Spontaneous and Protein-mediated Sterol Transfer between Intracellular Membranes

Abstract

Relatively little is known regarding intracellular cholesterol trafficking pathways. To resolve some of these potential pathways, spontaneous and protein-mediated sterol transfer was examined between different donor-acceptor membrane pairs in vitro using L-cell fibroblast plasma membrane (PM) and microsomal (MICRO) and mitochondrial (MITO) membranes. Several new exciting insights were provided. First, the initial rate of spontaneous molecular sterol transfer was more dependent on the type of acceptor than donor membrane, i.e. spontaneous intracellular sterol trafficking was vectorial. Therefore, the rate of sterol desorption from the donor membrane was not necessarily the rate-limiting step in molecular sterol transfer. Second, the rate of molecular sterol transfer was not obligatorily correlated with the direction of the cholesterol gradient. For example, although PM had a 3.2-fold higher cholesterol/phospholipid ratio than MITO, spontaneous sterol transfer was 4–5-fold faster up (MITO to PM) rather than down (PM to MITO) the concentration gradient. Third, sterol carrier protein-2 differentially stimulated the initial rate of sterol transfer for all donor-acceptor combinations, being most effective with PM donors: PM-MICRO, 27-fold; and PM-MITO, 12-fold. Sterol carrier protein-2 was less effective in enhancing sterol transfer in the reverse direction, i.e. MICRO-PM and MITO-PM (5- and 4-fold, respectively). Fourth, liver fatty acid-binding protein was limited in stimulating the initial rate of sterol transfer from PM to PM (1.5-fold), from PM to MITO (3-fold), and from MICRO to MITO (3-fold). In summary, these observations present important insights into potential sterol trafficking pathways between the major membrane components of the cell. Relatively little is known regarding intracellular cholesterol trafficking pathways. To resolve some of these potential pathways, spontaneous and protein-mediated sterol transfer was examined between different donor-acceptor membrane pairs in vitro using L-cell fibroblast plasma membrane (PM) and microsomal (MICRO) and mitochondrial (MITO) membranes. Several new exciting insights were provided. First, the initial rate of spontaneous molecular sterol transfer was more dependent on the type of acceptor than donor membrane, i.e. spontaneous intracellular sterol trafficking was vectorial. Therefore, the rate of sterol desorption from the donor membrane was not necessarily the rate-limiting step in molecular sterol transfer. Second, the rate of molecular sterol transfer was not obligatorily correlated with the direction of the cholesterol gradient. For example, although PM had a 3.2-fold higher cholesterol/phospholipid ratio than MITO, spontaneous sterol transfer was 4–5-fold faster up (MITO to PM) rather than down (PM to MITO) the concentration gradient. Third, sterol carrier protein-2 differentially stimulated the initial rate of sterol transfer for all donor-acceptor combinations, being most effective with PM donors: PM-MICRO, 27-fold; and PM-MITO, 12-fold. Sterol carrier protein-2 was less effective in enhancing sterol transfer in the reverse direction, i.e. MICRO-PM and MITO-PM (5- and 4-fold, respectively). Fourth, liver fatty acid-binding protein was limited in stimulating the initial rate of sterol transfer from PM to PM (1.5-fold), from PM to MITO (3-fold), and from MICRO to MITO (3-fold). In summary, these observations present important insights into potential sterol trafficking pathways between the major membrane