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The cholesterol affinities of many integral plasma membrane proteins have been estimated by molecular computation. However, these values lack experimental confirmation. We therefore developed a simple mathematical model to extract sterol affinity constants and stoichiometries from published isotherms for the dependence of the activity of such proteins on the membrane cholesterol concentration. The binding curves for these proteins are sigmoidal, with strongly lagged thresholds attributable to competition for the cholesterol by bilayer phospholipids. The model provided isotherms that matched the experimental data using published values for the sterol association constants and stoichiometries of the phospholipids. Three oligomeric transporters were found to bind cholesterol without cooperativity, with dimensionless association constants of 35 for Kir3.4* and 100 for both Kir2 and a GAT transporter. (The corresponding Δ G ° values were -8.8, -11.4, and -11.4 kJ/mol, respectively). These association constants are significantly lower than those for the phospholipids, which range from ∼100 to 6000. The BK channel, the nicotinic acetylcholine receptor, and the M192I mutant of Kir3.4* appear to bind multiple cholesterol molecules cooperatively ( n = 2 or 4), with subunit affinities of 563, 950, and 700, respectively. The model predicts that the three less avid transporters are approximately half-saturated in their native plasma membranes; hence, they are sensitive to variations in cholesterol in vivo. The more avid proteins would be nearly saturated in vivo. The method can be applied to any integral protein or other ligands in any bilayer for which there are reasonable estimates of the sterol affinities and stoichiometries of the phospholipids.

This review considers the hypothesis that a small portion of plasma membrane cholesterol regulates reverse cholesterol transport in coordination with overall cellular homeostasis. It appears that almost all of the plasma membrane cholesterol is held in stoichiometric complexes with bilayer phospholipids. The minor fraction of cholesterol that exceeds the complexation capacity of the phospholipids is called active cholesterol. It has an elevated chemical activity and circulates among the organelles. It also moves down its chemical activity gradient to plasma HDL, facilitated by the activity of ABCA1, ABCG1, and SR-BI. ABCA1 initiates this process by perturbing the organization of the plasma membrane bilayer, thereby priming its phospholipids for translocation to apoA-I to form nascent HDL. The active excess sterol and that activated by ABCA1 itself follow the phospholipids to the nascent HDL. ABCG1 similarly rearranges the bilayer and sends additional active cholesterol to nascent HDL, while SR-BI simply facilitates the equilibration of the active sterol between plasma membranes and plasma proteins. Active cholesterol also flows downhill to cytoplasmic membranes where it serves both as a feedback signal to homeostatic ER proteins and as the substrate for the synthesis of mitochondrial 27-hydroxycholesterol (27HC). 27HC binds the LXR and promotes the expression of the aforementioned transport proteins. 27HC-LXR also activates ABCA1 by competitively displacing its inhibitor, unliganded LXR. § Considerable indirect evidence suggests that active cholesterol serves as both a substrate and a feedback signal for reverse cholesterol transport. Direct tests of this novel hypothesis are proposed., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Almost all the cholesterol in cellular membranes is associated with phospholipids in simple stoichiometric complexes. This limits the binding of sterol ligands such as filipin and perfringolysin O (PFO) to a small fraction of the total. We offer a simple mathematical model that characterizes this complexity. It posits that the cholesterol accessible to ligands has two forms: active cholesterol, which is that not complexed with phospholipids; and extractable cholesterol, that which ligands can capture competitively from the phospholipid complexes. Simulations based on the model match published data for the association of PFO oligomers with liposomes, plasma membranes, and the isolated endoplasmic reticulum. The model shows how the binding of a probe greatly underestimates cholesterol abundance when its affinity for the sterol is so weak that it competes poorly with the membrane phospholipids. Two examples are the understaining of plasma membranes by filipin and the failure of domain D4 of PFO to label their cytoplasmic leaflets. Conversely, the exaggerated staining of endolysosomes suggests that their cholesterol, being uncomplexed, is readily available. The model is also applicable to the association of cholesterol with intrinsic membrane proteins. For example, it supports the hypothesis that the sharp threshold in the regulation of homeostatic endoplasmic reticulum proteins by cholesterol derives from the cooperativity of their binding to the sterol weakly held by the phospholipids. Thus, the model explicates the complexity inherent in the binding of ligands like PFO and filipin to the small accessible fraction of membrane cholesterol., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Cells manage their cholesterol by negative feedback using a battery of sterol-responsive proteins. How these activities are coordinated so as to specify the abundance and distribution of the sterol is unclear. We present a simple mathematical model that addresses this question. It assumes that almost all of the cholesterol is associated with phospholipids in stoichiometric complexes. A small fraction of the sterol is uncomplexed and thermodynamically active. It equilibrates among the organelles, setting their sterol level according to the affinity of their phospholipids. The activity of the homeostatic proteins in the cytoplasmic membranes is then set by their fractional saturation with uncomplexed cholesterol in competition with the phospholipids. The high-affinity phospholipids in the plasma membrane (PM) are filled to near stoichiometric equivalence, giving it most of the cell sterol. Notably, the affinity of the phospholipids in the endomembranes (EMs) is lower by orders of magnitude than that of the phospholipids in the PM. Thus, the small amount of sterol in the EMs rests far below stoichiometric capacity. Simulations match a variety of experimental data. The model captures the essence of cell cholesterol homeostasis, makes coherent a diverse set of experimental findings, provides a surprising prediction and suggests new experiments., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

This review considers the following hypotheses, some well-supported and some speculative. Almost all of the sterol molecules in plasma membranes are associated with bilayer phospholipids in complexes of varied strength and stoichiometry. These complexes underlie many of the material properties of the bilayer. The small fraction of cholesterol molecules exceeding the binding capacity of the phospholipids is thermodynamically active and serves diverse functions. It circulates briskly among the cell membranes, particularly through contact sites linking the organelles. Active cholesterol provides the upstream feedback signal to multiple mechanisms governing plasma membrane homeostasis, pegging the sterol level to a threshold set by its phospholipids. Active cholesterol could also be the cargo for various inter-organelle transporters and the form excreted from cells by reverse transport. Furthermore, it is integral to the function of caveolae; a mediator of Hedgehog regulation; and a ligand for the binding of cytolytic toxins to membranes. Active cholesterol modulates a variety of plasma membrane proteins-receptors, channels and transporters-at least in vitro., (© 2020 John Wiley & Sons A/S . Published by John Wiley & Sons Ltd.)

