In Saccharomyces cerevisiae, sterols are synthesized in the endoplasmic reticulum (ER) but are most abundant in the plasma membrane (PM), and the nonvesicular routes that move sterol between these membranes are only partially identified. The founding observation (Raychaudhuri, Im, Hurley and Prinz, 2006) was that movement of exogenous sterol from the PM to the ER requires the seven yeast oxysterol-binding protein–related (Osh) proteins and is stimulated by phosphoinositides (PIPs), and that Osh4p transfers sterol between membranes in vitro. Later work transformed the mechanistic picture: Osh proteins are phosphatidylinositol 4-phosphate (PI4P)-coupled lipid exchangers, Osh6/7p transport phosphatidylserine rather than sterol in vivo, and a second family (Lam/Ltc) and bridge-like proteins (BLTP2) were implicated in ER-PM sterol traffic, while another study found sterol movement between ER and PM to be Osh-independent. What remains unresolved is the quantitative in-vivo assignment: which protein families carry what fraction of the physiological PM-to-ER sterol flux and in which direction, how the PM ABC transporters Aus1/Pdr11 cooperate mechanistically with lipid-transfer proteins to mobilize PM sterol, and how these routes jointly shape organelle lipid composition.

Background

Sterols (ergosterol in fungi, cholesterol in mammals) are synthesized at the endoplasmic reticulum (ER) yet accumulate most strongly in the plasma membrane (PM); maintaining this gradient requires transport that is largely independent of vesicular traffic, so-called nonvesicular sterol transport. Nonvesicular lipid movement is believed to occur at membrane contact sites, regions where the ER comes within tens of nanometers of other organelles or the PM, and to be mediated by lipid transfer proteins (LTPs), soluble or tethered proteins that extract a lipid molecule from one bilayer and deliver it to the other. The yeast LTP family at the center of this problem is the Osh family (oxysterol-binding protein homologues; mammalian relatives are called ORPs, OSBP-related proteins). Yeast has seven Osh isoforms (Osh1-7p); each contains an OSBP-related domain (ORD), a hydrophobic pocket that binds one sterol or one phosphoinositide headgroup, and some isoforms carry targeting modules such as PH (pleckstrin homology) domains that bind specific phosphoinositides. Phosphoinositides are phosphorylated derivatives of phosphatidylinositol; the monophosphate PI4P (phosphatidylinositol 4-phosphate) is enriched at the trans-Golgi and the PM, where it is made by PI 4-kinases, and is dephosphorylated in the ER by the Sac1 phosphatase. In 2006, Raychaudhuri, Im, Hurley and Prinz showed that (i) movement of exogenous cholesterol from the PM to the ER in yeast slows dramatically when all seven Osh proteins are absent, (ii) purified Osh4p transfers sterol between liposomes in vitro - the first demonstration that any ORP is a lipid transfer protein - and (iii) PIPs stimulate sterol transfer by Osh4p at low concentration but inhibit it at high concentration. Together with the earlier finding of Li and Prinz (2004) that two PM ATP-binding cassette (ABC) transporters, Aus1p and Pdr11p, are required for nonvesicular, raft-modulated sterol movement from PM to ER, and the Osh4p sterol-pocket structure of Im et al. (2005), this established Osh proteins as PIP-regulated candidate sterol transporters, and the paper's Discussion posed the open questions that this problem addresses. Subsequent literature resolved much of the mechanism: de Saint-Jean et al. (2011) solved the Osh4p-PI4P complex structure and showed that Osh4p is a counter-exchanger that swaps sterol for PI4P between membranes, which explains PIP stimulation and inhibition as competition and exchange; Moser von Filseck et al. (2015, Nature Communications) quantified how a transmembrane PI4P gradient powers sterol gradient formation; Fuggetta et al. (2024) reconstituted the full Osh4p exchange cycle coupled to PI4P synthesis and Sac1p hydrolysis with a validated kinetic model. The in-vivo cargo question was also largely settled for one branch: Moser von Filseck et al. (2015, Science) showed that Osh6p/Osh7p transport phosphatidylserine (PS) to the PM in exchange for PI4P. However, the physiological sterol-flux question was reopened rather than closed: Georgiev et al. (2011), using dehydroergosterol (DHE, a fluorescent ergosterol analogue) imaging and pulse-chase of radiolabeled ergosterol, found that sterol movement between ER and PM proceeds robustly in cells lacking all Osh function, and concluded that Osh proteins regulate PM sterol organization rather than carrying the flux - an unreconciled contradiction with the 2006 assay. Meanwhile a second family of sterol-specific LTPs, the Lam/Ltc (Lipid transfer at contact site) proteins with StARkin domains (compact lipid-binding folds related to the StART domain of mammalian StAR-related sterol-transfer proteins, each cradling a single sterol molecule), was shown to operate at ER-PM and other contact sites and to affect ER-PM sterol transport (Gatta et al. 2015; structural basis by Tong et al. 2018); sterol biosensors of the D4H type (a genetically encoded domain that binds accessible cholesterol/ergosterol) have begun to visualize sterol flow in living cells (Marek et al. 2020); and bridge-like lipid transfer proteins (BLTPs, long hydrophobic-channel proteins related to VPS13 and ATG2 that are proposed to shuttle bulk lipid between apposed membranes) were recently targeted to ER-PM contacts (BLTP2/Fmp27-Hob1/2 with the Hoi1 adaptor, Dziurdzik et al. 2026). A 2025 review summarizes the field's current, explicitly tentative model - Osh proteins driving sterol from ER toward the PM, Lam proteins passively returning it - while stating that the mechanisms, and the reason Osh inactivation slows intramembrane sterol movement without lowering total PM sterol, remain unclear. The mammalian counterpart of PM-to-ER sterol transport was resolved by a different family (the Aster/GRAMD1 proteins, Sandhu et al. 2018), but budding yeast has no clear Aster ortholog, so the identity of the physiologically dominant yeast sterol route is still open. Whether and how the ABC transporters Aus1p/Pdr11p mobilize PM sterol for nonvesicular ER-directed transport - by translocating sterol across or within the bilayer, or by altering PM lipid organization - remains unknown; genetics has only shown substrate selectivity (Papay et al. 2020), and no structure or reconstitution exists.

