Subcellular localization of the mosquito sterol carrier protein-2 and sterol carrier protein-x

Subcellular distribution of Aedes aegypti sterol carrier protein-2 (AeSCP-2) and AeSCP-x was studied using electron microscopy. In both cultured A. aegypti cells and in the larval midgut, AeSCP-2 was detected mostly in the cytosol, with some labeling mitochondria and nucleus, but not in membranous vesicles. The widespread distribution of AeSCP-2 in the midgut epithelium is consistent with its potential lipid transfer function in all phases of cholesterol absorption. In contrast, AeSCP-x was found mostly in the peroxisome. Differences in the subcellular distribution of AeSCP-2 and AeSCP-x suggest that these two members of the SCP-2 gene family are functionally distinct. Overexpression of AeSCP-2 in A. aegypti cells showed increased localization of AeSCP-2 to cytosol, mitochondria, and nucleus. This is the first report on the nuclear distribution of an SCP. Overexpression of AeSCP-2 resulted in increased cholesterol incorporation in cells, suggesting that AeSCP-2 enhances cholesterol uptake.     

Overexpression of sterol carrier protein-2 differentially alters hepatic cholesterol accumulation in cholesterol-fed mice

Although in vitro studies suggest a role for sterol carrier protein-2 (SCP-2) in cholesterol trafficking and metabolism, the physiological significance of these observations remains unclear. This issue was addressed by examining the response of mice overexpressing physiologically relevant levels of SCP-2 to a cholesterol-rich diet. While neither SCP-2 overexpression nor cholesterol-rich diet altered food consumption, increased weight gain, hepatic lipid, and bile acid accumulation were observed in wild-type mice fed the cholesterol-rich diet. SCP-2 overexpression further exacerbated hepatic lipid accumulation in cholesterol-fed females (cholesterol/cholesteryl esters) and males (cholesterol/cholesteryl esters and triacyglycerol). Primarily in female mice, hepatic cholesterol accumulation induced by SCP-2 overexpression was associated with increased levels of LDL-receptor, HDL-receptor scavenger receptor-B1 (SR-B1) (as well as PDZK1 and/or membrane-associated protein 17 kDa), SCP-2, liver fatty acid binding protein (L-FABP), and 3α-hydroxysteroid dehydrogenase, without alteration of other proteins involved in cholesterol uptake (caveolin), esterification (ACAT2), efflux (ATP binding cassette A-1 receptor, ABCG5/8, and apolipoprotein A1), or oxidation/transport of bile salts (cholesterol 7α-hydroxylase, sterol 27α-hydroxylase, Na⁺/taurocholate cotransporter, Oatp1a1, and Oatp1a4). The effects of SCP-2 overexpression and cholesterol-rich diet was downregulation of proteins involved in cholesterol transport (L-FABP and SR-B1), cholesterol synthesis (related to sterol regulatory element binding protein 2 and HMG-CoA reductase), and bile acid oxidation/transport (via Oapt1a1, Oatp1a4, and SCP-x). Levels of serum and hepatic bile acids were decreased in cholesterol-fed SCP-2 overexpression mice, especially in females, while the total bile acid pool was minimally affected. Taken together, these findings support an important role for SCP-2 in hepatic cholesterol homeostasis.     

Sterol carrier protein-2 expression alters plasma membrane lipid distribution and cholesterol dynamics

