A potential role for sterol carrier protein-2 in cholesterol transfer to mitochondria

Chemistry and Physics of Lipids 105 (2000) 9 – 29 www.elsevier.com/locate/chemphyslip

A potential role for sterol carrier protein-2 in cholesterol transfer to mitochondria Adalberto M. Gallegos a, Jonathan K. Schoer a, Olga Starodub b, Ann B. Kier b, Jeffrey T. Billheimer c, Friedhelm Schroeder a,* a

Department of Physiology and Pharmacology, Texas A&M Uni6ersity, TVMC, College Station, TX 77843 -4466, USA b Department of Pathobiology, Texas A&M Uni6ersity, TVMC, College Station, TX 77843 -4466, USA c Cardio6ascular Department, DuPont Merck Pharmaceutical Company, Experimental Station 400 -3231, Wilmington, DE 19898 -0400, USA Received 12 May 1999; received in revised form 7 September 1999; accepted 18 October 1999

Abstract Mitochondrial cholesterol oxidation rapidly depletes cholesterol from the relatively cholesterol-poor mitochondrial membranes. However, almost nothing is known regarding potential mechanism(s) whereby the mitochondrial cholesterol pool is restored. Since most exogenous cholesterol enters the cell via the lysosomal pathway, this could be a source of mitochondrial cholesterol. In the present study, an in vitro fluorescent sterol transfer assay was used to examine whether the lysosomal membrane could be a putative cholesterol donor to mitochondria. First, it was shown that spontaneous sterol transfer from lysosomal to mitochondrial membranes was very slow (initial rate, 0.316 9 0.032 pmol/min). This was due, in part, to the fact that 90% of the lysosomal membrane sterol was not exchangeable, while the remaining 10% also had a relatively long half-time of exchange t1/2 = 202919 min. Second, the intracellular sterol carrier protein-2 (SCP-2) and its precursor (pro-SCP-2) increased the initial rate of sterol transfer from the lysosomal to mitochondrial membrane by 5.2- and 2.0-fold, respectively, but not in the reverse direction. The enhanced sterol transfer was due to a 3.5-fold increase in exchangeable sterol pool size and to induction of a very rapidly (t1/2 =4.190.6 min) exchangeable sterol pool. Confocal fluorescence imaging and indirect immunocytochemistry colocalized significant amounts of SCP-2 with the mitochondrial marker enzyme cytochrome oxidase in transfected L-cells overexpressing SCP-2. In summary, SCP-2 and pro-SCP-2 both stimulated molecular sterol transfer from lysosomal to mitochondrial membranes, suggesting a potential mechanism for replenishing mitochondrial cholesterol pools depleted by cholesterol oxidation. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Sterol carrier protein-2; Lysosome; Mitochondria; Membrane; Cholesterol

Abbre6iations: SCP-2, sterol carrier protein-2; pro-SCP-2, pro-sterol carrier protein-2; DHE, dehydroergosterol; LDL, low-density lipoprotein; StAR, steroidogenic acute regulatory protein. * Corresponding author. Tel.: + 1-409-862-1433; fax: + 1-409-862-4929. E-mail address: [email protected] (F. Schroeder) 0009-3084/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 3 0 8 4 ( 9 9 ) 0 0 1 2 8 - 0

