Intracellular sterol transport and distribution in Saccharomyces cerevisiae

Sterols, such as cholesterol in mammals and ergosterol in yeast, are important components of cellular membranes. Sterol biosynthesis is tightly regulated because deviations from normal sterol levels have deleterious effects on numerous cellular processes. Although their biosynthesis has been extensively studied, relatively little is known about the mechanisms controlling the intracellular transport and distribution of sterols. Recent work indicates sterols move between intracellular membranes by non-vesicular mechanisms.;To identify components of the non-vesicular transport pathway, we used a tritium suicide selection to isolate yeast mutants defective in sterol transport. The selection is based on the transport-dependent incorporation of [ 3H]cholesterol and utilized the Upc2-1 strain of yeast, which is capable of incorporating exogenous sterols under aerobic conditions. Incorporated [3H]cholesterol is transported from the PM to the endoplasmic reticulum (ER) where it is esterified and stored in lipid droplets. When stored at -80°C, cells that efficiently incorporated the exogenous sterol suffered radiation-induced DNA damage and died. Using this selection we identified a number of genes that, when disrupted, significantly reduced the accumulation of exogenous sterols and allowed the cells to survive. One mutant, disrupted in DET1, was defective in sterol transport between the ER and the PM. Lipid analyses of det1Delta showed that this defect was not due to altered sterol levels or localization, suggesting that Det1 plays a critical role in intracellular sterol transport.;We also examined the transbilayer distribution of sterol in live yeast. The hem1Delta strain is auxotrophic for sterols and consequently requires exogenous sterols for growth. hem1Delta efficiently incorporated the fluorescent sterol dehydroergosterol (DHE). DHE functionally replaced ergosterol and assumed a similar intracellular distribution with ∼70% present at the PM. Membrane impermeant quenchers added extracellularly reduced DHE fluorescence by ∼20%. When the quencher was able to access internal membranes, DHE fluorescence was reduced by ∼75%. Disruption of cellular ATP levels or the PM electrochemical gradient across had no additional effect on fluorescence reduction. Together these data indicate that the majority of PM sterol is localized to the inner leaflet and is maintained there by a process independent of energy and the PM electrochemical gradient.