© The Authors Journal compilation © 2015 Biochemical Society Essays Biochem. (2015) 57, 43–55: doi: 10.1042/BSE0570043
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Cholesterol trafficking and distribution David B. Iaea* † and Frederick R. Maxfield*1 *Department of Biochemistry, Weill Cornell Medical College, 1300 York Ave, New York, NY 10065, U.S.A. †Weill Cornell Medical College, Rockefeller University, and Memorial Sloan-Kettering Cancer Center Tri-Institutional Chemical Biology Program, New York, NY 10065, U.S.A.
Abstract Sterols are a critical component of cell membranes of eukaryotes. In mammalian cells there is approximately a six-fold range in the cholesterol content in various organelles. The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the physiochemical properties of membranes. Cholesterol trafficking among organelles is highly dynamic and is mediated by both vesicular and nonvesicular processes. Several proteins have been proposed to mediate inter- organelle trafficking of cholesterol. However, several aspects of the mechanisms involved in regulating trafficking and distribution of cholesterol remain to be elucidated. In the present chapter, we discuss the cellular mechanisms involved in cholesterol distribution and the trafficking processes involved in maintaining sterol homoeostasis.
Keywords: cholesterol, dehydroergosterol, endocytic recycling compartment, homoeostasis, non-vesicular transport, StARD4, sterol regulatory element-binding protein, trafficking.
Introduction Eukaryotic cells maintain a high rate of vesicular trafficking among the secretory and endocytic organelles and the plasma membrane. For instance, in cultured fibroblasts it has been To whom correspondence should be addressed (email [email protected]).
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estimated that lipids in the plasma membrane are internalized with t½ of 15–30 min [1]. Following endocytosis, the membrane components can be delivered to a variety of organelles such as the ERC (endocytic recycling compartment), the late endosome and lysosomes, or trafficked back to the cell surface [1,2]. In some cases, the endocytosed materials are transported to the ER (endoplasmic reticulum) [2]. Because of the rapid rate of vesicular transport among organelles, it is often challenging to characterize the means by which a cell sorts lipids and sterols. Understanding the intracellular dynamics of cholesterol is important because the proper distribution among cellular organelles is critical for cellular function [3–5]. Among the many lipids in eukaryotic cells, sterols have a unique chemical structure that allows them to be absorbed and extracted from membranes with relative ease. Cholesterol contains a hydroxy group as the sole polar component, four rings and a short alkyl chain [6]. The structure of cholesterol contrasts with most glycerophospholipids and sphingolipids, which are composed of large polar head groups and two long hydrocarbon chains. The structure of cholesterol and its biophysical properties have a substantial effect on the physiochemical characteristics of membranes, as it is able to pack against the fatty acyl chains of phospholipids, reduce membrane permeability, and facilitate the formation of Lo (liquid ordered) domains in membranes [3–5]. Cells have evolved complex mechanisms to regulate the abundance and distribution of cholesterol within membranes [7-9]. In the present chapter, we discuss how the distribution of cholesterol is regulated and the mechanisms by which cholesterol moves among membranes in a eukaryotic cell.
Models of sterol–phospholipid interactions The umbrella model [3] and the condensed complex model [10] are two of the descriptions used to analyse the interaction of sterols with phospholipids. The umbrella model is built upon structural mismatch of cholesterol with other lipids in the membrane bilayer: the single hydroxy group facing into aqueous solution only partially protects the hydrophobic core from water [3]. As a consequence, sterols preferentially associate with phospholipids with larger polar head groups, such as PC (phosphatidylcholine) and sphingomyelin, rather than phospholipids with smaller polar head groups, such as PE (phosphatidylethanolamine) [11]. As a result of differences in head group size, PC can provide coverage to two cholesterol molecules, but PE can provide coverage for only a single molecule [3]. In addition to the size of the polar head group, lipid acyl chain saturation is important in dictating the geometry of the bilayer [12]. As depicted in Figure 1, increased unsaturation leads to a conical shape because of the large cross-sectional area of the acyl chains in comparison with the head group. By contrast, saturated lipids tend to be more cylindrical. The ratio of head group:body size is a good indicator of sterol stability in membranes [12]. For instance, the unsaturated DOPC (dioleoylphosphatidylcholine) is not able to shield neighbouring sterols from the aqueous phase as well as the saturated DPPC (dipalmitoylphosphatidylcholine). In the condensed complex model, the effects of transient stoichiometric associations between sterols and phospholipids are taken into account [10]. Lipids with long saturated acyl chains, such as sphingomyelin and DPPC, associate with sterols in a reversible manner to form © The Authors Journal compilation © 2015 Biochemical Society
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Figure 1. Interactions between cholesterol and surrounding lipids Cholesterol stability in membranes depends on the size of the polar head group and acyl chain saturation from neighbouring lipids. (A) Lipids with large polar head groups, which provide increased protection from the aqueous environment, are the preferred neighbours for cholesterol. (B) Acyl chain saturation influences the stability of cholesterol. Lipids with unsaturated acyl chains, containing one or more double bonds, form a conical structure in the membrane. In comparison, lipids with saturated acyl chains form a cylindrical structure. Unsaturated acyl chains provide less stability and protection to cholesterol from the aqueous surroundings. (C) Cholesterol has a weak stability in membranes enriched in unsaturated phospholipids, such as DOPC-containing membranes. As a result, cholesterol will partition to a membrane environment where it can associate with the saturated acyl chains of phospholipids. DAG, diacylglycerol; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; SM, sphingomyelin. Adapted from [21].
complexes with a smaller molecular lateral area than when the sterols and lipids are apart [13,14]. Lipids that contain unsaturated acyl chains have a reduced tendency to form these sterol–lipid complexes. The tight packing of sterol and phospholipids in these condensed membrane domains leads to ordering of the lipid acyl chains and membrane thickening [15]. As a result of sterol–lipid condensation, the stability of sterol in a membrane depends on the composition of the bilayer. Despite their differences, the two models are not mutually exclusive. Both the umbrella and condensed complex describe the interaction between cholesterol, the saturated acyl chains and polar head group of phospholipids, and in both of the models the stability of cholesterol in the bilayer decreases as the amount of sterol exceeds the capacity of the phospholipids [3,10]. In cells the phospholipid composition of organelles varies greatly. If cholesterol can equilibrate among organelles, then the differences in lipid composition would be expected to lead to differences in their sterol content [6,16]. © 2015 Biochemical Society
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Distribution of sterols in mammalian cells As shown in Figure 2, significant differences in cholesterol distribution are maintained among cellular organelles. In the plasma membrane of mammalian cells, cholesterol is ~35 mol% of the lipids [17]. By contrast, the ER contains only 5 mol% cholesterol [18]. The cholesterol:phospholipid ratio in the Golgi is intermediate between the plasma membrane and the ER. From electron microscopy of filipin-labelled cells [19], it has been suggested that the concentration of cholesterol increases from the cis-Golgi to the trans-Golgi. In the endocytic pathway, cholesterol is highly enriched in the ERC [20]. In part, this unequal distribution of cholesterol among cellular membrane compartments can be attributed to the differences in the cholesterol–phospholipid interactions resulting from the differences in the organelle lipid composition [21,22]. The plasma membrane is relatively enriched in lipids that can stabilize cholesterol in the bilayer, whereas the ER is enriched in unsaturated lipids that provide weak stabilization of cholesterol. Because of these differences in cholesterol stabilization, these membranes can have unequal cholesterol concentration even if sterol transporters bring them close to chemical equilibrium with each other.
