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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.

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.

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.

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.

A general method for isolating plasma membranes is described. Vegetative amoebae of Dictyostelium discoideum were allowed to directly adsorb raw colloidal gold of particle diameter 10-20 nm. After quenching the gold surface, the cells were lysed and the lysates were diluted in 60% sucrose and centrifuged through a 65% sucrose cushion to selectively pellet the gold-laden membranes. Three generally applicable exogenous cell surface markers were used to follow the plasma membranes: intercalated [3H]cholesterol, octadecylrhodamine, and the adsorbed gold colloid itself. The isolates routinely contained approximately 60% of these tags, enriched approximately 15-fold with respect to protein. The recovery and degree of enrichment of contaminating markers in the plasma membrane fraction were lysosomes (3% and 1-fold); mitochondria (11% and 3-fold); rough endoplasmic reticulum, as reflected by RNA (3% and 0.7-fold); and DNA (9% and 4-fold). Membrane proteins and lipids were quantitatively solubilized from the gold by detergents. We conclude that this methodology provides an approach to the isolation of plasma membranes which compares favorably to existing techniques with respect to yield, purity, and ease.

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.

The kinetics of pyruvate transport across the isolated red blood cell membrane were studied by a simple and precise spectrophotometric method: following the oxidation of NADH via lactate dehydrogenase trapped within resealed ghosts. The initial rate of pyruvate entry was linear. Influx was limited by saturation at high pyruvate concentration. Pyruvate influx was greatly stimulated by increasing ionic strength in the outer but not the inner aqueous compartment. The Km ranged from 15.0 mM at mu = 0.05 to 3.7 mM at mu = 0.01, while the V went from 0.611 - 10(15) to 0.137 - 10(-15) mol - min-1 - ghost-1. Ionic strength was shown to affect the translocation step and not pyruvate binding. The energy of activation of pyruvate flux into resealed ghosts was 25 kcal/mol, similar to that found in intact red blood cells. Inhibitors of pyruvate influx included such anions as thiocyanate, chloride, bicarbonate, alpha-cyanocinnamate, salicylate and ketomalonate (but not acetate); noncompetitive inhibitors were phloretin, 1-fluoro-2,4-dinitrobenzene, 4-acetamido-4'-isothiocyanate-stilbene-2,2'-disulfonic acid and o-phenanthroline/CuSO4 mixtures. The last reagent, known to induce disulfide links in certain membrane proteins, blocked the ionic strength stimulation of pyruvate influx in this study.

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.

Band 3 is the major, membrane-spanning, approximately90 000 dalton polypeptide of the human erythrocyte membrane. To facilitate the analysis of its structural integration into the membrane, we have cleaved this protein in situ into large fragments and ascertained their disposition. Digestion of intact cells with chymotrypsin yielded band 3 fragments with apparent molecular weights of 38 000 and 55 000. Both fragments resisted elution by NaOH and acetic acid, suggesting that they are anchored in the apolar core of the membrane. Both pieces communicate with the extracellular space, and the 55 000 dalton species extends to the cytoplasmic surface as well. Digestion of unsealed ghosts with chymotrypsin produced a hydrophobic 17 000 dalton species, a segment of the 55 000 dalton fragment, which spans and is firmly anchored in the core of the membrane. Trypsin and papain at low concentration generated integral band 3 fragments of 52 000 daltons and released major band 3 fragments of less than or equal to 41 000 daltons from the cytoplasmic side of the membrane. The latter water-soluble polypeptides remained associated in discrete complexes which retained the capacity to bind glyceraldehyde-3-phosphate dehydrogenase. An interchain disulfide bond, which can be induced only at the cytoplasmic surface, cross-linked intact band 3, and certain of its water-soluble fragments. Finally, fragments of 23 000 daltons were generated from the innersurface domain by reacting disulfide-linked band 3 dimers with cyanide or reduced polypeptides with 2-nitro-5-thiocyanobenzoate. A provisional ordering of these fragments is proposed.

Plasma membranes purified from human red cells were Converted into small vesicleS by disruption in alkaline buffer of low ionic strength. Most of these vesicles were inside-out. The presence of divalent cations prevented this inversion. The inside-out vesicles were separatcd from right-side-out vesicles by centrifugration to equilibrium in dextran density gradients.