The transverse asymmetry (sidedness) of phospholipids in plasma membrane bilayers is well characterized, distinctive, actively maintained and functionally important. In contrast, numerous studies using a variety of techniques have concluded that plasma membrane bilayer cholesterol is either mostly in the outer leaflet or the inner leaflet or is fairly evenly distributed. Sterols might simply partition according to their differing affinities for the asymmetrically disposed phospholipids, but some studies have proposed that it is actively transported to the outer leaflet. Other work suggests that the sterol is enriched in the inner leaflet, driven by either positive interactions with the phosphatidylethanolamine on that side or by its exclusion from the outer leaflet by the long chain sphingomyelin molecules therein. This uncertainty raises three questions: is plasma membrane cholesterol sidedness fixed in a given cell or cell type; is it generally the same among mammalian species; and does it serve specific physiological functions? This review grapples with these issues., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Sterols associate preferentially with plasma membrane sphingolipids and saturated phospholipids to form stoichiometric complexes. Cholesterol in molar excess of the capacity of these polar bilayer lipids has a high accessibility and fugacity; we call this fraction active cholesterol. This review first considers how active cholesterol serves as an upstream regulator of cellular sterol homeostasis. The mechanism appears to utilize the redistribution of active cholesterol down its diffusional gradient to the endoplasmic reticulum and mitochondria, where it binds multiple effectors and directs their feedback activity. We have also reviewed a broad literature in search of a role for active cholesterol (as opposed to bulk cholesterol or lipid domains such as rafts) in the activity of diverse membrane proteins. Several systems provide such evidence, implicating, in particular, caveolin-1, various kinds of ABC-type cholesterol transporters, solute transporters, receptors and ion channels. We suggest that this larger role for active cholesterol warrants close attention and can be tested easily., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

It has been shown that modestly increasing plasma membrane cholesterol beyond its physiological set point greatly increases the endoplasmic reticulum and mitochondrial pools, thereby eliciting manifold feedback responses that return cell cholesterol to its resting state. The question arises whether this homeostatic mechanism reflects the targeting of cell surface cholesterol to specific intracellular sites or its general equilibration among the organelles. We now show that human fibroblast cholesterol can be increased as much as two-fold from 2-hydroxypropyl-β-cyclodextrin without changing the size of the cell surface pool. Rather, essentially all of the added cholesterol disperses rapidly among cytoplasmic membranes, increasing their overall cholesterol content by as much as five-fold. We conclude that the level of plasma membrane cholesterol is normally at capacity and that even small increments above this physiological set point redistribute essentially entirely to intracellular membranes, perhaps down their chemical activity gradients.

Is cholesterol distributed among intracellular compartments by passive equilibration down its chemical gradient? If so, its distribution should reflect the relative cholesterol affinity of the constituent membrane phospholipids as well as their capacity for association with the sterol. We examined this issue by analyzing the reactivity to cholesterol oxidase of large unilamellar vesicles (LUVs) containing phospholipids and varied levels of cholesterol. The rates of cholesterol oxidation differed among the various phospholipid environments by roughly 4 orders of magnitude. Furthermore, accessibility to the enzyme increased by orders of magnitude at cholesterol thresholds that suggested cholesterol:phospholipid association ratios of 1:1, 2:3, or 1:2 (moles:moles). The accessibility of cholesterol above these thresholds was still constrained by its particular phospholipid environment. One phospholipid, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidylserine, exhibited no threshold. The analysis suggested values for the stoichiometries of the putative cholesterol-phospholipid complexes, their relative stabilities, and the fractions of bilayer cholesterol not in complexes at the threshold equivalence points. Predictably, the saturated phosphorylcholine species had the lowest apparent stoichiometric ratios and the strongest associations with cholesterol. These results are in general agreement with the equilibrium distribution of cholesterol between the various LUVs and methyl-β-cyclodextrin. In addition, the behavior of the cholesterol in intact human red blood cells matched predictions made from LUVs of the corresponding composition. These results support a passive mechanism for the intracellular distribution of cholesterol that can provide a signal for its homeostatic regulation.