Figure 2. Heterogeneous distribution of cellular cholesterol The cholesterol content of membranes varies throughout the cell. Membrane cholesterol levels are displayed as a heat map with membranes enriched in cholesterol labelled red. Membrane compartments with relatively low cholesterol levels are labelled blue. Inset: transbilayer distribution of cholesterol at the plasma membrane. LD, lipid droplet; LE/Ly, late endosome/ lysosome. © The Authors Journal compilation © 2015 Biochemical Society
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Within the plasma membrane there is considerable evidence for small, dynamic nanodomains that are enriched in sphingomyelin and a subset of membrane proteins, including GPI (glycosylphosphatidylinositol)-anchored proteins [23,24]. These domains can play an important role in signal transduction processes, especially when they are clustered by protein crosslinking to form larger, more stable domains [25]. The enrichment of cholesterol in more ordered lipid domains of the plasma membrane is uncertain. In model membranes, cholesterol is usually enriched in Lo domains that are in equilibrium with Ld (liquid disordered) domains, but the degree of enrichment depends on the lipid composition of the model membranes [26]. A recent high-resolution secondary ion MS study reported that cholesterol is uniformly distributed in the plasma membrane and was not enriched in micrometre-scale domains that were enriched in sphingomyelin [27]. Further studies are required to better understand the distribution of cholesterol at the scale of 10–100 nm, which is now considered to be the size of membrane lipid nanodomains [28,29]. Although models such as the condensed complex model predict that cholesterol would preferentially interact with sphingomyelin [26], which is enriched in the exofacial leaflet of the plasma membrane, these models predict favorable interactions with PC species with saturated or monounsaturated acyl chains, which are also found in the cytoplasmic leaflet of the plasma membrane. Thus, sterols can be stabilized on both leaflets of the plasma membrane. Experimental studies using fluorescent sterol analogues such as DHE (dehydroergosterol) have shown that these sterols are highly enriched in the cytoplasmic leaflet of the plasma membrane [30]. Cholesterol can flip rapidly between the leaflets in a bilayer, and it has been shown that the t½ for cholesterol flipping is less than one second in erythrocytes [31,32]. The mechanism for enriching sterols on the cytoplasmic leaflet of the plasma membrane is not understood.
Regulatory/biosynthetic mechanisms The ER is the central organelle for maintaining sterol homoeostasis. Most steps of cellular cholesterol biosynthesis occur in the ER, which contains key metabolic enzymes, including HMG (3-hydroxy-3-methyl-glutaryl)-CoA reductase [7]. The proteins that regulate synthesis of cholesterol and LDL (low-density lipoprotein) receptors are also localized in the ER [9]. When ER sterols are abundant, SREBP (sterol regulatory element-binding protein) is retained in the ER in a complex with the cholesterol-sensing protein, SCAP (SREBP-cleavage-activating protein) and the ER retention proteins Insig-1 or -2 [18]. When cholesterol content in the ER is depleted, SCAP undergoes a conformational change and is released from Insig, allowing the SREBP–SCAP complex to translocate to the Golgi apparatus [33]. In the Golgi, SREBP-2 is proteolytically processed by the site 1 and site 2 proteases to release an N-terminal transcription factor, which translocates to the nucleus to activate the expression of genes implicated in sterol biosynthesis, uptake and metabolism [34]. The amount of unesterified cholesterol in a cell is further regulated by esterification of cholesterol by the enzyme ACAT (acyl-CoA:cholesterol acyltransferase), an ER enzyme [35]. The rate-limiting step in esterification of cholesterol by ACAT is delivery of cholesterol to the ER. In order for cholesterol levels sensed in the ER to reflect the cholesterol distribution in other organelles, such as the plasma membrane and endosomes, there must be a mechanism for rapid redistribution of cholesterol to these organelles. Depending on sterol abundance, a high fraction of total cellular cholesterol can be stored as cholesteryl esters in lipid droplets. © 2015 Biochemical Society
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The cholesterol stored in the lipid droplets can be used to maintain sterol homoeostasis when cholesterol levels in the cell become low [36].
Intracellular trafficking of lipids and sterols Cholesterol can move between membranes by vesicular and non-vesicular transport mechanisms [37,38]. Like other lipids, cholesterol can be incorporated into transport vesicles that carry membrane components between organelles. However, only a small fraction of membrane components internalized from the plasma membrane reach the ER, indicating that cholesterol sensing in the ER would be very slow and inefficient if it depended on vesicle transport. There is substantial evidence for high rates of non-vesicular sterol transport in cells [39]. Since cholesterol is very poorly soluble in water, non-vesicular transport requires binding to carrier proteins. Newly synthesized sterols are transported from the ER to the plasma membrane when vesicular trafficking is inhibited by either genetic or pharmacological intervention. For example, treatment with Brefeldin A, which causes Golgi disassembly and fusion with the ER, inhibits ~90% of protein traffic but diminishes cholesterol transport to the plasma membrane by ~20% [40]. Additionally, studies using DHE, a fluorescent cholesterol analogue, demonstrate that equilibration between the plasma membrane and the ERC occurs within minutes, and this transport is not drastically altered under ATP-depleted conditions that block vesicle transport [20]. This suggests that the majority of cholesterol transport is mediated by nonvesicular, carrier-mediated transport mechanisms (Figure 3).
Sterol transfer proteins in non-vesicular transport There are several protein families that are classified as sterol transfer proteins, which have been shown to be capable of transferring sterols between membranes [41,42]. Sterol transfer proteins can interact directly with membrane compartments to extract sterol and then diffuse in complex with the sterol to acceptor membranes. Sterol transfer proteins can significantly increase the rates of sterol transfer between liposomes in vitro [43,44]. Additionally, sterol transfer proteins may also function to shuttle sterol between adjacent membranes. In some cases, such as ER–plasma membrane contact sites, the membranes of two organelles are held in close proximity by protein complexes, facilitating rapid exchange of sterol between the membranes by reducing the distance that the sterol–protein complex must travel [45]. X-ray crystallographic studies of several sterol transfer proteins demonstrate that they each contain a hydrophobic ligand pocket capable of binding a single sterol molecule [46,47]. Molecular dynamics studies suggest that in several of these proteins, the sterol binding pocket is under ‘gated’ regulation that allows it to open and close upon interaction with the lipid bilayer, which facilitates sterol absorption and delivery to specific membranes [48]. Two major gene families of lipid transfer proteins that have been implicated in such trafficking are the START [StAR (steroidogenic acute regulatory protein)-related lipid transfer] domain family and the OSBP (oxysterol-binding protein) and ORP (OSBP-related protein) family, which have been discussed in detail elsewhere [41,49,50]. © The Authors Journal compilation © 2015 Biochemical Society
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Figure 3. Intracellular trafficking itinerary of cholesterol (1,2) LDL particles containing cholesteryl esters and free cholesterol are internalized by clathrin-mediated endocytosis of the LDL receptor [2]. (3,6) The low pH in early endosomes causes the LDL to be released from its receptor, and the empty LDL receptor returns to the cell surface for further rounds of endocytosis. (4) The LDL is retained in the early sorting endosomes, which mature into late endosomes where it encounters acid hydrolases including lysosomal acid lipase (LAL), which hydrolyses the cholesteryl esters in the core of the LDL particles. (5, inset) The free cholesterol is transported out of LE/Ly, mediated by NPC1 and NPC2, and delivered to other cellular membranes including the plasma membrane and ER by primarily non-vesicular mechanisms. (6) Cholesterol in the plasma membrane can traffic to the ERC and back by both vesicular and non-vesicular mechanisms. (7) Cholesterol transport from the plasma membrane to the ER informs the homoeostatic machinery about the free cholesterol levels in the cell. NPC1, Niemann-Pick C1 protein.
Fluorescent probes for cholesterol trafficking The intracellular transport of cholesterol between organelles is a fundamental homoeostatic mechanism, but these transport processes are not well understood. Cholesterol transport can be studied by introduction of radioactive sterols or precursors into cells, but this method requires stringent purification of organelles and the assumption that cholesterol does not redistribute during the purification process [20,51]. Minor contamination from membranes enriched in cholesterol can lead to significant errors in measuring cholesterol content of organelles such as the ER. Fluorescence microscopy is a powerful tool for studying intracellular transport processes. However, this method can be difficult for examining lipid molecules because addition of a © 2015 Biochemical Society
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Figure 4. Fluorescent probes and analogues for studying cholesterol Chemical structures of cholesterol (A), dehydroergosterol (B), cholestatrienol (C), cholesterolBODIPY (D) and filipin (E). Structural differences between cholesterol (A) and its fluorescent analogues (B-D) are indicated in red. BODIPY, boron-dipyrromethene.
fluorophore may greatly alter the physical properties of the molecule [52–54]. Two approaches have been used to examine cholesterol distribution in cells using fluorescence. One method is to use a sterol-binding fluorophore, such as filipin, to localize cholesterol in cells [55]. As depicted in Figure 4, filipin is a naturally fluorescent polyene antibiotic that binds to unesterified cholesterol [56]. This makes filipin a useful tool in determining the free cholesterol content and distribution in membranes. However, filipin photobleaches rapidly, and binding to cholesterol perturbs the membrane, thus making it unsuitable for live cell imaging. As alternative approaches for examining cholesterol trafficking in live cells, the introduction of either a boradiazaindacene moiety to cholesterol or the use of fluorescent sterol analogues such as DHE or cholestatrienol have been very successful [55]. These sterol analogues have been discussed in depth elsewhere [55]. These fluorescent sterol analogues can be incorporated into cells either by delivery to the plasma membrane or by reconstitution into lipoproteins [20,57]. Sterol trafficking can then be directly imaged or used to study trafficking kinetics or transbilayer distribution [55].
