Cyclopamine inhibition of Sonic hedgehog signal transduction is not mediated through effects on cholesterol transport.

Cyclopamine is a teratogenic steroidal alkaloid that causes cyclopia by blocking Sonic hedgehog (Shh) signal transduction. We have tested whether this activity of cyclopamine is related to disruption of cellular cholesterol transport and putative secondary effects on the Shh receptor, Patched (Ptc). First, we report that the potent antagonism of Shh signaling by cyclopamine is not a general property of steroidal alkaloids with similar structure. The structural features of steroidal alkaloids previously associated with the induction of holoprosencephaly in whole animals are also associated with inhibition of Shh signaling in vitro. Second, by comparing the effects of cyclopamine on Shh signaling with those of compounds known to block cholesterol transport, we show that the action of cyclopamine cannot be explained by inhibition of intracellular cholesterol transport. However, compounds that block cholesterol transport by affecting the vesicular trafficking of the Niemann-Pick C1 protein (NPC1), which is structurally similar to Ptc, are weak Shh antagonists. Rather than supporting a direct link between cholesterol homeostasis and Shh signaling, our findings suggest that the functions of both NPC1 and Ptc involve a common vesicular transport pathway. Consistent with this model, we find that Ptc and NPC1 colocalize extensively in a vesicular compartment in cotransfected cells.

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.

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.

Four cholesterol-sensing proteins.

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.

Circulation of cholesterol between lysosomes and the plasma membrane.

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.

Cell adhesion to fibronectin regulates membrane lipid biosynthesis through 5'-AMP-activated protein kinase.

We have shown that attachment to a fibronectin substrate stimulates two pathways of lipid biosynthesis in cultured human fibroblasts. Detachment of these cells (mechanically, with trypsin, or by RGDS peptides) caused a significant decrease in their 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and in their incorporation of [3H]acetate into fatty acids. This inhibition was substantially reversed by the reattachment of cells to fibronectin substrates, but not to poly-L-lysine substrates or to fibronectin in solution. Inhibiting phosphoprotein phosphatase activity with okadaic acid blocked the recovery of both biosynthetic activities. Both 3-hydroxy-3-methylglutaryl-coenzyme A reductase and fatty acid biosynthesis are known to be inhibited by the action of 5'-AMP-activated protein kinase, which is activated by an increase in the level of AMP relative to ATP. For example, in our system, sodium azide and 2-deoxy-D-glucose increased the ratio of cellular AMP to ATP and caused a decrease in lipid biosynthesis. We then verified the prediction that detachment of cells from substrates also caused an increase in the AMP/ATP ratio. We therefore conclude that the attachment of cells to fibronectin promotes lipid biosynthesis, presumably in coordination with the cellular growth response evoked by attachment to the extracellular matrix.

The fate of cholesterol exiting lysosomes.

Cholesterol released from ingested low density lipoproteins in lysosomes moves both to the plasma membrane and to the endoplasmic reticulum (ER) where it is re-esterified. Whether cholesterol can move directly from lysosomes to ER or first must traverse the plasma membrane has not been established. To examine this question, the endocytic pathway of rat hepatoma cells was loaded at 18 degrees C with low density lipoproteins (LDL) labeled with [3H]cholesteryl linoleate, and the label then was chased at 37 degrees C. The hydrolysis of the accumulated ester proceeded linearly for several hours. Almost all of the released [3H]cholesterol moved to the plasma membrane rapidly and without a discernable lag. In contrast, the re-esterification in the ER of the released [3H]cholesterol showed a characteristic lag of 0.5-1 h. These data are inconsistent with direct cholesterol transfer from lysosomes to ER; rather, they suggest movement through the plasma membrane. Furthermore, we found that progesterone, imipramine and 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A) strongly inhibited the re-esterification of lysosomal cholesterol in the ER. However, contrary to previous reports, they did not block transfer of [3H]cholesterol from lysosomes to the cell surface. Therefore, the site of action of these agents was not at the lysosomes. We suggest instead that their known ability to block cholesterol movement from the plasma membrane to the ER accounts for the inhibition of lysosomal cholesterol esterification. These findings are consistent with the hypothesis that cholesterol released from lysosomes passes through the plasma membrane on its way to the ER rather than proceeding there directly. As a result, ingested cholesterol is subject to the same homeostatic regulation as the bulk of cell cholesterol, which is located in the plasma membrane.

