The role of intracellular cholesterol transport in cholesterol homeostasis

How cholesterol is transported among the membranes of the cell is obscure. Similarly, the mechanisms governing the abundance of cell cholesterol are not entirely understood. It may be, however, that a link exists between the intracellular transport of cholesterol and its homeostasis. We propose that cholesterol circulates between the plasma membrane, which contains the bulk of the sterol, and organelle membranes, which contain only traces. A putative sensor translates small fluctuations in plasma membrane cholesterol into relatively large changes in this flux, thereby setting the magnitude of the intracellular pools. The cholesterol concentration in the endoplasmic reticulum and mitochondrial membranes then governs the activities of proteins embedded therein that mediate cholesterol transformations. This arrangement creates a feedback loop through which the intracellular effectors regulate the abundance of plasma membrane cholesterol.     

Plasma membranes contain half the phospholipid and 90% of the cholesterol and sphingomyelin in cultured human fibroblasts

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.     

Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies

The physiological importance of cholesterol in the cell plasma membrane has attracted increased attention in recent years. Consequently, the use of methods of controlled manipulation of membrane cholesterol content has also increased sharply, especially as a method of studying putative cholesterol-enriched cell membrane domains (rafts). The most common means of modifying the cholesterol content of cell membranes is the incubation of cells or model membranes with cyclodextrins, a family of compounds, which, due to the presence of relatively hydrophobic cavity, can be used to extract cholesterol from cell membranes. However, the mechanism of this activity of cyclodextrins is not completely established. Moreover, under conditions commonly used for cholesterol extraction, cyclodextrins may remove cholesterol from both raft and non-raft domains of the membrane as well as alter the distribution of cholesterol between plasma and intracellular membranes. In addition, other hydrophobic molecules such as phospholipids may also be extracted from the membranes by cyclodextrins. We review the evidence for the specific and non-specific effects of cyclodextrins and what is known about the mechanisms for cyclodextrin-induced cholesterol and phospholipid extraction. Finally, we discuss useful control strategies that may help to verify that the observed effects are due specifically to cyclodextrin-induced changes in cellular cholesterol.     

Cholesterol domains in biological membranes

Membrane cholesterol is distributed asymmetrically both within the cell or within cellular membranes. Elaboration of intracellular cholesterol trafficking, targeting and intramembrane distribution has been spurred by both molecular and structural approaches. The expression of recombinant sterol carrier proteins in L-cell fibroblasts has been especially useful in demonstrating for the first time that such proteins actually elicit intracellular and intra-plasma membrane redistribution of sterol. Additional advances in the use of native fluorescent sterols allowed resolution of transbilayer and lateral cholesterol domains in plasma membranes from cultured fibroblasts, brain synaptosomes and erythrocytes. In all three cell surface membranes, cholesterol is enriched in the inner, cytofacial leaflet. Up to three different cholesterol domains have been identified in the lateral plane of the plasma membrane: a fast exchanging domain comprising less than 10% of cholesterol, a slowly exchanging domain comprising about 30% of cholesterol, and a very slowly or non-exchangeable sterol domain comprising 50–60.

Of plasma membrane cholesterol. Factors modulating plasma membrane cholesterol domains include polyunsaturated fatty acids, expression of intracellular sterol carrier proteins, drugs such as ethanol, and several membrane pathologies (systemic lupus erythematosus, sickle cell anaemia and aging). Disturbances in plasma membrane cholesterol domains after transbilayer fluidity gradients in plasma membranes. Such changes are associated with decreased Ca²⁺ -ATPase and Na ⁺, K⁺ -ATPase activity. Thus, the size, dynamics and distribution of cholesterol domains within membranes not only regulate cholesterol efflux/influx but also modulate plasma membrane protein functions and receptor-effector coupled systems.     