Consequences of extensive non-vesicular transport As discussed in earlier sections, the plasma membrane has the largest pool of cholesterol, whereas the ER is relatively sterol poor [17,18]. Thus, it is important that transport pathways © The Authors Journal compilation © 2015 Biochemical Society
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between these organelles exist to facilitate cholesterol efflux to the plasma membrane and so that the ER can respond to alterations in sterol levels by modulating cholesterol synthesis and esterification. Sterol transport between the plasma membrane and the ER is substantial and occurs primarily by non-vesicular mechanisms [39,58]. In this perspective, the rate of sterol transport into and out of the ER serves to maintain a constant cholesterol level in the organelle, which in turn maintains sterol homoeostasis. StARD4 (START domain protein 4), a soluble sterol transporter, has been implicated in maintaining sterol homoeostasis and is transcriptionally regulated by SREBP-2 [59]. StARD4 plays an important role in delivery of cholesterol to the ER. A study using U2OS human osteosarcoma cells demonstrated that StARD4 overexpression increased cholesterol ester levels [44]. StARD4 silencing attenuated cholesterol-mediated regulation of SREBP-2 activation, whereas StARD4 overexpression amplified sterol sensing by the SCAP and SREBP-2 proteins in the ER. Silencing StARD4 increased the cellular cholesterol levels, presumably as a result of the slower delivery of cholesterol to the ER and reduction in SREBP-2 response. These data suggest that delivery of cholesterol to the ER is an important function of StARD4. Although it is clear that StARD4 delivers sterol to the ER, its expression must be finely tuned. Deregulation of StARD4 expression may result in desensitization of the ER homoeostatic machinery, which would then no longer be able to respond to variations in cellular cholesterol levels. Thus, modulation of StARD4 expression, by SREBP-2, ensures optimal sterol sensing and ER sensitivity to cholesterol abundance in organelles, including the plasma membrane. Further work is required to understand how the trafficking kinetics of sterols are integrated into maintaining sterol homoeostasis.
Conclusion Cholesterol is a highly dynamic lipid heterogeneously distributed throughout organelles and the membrane bilayer. It has a rapid lateral and transverse mobility with dramatic effects on the organization of its surrounding lipids. Additionally, there is increasing evidence that the majority of sterol trafficking and distribution is maintained by non-vesicular mechanisms. The presence of numerous sterol transport proteins demonstrates that cells can rapidly redistribute sterol throughout organelles and that cholesterol acts as a central lipid in sensing lipid homoeostasis. Although there is still work to be done in understanding the precise mechanisms of non- vesicular sterol transport, the development of fluorescent probes to analyse lipid and sterol transport and distribution makes this an exciting time in studying sterol trafficking. An important question for the next few years is to elucidate the trafficking kinetics of sterols into and out of organelles and how these transport steps are integrated into the overall cellular homoeostasis.
Summary • •
Cholesterol is a critical component of cell membranes in mammalian cells. The stability of cholesterol in the bilayer depends on its interactions with neighbouring phospholipids.
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• • • • • •
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Cholesterol is heterogeneously distributed throughout cell compartments as well as across the membrane bilayer. Cells have evolved complex pathways to maintain cholesterol homoeostasis. Trafficking and distribution of cholesterol in a cell is maintained primarily by non-vesicular mechanisms. Several protein families function as putative sterol transporters to maintain cholesterol homoeostasis. Fluorescent analogues and probes can be used to visualize cholesterol in fixed and living cells. Cholesterol homoeostasis and organelle levels are maintained by a complex network of non-vesicular trafficking processes.
Work in the Maxfield laboratory is supported by the National Institutes of Health [grant number R37-DK27083].
References 1. 2. 3. 4.
5.
6. 7. 8. 9. 10. 11. 12.
13. 14.
Maxfield, F.R. and McGraw, T.E. (2004) Endocytic recycling. Nat. Rev. Mol. Cell Biol. 5, 121– 132 Mukherjee, S., Ghosh, R.N. and Maxfield, F.R. (1997) Endocytosis. Physiol. Rev. 77, 759–803 Huang, J. and Feigenson, G.W. (1999) A microscopic interaction model of maximum solubility of cholesterol in lipid bilayers. Biophys. J. 76, 2142–2157 Pani, B., Ong, H.L., Liu, X., Rauser, K., Ambudkar, I.S. and Singh, B.B. (2008) Lipid rafts determine clustering of STIM1 in endoplasmic reticulum–plasma membrane junctions and regulation of store-operated Ca2+ entry (SOCE). J. Biol. Chem. 283, 17333–17340 Galan, C., Woodard, G.E., Dionisio, N., Salido, G.M. and Rosado, J.A. (2010) Lipid rafts modulate the activation but not the maintenance of store-operated Ca2+ entry. Biochim. Biophys. Acta 1803, 1083–1093 van Meer, G. (2005) Cellular lipidomics. EMBO J. 24, 3159–3165 Goldstein, J.L., DeBose-Boyd, R.A. and Brown, M.S. (2006) Protein sensors for membrane sterols. Cell 124, 35–46 Goldstein, J.L. and Brown, M.S. (2009) The LDL Receptor. Arterioscler. Thromb. Vasc. Biol. 29, 431–438 Brown, M.S. and Goldstein, J.L. (2009) Cholesterol feedback: from Schoenheimer’s bottle to Scap’s MELADL. J. Lipid Res. 50 (Suppl.), S15–S27 Radhakrishnan, A. and McConnell, H.M. (1999) Condensed complexes of cholesterol and phospholipids. Biophys. J. 77, 1507–1517 DiNitto, J.P., Cronin, T.C. and Lambright, D.G. (2003) Membrane recognition and targeting by lipid-binding domains. Sci. STKE 2003, re16 Ali, M.R., Cheng, K.H. and Huang, J. (2007) Assess the nature of cholesterol–lipid interactions through the chemical potential of cholesterol in phosphatidylcholine bilayers. Proc. Natl. Acad. Sci. U.S.A. 104, 5372–5377 McConnell, H.M. and Radhakrishnan, A. (2003) Condensed complexes of cholesterol and phospholipids. Biochim. Biophys. Acta 1610, 159–173 Radhakrishnan, A. and McConnell, H. (2005) Condensed complexes in vesicles containing cholesterol and phospholipids. Proc. Natl. Acad. Sci. U.S.A. 102, 12662–12666
© The Authors Journal compilation © 2015 Biochemical Society
D.B. Iaea and F.R. Maxfield