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1. Introduction Although the majority of cholesterol enters the cell via the low-density lipoprotein (LDL)-receptor-mediated lysosomal pathway, almost nothing is known regarding how unesterified cholesterol exits the lysosomal pathway (reviewed in Liscum and Dahl, 1992; Pfeifer et al., 1993; Liscum and Underwood, 1995; Edwards and Davis, 1996; Schroeder et al., 1996; Daum and Vance, 1997; Fielding and Fielding, 1997). Transfer of cholesterol to cholesterol poor organelles such as mitochondria and subsequent oxidation represents the major quantitative metabolite(s) of cholesterol (Edwards and Davis, 1996). The inner mitochondrial membrane (site of mitochondrial cholesterol oxidation) is devoid of cholesterol and cholesterol transfer from the outer to the inner mitochondrial membrane (reviewed in Pfeifer et al., 1993; Harmala et al., 1994; Schroeder et al., 1996; Daum and Vance, 1997; Schroeder et al., 1998) represents the rate-limiting step of cholesterol oxidation in several physiologically important processes (reviewed in Miller, 1988, 1995; Daum and Vance, 1997). First, in steroidogenic tissues, steroidogenic acute regulatory protein (StAR) appears to initiate a rapid burst of cholesterol transfer from outer to inner mitochondrial membranes resulting in formation of corticosteroids (reviewed in Pfeifer et al., 1993; Lin et al., 1995; Sugarawa et al., 1995a,b; Stocco, 1996; Arakane et al., 1998; Thomson, 1998). Second, not all steroidogenic tissues (e.g. placenta) contain StAR (Clark et al., 1995; Sugarawa et al., 1995a). Third, mitochondrial cholesterol oxidation giving rise to bile acids represents the single most important metabolite of cholesterol in mammals (Edwards and Davis, 1996). In liver, cholesterol oxidation to bile acids occurs in mitochondria (27a-hydroxylase), as well as peroxisomes (24a-hydroxylase), and endoplasmic reticulum (6a-, 7a-, 12a- and 6b-hydroxylase) (Edwards and Davis, 1996; Cooper, 1997; Xu et al., 1997). The 27a-hydroxylase is widely distributed in mitochondria of tissues other than liver (Edwards and Davis, 1996). Again, a mechanism for replenishing of mitochondrial cholesterol destined for bile acid production has not yet

been resolved. In summary, a mechanism for replenishment of mitochondrial cholesterol must exist (Pfeifer et al., 1993; Cooper, 1997; Thomson, 1998). One intracellular protein that may serve to replenish mitochondrial cholesterol is sterol carrier protein-2 (SCP-2). SCP-2 is found in all tissues examined and is especially rich in steroidogenic tissues, liver, intestine, and lung. Evidence consistent with SCP-2 transferring cholesterol to mitochondria for oxidation comes from studies with antibodies to SCP-2 which, when injected into adrenal cells, decrease steroid production (Chanderbhan et al., 1986, 1998). Overexpression of SCP-2 in transfected COS cells coexpressing P450 side chain cleavage enzyme stimulated cholesterol oxidation several fold, albeit not to the extent that StAR does (Yamamoto et al., 1991). Furthermore, immunogold electron microscopy of rat luteal cells stimulated with luteinizing hormone demonstrated increased labeling of mitochondria with gold beads bound to anti-SCP-2 antibodies (Mendis-Handagama et al., 1995). Human patients deficient in peroxisomes also present with deficiency in steroidogenesis and decreased SCP-2 (Magalhaes and Magalhaes, 1997). There is a paucity of information regarding the pathways and sources of this extramitochondrial cholesterol. In steroidogenic cells, the plasma membrane (Freeman, 1989 Choi and Freeman, 1998) or the lipid droplet (Chanderbhan et al., 1982; Bisgaier et al., 1985) appears to be the immediate source of unesterified cholesterol for mitochondrial steroidogenesis (Freeman, 1989; Choi and Freeman, 1998). However, this cholesterol is ultimately derived primarily from the LDL-receptor-mediated lysosomal pathway for internalization of exogenous cholesterol (reviewed in Brown and Goldstein, 1986). While the origin of most mitochondrial cholesterol in nonsteroidogenic cells is also the LDL-receptor-mediated lysosomal pathway, the immediate intracellular source of cholesterol for mitochondrial oxidation is not known. To date, the possibility that lysosomal membranes might directly transfer cholesterol to mitochondria has not been

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examined either in vitro or in intact cells. One of the major difficulties in resolving such a pathway in intact cells is the concomitant transfer of cholesterol from lysosomes to plasma membranes (Brasaemle and Attie, 1990; Liscum and Dahl, 1992; Liscum and Underwood, 1995; Lange et al., 1998), endoplasmic reticulum (Underwood et al., 1998), and golgi (reviewed in (Liscum and Dahl, 1992; Neufeld et al., 1996). Therefore, the present study was designed to test this possibility in vitro and to determine whether SCP-2 could mediate this transfer.

