DETERMINATION OF THE TRANSBILAYER DISTRIBUTION OF PLASMA MEMBRANE CHOLESTEROL

A novel determination of the transbilayer distribution of plasma membrane cholesterol | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results A novel determination of the transbilayer distribution of plasma membrane cholesterol View ORCID Profile Theodore L. Steck , View ORCID Profile Yvonne Lange doi: https://doi.org/10.1101/2025.11.13.687888 Theodore L. Steck a Department of Biochemistry and Molecular Biology, University of Chicago , Chicago, IL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Theodore L. Steck For correspondence: tsteck{at}bsd.uchicago.edu Yvonne Lange b Department of Pathology, Rush University Medical Center , Chicago, IL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yvonne Lange Abstract Full Text Info/History Metrics Preview PDF ABSTRACT The transbilayer distribution of plasma membrane cholesterol is unresolved; that is, several kinds of analyses have yielded contradictory results. We propose a new model for the sidedness of bilayer cholesterol. The underlying mechanism rests on two assumptions supported by a rich literature: (a) sterols associate with phospholipids in stoichiometric complexes, and (b) the complexes are filled to capacity in vivo. The sterol in each leaflet will then be the product of the abundance of its phospholipids and their sterol stoichiometry. We used literature values for the quantity of phospholipids in each leaflet and assigned a cholesterol:phospholipid stoichiometry of 1:1 for the exofacial leaflet and 1:2 for the endofacial leaflet. The model predicts that two thirds of the cholesterol in the human erythrocyte membrane is located in its outer leaflet, a 2:1 ratio. Previous simulations have inferred a similar sidedness based on the higher sterol affinity of the exofacial phospholipids. However, sterol affinity is irrelevant if the phospholipids are replete with sterol. The model also correctly predicts the overall cholesterol content of the bilayer to be ∼0.75 mole/mole phospholipid. Furthermore, it meets the requirement that the areas of the two leaflets be about equal. Generally speaking, in the absence of other factors, the sterol in one leaflet of any membrane bilayer will exceed that of the other when its phospholipids have a higher sterol stoichiometry and are fully complexed. Thus, the model not only estimates plasma bilayer cholesterol sidedness but provides an insight into how the plasma membrane is organized. HIGHLIGHTS The transbilayer distribution of plasma membrane cholesterol is unresolved. A new paradigm and model provide a novel mechanism to resolve this dilemma. Complexation of the cholesterol with phospholipids determines its sidedness. The model predicts that two-thirds of plasma membrane cholesterol is exofacial. The results meet the requirement that the areas of the leaflets are about equal. 1. Introduction The transbilayer distribution of the major lipids in the plasma membrane has been studied extensively, particularly in the human erythrocyte [ 1 - 8 ]. It is generally agreed that the polar lipids are asymmetrically disposed with almost all of the sphingomyelin, phosphatidylcholine and glycolipids located in the outer leaflet and the phosphatidylserine, phosphatidylethanolamine and phosphatidylinositides in the cytoplasmic leaflet [ 1 , 2 , 9 - 12 ]. The transbilayer distribution of the cholesterol remains unresolved. Various studies have suggested that the sterol is located mostly in one leaflet or the other or in a roughly even distribution [ 3 , 6 , 13 , 14 ]. For example, diametrically opposite asymmetries were found when different quenchers were applied to the surface of cells bearing fluorescent sterol surrogates [ 3 , 7 , 15 , 16 ]. Consensus is also lacking regarding the mechanism driving the sidedness of plasma membrane cholesterol. Some propose that the outer leaflet holds most of the cholesterol because it is enriched in saturated phospholipids with a relatively high sterol affinity [ 3 , 8 , 13 , 17 , 18 ]. It has also been argued that sterol is pumped from the inner to the outer leaflet [ 19 , 20 ]. Another contention is that a relative paucity of exofacial phospholipids is compensated for by extra cholesterol so as to equalize the surface area of the two leaflets and relieve interfacial tension [ 7 , 8 ]. Others have suggested a preference for the cytoplasmic leaflet [ 3 , 15 ]. In one study, it was proposed that the long chain sphingomyelins in the exofacial leaflet drive cholesterol to the contralateral side [ 21 ]. Another report inferred that phosphatidylserine holds the sterol in the endofacial leaflet [ 22 ]. In addition, theoretical modeling and simulations have implicated the material properties of the bilayer in determining cholesterol