Fatty acid uptake in Candida tropicalis: induction of a saturable process BERNARDO L. TRIGATTI, ANDREW D. BAKER, KRISHAN RAJARATNAM, RICHARD A. RACHUBINSKI, AND GERHARD E. GERBER' Department of Biochemistry, McMaster University, Hamilton, Ont., Canada L8N 325 Received July 5, 1991 TRIGATTI, B. L., BAKER, A. D., RAJARATNAM, K., RACHUBINSKI, R. A., and GERBER, G. E. 1992. Fatty acid uptake in Candida tropicalis: induction of a saturable process. Biochem. Cell Biol. 70: 76-80. The rates of oleate uptake by Candida tropicalis cells grown on a high oleate concentration (3.5 mM oleate in the presence of 0.50% Brij 58) were higher than those observed in cells grown on glucose; however, oleate uptake was not saturable with substrate concentration. Cells grown at a low oleate concentration (1.0 mM oleate and 2.5% Brij 58) grew to a lower density and at a slightly slower rate; these cells were found to take up oleate at a rate 43-fold higher than cells grown on high oleate concentration. Furthermore, oleate uptake by the cells grown in low oleate medium was a saturable process with Kt and V,, values of 56 pM and 15 nmol/(min.mg cell protein), respectively. The growth of C. tropicalis under low fatty acid concentration thus clearly results in the induction of a saturable process for its uptake. The total level of acyl-CoA synthetase activity in cells grown on the low oleate concentrations was only twofold higher than in high oleate or glucose grown cells; the level of this enzyme thus does not account for the saturable process and suggests that either the enzyme is regulated in vivo or else a hitherto unidentified enzyme is induced by growth in low concentrations of oleate. Key words: acyl-CoA synthetase, Candida tropicalis, induction, oleate, uptake. TRIGATTI, B. L., BAKER, A. D., RAJARATNAM, K., RACHUBINSKI, R. A., et GERBER, G. E. 1992. Fatty acid uptake in Candida tropicalis: induction of a saturable process. Biochem. Cell Biol. 70 : 76-80. Les taux d'incorporation de I'oltate par les cellules de Candida tropicalis croissant en presence d'une concentration elevte d'oltate (oltate 3,s mM en prtsence de Brij 58 0,50%) sont plus Clevis que ceux obsemts dans les cellules cultivtes sur le glucose; cependant, l'incorporation de I'oltate n'est pas saturable avec la concentration du substrat. Les cellules cultivtes en prtsence d'une faible concentration d'oltate (oltate 1,O mM et Brij 58 2,5%) croissent avec une densitt plus faible et a une vitesse ltgkrement plus lente; ces cellules absorbent I'oltate a un taux 43 fois plus Clevt que les cellules en prtsence d'une forte concentration d'oltate. De plus, l'absorption de I'oltate par les cellules cultivtes dans un milieu contenant peu d'oltate est un processus saturable avec une valeur de Kt de 56 ,uM et une V,, de 15 nmol/(min mg de prottine cellulaire). La croissance de C. tropicalis en prtsence d'une faible concentration d'acides gras entraine donc nettement l'induction d'un processus d'absorption saturable. Le taux global de l'activitt de I'acyl- CoA synthttase dans les cellules croissant en prtsence de faibles concentrations d'oltate est deux fois plus tlevt que dans les cellules en prtsence de beaucoup d'oltate ou de glucose. Le taux de cette enzyme n'est donc pas responsable du processus saturable et suggtre que l'enzyme pourrait Ctre contrSlte in vivo ou qu'une enzyme encore non identifite serait induite par la croissance en prtsence de faibles concentrations d'oltate. Mots clks : acyl-CoA synthttase, Candida tropicalis, induction, oltate, absorption. [Traduit par la rtdaction] The mechanism of cellular uptake of long-chain fatty acids is poorly understood. A major controversy is whether fatty acid permeation across the plasma membrane is protein mediated or simply a diffusive flip-flop process. In Escherichia coli, two gene products have been shown to be required for the process: the product of the fadL gene, which is required for the movement of fatty acids across the outer membrane which is normally impermeable to hydrophobic compounds (Black et al. 1987), and the fadD gene product, a long-chain acyl-CoA synthetase which recently has been implicated in facilitating the movement of long-chain fatty acids across the inner membrane (Mangroo and Gerber 1991). In mammalian cells, several lines of evidence support the proposal that the process is protein mediated. In hepatocytes, kinetic evidence indicates that uptake is saturable with substrate concentration (Stremmel et a/. 1986). A 40-kDa protein was identified and found to bind fatty acids with high affinity, and antibodies to this protein were reported to partially inhibit oleate uptake (Stremmel et a/. 1985, 1986). Recent evidence, however, indicates that this protein may be located in mitochondria, bringing its ABBREVIATIONS: Brij 58, polyoxyethylene 20 cetyl ether; ODm, optical density at 660 nm. '~uthor to whom all correspondence should be addressed. tinted in Canada / Imprime au Canada involvement in fatty acid movement across the plasma membrane into question (Berk et al. 1990). Furthermore, studies using model membrane systems have suggested that fatty acid movement across the plasma membrane may be rapid (Broring et al. 1989) and not protein mediated (Doody et al. 1980; Storch and Kleinfeld 1986). Thus while it is clear that fatty acid uptake generally occurs via a saturable pro- cess, it remains to be established whether this is due to pro- teins involved in translocation or some other metabolic process. The yeast Candida tropicalis grows on both n-alkanes and long-chain fatty acids, with a resultant induction of peroxi- somes containing the metabolic machinery for P-oxidation (Kawamoto et al. 1978; Dornrnes et al. 1983). A concomitant induction in long-chain fatty acid uptake would greatly facilitate the identification of components involved in the process; however, no studies of such inducible processes in oleate uptake have been reported. It was the purpose of this study to determine whether such inducible mechanisms for oleate uptake existed in C . tropicalis. It was found that the level of oleate uptake was dependent on the nature and concentration of the car- bon source. Cells grown on high concentrations of oleate (3.5 mM oleate and 0.50% Brij 58) had low levels of non- saturable oleate uptake, while growth of cells on a low oleate Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by China University of Petroleum on 06/05/13 For personal use only.