Use of cyclodextrins to manipulate plasma membrane cholesterol content: Evidence, misconceptions and control strategies

The physiological importance of cholesterol in the cell plasma membrane has attracted increased attention in recent years. Consequently, the use of methods of controlled manipulation of membrane cholesterol content has also increased sharply, especially as a method of studying putative cholesterol-enriched cell membrane domains (rafts). The most common means of modifying the cholesterol content of cell membranes is the incubation of cells or model membranes with cyclodextrins, a family of compounds, which, due to the presence of relatively hydrophobic cavity, can be used to extract cholesterol from cell membranes. However, the mechanism of this activity of cyclodextrins is not completely established. Moreover, under conditions commonly used for cholesterol extraction, cyclodextrins may remove cholesterol from both raft and non-raft domains of the membrane as well as alter the distribution of cholesterol between plasma and intracellular membranes. In addition, other hydrophobic molecules such as phospholipids may also be extracted from the membranes by cyclodextrins. We review the evidence for the specific and non-specific effects of cyclodextrins and what is known about the mechanisms for cyclodextrin-induced cholesterol and phospholipid extraction. Finally, we discuss useful control strategies that may help to verify that the observed effects are due specifically to cyclodextrin-induced changes in cellular cholesterol.     

Cholesterol homeostasis and the escape tendency (activity) of plasma membrane 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.     

Activation mobilizes the cholesterol in the late endosomes-lysosomes of Niemann Pick type C cells

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.     

Binding of high density lipoproteins to cell receptors promotes translocation of cholesterol from intracellular membranes to the cell surface

Cultured cells have on their cell surface a specific high-affinity binding site (receptor) for high density lipoproteins (HDL) which appears to promote cholesterol efflux. In this study we characterized the cellular mechanisms involved in HDL receptor-mediated transport of cholesterol from cultured human fibroblasts and bovine aortic endothelial cells. HDL3, chemically modified by tetranitromethane (TNM-HDL3), is not recognized by this receptor and was used as a control for efflux not mediated by HDL receptor binding. HDL3 and TNM-HDL3 were found to be equally effective in causing efflux of plasma membrane cholesterol radiolabeled with [3H]cholesterol. However, HDL3 was much more effective than TNM-HDL3 in causing efflux of [3H]cholesterol associated with intracellular membranes. By measuring movement of endogenously synthesized [3H]cholesterol to the plasma membrane, and into the medium, we found that HDL3 induced a rapid movement of [3H]cholesterol from a preplasma membrane compartment to the plasma membrane that preceded [3H]cholesterol efflux. This effect was not observed with TNM-HDL3. Thus, receptor binding of HDL3 appears to facilitate removal of cellular cholesterol from specific intracellular pools by initiation of translocation of intracellular cholesterol to the plasma membrane.     

Cholesterol-rich intracellular membranes: a precursor to the plasma membrane

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.     

Cholesterol movement in Niemann-Pick type C cells and in cells treated with amphiphiles

Cholesterol accumulates to massive levels in cells from Niemann-Pick type C (NP-C) patients and in cells treated with class 2 amphiphiles that mimic NP-C disease. This behavior has been attributed to the failure of cholesterol released from ingested low density lipoproteins to exit the lysosomes. However, we now show that the rate of movement of cholesterol from lysosomes to plasma membranes in NP-C cells is at least as great as normal, as was also found previously for amphiphile-treated cells. Furthermore, the lysosomes in these cells filled with plasma membrane cholesterol in the absence of lipoproteins. In addition, we showed that the size of the endoplasmic reticulum cholesterol pool and the set point of the homeostatic sensor of cell cholesterol were approximately normal in NP-C cells. The plasma membrane cholesterol pools in both NP-C and amphiphile-treated cells were also normal. Furthermore, the build up of cholesterol in NP-C lysosomes was not a physiological response to cholesterol overload. Rather, it appeared that the accumulation in NP-C lysosomes results from an imbalance in the brisk flow of cholesterol among membrane compartments. In related experiments, we found that NP-C cells did not respond to class 2 amphiphiles (e.g. trifluoperazine, imipramine, and U18666A); these agents may therefore act directly on the NPC1 protein or on its pathway. Finally, we showed that the lysosomal cholesterol pool in NP-C cells was substantially and preferentially reduced by incubating cells with the oxysterols, 25-hydroxycholesterol and 7-ketocholesterol; these findings suggest a new pharmacological approach to the treatment of NP-C disease.     