sidedness [ 8 , 13 , 14 , 17 , 23 , 24 ]. Still other factors have been considered [ 16 ]. We now propose a novel mechanism governing the transbilayer distribution of plasma membrane cholesterol: the phospholipids in the two leaflets are filled to capacity. There are two premises. One is that sterols form discrete associations with phospholipids. After McConnell et al. [ 25 ], these associations can be described as readily reversible noncovalent complexes, unitary or in high order clusters. A crucial attribute is their stoichiometric equivalence point, observed experimentally as a threshold in the titration of the phospholipid with the sterol [ 18 , 26 - 28 ]. The second premise is that the phospholipids in the plasma membrane are filled to capacity, i.e., to stoichiometric equivalence with the sterol. These assumptions are evaluated in the Discussion. We propose a new model based on this simple mechanism. If the phospholipids in each leaflet are essentially fully complexed with the sterol at their stoichiometric equivalence point, the distribution of the phospholipids will determine that of the sterol. The application of literature values leads to the prediction that two thirds of the cholesterol in the human erythrocyte bilayer is exofacial. This analysis is supported by two additional results. Unlike other mechanistic models, it correctly predicts the overall cholesterol concentration in the membrane. It also satisfies the expectation that the areas of the two leaflets are essentially equal. An early version of this study has been published as a preprint [ 29 ]. 2. Methods The stoichiometric complex model makes the following assumptions. (1) Cholesterol is in diffusional equilibrium within and between the liquid leaflets of the plasma membrane bilayer [ 28 , 30 ]. (2) Cholesterol associates stoichiometrically with bilayer phospholipids (see Discussion) [ 18 , 26 , 28 , 31 ]. (3) Plasma membrane cholesterol is maintained homeostatically at the stoichiometric equivalence point of the phospholipids [ 28 , 30 , 32 ]. The molar cholesterol/phospholipid ratio in each leaflet will therefore be equal to the overall stoichiometry characteristic of its complexes. (4) Consequently, the cholesterol in each leaflet is given by the product of the abundance of its phospholipid and the stoichiometry of its complexes. (Only when the phospholipids are incompletely filled with cholesterol do their affinities affect their fractional saturation and hence the distribution of the sterol among them.) We define C t , P t , C o , P o , C i and P i as the total (t) moles of cholesterol (C) and phospholipid (P) in the bilayer and in its outer (o) and inner (i) leaflets, respectively. The phospholipids in each compartment are fully complexed with cholesterol at stoichiometries n o and n i mole/mole. We express this requirement as C/P = C:P (mole/mole). It follows that the cholesterol in the leaflets are given by The overall bilayer cholesterol to phospholipid ratio, α, is given by: Then, substituting from Eqs. 1 and 2 , Rearranging gives Table 1 outlines how the stoichiometric complex model predicts the concentration and transbilayer distribution of sterols in the plasma membrane. Tables 2 - 4 then apply the model. Rows 1 and 2 in each table give the values assumed for the amount of the phospholipid in each leaflet and their sterol stoichiometries. The crux of the model is that, because the phospholipids are filled to capacity, their sterol stoichiometry (C:P) sets the cholesterol concentration (C/P) in each bilayer leaflet. It follows that the cholesterol content in each leaflet in row 3 is given by the corresponding phospholipid abundance in row 1 multiplied by its stoichiometry in row 2. Row 4 gives the cholesterol concentrations, C/P, obtained by dividing row 3 by row 1. The values for the bilayer as a whole (namely, C t and C t /P t ) are calculated from the values for the two leaflets. The small amounts of glycosphingolipids and minor phospholipids are ignored for want of sufficient data; however, their possible effect on the transbilayer distribution of cholesterol would not undermine the model. View this table: View inline View popup Download powerpoint Table 1: The stoichiometric complex model. Symbols refer to the equations given above. View this table: View inline View popup Download powerpoint Table 2: The stoichiometric complex model applied to the human erythrocyte bilayer 3. Results Table 2 applies the model to the human erythrocyte membrane. The abundance of lipids is expressed in moles, scaled to a total bilayer phospholipid, P t , of one mole. We estimated the distribution of phospholipids between the two leaflets in row 1 by assuming that the outer leaflet is composed entirely of sphingomyelin and phosphatidylcholine [ 1 , 5 ]. These two lipids contribute slightly more than half of the total phospholipid [ 4 , 33 ]. We therefore set the abundance of the phospholipid in the outer leaflet at P o = 0.50 moles. The inner leaflet will then contain the other phospholipid species plus a few percent each of the phosphatidylcholine and sphingomyelin [ 1 ]; they sum to P i = 0.50 moles. The stoichiometry of the outer leaflet lipids in row 2 of Table 2 was assigned the value of its major constituents, phosphatidylcholine and sphingomyelin; namely, C o :P o = 1.0 mole/mole [ 31 ]. The stoichiometry of the complexes in the cytoplasmic leaflet was given the value determined for dioleoylphosphatidylcholine, C i :P i = 0.5 mole/mole, reflecting the unsaturated phospholipids that predominate in this leaflet [ 4 , 31 ]. Since P o = P i , the overall stoichiometry of the bilayer is simply the average of C o :P o and C i :P i ; that is, C t :P t = 0.75 (row 2 in Table 2 ). The abundance of the cholesterol in each leaflet (row 3) was estimated by multiplying the abundance of the phospholipid in each compartment (row 1) by its corresponding sterol stoichiometry in row 2 ( Eqs. 1 and 2 ). This gives values for the cholesterol in the outer and inner leaflets of C o = 0.50 mole x 1.0 mole/mole = 0.50 moles and C i = 0.50 mole x 0.50 mole/mole = 0.25 moles. These values are also those calculated using Eqs. 7 and 8 . Row 4 gives the cholesterol concentrations (C/P) obtained by dividing the values in row 3 by those in row 1. Concentrations can be converted to mole fractions, C/(C+P). Row 3 in Table 2 delivers the result of interest. The predicted ratio of outer to inner leaflet cholesterol in the human erythrocyte bilayer is C o /C i = 2. That is, the two leaflets contain 67% and 33% of the cholesterol, respectively. The total bilayer cholesterol concentration is predicted to be C t /P t = 0.75. This value matches the experimental results for human red cells: C t /P t ≈ 0.70-0.80 [ 7 , 33 - 36 ]. Do the results in Table 2 predict an equal surface area for the two leaflets? Qualitatively, the outer leaflet has one third more total lipid than the contralateral leaflet but the greater saturation of its phospholipid chains make it more condensed by roughly that amount. We quantified their relative areas as follows. Two compositions were tested for the outer leaflet: either (a) 0.5 moles of dipalmitoylphosphatidylcholine (DPPC) plus 0.5 mole of cholesterol or (b) 0.25 moles of sphingomyelin and 0.25 moles of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) plus 0.5 moles of cholesterol. The molecular cross-section (area of total lipid) in each case would be ∼40 Å 2 /molecule at 30 mN/m, a surface pressure typical of plasma membranes [ 13 , 37 - 39 ]. The relative molecular area of the exofacial leaflet is therefore approximately (0.5 moles phospholipid + 0.5 moles cholesterol) x 40 Å 2 /molecule x NA (Avogadro’s number). That is, ∼40 x NA Å 2 . For the endofacial leaflet, we assumed 0.5 moles of the unsaturated phospholipid, dioleoylphosphatidylcholine (DOPC) plus 0.25 moles of cholesterol ( Table 2 ). This mixture has an overall molecular cross-section of ∼52 Å 2 /molecule at a surface pressure of 30 mN/m [ 13 , 40 , 41 ]. The relative molecular area of the inner leaflet is then approximately (0.5 moles phospholipid + 0.25 moles cholesterol) x 52 Å 2 /molecule x NA. That is, ∼39 x NA Å 2 . Thus, despite the uncertainties, idealizations and approximations of these estimates, our results meet the expectation that the two bilayer leaflets have closely similar areas. 3.1 Tests of the model The tidy outcome in Table 2 may not be accurate or apply to other cells. We therefore tested a few alternatives. In Table 3 , we changed the stoichiometry of the outer leaflet in row 2 to 0.90 mole/mole without changing the other parameters. The resulting bilayer cholesterol concentration was C t /P t = 0.70. This is within the physiological range of C t /P t ; namely, 0.70-0.80 [ 7 , 33 - 36 ]. In this scenario, 64% of the cholesterol is exofacial and the cholesterol asymmetry would be C o /C i = 1.8 instead of 2.0. Thus, the model tolerates modest variation in the stoichiometry of the complexes. View this table: View inline View popup Download powerpoint Table 3: The stoichiometric complex model for bilayers with an alternative stoichiometry The abundance of the phospholipids in the two leaflets of the plasma membrane might not always be equal. In this case, the cholesterol concentration in the bilayer, C t /P t , is given by a general expression derived from Eq. (3) , An illustration is provided in Table 4 . Here, the values of P o and P i in row 1 were changed to View this table: View inline View popup Download powerpoint Table 4: The stoichiometric complex model for bilayers with unequal P o and P i 0.55 and 0.45 moles. Because P o ≠ P i , the value for C t /P t in row 2 is not the average of C o /P o and C i /P i . As derived from Eq. (9) , it has the value C t /P t = 0.775 mole/mole. This prediction is well within the experimentally observed range of C t /P t ∼0.70-0.80 [ 7 , 33 - 36 ]. The ratio of outer to inner leaflet cholesterol in this case would be 2.4 with 71% of the cholesterol in the outer leaflet. Thus, the model yields results compatible with the experimentally determined erythrocyte membrane cholesterol concentration within a modest range in values for the stoichiometry and transbilayer distribution of the phospholipids. 