We recently showed that transport of ergosterol from the ER (endoplasmic reticulum) to the sterol-enriched PM (plasma membrane) in yeast occurs by a non-vesicular (Sec18p-independent) mechanism that results in the equilibration of sterol pools in the two organelles [Baumann, Sullivan, Ohvo-Rekilä, Simonot, Pottekat, Klaassen, Beh and Menon (2005) Biochemistry 44, 5816–5826]. To explore how this occurs, we tested the role of proteins that might act as sterol transporters. We chose to study oxysterol-binding protein homologues (Osh proteins), a family of seven proteins in yeast, all of which contain a putative sterol-binding pocket. Recent structural analyses of one of the Osh proteins [Im, Raychaudhuri, Prinz and Hurley (2005) Nature (London) 437, 154–158] suggested a possible transport cycle in which Osh proteins could act to equilibrate ER and PM pools of sterol. Our results indicate that the transport of newly synthesized ergosterol from the ER to the PM in an OSH deletion mutant lacking all seven Osh proteins is slowed only 5-fold relative to the isogenic wild-type strain. Our results suggest that the Osh proteins are not sterol transporters themselves, but affect sterol transport in vivo indirectly by affecting the ability of the PM to sequester sterols.
- endoplasmic reticulum
- Osh protein
- sterol trafficking
Lipids are trafficked between intracellular compartments via secretory vesicles that have been extensively characterized in studies of protein transport, as well as by non-vesicular processes that are less well understood. Many – but not all – lipids can be found in the lumenal as well as the cytoplasmic leaflets of bilayers enclosing intracellular organelles; this is so because of membrane transporters (flippases) that selectively promote transbilayer lipid flip-flop by ATP-independent and ATP-dependent mechanisms, and because of the natural propensity of certain lipids such as ceramide and sterols to flip-flop rapidly across bilayers without protein mediation. In contrast with vesicle-mediated lipid transport pathways that are topologically unselective, i.e. lipids can be transported regardless of whether they are located in the luminal or cytoplasmic leaflet of the vesicle-producing (donor) organelle, non-vesicular transport operates only for lipids that have access to the cytoplasmic leaflet of intracellular membranes. Such lipids can be exchanged between compartments by lipid transport proteins such as the recently identified ceramide transporter CERT, or possibly at subcellular sites where membranes are closely apposed. An example of the latter includes contact sites between the ER (endoplasmic reticulum) and mitochondria needed for phospholipid transport between these two organelles. These topics have been reviewed recently [1–5].
We are interested in the trafficking of sterols between the ER and the PM (plasma membrane). Cellular levels of sterol are sensed and controlled with a great degree of precision, mainly through the action of sterol-sensitive proteins localized to the ER. Although sterol biosynthesis occurs mainly in the ER, concentration of sterols in the ER membrane is low. In contrast, sterols are highly enriched in the PM where they have a low chemical activity as a result of their interaction with both sphingolipids and glycerophospholipids with saturated acyl chains . The low chemical activity of the PM sterols is reflected in their inability to be solubilized by cold non-ionic detergents in the form of DIMs (detergent-insoluble membranes) .