components of the cell. INTRODUCTIONSterols are essential components serving both structural and functional roles in mammalian cells. Sterols modulate membrane fluidity and thickness, thereby contributing to membrane passive permeability and activity of integral membrane proteins (1Yeagle P.L. Esfahani M. Swaney J.B. Advances In Cholesterol Research. Telford Press, Caldwell, NJ1990: 111Google Scholar, 2Bloch K. Vance D.E. Vance J. Biochemistry of Lipids, Lipoproteins, and Membranes. Elsevier Science Publishing Co., Inc., New York1991: 363Google Scholar). Sterols form both lateral and transbilayer sterol domains (for reviews, see 3Schroeder F. Nemecz G. Esfahani M. Swaney J.B. Advances In Cholesterol Research. Telford Press, Caldwell, NJ1990: 47Google Scholar, 4Schroeder F. Jefferson J.R. Kier A.B. Knittel J. Scallen T.J. Wood W.G. Hapala I. Proc. Soc. Exp. Biol. Med. 1991; 196: 235-252Crossref PubMed Scopus (239) Google Scholar, 5Bretscher M.S. Munro S. Science. 1993; 261: 1280-1281Crossref PubMed Scopus (731) Google Scholar), thereby providing a basis for unique membrane lipid-protein architecture. Sterols are required for cell growth (6Brown M.S. Goldstein J.L. J. Biol. Chem. 1974; 249: 7306-7314Abstract Full Text PDF PubMed Google Scholar, 7Kandutsch A.A. Chen H.W. J. Biol. Chem. 1977; 252: 409-415Abstract Full Text PDF PubMed Google Scholar) and for cell differentiation (8Volpe J.J. Obert K.A. J. Biol. Chem. 1981; 256: 2016-2021Abstract Full Text PDF PubMed Google Scholar). Furthermore, there appears to be synergism between cholesterol biosynthesis and synthesis of DNA, RNA, and phospholipids (9Cornell R.B. Horwitz A.F. J. Cell Biol. 1980; 86: 810-819Crossref PubMed Scopus (36) Google Scholar, 10Cornell R.B. Goldfein H. Biochim. Biophys. Acta. 1983; 750: 504-520Crossref PubMed Scopus (27) Google Scholar). In addition, cholesterol biosynthetic intermediates such as isoprenoids are required for glycoprotein synthesis and the synthesis of ubiquinones (11Maltese W.A. FASEB J. 1990; 4: 3319-3328Crossref PubMed Scopus (430) Google Scholar, 12Edwards P.A. Vance D.E. Vance J. Biochemistry of Lipids, Lipoproteins, and Membranes. Elsevier Science Publishing Co., Inc., New York1991: 383Google Scholar).Cholesterol is not uniformly distributed in cells. Sterol synthetic activity in eukaryotic cells is concentrated in the relatively cholesterol-poor endoplasmic reticulum and peroxisomes (12Edwards P.A. Vance D.E. Vance J. Biochemistry of Lipids, Lipoproteins, and Membranes. Elsevier Science Publishing Co., Inc., New York1991: 383Google Scholar), whereas the plasma membrane accommodates the major part of intracellular sterol content (13Colbeau A. Nachbauer J. Vignais P.M. Biochim. Biophys. Acta. 1971; 249: 462-492Crossref PubMed Scopus (493) Google Scholar, 14Schroeder F. Perlmutter J.F. Glaser M. Vagelos P.R. J. Biol. Chem. 1976; 251: 5015-5026Abstract Full Text PDF PubMed Google Scholar). Corticosteroid synthesis from cholesterol occurs in the cholesterol-deficient inner mitochondrial membrane (15Nestler J.E. Esfahani M. Swaney J.B. Advances In Cholesterol Research. Telford Press, Caldwell, NJ1990: 133Google Scholar). Consequently, extensive sterol transfer and targeting must occur between these intracellular membranes, either against or with the cholesterol gradient, respectively (4Schroeder F. Jefferson J.R. Kier A.B. Knittel J. Scallen T.J. Wood W.G. Hapala I. Proc. Soc. Exp. Biol. Med. 1991; 196: 235-252Crossref PubMed Scopus (239) Google Scholar, 16Dawidowicz E.A. Curr. Top. Membr. Transp. 1987; 29: 175-202Crossref Scopus (53) Google Scholar, 17Wirtz K.W.A. Gadella T.W.J. Experientia (Basel). 