4. Discussion There is no consensus in the literature concerning the transverse asymmetry of cholesterol in the plasma membrane or the mechanism by which it is established. We approached the issue with a new mechanistic model and tested it with experimental values from the literature. The central premise is that plasma membrane bilayer phospholipids are naturally fully complexed with cholesterol at their stoichiometric equivalence point. That this is the case for the human red cell plasma membrane can be inferred from the fact that the cholesterol concentration, C t /P t ∼0.70-0.80 [ 7 , 33 - 36 ], is just that given by experimental estimates of the sterol capacity of its phospholipids ( Table 2 ) [ 31 ]. Furthermore, that the red cell phospholipids are filled to capacity is shown by the dramatic rise in the accessibility of the cholesterol incremented slightly above its resting level [ 31 , 35 , 36 ]. The model predicts that two thirds of the cholesterol in the human red cell membrane bilayer resides in its outer leaflet ( Table 2 ). A similar value was obtained from a persuasive experiment based on the quenching of a fluorescent sterol surrogate [ 7 ]. Our analysis also accurately predicts the experimentally determined cholesterol concentration in the membrane, C/P ∼0.75 mole/mole. Furthermore, it concurs with the requirement that the surface areas of the two leaflets be about the same. 4.1. But do sterols form complexes with phospholipids? There is persuasive experimental evidence supporting this premise. Sterol-phospholipid associations are manifested by the well known phenomenon of membrane condensation [ 25 , 42 , 43 ]. They are promoted by multiple factors: the shielding of the sterol molecules from water under an umbrella of phospholipid head groups; favorable van der Waals interactions; hydrogen bonding; and polar contacts [ 26 , 44 ]. These factors have been assumed to create a nonspecific favorable energy of interaction or excess Gibbs free energy of solvation that drives the partition of sterols into bilayers [ 45 - 49 ]. However, sterols and phospholipids do not simply mingle in the bilayer; rather, they associate stoichiometrically with stereochemical specificity. This has been inferred from nuclear magnetic resonance spectroscopy [ 50 ], the phase behavior of binary mixtures in Langmuir monolayers [ 25 , 40 , 51 , 52 ], cryoelectron microscopy [ 53 ], computer simulations [ 42 , 54 , 55 ], X-ray lamellar diffraction [ 56 ], chemical cross-linking [ 43 ] and theoretical calculations [ 57 ]. Other evidence supporting complexes has been reviewed [ 26 , 41 , 53 , 58 , 59 ]. They could be short-lived, unitary or associated in clusters [ 25 , 57 , 60 ]. [Phospholipids can also form similar stoichiometric complexes with amphiphiles such as long-chain alcohols [ 61 - 64 ].] The following characteristics of sterol/phospholipid complexes are illustrated in Figure 1 . Download figure Open in new tab Fig. 1. Stoichiometric associations of cholesterol and phospholipids in bilayers. Hypothetical isotherms for the association of cholesterol with bilayer phospholipids were generated as previously described [ 66 , 69 ]. Chemically active (uncomplexed, available) cholesterol is plotted as a function of bilayer sterol concentration. Curve 4 depicts a bilayer with a relatively high phospholipid affinity for cholesterol and a stoichiometry of C:P = 1:1 such as observed for dipalmitoylphosphatidylcholine and sphingomyelin [ 31 ]. The sterol remains complexed until it nears stoichiometric equivalence. Curve 3 has the same 1:1 stoichiometry as curve 4 but a six-fold weaker affinity; hence, an earlier takeoff. It could represent 1-palmitoyl-2-oleoylphosphatidylcholine, POPC [ 31 ]. Curve 2 shows competitive displacement of the sterol from phosphatidylcholine complexes by a hypothetical amphiphile such as hexadecanol [ 63 , 64 ]. The stoichiometry of the association in this example is the same as that in curves 3 and 4 (C:P = 1:1) but the apparent affinity is much weaker. Curve 1 has a stoichiometry of C:P = 1:2, that reported for dioleoylphosphatidylcholine [ 31 ]. Its