In this study, we demonstrate that yeast ORPs can interact with two membranes simultaneously and facilitate regulated sterol transfer by a novel mechanism. Using Osh4p as a representative Osh protein, we identified a membrane-binding site on Osh4p that is distal to the membrane-binding surface near the mouth of the sterol-binding pocket. Our findings suggest that lipid content of the liposome interacting with the distal membrane–binding surface affects the rate of sterol extraction from and delivery to the second liposome. We confirmed the functional importance of the distal membrane–binding surface of Osh4p by altering it and showing that the mutant protein cannot replace the wild-type protein or facilitate sterol transfer in cells and that it is insensitive to the stimulatory effect of PI(4,5)P2 on sterol transfer in vitro. Because all yeast ORDs can bind two membranes simultaneously, they likely all have similar regulatory distal membrane–binding surfaces, although they almost certainly differ in terms of the preference for PIPs and other lipids.
The ability of the ORD's distal membrane–binding surface to regulate sterol extraction and delivery to a second membrane suggests a new mechanism for sterol transfer by ORPs. We have previously proposed that ORPs transport sterols by extracting a sterol from one membrane, diffusing through the aqueous phase, and delivering the bound sterol to a second membrane (Im et al., 2005
; Raychaudhuri et al., 2006
). Although our findings do not rule out this model, we now propose that ORPs extract or deliver sterols to a membrane most efficiently when they simultaneously interact with a second membrane via the distal membrane–binding surface of the ORD, which could occur at an MCS or any place where two membranes are closely apposed. Consistent with this model, we show that Osh4p transfers sterols most efficiently when the donor and acceptor membranes can come in close contact. In addition, we found that Osh4p transfers sterols as efficiently when it is covalently attached to a liposome as when it is free in solution. This was surprising because Osh4p probably cannot transfer a sterol between two membranes without detaching from both membranes; the movement of a sterol between a membrane and the hydrophobic binding pocket of an ORP probably only occurs when the entrance to the pocket is very close to or partially buried in the membrane. Thus, sterol transfer requires that ORPs detach from membranes. This is supported by our cross-linking data, which show that Osh4p still transfers sterols efficiently while covalently attached to one set of liposomes, but only if a cross-linker with a large arm length was used, indicating that the protein must be able to pivot between membranes to move sterols between them.
The ability of the distal membrane–binding surface of ORPs to regulate sterol extraction and deliver to a second membrane suggests how sterol transfer might be driven primarily in one direction between pairs of membranes at an MCS. The distribution of various PIP species in cellular membranes is highly regulated and membrane specific (Vicinanza et al., 2008
). For example, PI(4,5)P2
is highly enriched in the PM but largely absent from the ER. Thus, an asymmetric distribution of PIPs across the two membranes of an MCS could result in net sterol transport primarily in one direction because the net result would be a differential probability of sterol extraction/delivery at each organelle. However, this is difficult to demonstrate in vitro because both the donor and acceptor liposomes in a transfer assay can associate with the distal binding surface of ORDs; thus, PIPs in either donor or acceptor liposomes could regulate transfer. Interestingly, a recent study has demonstrated PIP-induced directional transport in vitro by the mammalian ORP9L and OSBP; transport was stimulated only when PI(4)P was in the acceptor but not donor liposomes (Ngo and Ridgway, 2009
). This ORP contains a PH domain that is not found in Osh4p. PI(4)P binding by the PH domain may position the ORD of these ORPs so that sterol transport is favored in one direction only.
We also found that four of the seven Osh proteins are enriched on regions of the ER that are closely apposed to the PM, which suggests that they may be enriched at PM–ER contact sites as has been previously reported (Levine and Munro, 2001
; Loewen et al., 2003
; Wang et al., 2005a
). Other yeast and mammalian ORPs have also been found at MCSs, suggesting that this may be a common feature of this protein family (Levine and Munro, 2001
; Rocha et al., 2009
). Because the ORD of Osh4p and probably other ORPs is ~6 nm in diameter, ORDs could only directly contact both membranes at an MCS when the bilayers are very closely apposed. This raises the question of whether the PM and ER can come close enough for ORDs to interact with both membranes simultaneously. The mean distance between the PM and ER in yeast is not well established. In Jurkat cells, the mean distance between these organelles is 17 ± 10 nm (Wu et al., 2006
), which is seemingly too large to be readily bridged by an ORD. However, other findings suggest that the PM and ER are not held at a fixed distance from each other at contact sites but are dynamic and can come within at least 6 nm. Expression of putative PM–ER cross-bridging proteins, the junctophilins, in embryonic amphibian cells induced the formation of structures in which PM and ER approached within a mean distance of ~7.6 nm (Takeshima et al., 2000
). Additionally, Várnai et al. (2007)
were able to cross-link proteins in the PM and ER in live mammalian cells using cross-linkers that require the membranes to be only ~4–6 nm apart. Thus, the PM and ER can come close enough, at least transiently, for ORDs to interact with both membranes simultaneously.
What determines the localization of Osh6p and Osh7p, which contain only ORDs, is unclear. Perhaps not surprisingly, we found that the ER-localized FFAT-binding protein Scs2p is not required to enrich these proteins at PM–ER junctions (unpublished data). We also found that depleting cells of acidic phospholipids PI(4)P, PI(4,5)P2, or PS did not affect the localization of the proteins (unpublished data). The ability of ORDs to interact with two membranes simultaneously could help promote the enrichment of Osh6p and Osh7p (and perhaps other ORPs) at MCSs.
The enrichment of ORPs at MCSs may serve several functions in the cell. First, it might facilitate the bulk transfer of sterols or perhaps other lipids between organelles. Although the transfer rate of Osh proteins in vitro suggests that they may not transfer sterols rapidly enough to significantly contribute to bulk sterol transfer, our findings indicate that they probably transfer sterols much more efficiently at MCSs. Second, it is also possible that the primary function of some ORPs is not bulk lipid transfer between organelles but rather a fine tuning of the sterol concentration of organelles or subdomains of organelles such as MCSs. Thus, they might transiently alter the sterol content of one organelle in response to a signal, such as a change in PIP levels, in the second organelle. Third, ORPs could also function as lipid sensors at MCSs rather than lipid transporters. Because ORPs can interact with two membranes simultaneously, they could sense not just a single lipid but respond to differences in the lipid composition of two organelles. Such a form of coincidence detection could be useful at MCSs, which may be highly specialized structures that are defined by their lipid composition as much as their protein components. ORPs could also more directly function in a signaling pathway by transmitting a signal directly between two organelles. For example, when positioned at MCSs, they might add or remove a signaling lipid from one membrane in response to binding PIPs in a second membrane.
In summary, we demonstrate a novel mechanism of interorganellar communication and lipid exchange between closely apposed membranes. The core lipid-binding ORD domain of ORPs can sense the lipid composition of one membrane and simultaneously modify the sterol content of a second membrane. Identifying other proteins that work in concert with ORPs will lead to a better understanding of how they transfer lipids and signals between cellular compartments or subdomains of organelles.