Quantitation of the pool of cholesterol associated with acyl-CoA:cholesterol acyltransferase in human fibroblasts.

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.

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.

Cholesterol homeostasis is modulated by amphiphiles at transcriptional and post-transcriptional loci.

A variety of amphiphiles inhibit plasma membrane cholesterol esterification and induce 3-hydroxy-3-methylglutaryl-coenzyme A reductase accumulation in cultured cells; among these are steroids, hydrophobic amines, phenothiazines, ionophores, colchicine, and lysophosphatides. It has been proposed that these amphiphiles signal a sterol deficiency to regulatory sites by blocking the movement of plasma membrane cholesterol into the cell (Lange, Y., and Steck, T. L. 1994. J. Biol. Chem. 269: 29371-29374). If this were the case, these agents also should enhance transcription of sterol responsive genes and stabilize 3-hydroxy-3-methylglutaryl-coenzyme A reductase. As a test of this hypothesis, the effect of the amphiphiles on such transcriptional and post-transcriptional events was assessed. A mouse embryo cell line was transfected with a construct containing the promoter for the human low density lipoprotein receptor upstream of the DNA sequence coding for chloramphenicol acyltransferase (CAT). Incubation of these cells for 7-18 h with the aforementioned agents caused the level of expression of the promoter/CAT construct to increase 2- to 9-fold. We showed further that the amphiphiles stimulated 3-hydroxy-3 methylglutaryl-coenzyme A reductase activity by increasing gene transcription as well as by decreasing degradation of the enzyme. These are the predicted homeostatic responses to cell cholesterol deficiency. These findings support the hypothesis that certain amphiphiles falsely signal a cholesterol deficiency to the intracellular sites regulating cholesterol homeostasis.

Movement of 25-hydroxycholesterol from the plasma membrane to the rough endoplasmic reticulum in cultured hepatoma cells.

Oxysterols serve as both substrates and signal molecules in the cholesterol-utilizing pathways of mammalian cells. Their distribution and movement within these cells, however, have not been well characterized; therefore we have undertaken such an analysis. Radiolabeled cholesterol and 25-hydroxycholesterol were pulsed into the cell surface membranes of rat hepatoma cells and their esterification was determined. The esterification of both probes was stimulated by feeding cells lipoproteins, even though lipoprotein cholesterol might be viewed as a competitor. Unlabeled 25-hydroxycholesterol, another potential competitor, also stimulated the esterification of the cell-surface probes. Esterification of both sterols was inhibited by a variety of amphiphilic agents. This inhibition was reversed by unlabeled 25-hydroxycholesterol. In cells incubated at 15 degrees C the fractional rate of esterification of the oxysterol was more than 100 times greater than that of cholesterol. Furthermore, the time course of esterification of plasma membrane cholesterol but not that of 25-hydroxycholesterol, was lagged. In contrast, the rate of esterification of the two probes was similar in broken cells supplied with saturating cholesterol. Finally, the transfer of 25-hydroxycholesterol from red blood cells to plasma lipoproteins was approximately 2000-fold faster than that of cholesterol. We conclude that 25-hydroxycholesterol and cholesterol are moved between the plasma membrane and endoplasmic reticulum by a common transport mechanism but that the oxysterol enters this pathway much more rapidly, possibly through a passive transfer step akin to its unmediated transfer from red cells to plasma.

Progesterone blocks intracellular translocation of free cholesterol derived from cholesteryl ester in macrophages.