2. Materials and methods

2.1. Materials Dehydroergosterol (DHE) was prepared as reported previously (Fischer et al., 1984, 1985). Human recombinant SCP-2 and pro-SCP-2 were isolated as described previously (Matsuura et al., 1993). Ovalbumin, Tween 20, Triton X-100, formaldehyde, methanol, and percoll were obtained from Sigma Chemical Co. (St. Louis, MO). Metrizamide was from Accurate Chemical and Scientific Corp. (Westbury, NY).

2.2. Cell culture Mouse L-cells (L aprt−tk−) were cultured in serum containing Higuchi medium, basically as previously described (Frolov et al., 1996a,b) with the following modifications: The cells were grown and incubated in 20 × 20 cm trays (Nalge-Nunc, Milwaukee, WI) in the absence or presence of DHE for a total of 4 days. They were serum deprived on the third day by removal of medium, washed with phosphate-buffered saline, followed by culture in serum-free Higuchi medium containing penicillin-streptomycin for 18 h. Dehydroergosterol (5 mg/ml ethanol stock solution) or cholesterol (5 mg/ml stock solution) were added initially to give a final sterol concentration of 15 mg sterol/ml medium into the trays that were intended to yield donor lysosomal and mitochondria membranes.

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2.3. Antibodies Purified SCP-2 was injected into female NZW rabbits to produce polyclonal antisera. The antibody was further purified first by affinity chromatography, and further by incubations with mouse liver homogenate from which the SCP-2 gene products had been removed. This minimized or eliminated nonspecific cross-reactivity as has previously been observed for a 30 kDa protein (Pu et al., 1998). The antibodies reacted positively with all SCP-2 gene products. A monoclonal antilysosomal membrane glycoprotein (LAMP-2) antibody was purchased from the Developmental Studies Hybridoma Bank (University of Iowa, IA). Antibodies to mitochondrial heat shock protein 70 and cytochrome oxidase were obtained from ABR, Inc. (Golden, CO). Polyclonal antibodies to microsomal 78 kDa glucose regulated protein antibody (StressGen, Victoria, Canada), polyclonal antibodies to peroxisomal catalase (Biodesign Intl., Kennebunk, ME), and monoclonal antibodies to plasma membrane Na+, K+ATPase (Developmental Studies Hybridoma Bank, University of Iowa, IA) were also obtained from the respective sources. Antibodies to PMP70 were from Zymed Laboratories (San Francisco, CA). FITC- and Texas Red conjugated goat anti-rabbit immunoglobulin (Ig)G were obtained from Sigma Chemical Co. (St. Louis, MO).

2.4. Indirect immunocytochemistry For immunocytochemistry, the working dilution of antisera used for double labeling was 1:20 (anti-SCP-2) and 1:40 (anti-cytochrome oxidase). For double labeling, FITC-conjugated and Texas Red-conjugated goat anti-rabbit IgG (1:100) were used as secondary antibodies. To identify mitochondria, monoclonal anti-cytochrome oxidase antibody (1:50) followed by FITC-conjugated goat anti-mouse polyvalent IgG (1:100) were used. L-Cell fibroblasts transfected with cDNA encoding pro-SCP-2 overexpress the protein that is post-translationally converted to mature 13.2 kDa SCP-2 (Moncecchi et al., 1996). The cells were fixed and permeabilized using one of two meth-

