mg protein was