Biochemical characterization of plasma membranes and intracellular membranes isolated from human platelets using Percoll gradients

Two kinds of membranes (plasma membranes and intracellular membranes) have been separated from human platelets by fractionation on Percoll gradients (successively at pH 7.4 and pH 9.6). On alkaline Percoll gradient, plasma membranes floated at low density, as shown with specific markers such as [3H]concanavalin A and monoacylglycerol lipase, whereas intracellular membranes sedimented in the higher densities and displayed a 5.6-12.4-fold enrichment in NADH diaphorase, antimycin insensitive NADH-cytochrome-c oxidoreductase and Ca2+-ATPase. Another criterion allowing differentiation of two membrane populations of human platelets was their lipid composition, which showed a cholesterol/phospholipid molar ratio of 0.5 in plasma membranes against 0.2 in intracellular membranes. Phospholipid analysis of the two kinds of membranes displayed also quite different profiles, since phosphatidylcholine increased from 30-32% in the plasma membrane to 52-66% in the intracellular membranes. This was at the expense of sphingomyelin (20-23% in plasma membrane, against 6.8-7.7% in intracellular membranes) and of phosphatidylserine (12-13% in plasma membrane, against 2-6% in intracellular membranes). Other striking differences between plasma membranes and intracellular membranes were obtained by SDS-polyacrylamide gel electrophoresis, which revealed the absence of actin and myosin in the intracellular membrane, whereas both proteins were present in significant amounts in plasma membranes. Finally, intracellular membranes but not plasma membranes were able to incorporate calcium. These results suggest that intracellular membrane fractions are derived from the dense tubular system and plasma membranes should correspond to the whole surface membrane of human platelets.     

Hexadecylphosphocholine alters nonvesicular cholesterol traffic from the plasma membrane to the endoplasmic reticulum and inhibits the synthesis of sphingomyelin in HepG2 cells

The synthetic lipid analogue, hexadecylphosphocholine is an antitumoral and antileishmanial agent that acts on cell membranes and can induce apoptosis. We have previously investigated the effect of hexadecylphosphocholine on the biosynthesis and intracellular transport of cholesterol in the human hepatoma HepG2 cell line. Here we show that the traffic of endocytosed-cholesterol from LDL to the plasma membrane and the transport of newly synthesized cholesterol from the endoplasmic reticulum to the plasma membrane were unaffected by alkylphosphocholine exposure. On the contrary, cholesterol traffic from the plasma membrane to the endoplasmic reticulum was drastically interrupted after 1 h of cell exposition to HePC and, consequently, the intracellular esterification of cholesterol was substantially decreased. Our results also demonstrate that this alkylphosphocholine exclusively affected the nonvesicular, energy-independent cholesterol traffic, without altering the vesicular transport. In addition, hydrolysis of plasma membrane sphingomyelin by exogenously added sphingomyelinase resulted in enhanced plasma-membrane cholesterol esterification, but sphingomyelinase treatment did not prevent the inhibition in cholesteryl ester formation caused by hexadecylphosphocholine. We also found that sphingomyelin synthesis was significantly inhibited in HepG2 cells after exposure to hexadecylphosphocholine. Since sphingomyelin and cholesterol are major lipid constituents of membrane raft microdomains, these results suggest that hexadecylphosphocholine could disturb membrane raft integrity and thence its functionality.     

