A detour for yeast oxysterol binding proteins

Abstract

Oxysterol binding protein-related proteins, including the yeast proteins encoded by the OSH gene family (OSH1-OSH7), are implicated in the non-vesicular transfer of sterols between intracellular membranes and the plasma membrane. In light of recent studies, we revisited the proposal that Osh proteins are sterol transfer proteins and present new models consistent with known Osh protein functions. These models focus on the role of Osh proteins as sterol-dependent regulators of phosphoinositide and sphingolipid pathways. In contrast to their posited role as non-vesicular sterol transfer proteins, we propose that Osh proteins coordinate lipid signaling and membrane reorganization with the assembly of tethering complexes to promote molecular exchanges at membrane contact sites.

FIGURE 1.

Non-vesicular sterol transport. Sterols are largely insoluble in water (<100 nm) (69), and their non-vesicular transport between the ER and PM is proposed to require STPs that either mediate sterol transfer while attached to membranes at MCSs (A) or carry sterols through the cytoplasm (B). Although sterols represent ∼30–40 mol % of all lipids at the PM (70) but only ∼5% of ER lipids (71), the two membranes are at equilibrium with respect to sterol content, i.e. the chemical activity (a) of sterols is similar in both membranes. Chemical activity coefficients (γ) for sterols (where a = γ·c, and c = sterol concentration) in the PM are lower than in the ER due to interactions between sterols and lipids with saturated acyl chains, which are more abundant in the PM (72). The probability that a STP abstracts a sterol from a membrane is proportional to γ. Each membrane is shown as having two pools of sterols characterized by high (open sterols) and low (filled sterols) γ values. The majority of sterol in the PM has low γ. This figure was adapted from Ref. . The ergosterol transport rate needed for cell growth is calculated as follows (55). A yeast cell has 10⁸ ergosterol molecules, of which ∼60% are in the PM. A pulse of [³H]ergosterol in the ER equilibrates with the entire PM ergosterol pool with t½ < 4 min (29). This value corresponds to an exchange rate constant (k) > 0.003 s⁻¹ and a rate of ergosterol transport into and out of the PM of >6.5 × 10⁸ molecules/cell/h. There are 40,990 Osh proteins/cell (33), and if all ergosterol transport is due to Osh proteins, then the transport rate is >16,000 ergosterol molecules transferred per Osh protein/h. The ergosterol transport rate needed for one yeast cell doubling is the equivalent of one new PM (6 × 10⁷ ergosterol molecules) per 90 min, which is ∼10⁴ s⁻¹.

FIGURE 2.

Yeast Osh protein domains and structure of Osh4p. Upper, domains of the canonical mammalian OSBP compared with all seven yeast Osh proteins. ORPs are defined by the ORD (highlighted in yellow), within which is the SEQVSHHPP signature motif found in all ORPs. The ORP superfamily can be divided into short and long subgroups. The latter contains protein-binding domains including a FFAT motif, ankyrin repeats (ANK), or a Golgi dynamics (GOLD) domain. Long Osh proteins also contain a PH domain that binds PIPs. In Osh4p (green), a conserved region (orange) corresponds to the surface region that associates with anionic lipids, such as PI(4,5)P₂. The corresponding sterol-bound (red) and PI4P-bound (purple) structures of Osh4p show the relative positions of these domains on the folded protein (lower).

FIGURE 3.

Roles of Osh proteins in membrane trafficking. A, at PM/ER MCSs, Osh3p is proposed to activate Sac1p-dependent dephosphorylation of PI4P (purple) to coordinate PIP signaling in both membranes (41). B, in the Golgi, Sec14p-PC and Sec14p-PI act on separate pathways to increase diacylglycerol (DAG) levels for secretory vesicle biogenesis (54). Opposing this positive regulatory control of vesicle biogenesis, Osh4p stimulates PI4P dephosphorylation by Sac1p, and the resulting PI is consumed in the production of complex sphingolipids (inositol phosphoceramide/mannosylinositol phosphoceramide (IPC/MIPC) and mannosyldiinositol phosphoceramide (M(IP)₂C)) at the expense of diacylglycerol synthesis (see Footnote 6). Sterols (red) are enriched with complex sphingolipids in membrane domains that are sorted into nascent vesicles (56). The binding and inactivation of Osh4p by sterols enriched in sterol/sphingolipid membrane domains might lead to Sac1p feedback inhibition. C, as newly formed exocytic vesicles move to the PM, PI4P levels in their membranes decrease, triggering an exchange of Rab GTPases that bind Sec2p wherein Ypt32p is swapped for Sec4p (58). As the GEF for Sec4p, Sec2p activates Sec4p, and vesicle-bound exocyst complex subunits are recruited for later docking with their counterparts on the PM. To reduce PI4P levels on vesicles, Osh proteins might sequester PI4P or activate a PI4P phosphatase for PI4P turnover and Ypt32p/Sec4p exchange.