1990; 46: 592-599Crossref PubMed Scopus (71) Google Scholar, 18Reinhart M.P. Experientia (Basel). 1990; 46: 599-611Crossref PubMed Scopus (59) Google Scholar). Despite their importance to sterol homeostasis in the cell, the major pathways of intracellular sterol trafficking are still unclear.The vast majority of information on sterol transfer/exchange has focused on model membranes (4Schroeder F. Jefferson J.R. Kier A.B. Knittel J. Scallen T.J. Wood W.G. Hapala I. Proc. Soc. Exp. Biol. Med. 1991; 196: 235-252Crossref PubMed Scopus (239) Google Scholar, 16Dawidowicz E.A. Curr. Top. Membr. Transp. 1987; 29: 175-202Crossref Scopus (53) Google Scholar, 17Wirtz K.W.A. Gadella T.W.J. Experientia (Basel). 1990; 46: 592-599Crossref PubMed Scopus (71) Google Scholar, 18Reinhart M.P. Experientia (Basel). 1990; 46: 599-611Crossref PubMed Scopus (59) Google Scholar). With regard to biological membranes, what little is known relates to erythrocytes (for review, see 19Phillips M.C. Johnson W.J. Rothblat G.H. Biochim. Biophys. Acta. 1987; 906: 223-276Crossref PubMed Scopus (423) Google Scholar and 20Kavecansky J. Joiner C.H. Schroeder F. Biochemistry. 1994; 33: 2880-2890Crossref PubMed Scopus (25) Google Scholar) and cell-surface membranes (4Schroeder F. Jefferson J.R. Kier A.B. Knittel J. Scallen T.J. Wood W.G. Hapala I. Proc. Soc. Exp. Biol. Med. 1991; 196: 235-252Crossref PubMed Scopus (239) Google Scholar, 21Woodford J.K. Jefferson J.R. Wood W.G. Hubbell T. Schroeder F. Biochim. Biophys. Acta. 1993; 1145: 257-265Crossref PubMed Scopus (28) Google Scholar, 22Woodford J.K. Hapala I. Jefferson J.R. Knittel J.J. Kavecansky J. Powell D. Scallen T.J. Schroeder F. Biochim. Biophys. Acta. 1994; 1189: 52-60Crossref PubMed Scopus (23) Google Scholar). Pathways of sterol exchange between intracellular membranes (microsomes, mitochondria, etc.) remain unresolved. Thus, the first objective of this study was to explore spontaneous sterol trafficking between cellular components such as plasma, microsomal, and mitochondrial membranes. The second objective was to investigate the role of SCP-2 1The abbreviations used are: SCP-2sterol carrier protein-2L-FABPliver fatty acid-binding proteinDHEdehydroergosterol (Δ5,7,9,(11),22-ergostatetraen-3β-ol)HPLChigh performance liquid chromatographyPIPESpiperazine-N,N′-bis(2-ethanesulfonic acid)PMplasma membrane(s)MICROmicrosomal membrane(s)MITOmitochondrial membrane(s). and L-FABP, cytosolic lipid transfer proteins, in targeting potential pathways of intracellular sterol trafficking. These specialized cytosolic proteins modulate specific reactions of sterol metabolism in vitro (23Noland B.J. Arebalo R.E. Hansbury E. Scallen T.J. J. Biol. Chem. 1980; 255: 4282-4289Abstract Full Text PDF PubMed Google Scholar, 24Gavey K.L. Noland B.J. Scallen T.J. J. Biol. Chem. 1981; 256: 2993-2999Abstract Full Text PDF PubMed Google Scholar, 25Trzaskos J.M. Gaylor J.L. Biochim. Biophys. Acta. 1983; 751: 52-65Crossref PubMed Scopus (82) Google Scholar) and enhance sterol transfer between model membranes (25Trzaskos J.M. Gaylor J.L. Biochim. Biophys. Acta. 1983; 751: 52-65Crossref PubMed Scopus (82) Google Scholar, 26Chanderabhan R. Noland B.J. Scallen T.J. Vahouny G.V. J. Biol. Chem. 1982; 257: 8928-8934Abstract Full Text PDF PubMed Google Scholar, 27Schroeder F. Butko P. Hapala I. Scallen T.J. Lipids. 1990; 25: 664-674Crossref Scopus (36) Google Scholar, 28Muczynski K.A. Stahl W.L. Biochemistry. 1983; 22: 6037-6048Crossref PubMed Scopus (29) Google Scholar, 29Hapala I. Kavecansky J. Butko P. Scallen T.J. Joiner C.H. Schroeder F. Biochemistry. 