predicted threshold is therefore C/P = 0.50 but its takeoff starts earlier due to the weak affinity of the complex. Sterol-phospholipid affinity. The relative strength of these associations varies with the fatty acyl chains and head groups of the phospholipids as well as with the structure of the sterol [ 13 , 31 , 42 , 65 ]. How affinity shapes the concentration dependence of the association of sterols with phospholipids is illustrated by curves 3 and 4 in Figure 1 . These isotherms are taken to reflect two states of the sterol: that associated with phospholipids and the uncomplexed excess that accumulates in the bilayer beyond the stoichiometric equivalence point. That phospholipids are avid for cholesterol is also suggested by the threshold in the cholesterol dependence isotherms of sterol binding proteins [ 66 ]. Sterol-phospholipid stoichiometry. Turning points in isotherms such as those pictured in Figure 1 are invariably found in the titration by sterols of the phospholipids in monolayers, liposomes and natural membranes. These thresholds signify that complexed sterol is poorly reactive to probes and ligands while a super-stoichiometric excess is increasingly available [ 18 , 26 ]. Such probes include cholesterol oxidase [ 31 , 35 , 58 ], methyl-β-cyclodextrin [ 41 , 67 ], bacterial toxins [ 36 , 68 , 69 ] and a variety of membrane proteins [ 32 , 66 , 70 - 72 ]. The stoichiometries suggested by such thresholds are typically one or two phospholipids per cholesterol [ 25 , 31 , 41 , 58 ]. [However, high affinity ligands can pull the sterol from the phospholipids somewhat below their stoichiometric equivalence point and thereby lower the threshold of their binding curve [ 66 , 69 ].] Curves 1 and 4 in Figure 1 illustrate isotherms for complexes with stoichiometries of C:P = 1:2 and 1:1 respectively. Competition and displacement. Many intercalating amphiphiles increase the availability of bilayer cholesterol to probes [ 62 - 64 , 73 , 74 ]. They apparently associate with the phospholipids, displacing the sterol from the complexes mole for mole. A comparison of curves 2 and 4 in Figure 1 illustrates such competitive displacement. The binding of cholesterol by sterol-specific proteins must similarly compete with the bilayer phospholipids, as seen in their sigmoidal sterol-dependence isotherms [ 66 ]. Such competition would not occur if the sterol were not complexed. Uncomplexed cholesterol has a high chemical potential. The thresholds in isotherms like those in Figure 1 are taken to manifest the transition of the sterol in phospholipid membranes from a low to a high chemical activity near their stoichiometric equivalence point [ 18 , 25 , 26 , 31 , 41 , 57 ]. That this behavior in fact reflects a change in the chemical activity of cholesterol has been demonstrated directly [ 67 ]. Thresholds in chemical activity are characteristic of complexes but not of simple nonideal solutions. It appears that stoichiometric complexes are the most likely mechanism underlying the observed cholesterol dependence isotherms [ 41 ]. In particular, thresholds such as illustrated in Figure 1 do not reflect a phase change, given that they are observed in monolayers and vesicles containing a single phospholipid above its melting temperature [ 25 , 31 , 41 ]. Nor could such thresholds be a manifestation of sterol insolubility or crystallization from a super-saturated bilayer solution because the thresholds in chemical activity occur well below the solubility limit for bilayer cholesterol; typically, a mole fraction ∼0.67 [ 41 , 74 ]. Furthermore, sterol crystals have a low chemical activity while super-stoichiometric cholesterol has a high chemical activity. We conclude that isotherms like those in Figure 1 are a manifestation of stoichiometric complexes. 4.2. Implications of complexation If, as postulated here, the sterols and phospholipids in plasma membrane bilayers are near stoichiometric equivalence, their complexes will fill both phase-separated domains (rafts) and the continuum surrounding them. Complexes would therefore contribute significantly to the material properties of the membrane: its condensation, molecular order, thickness, permeability, viscosity, compressibility, deformability, various elasticities, intrinsic curvature and lateral phase heterogeneity [ 7 , 8 , 42 , 75 - 79 ]. Specific benefits conferred by the saturation of plasma membrane bilayers with sterols include a low water permeability (especially important for osmotically-vulnerable fresh water eukaryotes such as protozoa) and the imposition of a barrier against the entry of potentially deleterious amphiphiles such as xenobiotics [ 77 , 79 , 80 ]. The uncomplexed fraction of plasma membrane cholesterol, although only a few percent of the total, is chemically active