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15. Rog, T., Pasenkiewicz-Gierula, M., Vattulainen, I. and Karttunen, M. (2009) Ordering effects of cholesterol and its analogues. Biochim. Biophys. Acta 1788, 97–121 16. van Meer, G., Voelker, D.R. and Feigenson, G.W. (2008) Membrane lipids: where they are and how they behave. Nat. Rev. Mol. Cell Biol. 9, 112–124 17. Ikonen, E. (2008) Cellular cholesterol trafficking and compartmentalization. Nat. Rev. Mol. Cell Biol. 9, 125–138 18. Radhakrishnan, A., Goldstein, J.L., McDonald, J.G. and Brown, M.S. (2008) Switch-like control of SREBP-2 transport triggered by small changes in ER cholesterol: a delicate balance. Cell Metab. 8, 512–521 19. Blanchette-Mackie, E.J. and Pentchev, P.G. (1998) Cholesterol distribution in Golgi, lysosomes, and endoplasmic reticulum. In Intracellular Cholesterol Trafficking (Chang, T.Y. and Freeman, D.A., eds), pp. 53–74, Kluwer Academic Publishers, Boston 20. Hao, M., Lin, S.X., Karylowski, O.J., Wustner, D., McGraw, T.E. and Maxfield, F.R. (2002) Vesicular and non-vesicular sterol transport in living cells. The endocytic recycling compartment is a major sterol storage organelle. J. Biol. Chem. 277, 609–617 21. Mesmin, B. and Maxfield, F.R. (2009) Intracellular sterol dynamics. Biochim. Biophys. Acta 1791, 636–645 22. Lange, Y. and Steck, T.L. (2008) Cholesterol homeostasis and the escape tendency (activity) of plasma membrane cholesterol. Prog. Lipid Res. 47, 319–332 23. Simons, K. and Ikonen, E. (1997) Functional rafts in cell membranes. Nature 387, 569–572 24. Mukherjee, S. and Maxfield, F.R. (2004) Membrane domains. Annu. Rev. Cell Dev. Biol. 20, 839–866 25. Holowka, D., Sheets, E.D. and Baird, B. (2000) Interactions between FcεRI and lipid raft components are regulated by the actin cytoskeleton. J. Cell Sci. 113, 1009–1019 26. Veatch, S.L. and Keller, S.L. (2005) Miscibility phase diagrams of giant vesicles containing sphingomyelin. Phys. Rev. Lett. 94, 148101 27. Frisz, J.F., Klitzing, H.A., Lou, K., Hutcheon, I.D., Weber, P.K., Zimmerberg, J. and Kraft, M.L. (2013) Sphingolipid domains in the plasma membranes of fibroblasts are not enriched with cholesterol. J. Biol. Chem. 288, 16855–16861 28. Dietrich, C., Yang, B., Fujiwara, T., Kusumi, A. and Jacobson, K. (2002) Relationship of lipid rafts to transient confinement zones detected by single particle tracking. Biophys. J. 82, 274–284 29. Eggeling, C., Ringemann, C., Medda, R., Schwarzmann, G., Sandhoff, K., Polyakova, S., Belov, V.N., Hein, B., von Middendorff, C., Schonle, A. and Hell, S.W. (2009) Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159–1162 30. Mondal, M., Mesmin, B., Mukherjee, S. and Maxfield, F.R. (2009) Sterols are mainly in the cytoplasmic leaflet of the plasma membrane and the endocytic recycling compartment in CHO cells. Mol. Biol. Cell 20, 581–588 31. Steck, T.L., Ye, J. and Lange, Y. (2002) Probing red cell membrane cholesterol movement with cyclodextrin. Biophys. J. 83, 2118–2125 32. Lange, Y., Dolde, J. and Steck, T.L. (1981) The rate of transmembrane movement of cholesterol in the human erythrocyte. J. Biol. Chem. 256, 5321–5323 33. Yang, T., Espenshade, P.J., Wright, M.E., Yabe, D., Gong, Y., Aebersold, R., Goldstein, J.L. and Brown, M.S. (2002) Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER. Cell 110, 489–500 34. Horton, J.D., Shah, N.A., Warrington, J.A., Anderson, N. N., Park, S.W., Brown, M.S. and Goldstein, J.L. (2003) Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes. Proc. Natl. Acad. Sci. U.S.A. 100, 12027–12032 35. Chang, T.Y., Chang, C.C. and Cheng, D. (1997) Acyl-coenzyme A: cholesterol acyltransferase. Annu. Rev. Biochem. 66, 613–638 © 2015 Biochemical Society
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36. Mahammad, S. and Parmryd, I. (2008) Cholesterol homeostasis in T cells. Methyl-β-cyclodextrin treatment results in equal loss of cholesterol from Triton X-100 soluble and insoluble fractions. Biochim. Biophys. Acta 1778, 1251–1258 37. Maxfield, F.R. and Menon, A.K. (2006) Intracellular sterol transport and distribution. Curr. Opin. Cell Biol. 18, 379–385 38. Maxfield, F.R. and Wustner, D. (2002) Intracellular cholesterol transport. J. Clin. Invest. 110, 891–898 39. Liscum, L. and Faust, J.R. (1989) The intracellular transport of low density lipoprotein-derived cholesterol is inhibited in Chinese hamster ovary cells cultured with 3-beta-[2-(diethylamino) ethoxy]androst-5-en-17-one. J. Biol. Chem. 264, 11796–11806 40. Urbani, L. and Simoni, R.D. (1990) Cholesterol and vesicular stomatitis virus G protein take separate routes from the endoplasmic reticulum to the plasma membrane. J. Biol. Chem. 265, 1919–1923 41. Beh, C.T., McMaster, C.R., Kozminski, K.G. and Menon, A.K. (2012) A detour for yeast oxysterol binding proteins. J. Biol. Chem. 287, 11481–11488 42. Soccio, R.E. and Breslow, J.L. (2003) StAR-related lipid transfer (START) proteins: mediators of intracellular lipid metabolism. J. Biol. Chem. 278, 22183–22186 43. Lusa, S., Heino, S. and Ikonen, E. (2003) Differential mobilization of newly synthesized cholesterol and biosynthetic sterol precursors from cells. J. Biol. Chem. 278, 19844–19851 44. Mesmin, B., Pipalia, N.H., Lund, F.W., Ramlall, T.F., Sokolov, A., Eliezer, D. and Maxfield, F.R. (2011) STARD4 abundance regulates sterol transport and sensing. Mol. Biol. Cell 22, 4004–4015 45. Levine, T. (2004) Short-range intracellular trafficking of small molecules across endoplasmic reticulum junctions. Trends Cell Biol. 14, 483–490 46. Romanowski, M.J., Soccio, R.E., Breslow, J.L. and Burley, S.K. (2002) Crystal structure of the Mus musculus cholesterol-regulated START protein 4 (StarD4) containing a StAR-related lipid transfer domain. Proc. Natl. Acad. Sci. U.S.A. 99, 6949–6954 47. Im, Y.J., Raychaudhuri, S., Prinz, W.A. and Hurley, J.H. (2005) Structural mechanism for sterol sensing and transport by OSBP-related proteins. Nature 437, 154–158 48. Murcia, M., Faraldo-Gomez, J.D., Maxfield, F.R. and Roux, B. (2006) Modeling the structure of the StART domains of MLN64 and StAR proteins in complex with cholesterol. J. Lipid Res. 47, 2614–2630 49. Prinz, W.A. (2007) Non-vesicular sterol transport in cells. Prog. Lipid Res. 46, 297–314 50. Clark, B.J. (2012) The mammalian START domain protein family in lipid transport in health and disease. J. Endocrinol. 212, 257–275 51. Frolov, A., Woodford, J.K., Murphy, E.J., Billheimer, J.T. and Schroeder, F. (1996) Spontaneous and protein-mediated sterol transfer between intracellular membranes. J. Biol. Chem. 271, 16075–16083 52. Mukherjee, S., Zha, X., Tabas, I. and Maxfield, F.R. (1998) Cholesterol distribution in living cells: fluorescence imaging using dehydroergosterol as a fluorescent cholesterol analog. Biophys. J. 75, 1915–1925 53. Scheidt, H.A., Muller, P., Herrmann, A. and Huster, D. (2003) The potential of fluorescent and spin-labeled steroid analogs to mimic natural cholesterol. J. Biol. Chem. 278, 45563–45569 54. Shrivastava, S., Haldar, S., Gimpl, G. and Chattopadhyay, A. (2009) Orientation and dynamics of a novel fluorescent cholesterol analogue in membranes of varying phase. J. Phys. Chem. B 113, 4475–4481 55. Maxfield, F.R. and Wustner, D. (2012) Analysis of cholesterol trafficking with fluorescent probes. Meth. Cell Biol. 108, 367–393 56. Schroeder, F., Holland, J.F. and Bieber, L.L. (1971) Fluorometric evidence for the binding of cholesterol to the filipin complex. J. Antibiot. (Tokyo) 24, 846–849 57. Wustner, D., Mondal, M., Tabas, I. and Maxfield, F.R. (2005) Direct observation of rapid internalization and intracellular transport of sterol by macrophage foam cells. Traffic 6, 396–412 © The Authors Journal compilation © 2015 Biochemical Society
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58. Skiba, P.J., Zha, X., Maxfield, F.R., Schissel, S.L. and Tabas, I. (1996) The distal pathway of lipoprotein-induced cholesterol esterification, but not sphingomyelinase-induced cholesterol esterification, is energy-dependent. J. Biol. Chem. 271, 13392–13400 59. Soccio, R.E., Adams, R.M., Romanowski, M.J., Sehayek, E., Burley, S.K. and Breslow, J.L. (2002) The cholesterol-regulated StarD4 gene encodes a StAR-related lipid transfer protein with two closely related homologues, StarD5 and StarD6. Proc. Natl. Acad. Sci. U.S.A. 99, 6943–6948
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Cholesterol trafficking and distribution David B. Iaea* † and Frederick R. Maxfield*1 *Department of Biochemistry, Weill Cornell Medical College, 1300 York Ave, New York, NY 10065, U.S.A. †Weill Cornell Medical College, Rockefeller University, and Memorial Sloan-Kettering Cancer Center Tri-Institutional Chemical Biology Program, New York, NY 10065, U.S.A.