1994; 33: 7682-7690Crossref PubMed Scopus (41) Google Scholar) and cell plasma membranes in vitro (4Schroeder F. Jefferson J.R. Kier A.B. Knittel J. Scallen T.J. Wood W.G. Hapala I. Proc. Soc. Exp. Biol. Med. 1991; 196: 235-252Crossref PubMed Scopus (239) Google Scholar, 22Woodford J.K. Hapala I. Jefferson J.R. Knittel J.J. Kavecansky J. Powell D. Scallen T.J. Schroeder F. Biochim. Biophys. Acta. 1994; 1189: 52-60Crossref PubMed Scopus (23) Google Scholar).DISCUSSIONDespite the substantial advances made in our understanding of cholesterol transport in the vasculature, relatively little is known regarding intracellular cholesterol trafficking pathways (4Schroeder F. Jefferson J.R. Kier A.B. Knittel J. Scallen T.J. Wood W.G. Hapala I. Proc. Soc. Exp. Biol. Med. 1991; 196: 235-252Crossref PubMed Scopus (239) Google Scholar, 43Liscum L. Underwood K.W. J. Biol. Chem. 1995; 270: 15443-15446Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). One potential complication in studying cholesterol trafficking in intact cells is that multiple types of donor and acceptor membranes coexist and compete, thereby making it difficult to resolve individual components. In contrast, herein, an in vitro approach with different combinations of specific membrane marker-enriched membrane fractions isolated from L-cell fibroblasts in conjunction with a fluorescent molecular sterol transfer assay yielded several exciting new observations.Spontaneous sterol transfer occurs most rapidly between the cholesterol-rich plasma membranes as compared with relatively cholesterol-poor mitochondria. This surprising finding is complemented by data showing for the first time the existence and vectorial nature, at least in vitro, of the spontaneous intracellular sterol trafficking process. For example, sterol spontaneously moved down the cholesterol gradient from plasma membranes to microsomes almost three times faster than in the opposite direction. However, this trend of spontaneous sterol transfer down a concentration gradient cannot be applied uniformly. Spontaneous sterol transfer between mitochondrial and plasma membranes (MITO-PM), up the cholesterol gradient, was faster than down the gradient (PM-MITO). In short, spontaneous sterol transfer between dissimilar donor-acceptor pairs was obligatorily correlated with direction of the cholesterol gradient (e.g. PM-MITO versus MITO-PM and MICRO-MITO versus MITO-MICRO). Finally, not all intracellular pathways are equally available for rapid spontaneous movement of sterol. The PM-MITO, MICRO-PM, and MICRO-MITO transfer rates were extremely slow compared with the velocity observed for the other donor-acceptor combinations (Fig. 3).One of the classic assumptions in cholesterol transfer, based primarily on model membrane data, is that the rate of sterol transfer between membranes is limited by the rate of sterol desorption from the donor membrane, followed by diffusion of the sterol molecule through an aqueous space and its subsequent incorporation into the acceptor membrane (for review, see 19Phillips M.C. Johnson W.J. Rothblat G.H. Biochim. Biophys. Acta. 1987; 906: 223-276Crossref PubMed Scopus (423) Google Scholar and 44Brown R.E. Biochim. Biophys. Acta. 1992; 1113: 3375-3389Crossref Scopus (83) Google Scholar). If this were true for biomembranes, then different acceptors should not affect the rate of sterol transfer from the donor membrane. However, analysis of sterol movement in heterogeneous donor-acceptor pairs showed that the initial rate of sterol transfer was affected greatly by the of the For example, of