and performs important complementary physiological functions [ 18 , 28 , 32 , 66 , 72 , 81 - 83 ]. It is the form that rapidly diffuses across the bilayer so as to equalize the chemical activity of the cholesterol in the two leaflets. In the case of the erythrocyte, it is the equilibration of plasma membrane cholesterol with plasma (lipo)proteins that keeps the bilayer phospholipids replete at their stoichiometric equivalence point over months in circulation [ 7 , 30 , 33 - 36 ]. The cholesterol in the plasma membrane of nucleated cells is similarly buffered by the equilibration of their active cholesterol with plasma (lipo)proteins. Just as important, there are elaborate metabolic mechanisms that serve to maintain the plasma membrane cholesterol of nucleated cells at stoichiometric equivalence with its phospholipids [ 28 , 32 , 68 ]. In particular, super-stoichiometric plasma membrane cholesterol serves as a feedback signal that circulates to the cell interior to regulate homeostatic elements keeping the plasma membrane phospholipids replete with cholesterol while minimizing any excess. These considerations suggest that the stoichiometric equivalence point of plasma membrane sterol and phospholipid is a sweet spot. Less sterol undermines the functions mentioned above while the persistence of a super-stoichiometric excess can be disruptive [ 34 , 77 , 84 - 88 ]. 4.3. Consideration of other mechanisms for sterol sidedness Various theoretical treatments have evaluated the influence of membrane stress and material properties such as intrinsic and spontaneous curvature, inter-leaflet coupling or differential leaflet area on the transbilayer distribution of cholesterol [ 8 , 13 , 14 , 17 , 23 , 24 ]. While our parsimonious model does not invoke these factors, their imposition could work against the sterol sidedness created by its complexation. On the other hand, such factors do not appear to have much effect in the case of human erythrocytes [ 78 ]. Some models have invoked the higher affinity of the exofacial phospholipids to predict the preponderance of the sterol in that leaflet [ 3 , 4 , 7 , 8 , 13 , 17 , 47 ]. There is an important difference, however, between an affinity mechanism and the stoichiometric complex mechanism proposed here, even though the sterol asymmetry in both cases is driven by the abundance of saturated phospholipids in the outer leaflet. A differential affinity mechanism can impose an asymmetrical transbilayer distribution but it does not set the concentration of the sterol in the membrane as does the stoichiometric complex model. Furthermore, the affinity of the phospholipids will not influence the cholesterol distribution in bilayers filled to their stoichiometric equivalence point. A recent publication proposed a novel mechanism that drives cholesterol to the exofacial leaflet of the plasma membrane [ 7 ]. It postulates a substantial deficit in the phospholipids in the outer leaflet with the consequent gap in its surface area filled by excess cholesterol. This model has been critiqued [ 11 , 89 , 90 ]. 4.4. Plasma membrane sterol sidedness in other cells While the profile of plasma membrane phospholipids varies among nucleated cells [ 91 ], their sidedness is well-regulated [ 92 ] and resembles that of human red cells [ 2 , 4 - 6 , 9 , 10 , 93 , 94 ]. Furthermore, the cholesterol concentration in the plasma membranes of nucleated cells [ 28 , 95 ] is similar to that in red cells, C t /P t ∼0.7-0.8 [ 7 , 33 - 36 ]. This accords with our view that the homeostatic setpoint in all of these cells is near the threshold in their sterol-binding curves; that is, near the stoichiometric capacity of their phospholipids [ 28 , 32 ]. Intracellular membranes differ from plasma membranes in that homeostatic mechanisms hold their phospholipids well below capacity [ 28 , 32 ]. The distribution of the sterol in the cytoplasmic membranes therefore reflects the affinity of their phospholipids rather than their stoichiometry, and the activity of their regulatory elements will vary with the fractional saturation of their membranes with the sterol. 5. Conclusion We anticipate that, in the absence of other influences, the cholesterol in any membrane bilayer will favor the side with the higher stoichiometry if the phospholipids in the two leaflets are fully complexed and are of similar abundance. This will generally be the exofacial side. Clearly, more detailed data for a variety of plasma membranes are needed to test the validity of the model. Authors contributions TLS and YL contributed to every phase of the work from its conception to its publication. Funding There is no external funding. Conflict of interest The authors declare no competing or financial interests. 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