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ods: (i) 3.7% formaldehyde and 1.4% methanol in phosphate-buffered saline (PBS) (pH 7.2) for 15 min, and 1% Triton X-100 in PBS for 5 min, followed by extensive washing (five times in 30 min) with 0.1% Tween 20 in PBS; (ii) methanol for 5 min at − 20°C and 57 mM borate buffer (pH 8.2) for rehydration and subsequent steps. Autofluorescence of residual aldehyde groups was quenched with 100 mM NH4Cl in PBS. Nonspecific reactivity was blocked using 2% ovalbumin in PBS, and borate buffer, respectively. Incubations with primary antibodies diluted in 1% ovalbumin in buffer, as well as with secondary antibodies (diluted in buffer only), were performed in a humid chamber for 30 min at 37°C, with subsequent extensive washing. For colocalization experiments, mixtures of primary and secondary antibodies were used Coverglasses were mounted with anti-fade medium on slides. Immunostained L-cell fibroblasts were examined by confocal microscopy using a MRC-1024 Laser Scanning system with a 15 mW Kr – Ar laser (Bio-Rad Inc., Hercules, CA). FITC was visualized using a 488 nm excitation band and an OG515 long pass filter. Texas Red was visualized using the 568 nm laser line and a 680/32 band pass emission filter. An Axiovert 135 inverted microscope, fitted with a 63×, 1.4 N.A. oil immersion lens (Zeiss Inc., New York, NY), was used to visualize the cells. Image acquisition, processing and editing for printing used the following software: LASER-SHARP (Bio-Rad Inc., Hercules, CA), META MORPH (Universal Imaging Corp., West Chester, PA), PHOTOSHOP (Adobe Systems Corp., Seattle, WA), and CLARIS DRAW (Claris Corp./Filemaker Inc., Santa Clara, CA).

2.5. Lysosomal and mitochondrial membrane isolation Lysosomal and mitochondrial membranes were isolated from control nontransfected L-cell fibroblasts, as follows in four general steps, i.e.: (a) the harvest of the cells through a series of centrifugations (Frolov et al., 1996a,b); (b) the preparation of postnuclear supernatant (PNS), in which the cells were first counted and then ruptured by low-pressure homogenization to yield a ho-

mogenate, which was further centrifuged (Frolov et al., 1996a,b); (c) the isolation of lysosomes and mitochondria through the use of discontinuous Storrie gradients of 6% w/v Percoll and varying concentrations of metrizamide (Madden and Storrie, 1987; Madden et al., 1987); (d) hypotonic lysis of lysosomes to obtain lysosomal membranes. Protein concentration was by the method of Lowry et al. (1951). Purification yield for mitochondria and lysosomal membrane fractions was estimated by Western blotting (Moncecchi et al., 1996). A monoclonal anti-lysosomal membrane glycoprotein (LAMP-2) antibody was applied in a quantitative Western blotting assay to the lysosomal membrane purification as compared with cell homogenate, basically as described earlier for other L-cell proteins (Atshaves et al., 1998; Gossett et al., 1998). Quantitation was performed by densitometric analysis using the NIH Image program as described therein. Mitochondrial purification was estimated by using a monoclonal anti-mitochondrial heat shock protein 70. Contamination with microsomes, peroxisomes, and plasma membranes was also tested using anti-78 kDa glucose regulated protein polyclonal antibody, anti-bovine PMP-70, and monoclonal antiNa+ , K+ , ATPase, respectively.

2.6. Lipid extraction and quantitation Lysosomal and mitochondrial membrane lipids were extracted and characterized for dehydroergosterol and cholesterol content as described previously (Frolov et al., 1996a,b).

2.7. Fluorescence measurement and sterol exchange assays Steady-state fluorescence measurements (i.e. intensity, polarization) were obtained with an ISS Photon Counting Fluorimeter (ISS, Champaign, IL) in the T format. The instrument light source was a 300 W Xenon arc lamp and was equipped with a Neslab Instruments cooling system (Plymouth, NH). The sample excitation was at 324, and light scatter was reduced by the use of dilute samples and KV 389 low fluorescence cut-off