the PM acceptor in the by MICRO or MITO to and in the initial sterol transfer respectively If the desorption of sterol from the donor membrane was the rate-limiting the of the acceptor membranes should on the of sterol transfer. These observations are with model membrane data showing that of both the donor and acceptor model membrane the rate of sterol and transfer F. Nemecz G. Biochemistry. PubMed Scopus Google Scholar, T.W.J. K.W.A. J. 1994; PubMed Scopus Google Scholar). Thus, these data not the that the rate-limiting step in the of sterol exchange between is necessarily the rate of sterol desorption from a donor membrane. Therefore, in to the desorption the of the acceptor membrane a role in this important made from this study is that SCP-2 is more effective and has different than in enhancing the initial rates of sterol trafficking in The concentration of SCP-2 used in this study in the of that is present in Biochemistry. PubMed Scopus Google Scholar, Biochim. Biophys. Acta. PubMed Scopus Google Scholar). This concentration was effective in stimulating sterol transfer in both and dissimilar donor-acceptor pairs and The of SCP-2 was in sterol transfer from plasma membranes to the rate was and from plasma membranes to The rate by SCP-2 was observed in sterol exchange between microsomes and SCP-2 sterol trafficking from PM to MICRO and from PM to MITO to a than in the reverse versus and versus 4-fold, respectively). Thus, SCP-2 sterol transfer down a concentration gradient from cholesterol-rich PM to relatively cholesterol-poor MICRO and the a in the initial sterol transfer rate in a donor-acceptor PM-MITO and MICRO-MITO it appears that was less effective in stimulating sterol transfer, it must be that the cytosolic concentration in liver and is up to higher than that of SCP-2 Biochim. Biophys. Acta. PubMed Scopus Google Scholar, 1990; PubMed Scopus Google Scholar). Furthermore, of SCP-2 from that of SCP-2 is present not in the in peroxisomes Scallen T.J. D. P.A. J. Cell Biol. PubMed Scopus Google Scholar). In contrast, is primarily with some the endoplasmic reticulum 1990; PubMed Scopus Google Scholar, M. T. H. F. Biol. Chem. PubMed Scopus Google Scholar, T. H. H. F. Biochim. Biophys. Acta. PubMed Scopus Google should be that in with L-FABP, the role of SCP-2 in sterol transfer has more To our there are a (4Schroeder F. Jefferson J.R. Kier A.B. Knittel J. Scallen T.J. Wood W.G. Hapala I. Proc. Soc. Exp. Biol. Med. 1991; 196: 235-252Crossref PubMed Scopus (239) Google Scholar, 21Woodford J.K. Jefferson J.R. Wood W.G. Hubbell T. Schroeder F. Biochim. Biophys. Acta. 1993; 1145: 257-265Crossref PubMed Scopus (28) Google Scholar, 29Hapala I. Kavecansky J. Butko P. Scallen T.J. Joiner C.H. Schroeder F. Biochemistry. 1994; 33: 7682-7690Crossref PubMed Scopus (41) Google Scholar) an of on sterol transfer was appears that sterol exchange between plasma membranes (4Schroeder F. Jefferson J.R. Kier A.B. Knittel J. Scallen T.J. Wood W.G. Hapala I. Proc. Soc. Exp. Biol. Med. 1991; 196: 235-252Crossref PubMed Scopus (239) Google Scholar), has on sterol transfer between model lipid I. Kavecansky J. Butko P. Scallen T.J. Joiner C.H. Schroeder F. Biochemistry. 