Abstract Sterols are a critical component of cell membranes of eukaryotes. In mammalian cells there is approximately a six-fold range in the cholesterol content in various organelles. The cholesterol content of membranes plays an important role in organizing membranes for signal transduction and protein trafficking as well as in modulating the physiochemical properties of membranes. Cholesterol trafficking among organelles is highly dynamic and is mediated by both vesicular and nonvesicular processes. Several proteins have been proposed to mediate inter- organelle trafficking of cholesterol. However, several aspects of the mechanisms involved in regulating trafficking and distribution of cholesterol remain to be elucidated. In the present chapter, we discuss the cellular mechanisms involved in cholesterol distribution and the trafficking processes involved in maintaining sterol homoeostasis.
Keywords: cholesterol, dehydroergosterol, endocytic recycling compartment, homoeostasis, non-vesicular transport, StARD4, sterol regulatory element-binding protein, trafficking.
Introduction Eukaryotic cells maintain a high rate of vesicular trafficking among the secretory and endocytic organelles and the plasma membrane. For instance, in cultured fibroblasts it has been To whom correspondence should be addressed (email [email protected]).
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estimated that lipids in the plasma membrane are internalized with t½ of 15–30 min [1]. Following endocytosis, the membrane components can be delivered to a variety of organelles such as the ERC (endocytic recycling compartment), the late endosome and lysosomes, or trafficked back to the cell surface [1,2]. In some cases, the endocytosed materials are transported to the ER (endoplasmic reticulum) [2]. Because of the rapid rate of vesicular transport among organelles, it is often challenging to characterize the means by which a cell sorts lipids and sterols. Understanding the intracellular dynamics of cholesterol is important because the proper distribution among cellular organelles is critical for cellular function [3–5]. Among the many lipids in eukaryotic cells, sterols have a unique chemical structure that allows them to be absorbed and extracted from membranes with relative ease. Cholesterol contains a hydroxy group as the sole polar component, four rings and a short alkyl chain [6]. The structure of cholesterol contrasts with most glycerophospholipids and sphingolipids, which are composed of large polar head groups and two long hydrocarbon chains. The structure of cholesterol and its biophysical properties have a substantial effect on the physiochemical characteristics of membranes, as it is able to pack against the fatty acyl chains of phospholipids, reduce membrane permeability, and facilitate the formation of Lo (liquid ordered) domains in membranes [3–5]. Cells have evolved complex mechanisms to regulate the abundance and distribution of cholesterol within membranes [7-9]. In the present chapter, we discuss how the distribution of cholesterol is regulated and the mechanisms by which cholesterol moves among membranes in a eukaryotic cell.
Models of sterol–phospholipid interactions The umbrella model [3] and the condensed complex model [10] are two of the descriptions used to analyse the interaction of sterols with phospholipids. The umbrella model is built upon structural mismatch of cholesterol with other lipids in the membrane bilayer: the single hydroxy group facing into aqueous solution only partially protects the hydrophobic core from water [3]. As a consequence, sterols preferentially associate with phospholipids with larger polar head groups, such as PC (phosphatidylcholine) and sphingomyelin, rather than phospholipids with smaller polar head groups, such as PE (phosphatidylethanolamine) [11]. As a result of differences in head group size, PC can provide coverage to two cholesterol molecules, but PE can provide coverage for only a single molecule [3]. In addition to the size of the polar head group, lipid acyl chain saturation is important in dictating the geometry of the bilayer [12]. As depicted in Figure 1, increased unsaturation leads to a conical shape because of the large cross-sectional area of the acyl chains in comparison with the head group. By contrast, saturated lipids tend to be more cylindrical. The ratio of head group:body size is a good indicator of sterol stability in membranes [12]. For instance, the unsaturated DOPC (dioleoylphosphatidylcholine) is not able to shield neighbouring sterols from the aqueous phase as well as the saturated DPPC (dipalmitoylphosphatidylcholine). In the condensed complex model, the effects of transient stoichiometric associations between sterols and phospholipids are taken into account [10]. Lipids with long saturated acyl chains, such as sphingomyelin and DPPC, associate with sterols in a reversible manner to form © The Authors Journal compilation © 2015 Biochemical Society
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Figure 1. Interactions between cholesterol and surrounding lipids Cholesterol stability in membranes depends on the size of the polar head group and acyl chain saturation from neighbouring lipids. (A) Lipids with large polar head groups, which provide increased protection from the aqueous environment, are the preferred neighbours for cholesterol. (B) Acyl chain saturation influences the stability of cholesterol. Lipids with unsaturated acyl chains, containing one or more double bonds, form a conical structure in the membrane. In comparison, lipids with saturated acyl chains form a cylindrical structure. Unsaturated acyl chains provide less stability and protection to cholesterol from the aqueous surroundings. (C) Cholesterol has a weak stability in membranes enriched in unsaturated phospholipids, such as DOPC-containing membranes. As a result, cholesterol will partition to a membrane environment where it can associate with the saturated acyl chains of phospholipids. DAG, diacylglycerol; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; SM, sphingomyelin. Adapted from [21].
complexes with a smaller molecular lateral area than when the sterols and lipids are apart [13,14]. Lipids that contain unsaturated acyl chains have a reduced tendency to form these sterol–lipid complexes. The tight packing of sterol and phospholipids in these condensed membrane domains leads to ordering of the lipid acyl chains and membrane thickening [15]. As a result of sterol–lipid condensation, the stability of sterol in a membrane depends on the composition of the bilayer. Despite their differences, the two models are not mutually exclusive. Both the umbrella and condensed complex describe the interaction between cholesterol, the saturated acyl chains and polar head group of phospholipids, and in both of the models the stability of cholesterol in the bilayer decreases as the amount of sterol exceeds the capacity of the phospholipids [3,10]. In cells the phospholipid composition of organelles varies greatly. If cholesterol can equilibrate among organelles, then the differences in lipid composition would be expected to lead to differences in their sterol content [6,16]. © 2015 Biochemical Society
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Distribution of sterols in mammalian cells As shown in Figure 2, significant differences in cholesterol distribution are maintained among cellular organelles. In the plasma membrane of mammalian cells, cholesterol is ~35 mol% of the lipids [17]. By contrast, the ER contains only 5 mol% cholesterol [18]. The cholesterol:phospholipid ratio in the Golgi is intermediate between the plasma membrane and the ER. From electron microscopy of filipin-labelled cells [19], it has been suggested that the concentration of cholesterol increases from the cis-Golgi to the trans-Golgi. In the endocytic pathway, cholesterol is highly enriched in the ERC [20]. In part, this unequal distribution of cholesterol among cellular membrane compartments can be attributed to the differences in the cholesterol–phospholipid interactions resulting from the differences in the organelle lipid composition [21,22]. The plasma membrane is relatively enriched in lipids that can stabilize cholesterol in the bilayer, whereas the ER is enriched in unsaturated lipids that provide weak stabilization of cholesterol. Because of these differences in cholesterol stabilization, these membranes can have unequal cholesterol concentration even if sterol transporters bring them close to chemical equilibrium with each other.