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filters in the emission paths. The total absorbance at the 324 nm excitation wavelength was kept below 0.15 in order to avoid the inner filter effect. Sterol exchange between (i) donor lysosomal membranes and acceptor mitochondrial membranes, or (ii) donor mitochondria and acceptor lysosome membranes was monitored by fluorescence polarization of DHE as described earlier for other L-cell membranes (Frolov et al., 1996a,b). Three types of experiments were performed for each of these two types of exchanges. In the first type of experiment, the polarization of DHE in a donor membrane (7 mg protein/ml in 2 ml of 10 mM PIPES buffer (pH 7.4)) was measured in the absence of acceptor membranes for 4 h at a temperature of 37°C. In the second type of experiment, the polarization of the donor membrane was measured for 10 min to obtain a baseline. Then, an acceptor membrane was added (total concentration, 70 mg/ml in the same PIPES buffer) to the donor sample, and the fluorescence polarization of the spontaneous exchange of sterol was measured for 4 h. In the third type of experiment, the fluorescence polarization of a donor membrane and a mediating protein (either 1.5 mM SCP-2 or 1.5 mM pro-SCP-2) was measured for a 10 min period to establish a baseline, after which the acceptor membrane was added and the polarization measured for the rest of the 4 h.

2.8. Standard cur6es Standard curves have been shown to be of extreme importance in determination of intermembrane molecular sterol transfer for both model membranes (Butko et al., 1992) and biological membranes (Kavecansky et al., 1994). The standard curves were constructed as means to convert the DHE polarization change from sterol exchange/transfer into molecular sterol transfer. The standard curve showing polarization of dehydroergosterol versus mole fraction of DHE in L-cell mitochondria donors was determined previously (Frolov et al., 1996a,b). The standard curve for L-cell lysosomal membranes was obtained similarly by culturing the cells with increasing DHE (1–30 mg/ml medium) and isolating lysosomal membranes as already described. An aliquot

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of each lysosomal membrane fraction was retained for DHE polarization measurement. The remainder of each lysosomal membrane fraction was subjected to lipid extraction followed by highperformance liquid chromatography to determine the fractional contribution of DHE and cholesterol in each lysosomal membrane preparation as previously described (Frolov et al., 1996a,b). This provided the values (DHE polarization and mole fraction DHE) necessary to construct the standard curve of polarization of DHE in lysosome donors versus mole fraction of dehydroergosterol. The lysosomal membrane standard curve and the mitochondrial membrane standard curve were then used to construct combined standard curves as described earlier (Frolov et al., 1996a,b). This allowed our current molecular sterol transfer analysis consisting of lysosome donors and mitochondria acceptor acceptors, and also another standard curve for the reverse direction sterol exchange.

2.9. Calculation of molecular sterol exchange Molecular sterol transfer between lysosomal and mitochondrial membrane donor/acceptor pairs, as well as mitochondrial and lysosomal membrane donor/acceptor pairs, was based on that described earlier for other dissimilar donor– acceptor pairs (Frolov et al., 1996a,b). A plot of DHE polarization as function of molar fraction of the DHE in donor membranes was fitted to Eq. (1), with the parameters P0 = 0.2499, B= 0.0686, and r 2 = 0.949 in the lysosomal membrane. P=PoC/(B + C)

(1)

where P is the measured fluorescence polarization and C is the reciprocal of the DHE concentration in the membranes. Po represents the DHE fluorescence polarization at its infinite dilution. Eq. (1) describes polarization dependence in solutions with only donor membranes. The relative concentration of DHE present in the donor (Xd) and acceptor (Xa) fractions can be described by Eqs. (2) and (3). Xd = Cd/Ct

(2)

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Xa = 1− Xd = 1−(Cd/Ct)

(3)

where Ct and Cd are the initial and current DHE concentrations in the donor membranes, respectively. The anisotropy of any mixture of donor and acceptor can then be expressed as: r = r = ro{Xd/(1+DlysoXd)}

The lysosomal membrane donor–mitochondrial membrane acceptor standard curve was computer best fit to Eq. (10) in order to obtain calculated DHE fluorescence polarization dependencies on the remaining DHE fraction in the lysosomal membrane donors, and thereby obtain the bn values P= %bnx n

+ro{(1−Xd)/[1 + (Dmito/10)(1 −Xd)]}

(10)

(4) where r is the measured fluorescence anisotropy and ro is the anisotropy at infinite dilution of the DHE in the membranes. The value of ro can be obtained from Po through Eq. (5). ro = 2Po/(3−Po)