1994; 33: 7682-7690Crossref PubMed Scopus (41) Google Scholar). was that spontaneous sterol exchange between plasma membranes from cells with for is faster in with cell J.K. Jefferson J.R. Wood W.G. Hubbell T. Schroeder F. Biochim. Biophys. Acta. 1993; 1145: 257-265Crossref PubMed Scopus (28) Google Scholar). These observations as that of the of sterol between biological membranes and thereby the intracellular sterol with cells SCP-2 remain to be by cytosolic lipid carrier proteins sterol transfer between biological membranes is not of data that SCP-2 or to the membrane and then with sterol in the membrane, thereby stimulating sterol desorption from the membrane T.W.J. K.W.A. J. 1994; PubMed Scopus Google Scholar, Gaylor J.L. Biochim. Biophys. Acta. 1990; PubMed Scopus (36) Google Scholar, J.K. Schroeder F. Chem. Lipids. 1995; PubMed Scopus Google Scholar, J.K. Schroeder F. 1995; Google Scholar). in the between donor or acceptor model membranes modulate the and direction of sterol transfer T.W.J. K.W.A. J. 1994; PubMed Scopus Google data in this not of a unique role to SCP-2 in of the membrane combinations in vitro for intracellular protein-mediated sterol trafficking. However, the new observations made be to sterol metabolism as Sterol movement a membrane for the majority of sterol synthesis and cholesterol was observed to be However, sterol synthesis not occur in a microsomal a of must be to the step in microsomal to cholesterol synthesis J. Biol. Chem. Full Text PDF PubMed Google Scholar, F. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). be that this sterol trafficking an important role in sterol This that the of sterol transfer in this microsomal sterol synthetic is by a cytosolic SCP-2 is an protein that this sterol MICRO-PM and MITO-PM pathways an important role in reverse cholesterol i.e. cholesterol from not or The data that SCP-2 MICRO-PM sterol transfer by in with intact fibroblasts with SCP-2 showed a in rapid cholesterol transfer from the endoplasmic reticulum to plasma membranes L. A. M. F. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). This is with the present with isolated membrane fractions in MICRO-MITO and PM-MITO pathways be in cholesterol to for to in cells T. H. J.F. J. Biol. 1993; PubMed Scopus Google Scholar, B.J. J. J. Biol. Chem. 1994; Full Text PDF PubMed Google cholesterol transfer from PM to MICRO be an important rapid step in the PM microsomal sterol trafficking in cholesterol either by microsomal or is to SCP-2 is a of microsomal in vitro P. Hapala I. Scallen T.J. Schroeder F. Biochemistry. 1990; 29: PubMed Scopus Google Scholar, M.P. 1990; PubMed Scopus (36) Google Scholar, D. Nemecz G. Schroeder F. Scallen T.J. and Biochemistry of Scholar). However, the of SCP-2 to cholesterol in is of plasma cholesterol is in L-cell fibroblasts J. and F. In contrast, W.J. M.P. J. 1994; Full Text PDF PubMed Google Scholar) and J. K.W.A. Biochim. Biophys. Acta. PubMed Scopus (27) Google Scholar) cell some as to SCP-2 in enhancing cholesterol in it appears that the in the cells or cells is in the SCP-2 in SCP-2 in the and cells In addition, L. A. M. F. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar) that both a and an sterol transfer are in Thus, in SCP-2 W.J. M.P. J. 1994; Full Text PDF PubMed Google Scholar, J. K.W.A. Biochim. Biophys. Acta. PubMed Scopus (27) Google Scholar) be by cholesterol transport L. A. M. F. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). Consequently, it is difficult to from between SCP-2 and cholesterol in the cell INTRODUCTIONSterols are essential components serving both structural and functional roles in mammalian cells. Sterols modulate membrane fluidity and thickness, thereby contributing to membrane passive permeability and activity of integral membrane proteins (1Yeagle P.L. Esfahani M. Swaney J.B. Advances In Cholesterol Research. Telford Press, Caldwell, NJ1990: 111Google Scholar, 2Bloch K. Vance D.E. Vance J. Biochemistry of Lipids, Lipoproteins, and Membranes. Elsevier Science Publishing Co., Inc., New York1991: 363Google Scholar). Sterols form both lateral and transbilayer sterol domains (for reviews, see 3Schroeder F. Nemecz G. Esfahani M. Swaney J.B. Advances In Cholesterol Research. Telford Press, Caldwell, NJ1990: 47Google Scholar, 4Schroeder F. Jefferson J.R. Kier A.B. Knittel J. Scallen T.J. Wood W.G. Hapala I. Proc. Soc. Exp. Biol. Med. 1991; 196: 235-252Crossref PubMed Scopus (239) Google Scholar, 5Bretscher M.S. Munro S. Science. 