Although sterol carrier protein-2 (SCP-2) binds, transfers, and/or enhances the metabolism of many membrane lipid species (fatty acids, cholesterol, phospholipids), it is not known if SCP-2 expression actually alters the membrane distribution of lipids in living cells or tissues. As shown herein for the first time, expression of SCP-2 in transfected L-cell fibroblasts reduced the plasma membrane levels of lipid species known to traffic through the HDL-receptor-mediated efflux pathway: cholesterol, cholesteryl esters, and phospholipids. While the ratio of cholesterol/phospholipid in plasma membranes of intact cells was not changed by SCP-2 expression, phosphatidylinositol, a molecule important to intracellular signaling and vesicular trafficking, and anionic phospholipids were selectively retained. Only modest alterations in plasma membrane phospholipid percent fatty acid composition but no overall change in the proportion of saturated, unsaturated, monounsaturated, or polyunsaturated fatty acids were observed. The reduced plasma membrane content of cholesterol was not due to SCP-2 inhibition of sterol transfer from the lysosomes to the plasma membranes. SCP-2 dramatically enhanced sterol transfer from isolated lysosomal membranes to plasma membranes by eliciting detectable sterol transfer within 30 s, decreasing the t(1/2) for sterol transfer 364-fold from >4 days to 7-15 min, and inducing formation of rapidly transferable sterol domains. In summary, data obtained with intact transfected cells and in vitro sterol transfer assays showed that SCP-2 expression (i) selectively modulated plasma membrane lipid composition and (ii) decreased the plasma membrane content cholesterol, an effect potentially due to more rapid SCP-2-mediated cholesterol transfer from versus to the plasma membrane.     

Sterol carrier protein-2 (SCP-2) involvement in cholesterol hydroperoxide cytotoxicity as revealed by SCP-2 inhibitor effects

Sterol carrier protein-2 (SCP-2) plays an important role in cholesterol trafficking and metabolism in mammalian cells. The purpose of this study was to determine whether SCP-2, under oxidative stress conditions, might also traffic hydroperoxides of cholesterol, thereby disseminating their cytotoxic effects. Two inhibitors, SCPI-1 and SCPI-3, known to block cholesterol binding by an insect SCP-2, were used to investigate this. A mouse fibroblast transfectant clone (SC2F) overexpressing SCP-2 was found to be substantially more sensitive to apoptotic killing induced by liposomal 7α-hydroperoxycholesterol (7α-OOH) than a wild-type control. 7α-OOH uptake by SC2F cells and resulting apoptosis were both inhibited by SCPI-1 or SCPI-3 at a subtoxic concentration. Preceding cell death, reactive oxidant accumulation and loss of mitochondrial membrane potential were also strongly inhibited. Similar SCPI protection against 7α-OOH was observed with two other types of SCP-2-expressing mammalian cells. In striking contrast, neither inhibitor had any effect on H₂O₂-induced cell killing. To learn whether 7α-OOH cytotoxicity is due to uptake/transport by SCP-2, we used a fluorescence-based competitive binding assay involving recombinant SCP-2, NBD-cholesterol, and SCPI-1/SCPI-3 or 7α-OOH. The results clearly showed that 7α-OOH binds to SCP-2 in SCPI-inhibitable fashion. Our findings suggest that cellular SCP-2 not only binds and translocates cholesterol but also cholesterol hydroperoxides, thus expanding their redox toxicity and signaling ranges under oxidative stress conditions.     

Role of an Ancestral D-Bifunctional Protein Containing Two Sterol-Carrier Protein-2 Domains in Lipid Uptake and Trafficking in Toxoplasma

The inability to synthesize cholesterol is universal among protozoa. The intracellular pathogen Toxoplasma depends on host lipoprotein-derived cholesterol to replicate in mammalian cells. Mechanisms of cholesterol trafficking in this parasite must be important for delivery to proper organelles. We characterized a unique d-bifunctional protein variant expressed by Toxoplasma consisting of one N-terminal d-3-hydroxyacyl-CoA dehydrogenase domain fused to two tandem sterol carrier protein-2 (SCP-2) domains. This multidomain protein undergoes multiple cleavage steps to release free SCP-2. The most C-terminal SCP-2 carries a PTS1 that directs the protein to vesicles before processing. Abrogation of this signal results in SCP-2 accumulation in the cytoplasm. Cholesterol specifically binds to parasite SCP-2 but with 10-fold lower affinity than phosphatidylcholine. In mammalian cells and Toxoplasma, the two parasite SCP-2 domains promote the circulation of various lipids between organelles and to the surface. Compared with wild-type parasites, TgHAD-2SCP-2–transfected parasites replicate faster and show enhanced uptake of cholesterol and oleate, which are incorporated into neutral lipids that accumulate at the basal end of Toxoplasma. This work provides the first evidence that the lipid transfer capability of an ancestral eukaryotic SCP-2 domain can influence the lipid metabolism of an intracellular pathogen to promote its multiplication in mammalian cells.     