A variety of intercalating amphipaths increase the chemical activity of plasma membrane cholesterol. To test whether intracellular cholesterol can be similarly activated, we examined NPC1 and NPC2 fibroblasts, since they accumulate large amounts of cholesterol in their late endosomes and lysosomes (LE/L). We gauged the mobility of intracellular sterol from its appearance at the surface of the intact cells, as determined by its susceptibility to cholesterol oxidase and its isotope exchange with extracellular 2-(hydroxypropyl)-β-cyclodextrin-cholesterol. The entire cytoplasmic cholesterol pool in these cells was mobile, exchanging with the plasma membrane with an apparent half-time of ∼3-4 hours, ∼4-5 times slower than that for wild type human fibroblasts (half-time ∼0.75 hours). The mobility of the intracellular cholesterol was increased by the membrane-intercalating amphipaths chlorpromazine and 1-octanol. Chlorpromazine also promoted the net transfer of LE/L cholesterol to serum and cyclodextrin. Surprisingly, the mobility of LE/L cholesterol was greatly stimulated by treating intact NPC cells with glutaraldehyde or formaldehyde. Similar effects were seen with wild type fibroblasts in which the LE/L cholesterol pool had been expanded using U18666A. We also showed that the cholesterol in the intracellular membranes of fixed wild-type fibroblasts was mobile; it was rapidly oxidized by cholesterol oxidase and was rapidly replenished by exogenous sterol. We conclude that a) the cholesterol in NPC cells can exit the LE/L (and the extensive membranous inclusions therein) over a few hours; b) this mobility is stimulated by the activation of the cholesterol with intercalating amphipaths; c) intracellular cholesterol is even more mobile in fixed cells; and d) amphipaths that activate cholesterol might be useful in treating NPC disease.

Recent evidence suggests that the major pathways mediating cell cholesterol homeostasis respond to a common signal: active membrane cholesterol. Active cholesterol is the fraction that exceeds the complexing capacity of the polar bilayer lipids. Increments in plasma membrane cholesterol exceeding this threshold have an elevated chemical activity (escape tendency) and redistribute via diverse transport proteins to both circulating plasma lipoproteins and intracellular organelles. Active cholesterol thereby prompts several feedback responses. It is the substrate for its own esterification and for the synthesis of regulatory side-chain oxysterols. It also stimulates manifold pathways that down-regulate the biosynthesis, curtail the ingestion and increase the export of cholesterol. Thus, the abundance of cell cholesterol is tightly coupled to that of its polar lipid partners through active cholesterol., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A few membrane-intercalating amphipaths have been observed to stimulate the interaction of cholesterol with cholesterol oxidase, saponin and cyclodextrin, presumably by displacing cholesterol laterally from its phospholipid complexes. We now report that this effect, referred to as cholesterol activation, occurs with dozens of other amphipaths, including alkanols, saturated and cis- and trans-unsaturated fatty acids, fatty acid methyl esters, sphingosine derivatives, terpenes, alkyl ethers, ketones, aromatics and cyclic alkyl derivatives. The apparent potency of the agents tested ranged from 3 microM to 7 mM and generally paralleled their octanol/water partition coefficients, except that relative potency declined for compounds with >10 carbons. Some small amphipaths activated cholesterol at a membrane concentration of approximately 3 mol per 100 mol of bilayer lipids, about equimolar with the cholesterol they displaced. Lysophosphatidylserine countered the effects of all these agents, consistent with its ability to reduce the pool of active membrane cholesterol. Various amphipaths stabilized red cells against the hemolysis elicited by cholesterol depletion, presumably by substituting for the extracted sterol. The number and location of cis and trans fatty acid unsaturations and the absolute stereochemistry of enantiomer pairs had only small effects on amphipath potency. Nevertheless, potency varied approximately 7-fold within a group of diverse agents with similar partition coefficients. We infer that a wide variety of amphipaths can displace membrane cholesterol by competing stoichiometrically but with only limited specificity for weak association with phospholipids. Any number of other drugs and experimental agents might do the same.

Side chain oxysterols are cholesterol derivatives thought to signal the abundance of cell cholesterol to homeostatic effector proteins. Here, we investigated how plasma membrane (PM) cholesterol might regulate 27-hydroxycholesterol (HC) biosynthesis in cultured fibroblasts. We showed that PM cholesterol was a major substrate for 27-HC production. Biosynthesis commenced within minutes of loading depleted cells with cholesterol, concurrent with the rapid inactivation of hydroxy-3-methylglutaryl CoA reductase (HMGR). 27-HC production rose approximately 30-fold in normal and Niemann-Pick C1 fibroblasts when PM cholesterol was increased by approximately 60%. 27-HC production was also stimulated by 1-octanol, which displaces PM cholesterol from its phospholipid complexes and thereby increases its activity (escape tendency) and elevates its intracellular abundance. Conversely, lysophosphatidylserine and U18666A inhibited 27-HC biosynthesis and the inactivation of HMGR, presumably by reducing the activity of PM cholesterol and, therefore, its circulation to mitochondria. We conclude that, in this in vitro system, excess (active) PM cholesterol rapidly reaches mitochondria where, as the rate-limiting substrate, it stimulates 27-HC biosynthesis. The oxysterol product then promotes the rapid degradation of HMGR, along with other homeostatic effects. The regulation of 27-HC production by the active excess of PM cholesterol can thus provide a feedback mechanism in the homeostasis of PM cholesterol.

We review evidence that sterols can form stoichiometric complexes with certain bilayer phospholipids, and sphingomyelin in particular. These complexes appear to be the basis for the formation of condensed and ordered liquid phases, (micro)domains and/or rafts in both artificial and biological membranes. The sterol content of a membrane can exceed the complexing capacity of its phospholipids. The excess, uncomplexed membrane sterol molecules have a relatively high escape tendency, also referred to as fugacity or chemical activity (and, here, simply activity). Cholesterol is also activated when certain membrane intercalating amphipaths displace it from the phospholipid complexes. Active cholesterol projects from the bilayer and is therefore highly susceptible to attack by cholesterol oxidase. Similarly, active cholesterol rapidly exits the plasma membrane to extracellular acceptors such as cyclodextrin and high-density lipoproteins. For the same reason, the pool of cholesterol in the ER (endoplasmic reticulum) increases sharply when cell surface cholesterol is incremented above the physiological set-point; i.e., equivalence with the complexing phospholipids. As a result, the escape tendency of the excess cholesterol not only returns the plasma membrane bilayer to its set-point but also serves as a feedback signal to intracellular homeostatic elements to down-regulate cholesterol accretion.