Figure 2. Heterogeneous distribution of cellular cholesterol The cholesterol content of membranes varies throughout the cell. Membrane cholesterol levels are displayed as a heat map with membranes enriched in cholesterol labelled red. Membrane compartments with relatively low cholesterol levels are labelled blue. Inset: transbilayer distribution of cholesterol at the plasma membrane. LD, lipid droplet; LE/Ly, late endosome/ lysosome. © The Authors Journal compilation © 2015 Biochemical Society
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Within the plasma membrane there is considerable evidence for small, dynamic nanodomains that are enriched in sphingomyelin and a subset of membrane proteins, including GPI (glycosylphosphatidylinositol)-anchored proteins [23,24]. These domains can play an important role in signal transduction processes, especially when they are clustered by protein crosslinking to form larger, more stable domains [25]. The enrichment of cholesterol in more ordered lipid domains of the plasma membrane is uncertain. In model membranes, cholesterol is usually enriched in Lo domains that are in equilibrium with Ld (liquid disordered) domains, but the degree of enrichment depends on the lipid composition of the model membranes [26]. A recent high-resolution secondary ion MS study reported that cholesterol is uniformly distributed in the plasma membrane and was not enriched in micrometre-scale domains that were enriched in sphingomyelin [27]. Further studies are required to better understand the distribution of cholesterol at the scale of 10–100 nm, which is now considered to be the size of membrane lipid nanodomains [28,29]. Although models such as the condensed complex model predict that cholesterol would preferentially interact with sphingomyelin [26], which is enriched in the exofacial leaflet of the plasma membrane, these models predict favorable interactions with PC species with saturated or monounsaturated acyl chains, which are also found in the cytoplasmic leaflet of the plasma membrane. Thus, sterols can be stabilized on both leaflets of the plasma membrane. Experimental studies using fluorescent sterol analogues such as DHE (dehydroergosterol) have shown that these sterols are highly enriched in the cytoplasmic leaflet of the plasma membrane [30]. Cholesterol can flip rapidly between the leaflets in a bilayer, and it has been shown that the t½ for cholesterol flipping is less than one second in erythrocytes [31,32]. The mechanism for enriching sterols on the cytoplasmic leaflet of the plasma membrane is not understood.
Regulatory/biosynthetic mechanisms The ER is the central organelle for maintaining sterol homoeostasis. Most steps of cellular cholesterol biosynthesis occur in the ER, which contains key metabolic enzymes, including HMG (3-hydroxy-3-methyl-glutaryl)-CoA reductase [7]. The proteins that regulate synthesis of cholesterol and LDL (low-density lipoprotein) receptors are also localized in the ER [9]. When ER sterols are abundant, SREBP (sterol regulatory element-binding protein) is retained in the ER in a complex with the cholesterol-sensing protein, SCAP (SREBP-cleavage-activating protein) and the ER retention proteins Insig-1 or -2 [18]. When cholesterol content in the ER is depleted, SCAP undergoes a conformational change and is released from Insig, allowing the SREBP–SCAP complex to translocate to the Golgi apparatus [33]. In the Golgi, SREBP-2 is proteolytically processed by the site 1 and site 2 proteases to release an N-terminal transcription factor, which translocates to the nucleus to activate the expression of genes implicated in sterol biosynthesis, uptake and metabolism [34]. The amount of unesterified cholesterol in a cell is further regulated by esterification of cholesterol by the enzyme ACAT (acyl-CoA:cholesterol acyltransferase), an ER enzyme [35]. The rate-limiting step in esterification of cholesterol by ACAT is delivery of cholesterol to the ER. In order for cholesterol levels sensed in the ER to reflect the cholesterol distribution in other organelles, such as the plasma membrane and endosomes, there must be a mechanism for rapid redistribution of cholesterol to these organelles. Depending on sterol abundance, a high fraction of total cellular cholesterol can be stored as cholesteryl esters in lipid droplets. © 2015 Biochemical Society
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The cholesterol stored in the lipid droplets can be used to maintain sterol homoeostasis when cholesterol levels in the cell become low [36].
Intracellular trafficking of lipids and sterols Cholesterol can move between membranes by vesicular and non-vesicular transport mechanisms [37,38]. Like other lipids, cholesterol can be incorporated into transport vesicles that carry membrane components between organelles. However, only a small fraction of membrane components internalized from the plasma membrane reach the ER, indicating that cholesterol sensing in the ER would be very slow and inefficient if it depended on vesicle transport. There is substantial evidence for high rates of non-vesicular sterol transport in cells [39]. Since cholesterol is very poorly soluble in water, non-vesicular transport requires binding to carrier proteins. Newly synthesized sterols are transported from the ER to the plasma membrane when vesicular trafficking is inhibited by either genetic or pharmacological intervention. For example, treatment with Brefeldin A, which causes Golgi disassembly and fusion with the ER, inhibits ~90% of protein traffic but diminishes cholesterol transport to the plasma membrane by ~20% [40]. Additionally, studies using DHE, a fluorescent cholesterol analogue, demonstrate that equilibration between the plasma membrane and the ERC occurs within minutes, and this transport is not drastically altered under ATP-depleted conditions that block vesicle transport [20]. This suggests that the majority of cholesterol transport is mediated by nonvesicular, carrier-mediated transport mechanisms (Figure 3).
Sterol transfer proteins in non-vesicular transport There are several protein families that are classified as sterol transfer proteins, which have been shown to be capable of transferring sterols between membranes [41,42]. Sterol transfer proteins can interact directly with membrane compartments to extract sterol and then diffuse in complex with the sterol to acceptor membranes. Sterol transfer proteins can significantly increase the rates of sterol transfer between liposomes in vitro [43,44]. Additionally, sterol transfer proteins may also function to shuttle sterol between adjacent membranes. In some cases, such as ER–plasma membrane contact sites, the membranes of two organelles are held in close proximity by protein complexes, facilitating rapid exchange of sterol between the membranes by reducing the distance that the sterol–protein complex must travel [45]. X-ray crystallographic studies of several sterol transfer proteins demonstrate that they each contain a hydrophobic ligand pocket capable of binding a single sterol molecule [46,47]. Molecular dynamics studies suggest that in several of these proteins, the sterol binding pocket is under ‘gated’ regulation that allows it to open and close upon interaction with the lipid bilayer, which facilitates sterol absorption and delivery to specific membranes [48]. Two major gene families of lipid transfer proteins that have been implicated in such trafficking are the START [StAR (steroidogenic acute regulatory protein)-related lipid transfer] domain family and the OSBP (oxysterol-binding protein) and ORP (OSBP-related protein) family, which have been discussed in detail elsewhere [41,49,50]. © The Authors Journal compilation © 2015 Biochemical Society
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Figure 3. Intracellular trafficking itinerary of cholesterol (1,2) LDL particles containing cholesteryl esters and free cholesterol are internalized by clathrin-mediated endocytosis of the LDL receptor [2]. (3,6) The low pH in early endosomes causes the LDL to be released from its receptor, and the empty LDL receptor returns to the cell surface for further rounds of endocytosis. (4) The LDL is retained in the early sorting endosomes, which mature into late endosomes where it encounters acid hydrolases including lysosomal acid lipase (LAL), which hydrolyses the cholesteryl esters in the core of the LDL particles. (5, inset) The free cholesterol is transported out of LE/Ly, mediated by NPC1 and NPC2, and delivered to other cellular membranes including the plasma membrane and ER by primarily non-vesicular mechanisms. (6) Cholesterol in the plasma membrane can traffic to the ERC and back by both vesicular and non-vesicular mechanisms. (7) Cholesterol transport from the plasma membrane to the ER informs the homoeostatic machinery about the free cholesterol levels in the cell. NPC1, Niemann-Pick C1 protein.
Fluorescent probes for cholesterol trafficking The intracellular transport of cholesterol between organelles is a fundamental homoeostatic mechanism, but these transport processes are not well understood. Cholesterol transport can be studied by introduction of radioactive sterols or precursors into cells, but this method requires stringent purification of organelles and the assumption that cholesterol does not redistribute during the purification process [20,51]. Minor contamination from membranes enriched in cholesterol can lead to significant errors in measuring cholesterol content of organelles such as the ER. Fluorescence microscopy is a powerful tool for studying intracellular transport processes. However, this method can be difficult for examining lipid molecules because addition of a © 2015 Biochemical Society
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Figure 4. Fluorescent probes and analogues for studying cholesterol Chemical structures of cholesterol (A), dehydroergosterol (B), cholestatrienol (C), cholesterolBODIPY (D) and filipin (E). Structural differences between cholesterol (A) and its fluorescent analogues (B-D) are indicated in red. BODIPY, boron-dipyrromethene.
fluorophore may greatly alter the physical properties of the molecule [52–54]. Two approaches have been used to examine cholesterol distribution in cells using fluorescence. One method is to use a sterol-binding fluorophore, such as filipin, to localize cholesterol in cells [55]. As depicted in Figure 4, filipin is a naturally fluorescent polyene antibiotic that binds to unesterified cholesterol [56]. This makes filipin a useful tool in determining the free cholesterol content and distribution in membranes. However, filipin photobleaches rapidly, and binding to cholesterol perturbs the membrane, thus making it unsuitable for live cell imaging. As alternative approaches for examining cholesterol trafficking in live cells, the introduction of either a boradiazaindacene moiety to cholesterol or the use of fluorescent sterol analogues such as DHE or cholestatrienol have been very successful [55]. These sterol analogues have been discussed in depth elsewhere [55]. These fluorescent sterol analogues can be incorporated into cells either by delivery to the plasma membrane or by reconstitution into lipoproteins [20,57]. Sterol trafficking can then be directly imaged or used to study trafficking kinetics or transbilayer distribution [55].