Holo-sterol carrier protein-2. (13)C NMR investigation of cholesterol and fatty acid binding sites

Although sterol carrier protein-2 (SCP-2) stimulates sterol transfer in vitro, almost nothing is known regarding the identity of the putative cholesterol binding site. Furthermore, the interrelationship(s) between this SCP-2 ligand binding site and the recently reported SCP-2 long chain fatty acid (LCFA) and long chain fatty acyl-CoA (LCFA-CoA) binding site(s) remains to be established. In the present work, two SCP-2 ligand binding sites were identified. First, both [4-(13)C]cholesterol and 22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBD-cholesterol) binding assays were consistent with a single cholesterol binding site in SCP-2. This ligand binding site had high affinity for NBD-cholesterol, K(d) = 4.15 +/- 0.71 nM. (13)C NMR-labeled ligand competition studies demonstrated that the SCP-2 high affinity cholesterol binding site also bound LCFA or LCFA-CoA. However, only the LCFA-CoA was able to effectively displace the SCP-2-bound [4-(13)C]cholesterol. Thus, the ligand affinities at this SCP-2 binding site were in the relative order cholesterol = LCFA-CoA > LCFA. Second, (13)C NMR studies demonstrated the presence of another ligand binding site on SCP-2 that bound either LCFA or LCFA-CoA but not cholesterol. Photon correlation spectroscopy was consistent with SCP-2 being monomeric in both liganded and unliganded states. In summary, both (13)C NMR and fluorescence techniques demonstrated for the first time that SCP-2 had a single high affinity binding site that bound cholesterol, LCFA, or LCFA-CoA. Furthermore, results with (13)C NMR supported the presence of a second SCP-2 ligand binding site that bound either LCFA or LCFA-CoA but not cholesterol. These data contribute to our understanding of a role for SCP-2 in both cellular cholesterol and LCFA metabolism.     

Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes

A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in <1 min and initial rate and maximal uptake of HDL free cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2/SCP-x/L-FABP null mice and hepatocytes. Taken together, these results suggest that L-FABP, particularly in the absence of SCP-2, plays a significant role in HDL-mediated cholesterol uptake in cultured primary hepatocytes.     

Identification and expression profile of a putative basement membrane protein gene in the midgut of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Background  

The midgut undergoes histolysis and remodeling during the larval to adult transition in holometabolous insects, but the molecular mechanisms underlying this process are not well understood.     

Sterol carrier protein-2/sterol carrier protein-x expression differentially alters fatty acid metabolism in L cell fibroblasts

Sterol carrier protein-2 (SCP-2) and SCP-x are ubiquitous proteins found in all mammalian tissues. Although both proteins interact with fatty acids, their relative contributions to the uptake, oxidation, and esterification of straight-chain (palmitic) and branched-chain (phytanic) fatty acids in living cells has not been resolved. Therefore, the effects of each gene product on fatty acid metabolism was individually examined. Based on the following, SCP-2 and SCP-x did not enhance the uptake/translocation of fatty acids across the plasma membrane into the cell: i) a 2-fold increase in phytanic and palmitic acid uptake was observed at long incubation times in SCP-2- and SCP-x-expressing cells, but no differences were observed at initial time points; ii) uptake of 2-bromo-palmitate, a nonoxidizable, poorly metabolizable fatty acid analog, was unaffected by SCP-2 or SCP-x overexpression; and iii) SCP-2 and SCP-x expression did not increase targeting of radiolabeled phytanic and palmitic acid to the unesterified fatty acid pool. Moreover, SCP-2 and SCP-x expression enhanced fatty acid uptake by stimulating the intracellular metabolism via fatty acid oxidation and esterification. In summary, these data showed for the first time that SCP-2 and SCP-x stimulate oxidation and esterification of branched-chain as well as straight-chain fatty acids in intact cells.     