(5)

The constant D in Eq. (4) is related to the constant B in Eq. (1) in the following manner: D = ZB[1+ (ro/2)]

(6)

where Z is the mole percent of DHE in the total membrane lipid. But Z can in turn be estimated with the following relation: Z= (Po − P)/(PB)

(7)

in which P is the DHE fluorescence polarization in the donor membranes in the absence of an acceptor, and Po and B parameters from Eq. (1). The first term in Eq. (4) represents the DHE fluorescence anisotropy of the donor component, while the second term describes the acceptor fraction for the total fluorescence anisotropy in the particular donor–acceptor combination. Thus, standard curves for dissimilar donor – acceptor pairs can be designed on the basis of Eq. (4) with the corresponding combination of D values in its two terms. For example, for the lysosome – mitochondria standard curve, Eq. (4) was rewritten as r = roXd/(1+XdDlyso) + (1− Xd)/[1 +10(1 −Xd/Dmito)]



n

Xd = %fi exp(− ki t) + F



This can be rewritten as: P= %bn %fi exp(− ki t)+ Fn

(11)

n

n

(12)

where fi represents the exchangeable sterol fraction domain in the donor membrane and ki is the respective rate constant. F is the sterol fraction that is not available for transfer. This algorithm was applied to the donor–acceptor pairs. The single exponential model is Xd = f1 exp(− kt)+ f2

(13)

P= %bn [ f1 exp(− kt)+ f2]n

(14)

which is consistent with one exchangeable ( f1) and one nonexchangeable ( f2) sterol domain in the donor membranes. Additionally, the velocity of the sterol transfer is described as the half-life, i.e. the time for transfer of 50% sterol from the donor membrane exchangeable domain to acceptor membrane. The half-life (t1/2) is related to the rate constant k as t1/2 = (ln 0.5)/k.

2.10. Initial rate of sterol transfer (8)

The fluorescence anisotropy was determined for Xd values of 0–1 and converted to polarization through the use of the familiar expression P= 3r/(2+ r)

The resultant data are presented in the Section 3. If the sterol transfer between donor and acceptor membranes follows multiexponential kinetics, then the following condition holds.

(9)

The initial rate of DHE exchange was determined according to a previously described method (Frolov et al., 1996a). Essentially, the precise equation that links the initial fluorescence polarization rate to the initial rate of molecular DHE

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transfer between biological membranes was derived from the respective standard curves. For example, exchange between lysosome donor and mitochondrial acceptor is described by the following equation: F = b0 +b1Xd +b2X 2d

(15)

The time derivatives for both parts of Eq. (15) yield the following (dP/dt)= b1(dXd/dt) +2b2(dXd/dt)

(16)

When t approaches one and Xd approaches one, then 1/(b1 + 2b2)(dP/dt) t “ 0 = − 0.29(dP/dt) t “ 0

(17)

Now, taking into account the donor membrane concentration (7 mg protein/ml), the lysosomal sterol concentration (153 pmol/mg protein), the initial concentration of DHE in the donor membranes (7.5 mol%) and the values of b1 and b2 ( − 9.562× 10 − 2 and − 9.065 ×10 − 2, respectively), Eq. (17) can now be rearranged into its final form: (d[DHE]/dt) t “ 0 = −0.29(dP/dt) t “ 0

(18)

This provides initial rates of molecular DHE transfer in pmol/min.

3. Results

3.1. Indirect immunofluorescence confocal imaging of transfected L-cells expressing SCP-2 Although immunogold labeling of a variety of tissues revealed the highest concentration of SCP2 in peroxisomes, significant immunogold labeling is detectable in mitochondria (reviewed in Schroeder et al., 1998). However, this pattern of expression has not yet been completely demonstrated in L-cell fibroblasts, the source of isolated membrane fractions used for in vitro sterol exchange assays in the following sections. Therefore, in order to establish the significance of the in vitro sterol intermembrane sterol exchange assays (see later), it was important to establish whether SCP2 distributed in intracellularly in L-cells in a pattern similar to that in normal tissues.