The cholesterol content of the endoplasmic reticulum (ER) and the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) imbedded therein respond homeostatically within minutes to changes in the level of plasma membrane cholesterol. We have now examined the roles of sterol regulatory element-binding protein (SREBP)-dependent gene expression, side chain oxysterol biosynthesis, and cholesterol precursors in the short term regulation of ER cholesterol levels and HMGR activity. We found that SREBP-dependent gene expression is not required for the response to changes in cell cholesterol of either the pool of ER cholesterol or the rate of cholesterol esterification. It was also found that the acute proteolytic inactivation of HMGR triggered by cholesterol loading required the conversion of cholesterol to 27-hydroxycholesterol. High levels of exogenous 24,25-dihydrolanosterol drove the inactivation of HMGR; lanosterol did not. However, purging endogenous 24,25-dihydrolanosterol, lanosterol, and other biosynthetic sterol intermediates by treating cells with NB-598 did not greatly affect either the setting of their ER cholesterol pool or the inactivation of their HMGR. In summary, neither SREBP-regulated genes nor 27-hydroxycholesterol is involved in setting the ER cholesterol pool. On the other hand, 27-hydroxycholesterol, rather than cholesterol itself or biosynthetic precursors of cholesterol, stimulates the rapid inactivation of HMGR in response to high levels of cholesterol.

Adding cholesterol to monolayers of certain phospholipids drives the separation of liquid-ordered from liquid-disordered domains. The ordered phases appear to contain stoichiometric complexes of cholesterol and phospholipid. Furthermore, it has been suggested that the cholesterol in these complexes has a low chemical activity compared to that of the free sterol; i.e., that in excess of the phospholipid binding capacity. We have now tested the hypothesis that the membrane intercalator 1-hexadecanol (HD) similarly associates with phospholipids and thereby displaces the complexed cholesterol. HD introduced into monolayers of pure dimyristoylphosphatidylcholine generated highly condensed (stable and solid) domains. In contrast, the phase behavior of mixed monolayers of the phospholipid, sterol, and alcohol suggested that HD could substitute for cholesterol mole for mole in promoting liquid-ordered domains. We also found that the transfer of cholesterol from mixed monolayers to aqueous cyclodextrin was greatly stimulated by the presence of HD, but only at levels sufficient to competitively displace the sterol from the phospholipid. This enhanced efflux was interpreted to reflect an increase in uncomplexed cholesterol. We conclude that HD forms complexes with dimyristoylphosphatidylcholine that are surprisingly similar to those of cholesterol. HD competitively displaces cholesterol from the phospholipid and thereby increases its chemical activity.

Cholesterol is predicted to associate more strongly with the outer than the inner leaflet of plasma membrane bilayers based on the relative in vitro affinities of their phospholipids. Complex formation with the high-affinity species (especially saturated sphingomyelins) is said to reduce the chemical activity (escape potential or fugacity) of the sterol. We therefore tested the hypothesis that scrambling the sidedness of plasma membrane phospholipids of intact cells will increase the chemical activity of outer surface cholesterol. Upon activating the plasma membrane scramblase in intact human red cells by introducing ionomycin to raise cytoplasmic Ca++, phosphatidylserine became exposed and, concomitantly, the chemical activity of exofacial cholesterol was increased. (This was gauged by its susceptibility to cholesterol oxidase and its rate of transfer to cyclodextrin.) Similar behavior was observed in human fibroblasts. Two other treatments known to activate cell surface cholesterol (namely, exposure to glutaraldehyde and to low-ionic-strength buffer) also brought phosphatidylserine to the cell surface but by a Ca++-independent mechanism. Given that phospholipid scrambling is important in blood coagulation and apoptosis, the concomitant activation of cell surface cholesterol could contribute to these and other pathophysiological signaling processes.

We tested the hypothesis that certain membrane-intercalating agents increase the chemical activity of cholesterol by displacing it from its low activity association with phospholipids. Octanol, 1,2-dioctanoyl-sn-glycerol (a diglyceride), and N-hexanoyl-D-erythrosphingosine (a ceramide) were shown to increase both the rate of transfer and the extent of equilibrium partition of human red blood cell cholesterol to methyl-beta-cyclodextrin. These agents also promoted the interaction of the sterol with two cholesterol-specific probes, cholesterol oxidase and saponin. Expanding the pool of bilayer phospholipids with lysophosphatides countered these effects. The three intercalators also protected the red cells against lysis by cholesterol depletion as if substituting for the extracted sterol. As is the case for excess plasma membrane cholesterol, treating human fibroblasts with octanol, diglyceride, or ceramide stimulated the rapid inactivation of their hydroxymethylglutaryl-CoA reductase, presumably through an increase in the pool of endoplasmic reticulum cholesterol. These data supported the stated hypothesis and point to competition between cholesterol and endogenous and exogenous intercalators for association with membrane phospholipids. We also describe simple screens using red cells in a microtiter well format to identify intercalating agents that increase or decrease the activity of membrane cholesterol.