The mechanism by which sterols are transported from the ER to the PM is unknown. Experiments with mammalian cells indicate that delivery of newly synthesized cholesterol to the PM is ATP-dependent and Brefeldin A-insensitive, suggestive of a novel vesicular mechanism or a non-vesicular pathway [8,9]. To explore the molecular basis of biosynthetic sterol transport, we recently developed methods to study the transport of ergosterol from the ER to the PM in the budding yeast Saccharomyces cerevisiae . We metabolically labelled cells with [3H]methionine to generate a pulse of radiolabelled ergosterol in the ER, then tracked [3H]ergosterol transport to the PM by removing cell aliquots at various chase times and incubating them with MβCD (methyl-β-cyclodextrin) on ice. Using reversed-phase HPLC, we determined the specific radioactivity of ergosterol in both the MβCD-extracted material and the cell sample as a whole. Our results indicate that transport of a pulse of metabolically radiolabelled ergosterol from the ER to the outer leaflet of the PM occurs via an equilibration mechanism with a half-time of approx. 10 min. This corresponds to the trafficking of approx. 105 ergosterol molecules into and out of the PM per second (see Appendix). Our results also indicate that transport of newly synthesized ergosterol to the PM is ATP-dependent and proceeds unabated in the absence of the classical secretory pathway. Sec18p [or NSF (N-ethylmaleimide-sensitive fusion protein)], an ATPase involved in vesicle fusion, is required at multiple stages of classical vesicle-mediated transport . In a temperature-sensitive sec18-1 yeast mutant, secretory protein transport is shut down at the non-permissive temperature, as is the ER–PM transport of inositol phosphosphingolipids. However, under the same conditions, ergosterol transport from the ER to the PM is indistinguishable from that seen in the sec18-1 isogenic wild-type strain. Our results differ from those of Schnabl et al.  who reported that sec blocks caused only partial reduction of protein and lipid transport to the PM in yeast. This indicates that ergosterol transport between the ER and PM occurs via a non-vesicular (Sec18p-independent) transport mechanism [10,13,14]; a Sec18p-independent mechanism has also been described for the transport of exogenously supplied sterol from the PM to the ER . Although we infer a non-vesicular transport mechanism based on the non-requirement for Sec18p in ergosterol transport, we note a recent report describing delivery of a secretory protein from the ER to the PM in yeast via a Sec18p-independent mechanism . Thus it remains a formal possibility that ergosterol is transported between the ER and PM by a vesicular pathway that operates independently of Sec18p.
Our results are consistent with the proposal that two pools of sterol co-exist at the PM. One pool – corresponding to most of the sterol in the PM – is held at low chemical activity as a result of sterol–phospholipid interactions. The other, much smaller pool, has a high chemical activity because it corresponds to sterols not partnered with PM phospholipids. We propose that this free (high-chemical-activity) sterol pool in the PM is in equilibrium with ER sterol, and that it is sterol molecules from the free sterol pools in the ER and PM that are transported between the two organelles. Experiments in which the cellular sphingolipid content was reduced resulted in an increase in the free sterol pool, which was reflected in (i) a decrease in PM sterol levels, (ii) an increase in intracellular sterol levels, (iii) a reduction in the fraction of sterol molecules recovered in DIMs, and (iv) a decrease in the rate at which a pulse of newly synthesized ergosterol equilibrates with the PM pool. The latter result indicates that the transport machinery is saturated in wild-type cells .
Sterol transport mechanisms?
To understand how ergosterol is physically transported between the ER and PM, we adopted two approaches. In the first approach, we used a tritium suicide selection strategy to identify yeast mutants defective in ergosterol transport (det mutants). This unbiased approach is distinct from that taken by Reiner et al.  who screened the yeast deletion mutant collection for genes required for anaerobic growth; some of the genes thus identified would be predicted to affect sterol uptake and/or intracellular trafficking. With our approach, we reasoned that we would recover some mutants where sterol transport was directly affected, as well as other transport mutants defective for sphingolipids or other factors that affect the chemical activity of sterol pools. We have currently identified three complementation groups of det mutants. At least one of these complementation groups of mutants has a defect in the ergosterol transport machinery. Work is under way to identify the putative transport protein or factor that is defective in this class of mutants.
In a second ‘reverse genetics’ approach, we tested the role of candidate transport proteins in ER–PM sterol transport. It occurred to us that proteins analogous to the recently discovered CERT protein  are likely to be sterol transporters – such transporters would perhaps have a lipid-binding domain, as well as sequence motifs that would allow them to engage the ER and the PM simultaneously, or alternately. Candidate transporters that fit this description include the yeast Osh protein family (homologues of the mammalian oxysterol-binding proteins) for testing. S. cerevisiae has seven Osh proteins. Although no single Osh gene is essential and any one can substitute for the other six, elimination of all seven Osh genes is lethal, suggesting that the Osh proteins have a common essential function [19,20]. We hypothesized that this common essential function might involve sterol transport between the ER and PM. Recent X-ray crystallographic analyses of Osh4p (also known as Kes1p) suggest that Osh proteins, like other lipid transfer proteins, have a deep lipid-binding pocket covered with a regulatory flap that opens when the protein is membrane-associated . This structural model is consistent with the possibility that Osh proteins engage in transport cycles in which they pick up sterol from one membrane compartment, sequester it within the protein, and then offload it at another membrane. We examined the transport of newly synthesized ergosterol from the ER to the PM in a yeast strain bearing only a single OSH gene in the form of the osh4-1 temperature-sensitive allele. On shifting the growth temperature to 37 °C, Osh protein function is rapidly lost in the oshΔ,osh4-1 strain. However, ER–PM ergosterol transport is reduced only 5-fold compared with the isogenic wild-type strain. The inability to eliminate sterol transport in the oshΔ cells suggests that if Osh proteins are sterol transporters, then other transporters or transport mechanisms must operate as well. It is also possible that Osh proteins affect sterol transport only indirectly by affecting the size of the free (high-chemical-activity) sterol pool. Indeed, examination of the properties of the oshΔ,osh4-1 strain indicate an increase in the pool of free sterol, as reflected in lower recovery of sterols in DIMs and higher concentrations of intracellular sterol, as detected by filipin staining. These results suggest that elimination of Osh proteins has an impact on the ability of the PM to sequester sterols but does not directly affect the transport of sterols between the two compartments. Work is under way to confirm these findings.