Consequences of extensive non-vesicular transport As discussed in earlier sections, the plasma membrane has the largest pool of cholesterol, whereas the ER is relatively sterol poor [17,18]. Thus, it is important that transport pathways © The Authors Journal compilation © 2015 Biochemical Society
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between these organelles exist to facilitate cholesterol efflux to the plasma membrane and so that the ER can respond to alterations in sterol levels by modulating cholesterol synthesis and esterification. Sterol transport between the plasma membrane and the ER is substantial and occurs primarily by non-vesicular mechanisms [39,58]. In this perspective, the rate of sterol transport into and out of the ER serves to maintain a constant cholesterol level in the organelle, which in turn maintains sterol homoeostasis. StARD4 (START domain protein 4), a soluble sterol transporter, has been implicated in maintaining sterol homoeostasis and is transcriptionally regulated by SREBP-2 [59]. StARD4 plays an important role in delivery of cholesterol to the ER. A study using U2OS human osteosarcoma cells demonstrated that StARD4 overexpression increased cholesterol ester levels [44]. StARD4 silencing attenuated cholesterol-mediated regulation of SREBP-2 activation, whereas StARD4 overexpression amplified sterol sensing by the SCAP and SREBP-2 proteins in the ER. Silencing StARD4 increased the cellular cholesterol levels, presumably as a result of the slower delivery of cholesterol to the ER and reduction in SREBP-2 response. These data suggest that delivery of cholesterol to the ER is an important function of StARD4. Although it is clear that StARD4 delivers sterol to the ER, its expression must be finely tuned. Deregulation of StARD4 expression may result in desensitization of the ER homoeostatic machinery, which would then no longer be able to respond to variations in cellular cholesterol levels. Thus, modulation of StARD4 expression, by SREBP-2, ensures optimal sterol sensing and ER sensitivity to cholesterol abundance in organelles, including the plasma membrane. Further work is required to understand how the trafficking kinetics of sterols are integrated into maintaining sterol homoeostasis.
Conclusion Cholesterol is a highly dynamic lipid heterogeneously distributed throughout organelles and the membrane bilayer. It has a rapid lateral and transverse mobility with dramatic effects on the organization of its surrounding lipids. Additionally, there is increasing evidence that the majority of sterol trafficking and distribution is maintained by non-vesicular mechanisms. The presence of numerous sterol transport proteins demonstrates that cells can rapidly redistribute sterol throughout organelles and that cholesterol acts as a central lipid in sensing lipid homoeostasis. Although there is still work to be done in understanding the precise mechanisms of non- vesicular sterol transport, the development of fluorescent probes to analyse lipid and sterol transport and distribution makes this an exciting time in studying sterol trafficking. An important question for the next few years is to elucidate the trafficking kinetics of sterols into and out of organelles and how these transport steps are integrated into the overall cellular homoeostasis.
Summary • •
Cholesterol is a critical component of cell membranes in mammalian cells. The stability of cholesterol in the bilayer depends on its interactions with neighbouring phospholipids.
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• • • • • •
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Cholesterol is heterogeneously distributed throughout cell compartments as well as across the membrane bilayer. Cells have evolved complex pathways to maintain cholesterol homoeostasis. Trafficking and distribution of cholesterol in a cell is maintained primarily by non-vesicular mechanisms. Several protein families function as putative sterol transporters to maintain cholesterol homoeostasis. Fluorescent analogues and probes can be used to visualize cholesterol in fixed and living cells. Cholesterol homoeostasis and organelle levels are maintained by a complex network of non-vesicular trafficking processes.
Work in the Maxfield laboratory is supported by the National Institutes of Health [grant number R37-DK27083].
References 1. 2. 3. 4.
5.
6. 7. 8. 9. 10. 11. 12.
13. 14.
Maxfield, F.R. and McGraw, T.E. (2004) Endocytic recycling. Nat. Rev. Mol. Cell Biol. 5, 121– 132 Mukherjee, S., Ghosh, R.N. and Maxfield, F.R. (1997) Endocytosis. Physiol. Rev. 77, 759–803 Huang, J. and Feigenson, G.W. (1999) A microscopic interaction model of maximum solubility of cholesterol in lipid bilayers. Biophys. J. 76, 2142–2157 Pani, B., Ong, H.L., Liu, X., Rauser, K., Ambudkar, I.S. and Singh, B.B. (2008) Lipid rafts determine clustering of STIM1 in endoplasmic reticulum–plasma membrane junctions and regulation of store-operated Ca2+ entry (SOCE). J. Biol. Chem. 283, 17333–17340 Galan, C., Woodard, G.E., Dionisio, N., Salido, G.M. and Rosado, J.A. (2010) Lipid rafts modulate the activation but not the maintenance of store-operated Ca2+ entry. Biochim. Biophys. Acta 1803, 1083–1093 van Meer, G. (2005) Cellular lipidomics. EMBO J. 24, 3159–3165 Goldstein, J.L., DeBose-Boyd, R.A. and Brown, M.S. (2006) Protein sensors for membrane sterols. Cell 124, 35–46 Goldstein, J.L. and Brown, M.S. (2009) The LDL Receptor. Arterioscler. Thromb. Vasc. Biol. 29, 431–438 Brown, M.S. and Goldstein, J.L. (2009) Cholesterol feedback: from Schoenheimer’s bottle to Scap’s MELADL. J. Lipid Res. 50 (Suppl.), S15–S27 Radhakrishnan, A. and McConnell, H.M. (1999) Condensed complexes of cholesterol and phospholipids. Biophys. J. 77, 1507–1517 DiNitto, J.P., Cronin, T.C. and Lambright, D.G. (2003) Membrane recognition and targeting by lipid-binding domains. Sci. STKE 2003, re16 Ali, M.R., Cheng, K.H. and Huang, J. (2007) Assess the nature of cholesterol–lipid interactions through the chemical potential of cholesterol in phosphatidylcholine bilayers. Proc. Natl. Acad. Sci. U.S.A. 104, 5372–5377 McConnell, H.M. and Radhakrishnan, A. (2003) Condensed complexes of cholesterol and phospholipids. Biochim. Biophys. Acta 1610, 159–173 Radhakrishnan, A. and McConnell, H. (2005) Condensed complexes in vesicles containing cholesterol and phospholipids. Proc. Natl. Acad. Sci. U.S.A. 102, 12662–12666
