STEROL UPTAKE IN SACCHAROMYCES-CEREVISIAE HEME AUXOTROPHIC MUTANTS ISAFFECTED BY ERGOSTEROL AND OLEATE BUT NOT BY PALMITOLEATE OR BY STEROL ESTERIFICATION

The relationship between sterol uptake and heme competence in two yeas t strains impaired in heme synthesis, namely, G204 and H12-6A, was ana lyzed. To evaluate heme availability, a heterologous 17 alpha-hydroxyl ase cytochrome P-450 cDNA (P-450c17) was expressed in these strains, a nd its activity was measured in vivo. Heme deficiency in G204 led to a ccumulation of squalene and lethality. The heterologous cytochrome P-4 50 was inactive in this strain. The leaky H12-6A strain presented a sl ightly 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 towar d 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 endo genous sterol levels during growth by replacement with sterol from the outside medium. Endogenous sterol biosynthesis appears to be the prim ary factor capable of excluding exogenous sterol. Oleate but not palmi toleate 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 potent ial cytotoxicity of the early intermediates of the mevalonate pathway could be achieved by a secondary heme mutation in erg auxotrophs.