Sterol carrier protein‐2:Not just for cholesterol any more

Although sterol carrier protein-2 (SCP-2) mediates cholesterol esterification in L-cell fibroblasts and stimulates an accumulation of cholesterol in these cells, a potential role for SCP-2 in fatty acid uptake and trafficking has not been appreciated. Certainly, recent experiments have shown that SCP-2 binds fatty acids in vitro with an affinity similar to that observed for fatty acid binding proteins. Because of the ubiquitous tissue distribution of SCP-2, as opposed to the specific distribution of fatty acid binding proteins, as well as the need for fatty acid trafficking in all cells, I have recently proposed that SCP-2 is the universal fatty acid trafficking protein. This supposition is based on a number of observations made with L-cell fibroblasts expressing either the 13.2 kDa SCP-2 or the 15 kDa proSCP-2. In L-cells expressing the 13.2 kDa SCP-2, fluorescent fatty acid uptake was increased by 10–30% depending upon the probe used. In 15 kDa proSCP-2 expressing cells, fluorescent fatty acid uptake was increased 20–40% depending upon the probe used. However, only expression of the 15 kDa pro-SCP-2 increased the cytoplasmic diffusion of the fluorescent fatty acid. Expression of either protein increased the uptake of [³H]-oleic acid 1.9-fold compared to control, with targeting of [³H]-oleic acid for esterification into cholesteryl esters. The 13.2 kDa SCP-2 did target a significant amount of [³H]-oleic acid for esterification into the triacylglycerol pool. Expression of either protein markedly reduced total cellular phospholipid levels, however both proteins increased cholesteryl ester levels. Interestingly, expression of the 15 kDa proSCP-2 decreased ethanolamine plasmalogen levels with a concomitant increase in choline plasmalogen. Expression of both proteins increased PUFA content of the phospholipids, although this effect was greater in 15 kDa proSCP-2 expressing cells. Hence, expression of SCP-2 increased fatty acid uptake and targeted fatty acid to unique lipid pools, suggesting that SCP-2 may effectively serve as universal fatty acid binding and trafficking protein.     

Interaction of fluorescent delta 5,7,9(11),22-ergostatetraen-3 beta-ol with sterol carrier protein-2

The fluorescent sterol delta 5,7,9(11)-dehydroergostatetraen-3 beta-ol (dehydroergosterol) was used as an analogue of cholesterol to examine the molecular interaction of purified rat liver sterol carrier protein-2 (SCP-2) with sterol. The binding of dehydroergosterol to SCP-2 was evidenced by light scatter and by fluorescence polarization, lifetime, limiting anisotropy, and rotational relaxation time of dehydroergosterol. In addition, energy transfer efficiency from SCP-2 tryptophan to dehydroergosterol was 96%, indicating that the apparent distance, R, between the SCP-2 tryptophan (energy donor) and the dehydroergosterol (energy acceptor) was 13.7 A. Scatchard binding analysis of light scatter, lifetime, and energy transfer data all indicated a 1:1 molar stoichiometry with Kd = 1.2, 1.6, and 1.3 microM, respectively. SCP-2 enhanced the activity of microsomal acyl-CoA:cholesterol acyltransferase through transfer of [3H]cholesterol from donor palmitoyloleoyl phosphatidylcholine/cholesterol small unilamellar vesicles to rat liver microsomes containing the enzyme. A recently developed fluorescence assay utilizing dehydroergosterol fluorescence polarization (Nemecz, G., Fontaine, R. N., and Schroeder, F. (1988) Biochim. Biophys. Acta 948, 511-521; Nemecz, G., and Schroeder, F. (1988) Biochemistry 27, 7740-7749) was applied to examine the effect of SCP-2 on sterol exchange. In the absence of SCP-2, two spontaneously exchangeable sterol domains were observed in palmitoyloleoyl phosphatidylcholine/sterol (65:35 molar ratio) small unilamellar vesicles. SCP-2 enhanced the rate of exchange of the faster exchanging domain 2-fold. The transfer rate of the more slowly exchangeable sterol domain and the fraction of cholesterol represented by each domain were not affected. These results demonstrate the utility of dehydroergosterol to probe SCP-2 interactions with sterols and are indicative of a physiological role for SCP-2 as a soluble sterol carrier.     