How do cells sense and control their cholesterol levels? Whereas most of the cell cholesterol is located in the plasma membrane, the effectors of its abundance are regulated by a small pool of cholesterol in the endoplasmic reticulum (ER). The size of the ER compartment responds rapidly and dramatically to small changes in plasma membrane cholesterol around the normal level. Consequently, increasing plasma membrane cholesterol in vivo from just below to just above the basal level evoked an acute (<2 h) and profound ( approximately 20-fold) decrease in ER 3-hydroxy-3-methylglutaryl-CoA reductase activity in vitro. We tested the hypothesis that the sharply inflected ER response to cholesterol is governed by the thermodynamic activity (fugacity) of plasma membrane cholesterol. The following two independent measures of plasma membrane cholesterol activity in human red cells and fibroblasts were used: susceptibility to cholesterol oxidase and cholesterol transfer to cyclodextrin. Both indicators revealed a threshold at the physiologic set point of plasma membrane cholesterol. Incrementing the phospholipid compartment in the plasma membrane with lysophosphatidylcholine, previously shown to decrease cholesterol oxidase susceptibility, reduced the transfer of plasma membrane cholesterol to cyclodextrin and to the ER. Conversely, the membrane intercalator, n-octanol, increased cholesterol oxidation, transfer, and ER pool size, perhaps by displacing cholesterol from plasma membrane phospholipids. We conclude that the activity of the fraction of cholesterol in excess of other plasma membrane lipids sets the cholesterol level in the ER. Cholesterol-sensitive elements therein respond by nulling the active plasma membrane pool, thereby keeping the cholesterol matched to the other plasma membrane lipids.

We probed the kinetics with which cholesterol moves across the human red cell bilayer and exits the membrane using methyl-beta-cyclodextrin as an acceptor. The fractional rate of cholesterol transfer (% s(-1)) was unprecedented, the half-time at 37 degrees C being ~1 s. The kinetics observed under typical conditions were independent of donor concentration and directly proportional to acceptor concentration. The rate of exit of membrane cholesterol fell hyperbolically to zero with increasing dilution. The energy of activation for cholesterol transfer was the same at high and low dilution; namely, 27-28 Kcal/mol. This behavior is not consistent with an exit pathway involving desorption followed by aqueous diffusion to acceptors nor with a simple one-step collision mechanism. Rather, it is that predicted for an activation-collision mechanism in which the reversible partial projection of cholesterol molecules out of the bilayer precedes their collisional capture by cyclodextrin. Because the entire membrane pool was transferred in a single first-order process under all conditions, we infer that the transbilayer diffusion (flip-flop) of cholesterol must have proceeded faster than its exit, i.e., with a half-time of <1 s at 37 degrees C.

Cells keep their cholesterol in balance by sensing its level in the endoplasmic reticulum and transducing this information into the expression of multiple homeostatic genes. Two recent papers from the Brown and Goldstein laboratory provide important new insights into how an integral ER protein, SCAP, mediates this process.

The dynamics of endolysosomal cholesterol were investigated in Niemann-Pick type C (NPC) cells and in human fibroblasts treated with class 2 amphiphiles to mimic NPC cells. We showed through new approaches that the massive pools of endolysosomal cholesterol in these cells are not trapped but, rather, circulate to the cell surface at about the normal rate. This flux spared NPC and amphiphile-treated cells from disruption by the extraction of their plasma membrane cholesterol with cyclodextrin. Nocodazole, a microtubule-depolymerizing agent, reversed the resistance of NPC and U18666A-treated cells to cholesterol depletion, apparently by reducing the flux of endolysosomal cholesterol to the plasma membrane. Neither nocodazole nor bafilomycin A1 (an inhibitor of the vacuolar proton pump) acted in the same way as the NPC mutation or class 2 amphiphiles: both agents decreased plasma membrane cholesterol at the expense of the endolysosomal pool and both blocked the actions of the amphiphile, U18666A. Finally, the resistance of NPC cells to lysis by amphotericin B was shown not to reflect a reduction in plasma membrane cholesterol arising from a block in lysosomal cholesterol export but rather the diversion of the amphotericin B to cholesterol-rich endolysosomes. We conclude that the large pool of endolysosomal cholesterol in NPC and amphiphile-treated fibroblasts is dynamic and that its turnover, as in normal cells, is dependent on microtubules.

Plasma membrane cholesterol both regulates and is regulated by effector proteins in the endoplasmic reticulum (ER) through a feedback system that is poorly understood. We now show that ER cholesterol varies over a fivefold range in response to experimental agents that act upon protein kinase C (PKC). Agents that activate Ca(2+)-dependent PKC [phorbol-12-myristate-13-acetate (PMA) and bryostatin 1] increased the level of ER cholesterol; inhibitors such as staurosporine and calphostin C decreased it. Rottlerin, a selective inhibitor of the PKC-delta isoform, also increased ER cholesterol. The esterification of plasma membrane cholesterol was altered by protein kinase C-directed agents in a corresponding fashion. Furthermore, the regulatory effect of plasma membrane cholesterol on the esterification of ER cholesterol was blocked by PKC-directed agents. These findings suggest that multiple protein kinase C isoforms participate in the regulation of ER cholesterol and therefore in cholesterol homeostasis., ((c)2002 Elsevier Science.)

The abundance of cell cholesterol is governed by multiple regulatory proteins in the endoplasmic reticulum (ER) which, in turn, are under the control of the cholesterol in that organelle. But how does ER cholesterol reflect cell (mostly plasma membrane) cholesterol? We have systematically quantitated this relationship for the first time. We found that ER cholesterol in resting human fibroblasts comprised approximately 0.5% of the cell total. The ER pool rose by more than 10-fold in less than 1 h as cell cholesterol was increased by approximately 50% from below to above its physiological value. The curve describing the dependence of ER on plasma membrane cholesterol had a J shape. Its vertex was at the ambient level of cell cholesterol and thus could correspond to a threshold. A variety of class 2 amphiphiles (e.g., U18666A) rapidly reduced ER cholesterol but caused only minor alterations in the J-curve. In contrast, brief exposure of cells to the oxysterol, 25-hydroxycholesterol, elevated and linearized the J-curve, increasing ER cholesterol at all values of cell cholesterol. This finding can explain the rapid action of oxysterols on cholesterol homeostasis. Other functions have also been observed to depend acutely on the level of plasma membrane cholesterol near its physiological level, perhaps reflecting a cholesterol-dependent structural or organizational transition in the bilayer. Such a physical transition could serve as a set-point above which excess plasma membrane cholesterol is transported to the ER where it would signal regulatory proteins to down-regulate its further accumulation.

