Transport of newly synthesized sterol to the sterol-enriched plasma membrane occurs via nonvesicular equilibration. Biochemistry 44:5816–5826

ABSTRACT: The mechanism by which newly synthesized sterols are transported from their site of synthesis, the endoplasmic reticulum (ER), to the sterol-enriched plasma membrane (PM) is not fully understood. Studies in mammalian cells suggest that newly synthesized cholesterol is transported to the PM in Golgi-bypassing vesicles and/or via a nonvesicular process. Using the yeast Saccharomyces cereVisiae as a model system, we now rule out an essential role for known vesicular transport pathways in transporting the major yeast sterol, ergosterol, from its site of synthesis to the PM. We use a cyclodextrin-based sterol capture assay to show that transport of newly synthesized ergosterol to the PM is unaltered in cells defective in Sec18p, a protein required for almost all intracellular vesicular trafficking events; we also show that transport is not blocked in cells that are defective in formation of transport vesicles at the ER or in vesicle fusion with the PM. Our data suggest instead that transport occurs by equilibration (t1/2 10-15 min) of ER and PM ergosterol pools via a bidirectional, nonvesicular process that is saturated in wild-type exponentially growing yeast. To reconcile an equilibration process with the high ergosterol concentration of the PM relative to ER, we note that a large fraction of PM ergosterol is found condensed with sphingolipids in membrane rafts that coexist with free sterol. We propose that the concentration of free sterol is similar in the PM and ER and that only free (nonraft) sterol molecules have access to a nonvesicula