NMR and X-ray structures of the putative sterol carrier protein 2 from <Emphasis Type="Italic">Thermus thermophilus</Emphasis> HB8 show conformational changes

Sterol carrier protein 2 (SCP-2), also known as nonspecific lipid transfer protein, is a ubiquitous intracellular ~13 kDa protein found in mammals, insects, plants, archaea, and bacteria. Vertebrate SCP-2 has been implicated in a wide range of lipid-related functions in vitro, although its actual physiological role is still unknown. Tunnels in the protein serve as fatty acid binding vehicles. Here we report the first putative SCP-2 structure from a bacterium: specifically, the NMR and X-ray structures of the TTHA0401 protein (also designated as TT1886) from the extremely thermophilic bacterium Thermus thermophilus. The NMR structure and the two chain structures (chain A and chain B) of the asymmetric crystallographic unit (space group (P2₁2₁2₁)) revealed an internal cavity. However, this cavity is open to the outside, forming a tunnel, in only one of those structures (chain A, X-ray). The location of this tunnel is different from the one found in other SCP-2 proteins, and inaccessible cavities have not been seen before in SCP structures. We present evidence that at physiological concentrations, TTHA0401 likely exists as a monomer in equilibrium between open and closed conformations. This equilibrium is influenced by temperature-dependent dynamics, and is likely to be very different at the high temperatures preferred by this hyperthermophilic bacterium. Alternatively, another protein binding to TTHA0401 may induce a conformational change, which would constitute an intriguing metabolic regulation method in bacteria.     

Sterol carrier protein-2 alters high density lipoprotein-mediated cholesterol efflux

Although sterol carrier protein-2 (SCP-2) participates in the uptake and intracellular trafficking of cholesterol, its effect on "reverse cholesterol transport" has not been explored. As shown herein, SCP-2 expression inhibited high density lipoprotein (HDL)-mediated efflux of [(3)H]cholesterol and fluorescent 22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3b-ol (NBD-cholesterol) up to 61 and 157%, respectively. Confocal microscopy of living cells allowed kinetic analysis of two intracellular pools of HDL-mediated NBD-cholesterol efflux: the highly fluorescent lipid droplet pool and the less fluorescent pool outside the lipid droplets, designated the cytoplasmic compartment. Both the whole cell and the cytoplasmic compartment exhibited two similar kinetic pools, the half-times of which were consistent with protein (t(b)(12) near 1 min) and vesicular (t(d)(12) = 10-20 min) mediated sterol transfer. Although SCP-2 expression did not alter cytoplasmic sterol pool sizes, the rapid t(b)(12) decreased 36%, while the slower t(d)(12) increased 113%. Lipid droplets also exhibited two kinetic pools of NBD-cholesterol efflux but with half-times over 200% shorter than those of the cytoplasmic compartment. The lipid droplet slower effluxing pool size and t(d)(12) were increased 48% and 115%, respectively, in SCP-2-expressing cells. Concomitantly, the level of the lipid droplet-specific adipose differentiation-related protein decreased 70%. Overall, HDL-mediated sterol efflux from L-cell fibroblasts reflected that of the cytoplasmic rather than lipid droplet compartment. SCP-2 differentially modulated sterol efflux from the two cytoplasmic pools. However, net efflux was determined primarily by inhibition of the slowly effluxing pool rather than by acceleration of the rapid protein-mediated pool. Finally, SCP-2 expression also inhibited sterol efflux from lipid droplets, an effect related to decreased adipose differentiation-related protein, a lipid droplet surface protein that binds cholesterol with high affinity.     