Problem Statement

Determine, quantitatively and mechanistically, how nonvesicular sterol transport between the plasma membrane (PM) and the endoplasmic reticulum (ER) is executed in Saccharomyces cerevisiae in vivo. Specifically: (1) assign the physiological sterol flux between the PM and the ER to specific protein families - the seven Osh/ORP (oxysterol-binding-protein-related) isoforms, the Lam/Ltc proteins (sterol-specific lipid-transfer proteins at membrane contact sites whose StARkin domains, lipid-binding folds related to the StART domain, each cradle one sterol molecule), and the BLTP2-family bridge-like lipid transfer proteins (large hydrophobic-channel proteins proposed to shuttle bulk lipid between apposed membranes) - stating which proteins carry what fraction of the flux, in which direction (PM to ER, ER to PM), and under which growth conditions (the PM ABC transporters Aus1p/Pdr11p are not themselves assigned a sterol-flux fraction in (1), and their mobilizing role is treated in part (3)); (2) reconcile the conflicting assay outcomes in the literature, in which Osh proteins were reported both required (cholesterol pulse-chase in cells carrying upc2-1, a gain-of-function allele of the transcription factor Upc2p that permits aerobic uptake of exogenous sterol) and dispensable (dehydroergosterol and radiolabeled-ergosterol assays) for PM-ER sterol movement, and establish whether Osh proteins directly transport sterol in vivo or organize PM sterol so that other proteins carry the flux; (3) identify the molecular mechanism by which the plasma membrane ABC (ATP-binding cassette) transporters Aus1p and Pdr11p mobilize PM sterol for ER-directed nonvesicular transport - direct sterol translocation within or across the bilayer versus indirect effects on PM lipid organization - and whether and how they functionally cooperate with lipid-transfer proteins such as Osh or Lam proteins; and (4) quantify how altering these routes changes organelle-specific lipid composition and downstream physiology (for example phosphatidylinositol 4-phosphate (PI4P) and phosphatidylserine pools, autophagy, and stress signaling) in defined genetic backgrounds. The PIP-counter-exchange mechanism of Osh4p and the phosphatidylserine/PI4P cargo identity of Osh6p/Osh7p are already established and are not part of what must be determined; the problem is the in-vivo flux assignment, the assay-discrepancy resolution, the ABC-transporter mechanism, and the quantitative cellular consequences. The four parts are jointly posed and interrelated.

References

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