What is the connection among the following three medical conditions: Niemann-Pick type C disease (a cause of mental retardation and early death), systemic lipidosis (in which an obscure side effect of numerous drugs transforms lysosomes into lamellar bodies), and holoprosencephaly (a catastrophe in embryonic development)? Recent evidence suggests that the pathogenesis in each use involves impaired sensing of cellular cholesterol.

The cholesterol in the lysosomes of cultured human fibroblasts was determined to constitute approximately 6% of the cell total. This pool was enlarged by as much as 10-fold in Niemann-Pick type C cells. Certain amphiphiles (e.g. U18666A, progesterone, and imipramine) caused lysosomal cholesterol to increase to similarly high levels at a rate of approximately 0.8% of cell cholesterol/h. Lysosomal cholesterol accumulated even in the absence of exogenous lipoproteins. Furthermore, nearly all of the lysosomal cholesterol in both of the two perturbed systems was shown to be derived from the plasma membrane. Oxysterols known to alter cholesterol movement and homeostasis blocked lysosomal cholesterol accretion in amphiphile-treated cells, suggesting that this process is regulated physiologically. Treating cells with amphiphiles slightly reduced the efflux of cholesterol from lysosomes and slightly increased the influx from the plasma membrane, causing the lysosomal cholesterol compartment to double in size in approximately 15 h. After more prolonged amphiphile treatments, a population of buoyant lysosomes appeared that exchanged cholesterol with the plasma membrane completely but slowly. Niemann-Pick type C lysosomes were similarly buoyant and sluggish. We conclude that cholesterol circulates bidirectionally between the plasma membrane and lysosomes. The massive accumulation of lysosomal cholesterol in the perturbed cells does not appear to reflect disabled lysosomal transport but rather the formation of lysosomes modified for lipid storage, i.e. lamellar bodies.

The esterification of cholesterol in homogenates of human fibroblasts was explored as a means of estimating the size of the pool of cholesterol associated with the endoplasmic reticulum (ER) in vivo. The rationale was that the acyl-coenzyme A:cholesterol acyltransferase (ACAT) in homogenates should have access only to cholesterol associated with the (rough) ER membrane fragments in which it resides. Reacting whole homogenates to completion with an excess of [14C]oleoyl-CoA converted approximately 0.1-2% of total cell-free cholesterol to [14C]cholesteryl esters. Control studies indicated that membranes not associated with ACAT did not contribute cholesterol to this reaction. The extent of in vitro cholesterol esterification varied with pretreatment of the cells. Exposing intact cells to serum lipoproteins, oxysterols, or sphingomyelinase increased cholesterol esterification in homogenates severalfold; exposing the cells to mevinolin or cholesterol oxidase had the opposite effect. The variation in cholesterol esterification did not correlate with either the total cellular cholesterol or the intrinsic activity of ACAT, neither of which was changed significantly by the pretreatments. Rather, the total amount of cholesterol esterified in homogenates paralleled the rate of cholesterol esterification in the corresponding intact cells. The pool of cholesterol esterified in vitro therefore appears to reflect that associated with the ER in vivo. Since several of the mechanisms keeping cell cholesterol under tight feedback control are themselves located in the ER, this pool might not only be regulated physiologically, but could, in turn, help to regulate homeostatic effector pathways.

Diverse amphiphiles act on cellular cholesterol metabolism as if signaling regulatory sites. One class (oxysterols) mimics the homeostatic effects of excess cell cholesterol, inhibiting cholesterol biosynthesis and stimulating plasma membrane cholesterol esterification. A second class of amphiphiles has effects precisely opposite to the oxysterols, i.e. they immediately inhibit plasma membrane cholesterol esterification and progressively induce 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and cholesterol biosynthesis. This second class of agents includes steroids, hydrophobic amines, phenothiazines, ionophores, colchicine, cytochalasins, and lysophosphatides, most of which interact with P-glycoproteins. These data support a general hypothesis describing cellular cholesterol homeostasis. (a) Proteins regulating sterol metabolism are embedded in intracellular membranes where their activities are governed by the local level of cholesterol. (b) Excess plasma membrane and lysosomal cholesterol circulates through those intracellular membranes and sets the homeostatic activities therein. (c) The two classes of agents mentioned above affect cholesterol homeostasis by increasing or decreasing, respectively, the ambient level of cholesterol at the sites of regulation.