Sterol carrier protein-2-facilitated intermembrane transfer of cholesterol- and phospholipid-derived hydroperoxides

Sterol carrier protein-2 (SCP-2) facilitates cholesterol (Ch) and phospholipid (PL) transfer/exchange between membranes and appears to play a key role in intracellular lipid trafficking. Whether SCP-2 can also facilitate lipid hydroperoxide (LOOH) transfer between membranes and thereby potentially enhance dissemination of peroxidative damage was examined in this study. Transfer kinetics of photochemically generated cholesterol hydroperoxide (ChOOH) species (5alpha-OOH, 6alpha/6beta-OOH, 7alpha/7beta-OOH) and phospholipid hydroperoxide (PLOOH) families (PCOOH, PEOOH, PSOOH) were determined, using HPLC with electrochemical detection for peroxide analysis. LOOH donor/acceptor pairs employed in transfer experiments included (i) all liposomes (e.g., agglutinable SUVs/ nonagglutinable LUVs); (ii) photoperoxidized erythrocyte ghosts/SUVs or vice versa; and (iii) SUVs/mitochondria. In a SUV/ghost system at 37 degrees C, the rate constant for total ChOOH spontaneous transfer was approximately 8 times greater than that for unoxidized Ch. Purified bovine liver and human recombinant SCP-2 exhibited an identical ability to stimulate overall ChOOH transfer, 0.5 unit/mL (based on [(14)C]Ch transfer) increasing the first-order rate constant (k) approximately 7-fold. SCP-2-enhanced translocation of individual ChOOHs increased with increasing hydrophilicity in the following order: 6beta-OOH < 6alpha-OOH < 5alpha-OOH < 7alpha/7beta-OOH. Likewise, SCP-2 stimulated PCOOH, PEOOH, or PSOOH transfer approximately 6-fold, but the net k was 1/5 that of 5alpha-OOH and 1/10 that of 7alpha/7beta-OOH. Donor membrane properties favoring SCP-2-enhanced LOOH transfer included (i) increasing PL unsaturation and (ii) increasing net negative charge imposed by phosphatidylserine. Cytotoxic relevance was demonstrated by showing that SCP-2 accelerates 7alpha-OOH transfer from SUVs to isolated mitochondria and that this enhances peroxide-induced loss of the mitochondrial membrane potential. On the basis of these findings, we postulate that SCP-2, by trafficking ChOOHs and PLOOHs in addition to parent lipids, might exacerbate cell injury under oxidative stress conditions.     

Sterol carrier protein-2 directly interacts with caveolin-1 in vitro and in vivo

HDL-mediated reverse-cholesterol transport as well as phosphoinositide signaling are mediated through plasma membrane microdomains termed caveolae/lipid rafts. However, relatively little is known regarding mechanism(s) whereby these lipids traffic to or are targeted to caveolae/lipid rafts. Since sterol carrier protein-2 (SCP-2) binds both cholesterol and phosphatidylinositol, the possibility that SCP-2 might interact with caveolin-1 and caveolae was examined. Double immunolabeling and laser scanning fluorescence microscopy showed that a small but significant portion of SCP-2 colocalized with caveolin-1 primarily at the plasma membrane of L-cells and more so within intracellular punctuate structures in hepatoma cells. In SCP-2 overexpressing L-cells, SCP-2 was detected in close proximity to caveolin, 48 +/- 4 A, as determined by fluorescence resonance energy transfer (FRET) and immunogold electron microscopy. Cell fractionation of SCP-2 overexpressing L-cells and Western blotting detected SCP-2 in purified plasma membranes, especially in caveolae/ lipid rafts as compared to the nonraft fraction. SCP-2 and caveolin-1 were coimmunoprecipitated from cell lysates by anti-caveolin-1 and anti-SCP-2. Finally, a yeast two-hybrid assay demonstrated that SCP-2 directly interacts with caveolin-1 in vivo. These interactions of SCP-2 with caveolin-1 were specific since a functionally related protein, phosphatidyinositol transfer protein (PITP), colocalized much less well with caveolin-1, was not in close proximity to caveolin-1 (i.e., >120 A), and was not coimmunoprecipitated by anti-caveolin-1 from cell lysates. In summary, it was shown for the first time that SCP-2 (but not PITP) selectively interacted with caveolin-1, both within the cytoplasm and at the plasma membrane. These data contribute significantly to our understanding of the role of SCP-2 in cholesterol and phosphatidylinositol targeted from intracellular sites of synthesis in the endoplasmic reticulum to caveolae/lipid rafts at the cell surface plasma membrane.