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L-cells do not normally express significant levels of SCP-2 (Moncecchi et al., 1996). Therefore, transfected L-cells overexpressing SCP-2 (Moncecchi et al., 1996) and immunofluorescence imaging were used to determine the intracellular localization of SCP-2. Our laboratory previously used immunofluorescence and confocal imaging of these cells to establish that while a high concentration of SCP-2 was colocalized with peroxisomes, significant amounts of SCP-2 were extraperoxisomal at unknown site(s) Atshaves et al., 1999). To determine if SCP-2 was present in mitochondria, the transfected L-cells were simultaneously labeled for SCP-2 with Texas Red-conjugated secondary antibody (Fig. 1, panel A) and cytochrome oxidase with FITC-conjugated antibody (Fig. 1, panel B). The anti-SCP-2 confocal fluorescence imaging pattern was primarily punctate (peroxisomal pattern), while that of anti-anticytochrome oxidase was rod-like (mitochondria pattern). The rod-like pattern obtained with the mitochondria was weakly superimposable to that with SCP-2 on the same cells (Fig. 1, panel C). A pixel fluorogram of the latter merged image showed discrete/distinct red (SCP-2) and green (mitochondria) populations (Fig. 1, panel D). However, a smaller population of yellow to orange points fell along the diagonal line, i.e. the superimposition area. Correlation coefficients representing the red/green (0.2) and green/red (0.1) ratios confirmed this colocalization of some SCP-2 in mitochondria. Thus, these data were consistent with those obtained with normal animal tissues, indicating that SCP-2 is associated with mitochondria, albeit less strongly than with peroxisomes (reviewed in Schroeder et al., 1998).

3.2. Purification of lysosomal membranes and mitochondria Western blotting of lysosomal membrane and mitochondrial fractions purified from control Lcell fibroblasts was performed in order to determine the purity of these fractions needed for the fluorescent sterol exchange assay (Fig. 2). A monoclonal anti-lysosomal membrane glycoprotein (LAMP-2) antibody detected this protein in L-cell

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lysosomal membranes (Fig. 2A). Quantitative analysis of the immunoblot, as described in Section 2, revealed that the LAMP-2 was purified approximately 35-fold as compared with crude homogenate. Mitochondrial purification was estimated by using a monoclonal anti-mitochondrial heat shock protein 70 (Fig. 2B). Quantitative analysis of the blot, as described in Section 2, revealed that the mitochondrial heat shock protein 70 was purified approximately 8-fold as compared with crude homogenate.

Contamination of both fractions with microsomes, peroxisomes, and plasma membranes was also tested using anti-78 kDa glucose regulated protein polyclonal antibody (microsomal marker), anti-bovine catalase and anti-PMP70 (peroxisomal matrix and membrane markers, respectively), and monoclonal anti-Na+, K+-ATPase (plasma membrane marker). Neither catalase nor PMP70 were detected in purified lysosomal membranes or mitochondria. Weakly detectable levels of the microsomal and plasma membrane markers were observed in the lysosomal preparations, due to

Fig. 1. Double-label confocal analysis with pixel fluorogram of SCP-2 expressing transfected L-cell fibroblasts. Cells were simultaneously labeled for SCP-2 with Texas Red-conjugated secondary antibody (panel A) and cytochrome oxidase with FITC-conjugated antibody (panel B). The rod-like pattern obtained with the mitochondria was weakly superimposable to that with SCP-2 on the same cells (panel C). A pixel fluorogram of the latter image is shown in panel D.

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Fig. 2. Western blots of purified lysosomal membrane (A) and mitochondrial membrane (B) isolates. All procedures were as described in Section 2. In panel A, incubated with anti-LAMP2 antibodies, the lanes were: lane 1, 50 mg cell homogenate; lane 2, 50 mg postnuclear supernatant; lane 3, 10 mg lysosomal membrane; lane 4, 50 mg mitochondria; lane 5, molecular weight markers. In panel B, incubated with anti-HSP-70 antibodies, the lanes were: lane 1, 50 mg cell homogenate; lane 2, 50 mg postnuclear supernatant; lane 3, 50 mg lysosomal membrane; lane 4, 10 mg mitochondria; lane 5, molecular weight markers.

autophagy of intracellular organelles (Smythe, 1996) and to continual recycling of endocytosed plasma membrane into the lysosomes (Colbeau et al., 1971). Weakly detectable amounts of microsomal and peroxisomal markers were detected in the mitochondrial fraction. In summary, the fold-enrichment of L-cell lysosomal membrane and mitochondrial membrane markers achieved herein was similar to that observed by the methodologies cited in Section 2.