Macrophage foam cells must accommodate continuing fluxes of free cholesterol in spite of a greatly expanded store of cholesteryl ester. Though endogenous free cholesterol synthesis is suppressed, free cholesterol continues to enter the cell via endocytosis of oxidized/modified lipoproteins. It has been shown previously that this free cholesterol is released into the lysosomal compartment and rapidly transported to the plasma membrane prior to its esterification. A substantial amount of free cholesterol is also presented via the continuous hydrolysis of cholesteryl ester during the cholesteryl ester cycle. We addressed the question of whether the intracellular free cholesterol derived from the hydrolysis of cholesteryl ester formed a protected pool for rapid re-esterification. Incubation of macrophage foam cells with cyclic AMP to enhance cholesteryl ester hydrolysis, and with S58035 to inhibit acyl-CoA:cholesterol acyltransferase (ACAT) activity, led to conversion of cellular cholesteryl ester to free cholesterol and transport of this free cholesterol to the plasma membrane. Addition of progesterone, previously demonstrated to be an inhibitor of free cholesterol transport in other cell types, also led to conversion of cholesteryl ester to free cholesterol even though progesterone was only a weak inhibitor of ACAT activity. Free cholesterol in the plasma membrane was an important source of ACAT substrate to balance the constitutive hydrolysis of cholesteryl ester in cholesterol-loaded macrophages. Treatment of cells with progesterone, however, prevented free cholesterol derived from cholesteryl ester hydrolysis from moving to the plasma membrane. The sequestration of free cholesterol by progesterone could be reversed by incubation with human HDL3.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol homeostasis. Modulation by amphiphiles.

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.

Cholesterol movement from plasma membrane to rough endoplasmic reticulum. Inhibition by progesterone.

The effect of progesterone on the movement of sterols from the cell surface to the rough endoplasmic reticulum (ER) was examined in rat hepatoma cells. Plasma membranes were labeled exogenously with [3H]cholesterol or [3H]zymosterol. Translocation of the labeled sterols to the rough ER was inferred from their conversion to [3H]cholesteryl esters and [3H]cholesterol, respectively. Progesterone inhibited both of these reactions by more than 90%. The concentration for half-maximal inhibition was 0.7 microgram/ml. Progesterone did not inhibit acyl-CoA:cholesterol acyltransferase activity itself, since the steroid had no effect on the esterification of [3H]cholesterol synthesized in vitro in the rough ER from [3H]zymosterol. Moreover, the small amount of [3H]cholesterol synthesized from plasma membrane [3H]zymosterol in progesterone-treated intact cells was esterified at the same fractional rate as cholesterol in control cells. Subcellular fractionation of cells pulse-labeled with [3H]cholesterol and treated with progesterone suggested that the block in plasma membrane cholesterol transfer to the rough ER occurred at the level of the plasma membrane.

Role of the plasma membrane in cholesterol esterification in rat hepatoma cells.

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.

Isolation and function of a human endothelial cell C1q receptor.

It has been shown previously that cultured human venous and arterial endothelial cells (EC) bind C1q in a time- and dose-dependent manner. Cultured human endothelial cells express an average number of 5.2 x 10(5) binding sites/cell. In the present study the putative receptor for C1q (C1qR) was isolated from the membranes of 1-5 x 10(9) human umbilical cord EC by affinity chromatography on C1q-Sepharose. During isolation, C1qR was detected by its capacity to inhibit the lysis of EAC1q in C1q-deficient serum. The eluate from C1q-Sepharose was concentrated, dialysed and subjected to QAE-A50 chromatography and subsequently to gel filtration on HPLC-TSK 3000. C1qR filtered at an apparent molecular weight of 60 kDa. Purified C1qR exhibited an apparent molecular weight of 55-62 kDa in the unreduced state and a molecular weight of 64-68 kDa in reduced form. Two IgM monoclonal antibodies (mAb) D3 and D5 were raised following immunization of mice with purified receptor preparations. Both monoclonal antibodies increased the binding of (125)I-C1q to endothelial cells but F(ab')(2) anti-C1qR mAb inhibited the binding of a(125)I-C1q to EC in a dosedependent manner. The D3 mAb recognized a band of 54-60 kDa in Western blots of membranes of human EC and polymorphonuclear leukocytes. Previously, the authors showed that C1q induces the binding of IgM-containing immune complexes to EC. Therefore, it was hypothesized that during a primary immune response generation of IgM-IC may occur, resulting in binding and activation of C1, dissociation of activated C1 by C1 inhibitor and subsequent interaction of IgM-IC bearing C1q with EC-C1qR.