1993; 261: 1280-1281Crossref PubMed Scopus (731) Google Scholar), thereby providing a basis for unique membrane lipid-protein architecture. Sterols are required for cell growth (6Brown M.S. Goldstein J.L. J. Biol. Chem. 1974; 249: 7306-7314Abstract Full Text PDF PubMed Google Scholar, 7Kandutsch A.A. Chen H.W. J. Biol. Chem. 1977; 252: 409-415Abstract Full Text PDF PubMed Google Scholar) and for cell differentiation (8Volpe J.J. Obert K.A. J. Biol. Chem. 1981; 256: 2016-2021Abstract Full Text PDF PubMed Google Scholar). Furthermore, there appears to be synergism between cholesterol biosynthesis and synthesis of DNA, RNA, and phospholipids (9Cornell R.B. Horwitz A.F. J. Cell Biol. 1980; 86: 810-819Crossref PubMed Scopus (36) Google Scholar, 10Cornell R.B. Goldfein H. Biochim. Biophys. Acta. 1983; 750: 504-520Crossref PubMed Scopus (27) Google Scholar). In addition, cholesterol biosynthetic intermediates such as isoprenoids are required for glycoprotein synthesis and the synthesis of ubiquinones (11Maltese W.A. FASEB J. 1990; 4: 3319-3328Crossref PubMed Scopus (430) Google Scholar, 12Edwards P.A. Vance D.E. Vance J. Biochemistry of Lipids, Lipoproteins, and Membranes. Elsevier Science Publishing Co., Inc., New York1991: 383Google Scholar).Cholesterol is not uniformly distributed in cells. Sterol synthetic activity in eukaryotic cells is concentrated in the relatively cholesterol-poor endoplasmic reticulum and peroxisomes (12Edwards P.A. Vance D.E. Vance J. Biochemistry of Lipids, Lipoproteins, and Membranes. Elsevier Science Publishing Co., Inc., New York1991: 383Google Scholar), whereas the plasma membrane accommodates the major part of intracellular sterol content (13Colbeau A. Nachbauer J. Vignais P.M. Biochim. Biophys. Acta. 1971; 249: 462-492Crossref PubMed Scopus (493) Google Scholar, 14Schroeder F. Perlmutter J.F. Glaser M. Vagelos P.R. J. Biol. Chem. 1976; 251: 5015-5026Abstract Full Text PDF PubMed Google Scholar). Corticosteroid synthesis from cholesterol occurs in the cholesterol-deficient inner mitochondrial membrane (15Nestler J.E. Esfahani M. Swaney J.B. Advances In Cholesterol Research. Telford Press, Caldwell, NJ1990: 133Google Scholar). Consequently, extensive sterol transfer and targeting must occur between these intracellular membranes, either against or with the cholesterol gradient, respectively (4Schroeder F. Jefferson J.R. Kier A.B. Knittel J. Scallen T.J. Wood W.G. Hapala I. Proc. Soc. Exp. Biol. Med. 1991; 196: 235-252Crossref PubMed Scopus (239) Google Scholar, 16Dawidowicz E.A. Curr. Top. Membr. Transp. 1987; 29: 175-202Crossref Scopus (53) Google Scholar, 17Wirtz K.W.A. Gadella T.W.J. Experientia (Basel). 1990; 46: 592-599Crossref PubMed Scopus (71) Google Scholar, 18Reinhart M.P. Experientia (Basel). 1990; 46: 599-611Crossref PubMed Scopus (59) Google Scholar). Despite their importance to sterol homeostasis in the cell, the major pathways of intracellular sterol trafficking are still unclear.The vast majority of information on sterol transfer/exchange has focused on model membranes (4Schroeder F. Jefferson J.R. Kier A.B. Knittel J. Scallen T.J. Wood W.G. Hapala I. Proc. Soc. Exp. Biol. Med. 1991; 196: 235-252Crossref PubMed Scopus (239) Google Scholar, 16Dawidowicz E.A. Curr. Top. Membr. Transp. 