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J Bacteriol, April 1998, p. 1913-1919, Vol. 180, No. 7
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Sterol Uptake in Saccharomyces cerevisiae Heme Auxotrophic Mutants Is Affected by Ergosterol and Oleate but Not by Palmitoleate or by Sterol Esterification

Frédérique Ness,^1,2 Tilman Achstetter,¹ Catherine Duport,³ Francis Karst,² Roberto Spagnoli,⁴ and Eric Degryse^1,*

Yeast Department, Transgène S.A., 67082 Strasbourg Cedex,¹ Laboratoire de Génétique Physiologique et Moléculaire IBMIG, 86022 Poitiers Cedex,² Centre de Génétique Moléculaire du CNRS, 91198 Gif sur Yvette Cedex,³ and Department for Biotechnological Research, Hoechst Marion Roussel, 93235 Romainville Cedex,⁴ France

Received 10 November 1997/Accepted 26 January 1998

The relationship between sterol uptake and heme competence in two yeast strains impaired in heme synthesis, namely, G204 and H12-6A, was analyzed. To evaluate heme availability, a heterologous 17-hydroxylase cytochrome P-450 cDNA (P-450c17) was expressed in these strains, and its activity was measured in vivo. Heme deficiency in G204 led to accumulation of squalene and lethality. The heterologous cytochrome P-450 was inactive in this strain. The leaky H12-6A strain presented a slightly modified sterol content compared to that for the wild type, and the P-450c17 recovered partial activity. By analyzing sterol transfer on nongrowing cells, it was shown that the cells were permeable toward exogenous cholesterol when they were depleted of endogenous sterols, which was the case for G204 but not for H12-6A. It was concluded that the fully blocked heme mutant (G204) replenishes its diminishing endogenous sterol levels during growth by replacement with sterol from the outside medium. Endogenous sterol biosynthesis appears to be the primary factor capable of excluding exogenous sterol. Oleate but not palmitoleate was identified as a component that reduced but did not prevent sterol transfer. Sterol transfer was only slightly affected by a lack of esterification. It is described herein how avoidance of the potential cytotoxicity of the early intermediates of the mevalonate pathway could be achieved by a secondary heme mutation in erg auxotrophs.

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^* Corresponding author. Mailing address: Yeast Department, Transgène SA, 11 rue de Molsheim, 67082 Strasbourg Cédex. Phone: (33) 03 88 27 91 52. Fax: (33) 03 88 22 58 07. E-mail: degryse{at}transgene.fr.

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