Osh proteins regulate membrane sterol organization but are not required for sterol movement between the ER and PM

Sterols, such as cholesterol in mammalian cells and ergosterol in fungi (Fig. 1A), are important components of biological membranes (1). They are highly enriched in the plasma membrane (PM)1(2–5). For example, the PM of the budding yeast Saccharomyces cerevisiae contains ~108 ergosterol molecules, corresponding to ~40 mole percent of total PM lipids and ~70% of total cellular ergosterol (6–8). Ergosterol is synthesized in the endoplasmic reticulum (ER) and transported to the PM primarily by an ATP-requiring, non-vesicular mechanism (9, 10) (Fig. 1B). Retrograde transport also occurs, since exogenously supplied sterols can move from the PM to the ER where they are esterified by sterol acyl transferase enzymes Are1 and Are2 and encapsulated in lipid droplets (LDs)(11, 12) (Fig. 1B). Figure 1 Intracellular sterol transport and Osh proteins The non-vesicular transport of sterols between the ER and PM is poorly understood (13). Because sterols are largely insoluble in water, they must be shielded from the aqueous environment for transport to occur efficiently. In analogy with other lipid transport processes, this is likely to require a cytoplasmic lipid transport protein similar to the ceramide transport protein (CERT)(14) and the steroidogenic acute regulatory protein (StAR)(15, 16). The former catalyzes ATP-dependent, non-vesicular transport of ceramide from the ER to the Golgi, whereas the latter is involved in transporting cholesterol to the inner mitochondrial membrane. These proteins are presumed to extract lipids from a donor membrane, shield them during transport through the aqueous milieu of the cytoplasm and offload them when encountering an acceptor membrane. Transport is bi-directional (Fig. 1B) and so there is no net flux unless the lipid is consumed at the acceptor membrane (4). Sterol transport proteins (STPs) required for ER-PM sterol transport have not been identified. Budding yeast lack CERT and StAR homologues, but they possess seven homologues (Osh1–7) of the mammalian oxysterol-binding protein [OSBP] that have been proposed to function as STPs (17–19) (Fig. 1C). Although no single Osh protein is essential, and any one can substitute for the other six, disruption of all seven OSH genes is lethal (20). This suggests that Osh proteins have a common essential function. X-ray crystallographic analyses of Osh4 (also known as Kes1) show that it has a deep lipid-binding pocket covered with a regulatory flap that could open when the protein is membrane associated (19). The lipid-binding pocket accommodates a single sterol molecule, with its hydroxyl group pointing toward the bottom of the pocket(19). Based on this structural model it was suggested that Osh4 could extract sterol from one membrane, sequester it in its lipid-binding pocket, and offload it at another membrane (19). Consistent with this idea, Osh4, Osh5 and Osh2-ORD (the OSBP-related domain of Osh2) were shown to transport sterols between populations of liposomes (18, 21), and the retrograde transport of cholesterol was shown to be 7-fold slower in Osh-deficient yeast cells (18). Thus, the common essential function of Osh proteins could be to transport sterols between the ER and PM. To test this hypothesis, we compared sterol trafficking in wild-type cells and cells lacking functional Osh proteins. We introduced dehydroergosterol (DHE), a naturally occurring fluorescent sterol (22, 23), into the PM and observed its redistribution to LDs by fluorescence microscopy. Redistribution of DHE to LDs was correlated with the production of DHE esters. In the second approach we generated a pulse of radiolabeled ergosterol in the ER and monitored its arrival at the PM by measuring radioactivity in PM-enriched subcellular fractions and detergent resistant membranes (DRMs), as well as in the sterol fraction extracted from intact cells by methyl-β-cyclodextrin (MβCD). Our results indicate that (i) Osh proteins are dispensable for sterol movement between the PM, ER and LDs, and (ii) Osh protein family members regulate the organization of sterols at the PM.