Depletion of cellular cholesterol interferes with intracellular trafficking of liposome-encapsulated ovalbumin

Cholesterol is a major constituent of plasma cell membranes and influences the functions of proteins residing in the membrane. To assess the role of cholesterol in phagocytosis and intracellular trafficking of liposomal antigen, macrophages were treated with inhibitors of cholesterol biosynthesis for various time periods and levels of cholesterol depletion were assessed by thin layer chromatography. In control macrophages, cholesterol was present in the plasma membrane and in intracellular stores, as visualised by staining with the cholesterol-binding compound filipin, whereas macrophages treated with cholesterol inhibitors failed to stain with filipin. However, these macrophages were still capable of phagocytosis as evidenced by their internalisation of fluorescent-labelled bacteria and liposome-encapsulated Texas red labelled-ovalbumin, L(TR-OVA). While fluorescent ovalbumin (OVA) was consistently transported to the Golgi in macrophages incubated with L(TR-OVA), in cells treated with cholesterol inhibitors, OVA remained spread diffusely throughout the cytoplasm. Even though the mean fluorescence intensity of MHC class I molecules on cholesterol inhibitor-treated macrophages was equivalent to that of the control macrophages, the amount of MHC class I-liposomal OVA-peptide complex detected on the cell surface of cholesterol inhibitor-treated macrophages, was only 45.6 +/- 7.4% (n = 4, mean +/- SEM) of control levels after intracellular processing of L(OVA). We conclude that cholesterol depletion does not eliminate phagocytosis or MHC class I surface expression, but does affect the trafficking and consequently the MHC class I antigen-processing pathway.     

Leishmania donovani Infection Enhances Lateral Mobility of Macrophage Membrane Protein Which Is Reversed by Liposomal Cholesterol

BackgroundThe protozoan parasite Leishmania donovani (LD) reduces cellular cholesterol of the host possibly for its own benefit. Cholesterol is mostly present in the specialized compartment of the plasma membrane. The relation between mobility of membrane proteins and cholesterol depletion from membrane continues to be an important issue. The notion that leishmania infection alters the mobility of membrane proteins stems from our previous study where we showed that the distance between subunits of IFNγ receptor (R1 and R2) on the cell surface of LD infected cell is increased, but is restored to normal by liposomal cholesterol treatment.Conclusions/SignificancesTo our knowledge this is the first direct demonstration that LD parasites during their intracellular life cycle increases lateral mobility of membrane proteins and decreases F-actin level in infected macrophages. Such defects may contribute to ineffective intracellular signaling and other cellular functions.     

The isolation and partial characterization of the plasma membrane from Trypanosoma brucei.

Whole sheets of plasma membrane, each with their attached flagellum, were purified from Trypanosoma brucei. The method devised for their isolation included a new technique of cell breakage that used a combination of osmotic stress followed by mechanical sheer and avoided the problem of extreme vesiculation as well as the trapping of organelles in cell 'ghosts'. The purified membranes all contained the pellicular microtubular array. The antigenic surface coat was completely released from the plasma membrane during the isolation procedure. The membranes had a very high cholesterol/phospholipid ratio (1.54). A large proportion (42%) of the cellular DNA was recovered in the plasma-membrane fraction unless a step involving deoxyribonuclease treatment, which decreased the DNA content to less than 13%, was included before secrose-density gradient centrifugation. This step also aided the separation of plasma membranes from other cellular components. The ouabain-sensitive Na+ + K+-stimulated adenosine triphosphatase and adenylate cyclase co-purified with the plasma membranes. Although 5'-nucleotidase was thought to be a plasma-membrane component, it was easily detached from the membrane. The purified membranes were essentially free of L-alanine-alpha-oxoglutarate aminotransferase, L-asparte-alpha-oxoglutarate aminotransferase, malate dehydrogenase, oligomycin-sensitive adenosine triphosphatase, glucose 6-phosphatase, Mg2+-stimulated p-nitrophenyl phosphatase and catalase.     