The source of the cholesterol used for ester synthesis by cultured rat hepatoma cells was examined. The activities synthesizing and esterifying cholesterol co-distributed with RNA at a high buoyant density, presumably in the rough endoplasmic reticulum (RER). Cholesterol mass was undetectable in the RER, and the transfer of cholesterol synthesized in the RER to the cell surface was more than 100 times greater than was its esterification. Similarly, essentially all of the cholesterol liberated from ingested intracellular lipoproteins was recovered at the cell surface. The plasma membranes, which contained approximately 87% of cell cholesterol, provided > 100 times more cholesterol for esterification in the RER than did nascent cholesterol. The supply of cholesterol was rate-limiting for esterification in cell homogenates. Prior oxidation of plasma membrane cholesterol in intact cells reduced the acyl-CoA:cholesterol acyltransferase activity in isolates proportionately. Finally, cholesterol in hepatoma plasma membranes was a far better substrate for in vitro esterification than was that in fibroblast plasma membranes, red blood cell ghosts, or liposomes. We conclude that the level of saturation of acyl-CoA:cholesterol acyltransferase, controlled principally through the bidirectional movement of the substrate between plasma membranes and RER, plays a major role in the regulation of cholesterol esterification.

Where examined, cholesterol is synthesized in the endoplasmic reticulum; however, its precursor, zymosterol, is found mostly in the plasma membrane. The novel implication of these disparate findings is that zymosterol circulates within the cell. In tracing its movements, we have now established the following: (a) in human fibroblasts, zymosterol is converted to cholesterol solely in the rough ER. (b) Little or no zymosterol or cholesterol accumulates in the rough ER in vivo. (c) Newly synthesized zymosterol moves to the plasma membrane without a detectable lag and with a half-time of 9 min, about twice as fast as cholesterol. (d) The pool of radiolabeled zymosterol in the plasma membrane turns over rapidly, faster than does intracellular cholesterol. Thus, plasma membrane zymosterol is not stagnant. (e) [3H]Zymosterol pulsed into intact cells is initially found in the plasma membrane. It is rapidly internalized and is then converted to [3H] cholesterol. Half of the [3H]cholesterol produced returns to the plasma membrane within 30 min of the initial [3H]zymosterol pulse. (f) Nascent zymosterol accumulates in a buoyant sterol-rich intracellular membrane before it reaches the plasma membrane. This membrane also acquires nascent cholesterol, exogenous [3H]zymosterol pulsed into intact cells, and [3H]cholesterol synthesized from the exogenous [3H] zymosterol. These results suggest that at least one sterol moves rapidly and in both directions among the rough endoplasmic reticulum, a sterol-rich intracellular membrane bearing nascent cholesterol, and the plasma membrane.

The literature suggests that cholesterol and sphingomyelin might be essentially confined to plasma membranes in mammalian cells; however, this premise has thus far escaped a direct test. We explored the issue in three ways. First, we fractionated whole homogenates of cultured human fibroblasts by equilibrium sucrose density gradient centrifugation. We found that the profiles of cholesterol and sphingomyelin were indistinguishable from those of two plasma membrane markers, 5' nucleotidase and [3H]galactose, which was conjugated to the surface of intact cells from an exogenous donor by galactosyltransferase. Second, we determined the relative surface areas of intact cells from their uptake of 1-(4-trimethyl-amino)phenyl-6-phenylhexa-1,3,5-triene, a cationic fluorescent dye which partitions into but does not cross plasma membranes. Relative to human red cell ghosts, the apparent surface area of the fibroblasts was 17,500 microns2/cell while for canine hepatocytes, the value was 11,500 microns2/cell. The relative ratios of cell cholesterol to dye binding (hence, surface area) were quite similar in ghosts, fibroblasts, and liver cells; namely 1.0, 1.12, and 0.67, respectively. Finally, we found that the specific ratios of both cholesterol and sphingomyelin to 5' nucleotidase were only 10% less in gradient-purified plasma membranes than in whole homogenates. Similar results were obtained using an entirely different method of purification: two-phase aqueous partition. The cholesterol and sphingomyelin in fractions rich in other membranes was closely proportional to their 5' nucleotidase content, suggesting that the presence of these lipids reflected contamination by plasma membrane fragments. The 5' nucleotidase/phospholipid ratio in the purified plasma membrane fraction was roughly twice that in whole cells. We conclude that the compartment marked by 5' nucleotidase in cultured human fibroblasts contains approximately 90% of the two named lipids and half the cell phospholipid phosphorus.

We report the results of experiments which show that cholesterol transfer between membranes cannot proceed by aqueous diffusion, as widely held, but must involve a more complex mechanism. (a) The rate of transfer of [3H]cholesterol from red blood cells was found to vary inversely with the size of the acceptor particle (ghosts, vesicles of ghosts, liposomes, and plasma lipoproteins). (b) The transfer of [3H]cholesterol from red blood cells to ghosts was accelerated by the presence of plasma, even though the plasma competed with the ghosts as an acceptor. (c) The rate of transfer of [3H]cholesterol from red blood cells to ghosts decreased to zero with increasing dilution but was not simply second-order. (d) The cholesterol in retinal rod disc membranes is not at equilibrium with plasma lipoproteins in that disc cholesterol increased when the homogenates were incubated in vitro with plasma. (e) The kinetics of cholesterol transfer cannot be limited by unstirred layer effects since the transfer of lysolecithin in the same system was faster than that of cholesterol by 3 orders of magnitude. The simplest model compatible with all the data suggests a two-step pathway involving a first-order followed by a second-order process. The first step could be a unimolecular activation event, perhaps the movement of the sterol in the donor particle to a more exposed (hydrated) position. In the second step, the activated sterol would be transferred during transient collisions between donor and acceptor particles. When collision is not rate-limiting, the overall process would appear to be simply first-order, hence kinetically indistinguishable from the aqueous diffusion mechanism. The activation-collision model thus not only rationalizes our data but is also consistent with the simpler kinetics previously reported for the transfer of both membrane phospholipids and sterols.