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.

Disposition of intracellular cholesterol in human fibroblasts.

We have examined the intracellular distribution of unesterified cholesterol in cultured human fibroblasts. Intact cells were treated with cholesterol oxidase to selectively transform cell surface cholesterol to cholestenone. Isopycnic centrifugation of homogenates showed that the cholestenone had a peak buoyant density of 1.13 g/cm3. The approximately 10% of total cholesterol which remained unoxidized was distributed in two peaks of roughly equal size: a sharp peak at approximately 1.09 g/cm3 and a broad peak centered at 1.18 g/cm3. When intact cells were incubated with exogenous [3H]cholesterol, the radiolabel entered the nonoxidizable pool in a temperature-dependent fashion with a half time of 3 h at 37 degrees C. This label initially was associated with the dense but not the buoyant peak of nonoxidized cholesterol. After 40 h, the buoyant peak also became labeled; both peaks then had a specific activity slightly less than the surface cholestenone. The buoyant density of the unoxidized cholesterol did not coincide with markers for the Golgi apparatus, endoplasmic reticulum, or lysosomes. However, two ingested markers of pinocytosis, calcein and horseradish peroxidase, comigrated with the dense peak of unoxidized cholesterol. That the size of the unoxidized cholesterol pool was greater in cells deprived of serum lipoproteins than in fed cells suggested that none of the intracellular cholesterol need be ascribed to ingested sterols. The mass of unoxidizable cholesterol was not diminished when cholesterol biosynthesis was inhibited by lovastatin in lipoprotein-deprived cells. Furthermore, the newly synthesized radiolabeled cholesterol resistant to cholesterol oxidase did not migrate with intracellular cholesterol mass on sucrose density gradients. The newly synthesized cholesterol amounted to about 10% of the total unoxidized sterol. These data indicate that most of the intracellular cholesterol was not newly synthesized. We conclude that a) approximately 90% of fibroblast cholesterol is associated with the cell surface; b) the bulk of intracellular cholesterol, approximately 10% of total, is derived from internalized (endocytic) plasma membrane; and c) the most recently synthesized cholesterol, approximately 1% of the total, is in a discrete organelle.

Zymosterol is located in the plasma membrane of cultured human fibroblasts.

Zymosterol (5 alpha-cholesta-8(9),24-dien-3 beta-ol) comprised a negligible fraction of the mass of sterol in cultured human fibroblasts but was well labeled biosynthetically with radioactive acetate. Treatment of cells with triparanol, a potent inhibitor of sterol delta 24-reductase, led to a marked increase in labeled zymosterol while its mass rose to 1 mol% of total sterol. All of this sterol could be chased into cholesterol. Furthermore, cell homogenates converted exogenous radiolabeled zymosterol to cholesterol. Three lines of evidence suggested that biosynthetically labeled zymosterol was associated with the plasma membrane. 1) About 80% of radiolabeled zymosterol was oxidized by the impermeant enzyme, cholesterol oxidase, in glutaraldehyde-fixed intact cells. 2) Sucrose density gradient analysis of homogenates showed that the equilibrium buoyant density profile of newly synthesized zymosterol was identical with that of the plasma membrane. 3) Newly synthesized zymosterol was transferred as readily from fixed intact fibroblasts to exogenous acceptors as was cholesterol. Given that cholesterol is synthesized within the cell, it is unclear why most of the zymosterol is in the plasma membrane. The pathway of cholesterol biosynthesis may compel zymosterol to flux through the plasma membrane. Alternatively, plasma membrane zymosterol may represent a separate pool, in equilibrium with the zymosterol in the intracellular biosynthetic pool.