1987; 29: 175-202Crossref Scopus (53) Google Scholar, 17Wirtz K.W.A. Gadella T.W.J. Experientia (Basel). 1990; 46: 592-599Crossref PubMed Scopus (71) Google Scholar, 18Reinhart M.P. Experientia (Basel). 1990; 46: 599-611Crossref PubMed Scopus (59) Google Scholar). With regard to biological membranes, what little is known relates to erythrocytes (for review, see 19Phillips M.C. Johnson W.J. Rothblat G.H. Biochim. Biophys. Acta. 1987; 906: 223-276Crossref PubMed Scopus (423) Google Scholar and 20Kavecansky J. Joiner C.H. Schroeder F. Biochemistry. 1994; 33: 2880-2890Crossref PubMed Scopus (25) Google Scholar) and cell-surface membranes (4Schroeder F. Jefferson J.R. Kier A.B. Knittel J. Scallen T.J. Wood W.G. Hapala I. Proc. Soc. Exp. Biol. Med. 1991; 196: 235-252Crossref PubMed Scopus (239) Google Scholar, 21Woodford J.K. Jefferson J.R. Wood W.G. Hubbell T. Schroeder F. Biochim. Biophys. Acta. 1993; 1145: 257-265Crossref PubMed Scopus (28) Google Scholar, 22Woodford J.K. Hapala I. Jefferson J.R. Knittel J.J. Kavecansky J. Powell D. Scallen T.J. Schroeder F. Biochim. Biophys. Acta. 1994; 1189: 52-60Crossref PubMed Scopus (23) Google Scholar). Pathways of sterol exchange between intracellular membranes (microsomes, mitochondria, etc.) remain unresolved. Thus, the first objective of this study was to explore spontaneous sterol trafficking between cellular components such as plasma, microsomal, and mitochondrial membranes. The second objective was to investigate the role of SCP-2 1The abbreviations used are: SCP-2sterol carrier protein-2L-FABPliver fatty acid-binding proteinDHEdehydroergosterol (Δ5,7,9,(11),22-ergostatetraen-3β-ol)HPLChigh performance liquid chromatographyPIPESpiperazine-N,N′-bis(2-ethanesulfonic acid)PMplasma membrane(s)MICROmicrosomal membrane(s)MITOmitochondrial membrane(s). and L-FABP, cytosolic lipid transfer proteins, in targeting potential pathways of intracellular sterol trafficking. These specialized cytosolic proteins modulate specific reactions of sterol metabolism in vitro (23Noland B.J. Arebalo R.E. Hansbury E. Scallen T.J. J. Biol. Chem. 1980; 255: 4282-4289Abstract Full Text PDF PubMed Google Scholar, 24Gavey K.L. Noland B.J. Scallen T.J. J. Biol. Chem. 1981; 256: 2993-2999Abstract Full Text PDF PubMed Google Scholar, 25Trzaskos J.M. Gaylor J.L. Biochim. Biophys. Acta. 1983; 751: 52-65Crossref PubMed Scopus (82) Google Scholar) and enhance sterol transfer between model membranes (25Trzaskos J.M. Gaylor J.L. Biochim. Biophys. Acta. 1983; 751: 52-65Crossref PubMed Scopus (82) Google Scholar, 26Chanderabhan R. Noland B.J. Scallen T.J. Vahouny G.V. J. Biol. Chem. 1982; 257: 8928-8934Abstract Full Text PDF PubMed Google Scholar, 27Schroeder F. Butko P. Hapala I. Scallen T.J. Lipids. 1990; 25: 664-674Crossref Scopus (36) Google Scholar, 28Muczynski K.A. Stahl W.L. Biochemistry. 1983; 22: 6037-6048Crossref PubMed Scopus (29) Google Scholar, 29Hapala I. Kavecansky J. Butko P. Scallen T.J. Joiner C.H. Schroeder F. Biochemistry. 1994; 33: 7682-7690Crossref PubMed Scopus (41) Google Scholar) and cell plasma membranes in vitro (4Schroeder F. Jefferson J.R. Kier A.B. Knittel J. Scallen T.J. Wood W.G. Hapala I. Proc. Soc. Exp. Biol. Med. 1991; 196: 235-252Crossref PubMed Scopus (239) Google Scholar, 22Woodford J.K. Hapala I. Jefferson J.R. Knittel J.J. Kavecansky J. Powell D. Scallen T.J. Schroeder F. Biochim. Biophys. Acta. 1994; 1189: 52-60Crossref PubMed Scopus (23) Google Scholar).