We anticipate that the combination of the two approaches outlined above – the search for transport mutants and analyses of candidate transporters – will yield fresh insights concerning the mechanisms by which sterols are transported between intracellular compartments. Results obtained in these studies in yeast will probably be of relevance to investigations of sterol trafficking in mammalian cells where defects in sterol biosynthesis, transport and/or storage lead to a number of different diseases.
Flux of sterol into and out of the PM
The rate of sterol transport between the ER and PM in yeast can be estimated as follows [for the purposes of this calculation, we assume that only these two compartments – the biosynthetic compartment (ER) and the major sterol repository (PM) – are involved in sterol transport].
(1) The ergosterol content of yeast is approx. 4.5 mg/g CDW (cell dry weight) or 1.13×10−5 mol/g CDW . Since the dry weight of a yeast cell is 1.5×10−11 g , this implies an ergosterol content of approx. 108 molecules/cell.
(2) The ergosterol content of the PM can be estimated by assuming the volume of a yeast cell to be 70 μm3 , corresponding to a sphere of radius approx. 2.6 μm and surface area approx. 8.2×107 nm2. Assuming that the transmembrane segments of membrane proteins occupy approx. 30% of the PM and that the cross-sectional area of a phospholipid is approx. 0.6 nm2 , then the number of phospholipids in both leaflets of the PM will be approx. 1.9×108. Further assuming that sterol comprises approx. 30% of these lipids , then the PM has approx. 0.6×108 sterol molecules or approx. 60% of the total sterol of the cell.
(3) Our measurements indicate that a pulse of radiolabelled ergosterol generated in the ER equilibrates with the MβCD-extractable sterol pool at the PM with a half-time of approx. 10 min, corresponding to a first-order rate constant of approx. 0.001 s−1. This implies that approx. 6×104 ergosterol molecules traffic in and out of the PM per second.
(4) To estimate the ergosterol flux required to maintain a cell doubling time of approx. 2 h during which the equivalent of a new PM is synthesized, approx. 0.6×108 ergosterol molecules must be transported per 2 h, or approx. 0.8×104 molecules/s. Thus the rate of ergosterol transport is more than sufficient to allow for membrane synthesis during cell growth. We are grateful to Fred Maxfield (Department of Biochemistry, Weill Medical College of Cornell University, New York, NY, U.S.A.) for suggesting this calculation. A related calculation for sterol transport into and out of the mammalian endocytic recycling compartment is presented in .
This work was supported by NIH (National Institutes of Health; Bethesda, MD, U.S.A.) grant GM55427 (to A.K.M.), a fellowship from the Academy of Finland (to H.O.-R.) and a Canadian NSERC (Natural Sciences and Engineering Research Council) grant (to C.T.B.).
Non-Vesicular Intracellular Traffic: Biochemical Society Focused Meeting held at Goodenough College, London, U.K., 15–16 December 2005. Organized and edited by S. Cockcroft (University College London, U.K.) and T. Levine (Institute of Ophthalmology, London, U.K.).
Abbreviations: CDW, cell dry weight; CERT, ceramide transporter; DIM, detergent-insoluble membrane; ER, endoplasmic reticulum; MβCD, methyl-β-cyclodextrin; Osh, oxysterol-binding protein homologue; PM, plasma membrane
- © 2006 The Biochemical Society