Cholesterol appears to be abundant on both sides of the human erythrocyte membrane, but its precise distribution and rate of transmembrane movement are undetermined. Although the cholesterol in the intact cell normally was resistant to cholesterol oxidase, enrichment of cells with exogenous cholesterol or preincubation at very low ionic strength rendered the entire cholesterol pool susceptibility to attack. Under these experimental conditions, all of the membrane cholesterol was oxidized in a strictly first order fashion with a half-time as short as 10 s at 37 degrees C. Since the enzyme had access only to the outer membrane surface, these data suggest that the transmembrane movement (flip-flop) of cholesterol is extremely rapid. From an error analysis, we estimate an upper bound on the half-time of the transmembrane movement of cholesterol of 3 s at 37 degrees C. A physiological function for rapid sterol flip-flop is suggested.

The kinetics of transfer of [3H]cholesterol between human erythrocytes and plasma at 37 degrees C in physiological buffer had these features. 1) Cholesterol transfer was strikingly similar in both directions. 2) Transfer progressed to isotopic equilibrium in a monotonic, apparently first order fashion, except for a minor rapid component (approximately 15%) observed in the transfer of cholesterol from cells to plasma. 3) The mechanism of transfer was not via transient collisions in that the rate of the reaction was quite insensitive to the concentration of reactants over a wide range. 4) The mechanism of transfer did not involve specific, stable complex formation in that there was little difference in the behavior of erythrocytes and inside-out plasma membrane vesicles derived therefrom or between plasma and sonicated liposomes as acceptors. Furthermore, transfer was not affected by vigorous proteolysis of either the cells or the plasma. 5) The kinetics of transfer were fully compatible with diffusion of cholesterol through the aqueous compartment. This was shown by fitting our data to a rigorous model for diffusion equilibrium between three compartments. 6) The partition coefficient of [3H]cholesterol between red cells and buffer was shown to be 10(7). 7) The rate constants for cholesterol release from both red cells and plasma were approximately 1 X 10(-4) s-1 (t 1/2 approximately 2 h). The rate constant for cholesterol uptake into red cells was approximately 1 X 10(3) s-1 (t 1/2 approximately 1 ms). 8) The similarity of the corresponding kinetic constants among red cells, plasma, and liposomes suggests that phospholipids in a variety of physical forms are equivalent solvents for cholesterol. We conclude that despite its extremely low solubility in water, cholesterol moves between lipid compartments by aqueous diffusion.

The disposition of newly synthesized sterols in cultured human fibroblasts has been examined in this study. We began by demonstrating that cholesterol mass and exogenously added [3H]cholesterol both are markers for the plasma membrane, perhaps better than 5'-nucleotidase. Cells were incubated with radioactive acetate to label their endogenous sterols biosynthetically, treated with cholesterol oxidase to convert plasma membrane cholesterol to cholestenone, and then homogenized and spun to equilibrium on sucrose gradients. The density gradient profiles of the various organelles were monitored using these markers: plasma membrane, radioactive cholestenone; smooth endoplasmic reticulum, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase); and Golgi apparatus, galactosyltransferase. The buoyant density profiles of radioactive intracellular cholesterol and lanosterol both had a peak at 1.12 g/cm3, similar to 5'-nucleotidase and galactosyltransferase but not to HMG-CoA reductase. This result suggests that cholesterol biosynthesis is not taken to completion in the endoplasmic reticulum. Digitonin treatment shifted the profiles of both plasma membrane and intracellular cholesterol to higher densities. Pretreatment of intact cells with cholesterol oxidase abolished the digitonin shift of plasma membranes but not the intracellular cholesterol, indicating that these two membrane pools are not entirely physically associated. Because intracellular cholesterol was shifted more than any of the organelle markers, it must reside in a separate membrane. Since digitonin selectively shifts the density of membranes rich in cholesterol, we infer that newly synthesized cholesterol accumulates in such membranes prior to its delivery to the plasma membrane. Taken together, these results suggest that cholesterol may be concentrated for delivery to the plasma membrane by being synthesized from a sterol precursor such as lanosterol in a discrete but undefined intracellular membrane.

Three membrane properties were strikingly affected when the cholesterol of human erythrocytes, normally approximately 0.8 mol/mol of phospholipid (i.e. C/P approximately 0.8), was altered by equilibration with phospholipid dispersions of an appropriate cholesterol content. 1) While the sterol in intact red cell membranes of C/P less than or equal to 0.8 was resistant to cholesterol oxidase digestion, enrichment to C/P greater than or equal to 0.9 rendered the entire cholesterol pool sensitive to enzyme attack. Susceptibility to oxidation was reversed by removal of the excess cholesterol. Treatment of cells with 1 X 10(-4) M chlorpromazine also rendered the entire cholesterol pool susceptible to cholesterol oxidase. 2) Whereas ghosts with C/P less than or equal to 0.8 underwent invagination in low ionic strength, alkaline buffers to form inside-out vesicles, enrichment to C/P greater than or equal to 1.0 promoted right-side-out vesicle formation instead. This effect was mimicked by treatment with 2,4-dinitrophenol, an amphipath known to cause eversion of the membrane of red cells. In contrast, the presence of chlorpromazine, which promotes invagination of the intact red cell membrane, favored the formation of inside-out vesicles in ghosts. 3) The breakdown of ghosts into endocytic vesicles in dilute, alkaline media and the concomitant release and dissolution of the submembrane reticulum of spectrin and actin were retarded by excess cholesterol and promoted by its removal. This cholesterol effect was mimicked by exposing ghosts to chlorpromazine, but not dinitrophenol. Our data suggest that cholesterol may act physiologically both to stabilize the red cell membrane and to constrain its contour against invagination, and that red cell membrane cholesterol is maintained in vivo just below a critical level at which important organizational changes can occur.