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15. Rog, T., Pasenkiewicz-Gierula, M., Vattulainen, I. and Karttunen, M. (2009) Ordering effects of cholesterol and its analogues. Biochim. Biophys. Acta 1788, 97–121 16. van Meer, G., Voelker, D.R. and Feigenson, G.W. (2008) Membrane lipids: where they are and how they behave. Nat. Rev. Mol. Cell Biol. 9, 112–124 17. Ikonen, E. (2008) Cellular cholesterol trafficking and compartmentalization. Nat. Rev. Mol. Cell Biol. 9, 125–138 18. Radhakrishnan, A., Goldstein, J.L., McDonald, J.G. and Brown, M.S. (2008) Switch-like control of SREBP-2 transport triggered by small changes in ER cholesterol: a delicate balance. Cell Metab. 8, 512–521 19. Blanchette-Mackie, E.J. and Pentchev, P.G. (1998) Cholesterol distribution in Golgi, lysosomes, and endoplasmic reticulum. In Intracellular Cholesterol Trafficking (Chang, T.Y. and Freeman, D.A., eds), pp. 53–74, Kluwer Academic Publishers, Boston 20. Hao, M., Lin, S.X., Karylowski, O.J., Wustner, D., McGraw, T.E. and Maxfield, F.R. (2002) Vesicular and non-vesicular sterol transport in living cells. The endocytic recycling compartment is a major sterol storage organelle. J. Biol. Chem. 277, 609–617 21. Mesmin, B. and Maxfield, F.R. (2009) Intracellular sterol dynamics. Biochim. Biophys. Acta 1791, 636–645 22. Lange, Y. and Steck, T.L. (2008) Cholesterol homeostasis and the escape tendency (activity) of plasma membrane cholesterol. Prog. Lipid Res. 47, 319–332 23. Simons, K. and Ikonen, E. (1997) Functional rafts in cell membranes. Nature 387, 569–572 24. Mukherjee, S. and Maxfield, F.R. (2004) Membrane domains. Annu. Rev. Cell Dev. Biol. 20, 839–866 25. Holowka, D., Sheets, E.D. and Baird, B. (2000) Interactions between FcεRI and lipid raft components are regulated by the actin cytoskeleton. J. Cell Sci. 113, 1009–1019 26. Veatch, S.L. and Keller, S.L. (2005) Miscibility phase diagrams of giant vesicles containing sphingomyelin. Phys. Rev. Lett. 94, 148101 27. Frisz, J.F., Klitzing, H.A., Lou, K., Hutcheon, I.D., Weber, P.K., Zimmerberg, J. and Kraft, M.L. (2013) Sphingolipid domains in the plasma membranes of fibroblasts are not enriched with cholesterol. J. Biol. Chem. 288, 16855–16861 28. Dietrich, C., Yang, B., Fujiwara, T., Kusumi, A. and Jacobson, K. (2002) Relationship of lipid rafts to transient confinement zones detected by single particle tracking. Biophys. J. 82, 274–284 29. Eggeling, C., Ringemann, C., Medda, R., Schwarzmann, G., Sandhoff, K., Polyakova, S., Belov, V.N., Hein, B., von Middendorff, C., Schonle, A. and Hell, S.W. (2009) Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159–1162 30. Mondal, M., Mesmin, B., Mukherjee, S. and Maxfield, F.R. (2009) Sterols are mainly in the cytoplasmic leaflet of the plasma membrane and the endocytic recycling compartment in CHO cells. Mol. Biol. Cell 20, 581–588 31. Steck, T.L., Ye, J. and Lange, Y. (2002) Probing red cell membrane cholesterol movement with cyclodextrin. Biophys. J. 83, 2118–2125 32. Lange, Y., Dolde, J. and Steck, T.L. (1981) The rate of transmembrane movement of cholesterol in the human erythrocyte. J. Biol. Chem. 256, 5321–5323 33. Yang, T., Espenshade, P.J., Wright, M.E., Yabe, D., Gong, Y., Aebersold, R., Goldstein, J.L. and Brown, M.S. (2002) Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER. Cell 110, 489–500 34. Horton, J.D., Shah, N.A., Warrington, J.A., Anderson, N. N., Park, S.W., Brown, M.S. and Goldstein, J.L. (2003) Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes. Proc. Natl. Acad. Sci. U.S.A. 100, 12027–12032 35. Chang, T.Y., Chang, C.C. and Cheng, D. (1997) Acyl-coenzyme A: cholesterol acyltransferase. Annu. Rev. Biochem. 66, 613–638 © 2015 Biochemical Society
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36. Mahammad, S. and Parmryd, I. (2008) Cholesterol homeostasis in T cells. Methyl-β-cyclodextrin treatment results in equal loss of cholesterol from Triton X-100 soluble and insoluble fractions. Biochim. Biophys. Acta 1778, 1251–1258 37. Maxfield, F.R. and Menon, A.K. (2006) Intracellular sterol transport and distribution. Curr. Opin. Cell Biol. 18, 379–385 38. Maxfield, F.R. and Wustner, D. (2002) Intracellular cholesterol transport. J. Clin. Invest. 110, 891–898 39. Liscum, L. and Faust, J.R. (1989) The intracellular transport of low density lipoprotein-derived cholesterol is inhibited in Chinese hamster ovary cells cultured with 3-beta-[2-(diethylamino) ethoxy]androst-5-en-17-one. J. Biol. Chem. 264, 11796–11806 40. Urbani, L. and Simoni, R.D. (1990) Cholesterol and vesicular stomatitis virus G protein take separate routes from the endoplasmic reticulum to the plasma membrane. J. Biol. Chem. 265, 1919–1923 41. Beh, C.T., McMaster, C.R., Kozminski, K.G. and Menon, A.K. (2012) A detour for yeast oxysterol binding proteins. J. Biol. Chem. 287, 11481–11488 42. Soccio, R.E. and Breslow, J.L. (2003) StAR-related lipid transfer (START) proteins: mediators of intracellular lipid metabolism. J. Biol. Chem. 278, 22183–22186 43. Lusa, S., Heino, S. and Ikonen, E. (2003) Differential mobilization of newly synthesized cholesterol and biosynthetic sterol precursors from cells. J. Biol. Chem. 278, 19844–19851 44. Mesmin, B., Pipalia, N.H., Lund, F.W., Ramlall, T.F., Sokolov, A., Eliezer, D. and Maxfield, F.R. (2011) STARD4 abundance regulates sterol transport and sensing. Mol. Biol. Cell 22, 4004–4015 45. Levine, T. (2004) Short-range intracellular trafficking of small molecules across endoplasmic reticulum junctions. Trends Cell Biol. 14, 483–490 46. Romanowski, M.J., Soccio, R.E., Breslow, J.L. and Burley, S.K. (2002) Crystal structure of the Mus musculus cholesterol-regulated START protein 4 (StarD4) containing a StAR-related lipid transfer domain. Proc. Natl. Acad. Sci. U.S.A. 99, 6949–6954 47. Im, Y.J., Raychaudhuri, S., Prinz, W.A. and Hurley, J.H. (2005) Structural mechanism for sterol sensing and transport by OSBP-related proteins. Nature 437, 154–158 48. Murcia, M., Faraldo-Gomez, J.D., Maxfield, F.R. and Roux, B. (2006) Modeling the structure of the StART domains of MLN64 and StAR proteins in complex with cholesterol. J. Lipid Res. 47, 2614–2630 49. Prinz, W.A. (2007) Non-vesicular sterol transport in cells. Prog. Lipid Res. 46, 297–314 50. Clark, B.J. (2012) The mammalian START domain protein family in lipid transport in health and disease. J. Endocrinol. 212, 257–275 51. Frolov, A., Woodford, J.K., Murphy, E.J., Billheimer, J.T. and Schroeder, F. (1996) Spontaneous and protein-mediated sterol transfer between intracellular membranes. J. Biol. Chem. 271, 16075–16083 52. Mukherjee, S., Zha, X., Tabas, I. and Maxfield, F.R. (1998) Cholesterol distribution in living cells: fluorescence imaging using dehydroergosterol as a fluorescent cholesterol analog. Biophys. J. 75, 1915–1925 53. Scheidt, H.A., Muller, P., Herrmann, A. and Huster, D. (2003) The potential of fluorescent and spin-labeled steroid analogs to mimic natural cholesterol. J. Biol. Chem. 278, 45563–45569 54. Shrivastava, S., Haldar, S., Gimpl, G. and Chattopadhyay, A. (2009) Orientation and dynamics of a novel fluorescent cholesterol analogue in membranes of varying phase. J. Phys. Chem. B 113, 4475–4481 55. Maxfield, F.R. and Wustner, D. (2012) Analysis of cholesterol trafficking with fluorescent probes. Meth. Cell Biol. 108, 367–393 56. Schroeder, F., Holland, J.F. and Bieber, L.L. (1971) Fluorometric evidence for the binding of cholesterol to the filipin complex. J. Antibiot. (Tokyo) 24, 846–849 57. Wustner, D., Mondal, M., Tabas, I. and Maxfield, F.R. (2005) Direct observation of rapid internalization and intracellular transport of sterol by macrophage foam cells. Traffic 6, 396–412 © The Authors Journal compilation © 2015 Biochemical Society
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58. Skiba, P.J., Zha, X., Maxfield, F.R., Schissel, S.L. and Tabas, I. (1996) The distal pathway of lipoprotein-induced cholesterol esterification, but not sphingomyelinase-induced cholesterol esterification, is energy-dependent. J. Biol. Chem. 271, 13392–13400 59. Soccio, R.E., Adams, R.M., Romanowski, M.J., Sehayek, E., Burley, S.K. and Breslow, J.L. (2002) The cholesterol-regulated StarD4 gene encodes a StAR-related lipid transfer protein with two closely related homologues, StarD5 and StarD6. Proc. Natl. Acad. Sci. U.S.A. 99, 6943–6948
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