Lysosomal membrane cholesterol dynamics

Although the majority of exogenous cholesterol and cholesterol ester enters the cell by LDL-receptor-mediated endocytosis and the lysosomal pathway, the assumption that cholesterol transfers out of the lysosome by rapid (minutes), spontaneous diffusion has heretofore not been tested. As shown herein, lysosomal membranes were unique among known organellar membranes in terms of cholesterol content, cholesterol dynamics, and response to cholesterol-mobilizing proteins. First, the lysosomal membrane cholesterol:phospholipid molar ratio, 0.38, was intermediate between those of the plasma membrane and other organellar membranes. Second, a fluorescence sterol exchange assay showed that the initial rate of spontaneous sterol transfer out of lysosomes and purified lysosomal membranes was extremely slow, t(1/2) >4 days. This was >100-fold longer than that reported in intact cells (2 min) and 40-60-fold longer than from any other known intracellular membrane. Third, when probed with several cholesterol-binding proteins, the initial rate of sterol transfer was maximally increased nearly 80-fold and the organization of cholesterol in the lysosomal membrane was rapidly altered. Nearly half of the essentially nonexchangeable sterol in the lysosomal membrane was converted to rapidly (t(1/2) = 6 min; fraction = 0.06) and slowly (t(1/2) = 154 min; fraction = 0.36) exchangeable sterol domains/pools. In summary, the data revealed that spontaneous cholesterol transfer out of the lysosome and lysosomal membrane was extremely slow, inconsistent with rapid spontaneous diffusion across the lysosomal membrane. In contrast, the very slow spontaneous transfer of sterol out of the lysosome and lysosomal membrane was consistent with cholesterol leaving the lysosome earlier in the endocytic process and/or with cholesterol transfer out of the lysosome being mediated by additional process(es) extrinsic to the lysosome and lysosomal membrane.     

Regulation of endoplasmic reticulum cholesterol by plasma membrane cholesterol

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.     

Movement of zymosterol, a precursor of cholesterol, among three membranes in human fibroblasts

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 cell biology of glycosphingolipids

Glycosphingolipids, a family of heterogeneous lipids with biophysical properties conserved from fungi to mammals, are key components of cellular membranes. Because of their tightly packed backbone, they have the ability to associate with other sphingolipids and cholesterol to form microdomains called lipid rafts, with which a variety of proteins associate. These microdomains are thought to originate in the Golgi apparatus, where most sphingolipids are synthesized, and are enriched at the plasma membrane. They are involved in an increasing number of processes, including sorting of proteins by allowing selectivity in intracellular membrane transport. Apart from being involved in recognition and signaling on the cell surface, glycosphingolipids may fulfill unexpected roles on the cytosolic surface of cellular membranes.     

Analysis of the distribution of cholesterol in the intact cell

We have used the enzyme cholesterol oxidase, which catalyzes the oxidation of cholesterol to cholest-4-en-3-one, to examine the distribution of cholesterol in cultured fibroblasts, Chinese hamster ovary cells, and isolated rat liver hepatocytes. While the plasma membrane normally was not attacked by cholesterol oxidase, we found that treating cells with low ionic strength buffer and glutaraldehyde rendered their cholesterol highly susceptible to oxidation. Most of the cholesterol was oxidized in all three cell types: 94% in fibroblasts, 92% in Chinese hamster ovary cells, and 80% in hepatocytes. Given that the enzyme had access only to the outer surface of the cells and cholesterol can move rapidly across the fixed plasma membrane, these values are taken to reflect the fraction of cellular cholesterol present in the plasma membrane. Additional experiments confirmed this interpretation. Fibroblasts were labeled with [3H]cholesterol by brief exposure to exogenous radiolabel and incubated with [14C]mevalonic acid to label cholesterol biosynthetically. Cholesterol oxidase attacked at least 97% of the exogenous label but as little as 10% of the biosynthetically labeled cholesterol. These data suggest that the cholesterol oxidase did not reach the intracellular pool and that cholesterol in the plasma membrane is not in rapid equilibrium with internal membranes. A study of the transfer of cholesterol to plasma from cells labeled biosynthetically with [3H]cholesterol and exogenously with [14C]cholesterol confirmed the different subcellular distribution of the two labels. These studies demonstrate that an unexpectedly high proportion of cell cholesterol is associated with plasma membranes and that this cholesterol pool can be rapidly and selectively labeled and oxidized. These features make cholesterol a useful specific marker for the plasma membrane.