3.3. DHE fluorescence polarization in L-cell fibroblast lysosomal membranes: construction of the standard cur6e When L-cell fibroblasts take up increasing amounts of DHE into plasma membranes, microsomes, or mitochondria, the DHE molecules exhibit self-quenching, as shown by decreasing DHE polarization and by decreasing DHE fluorescence intensity (Frolov et al., 1996a,b). Similarly, selfquenching was observed with purified lysosomal membranes enriched with DHE (Fig. 3). With increasing concentration of DHE in the cell cul-

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ture medium, the fluorescence polarization of the DHE in the lysosomal membranes decreased suggestion of self-quenching, as has previously been demonstrated for other L-cell membrane fractions (Frolov et al., 1996a,b). In order to confirm this possibility, the lipids were extracted from the lysosomal membranes isolated from L-cells grown in the presence of varying concentrations of the fluorescent sterol. The relative DHE and total lipid mass were then measured in the lipid extract as described in Section 2. When the DHE mass as a fraction of total lipid was plotted versus the DHE polarization, the resulting curve showed DHE polarization as function of molar fraction of the DHE in lysosomal membrane (Fig. 3B). DHE fluorescence polarization in lysosomal membranes decreased with increasing DHE content. This was consistent with Weber’s theory of concentrationdependent fluorescence polarization (Weber, 1954). When the data in Fig. 3B were fit to Eq. (1), parameters P0 = 0.2499, B =0.0686, and r 2 = 0.949 were obtained. P0 represents the DHE fluorescence polarization at its infinite dilution, i.e. in the absence of self-quenching. Based on the presented data and previously determined standard curve for L-cell mitochondria, it was possible to construct a standard curve for the exchange assays between lysosomal membranes and mitochondrial membranes, as described in Section 2. The standard curve for the heterogeneous sterol exchanges involving lysosomal membrane donor–mitochondria acceptor pair (Fig. 4A) clearly showed that as the exchange progressed (decreasing mole fraction of DHE in the donor lysosomal membrane), the dehydroergosterol polarization increased. Likewise, the standard curve for the heterogeneous sterol exchanges in the opposite direction (involving mitochondrial membrane donor-lysosomal membrane acceptor pair) also showed that as the exchange progressed (decreasing mole fraction of DHE in the donor lysosomal membrane), the DHE polarization increased (Fig. 4B). However, due to the difference in sterol content of the mitochondrial as compared with lysosomal membranes, the shape of the two curves (back exchange versus forward exchange) differed. The acquisition of these two standard curves now permitted determi-

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Fig. 3.

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nation of molecular sterol exchange from DHE polarization exchanges curves as described in Section 2 and in the following sections.

3.4. Spontaneous and SCP-2 -mediated sterol exchange between lysosomal donor and mitochondrial acceptor membranes The basic characteristics of spontaneous sterol exchange between lysosomal membrane donors and mitochondrial acceptors are depicted in Fig. 5. In the absence of mitochondrial membrane acceptors, DHE polarization in lysosomal membrane donors was unaltered over the time period examined (not shown). However, addition of 10fold excess acceptor mitochondrial membranes elicited a slow spontaneous sterol exchange from lysosomal membranes to mitochondrial membranes (Fig. 5, bottom curve). SCP-2 stimulated sterol exchange between lysosomal membrane donors and mitochondrial acceptors. Addition of SCP-2 or pro-SCP-2 to lysosomal membrane donors alone did not significantly alter DHE polarization over the time period examined (not shown). However, in the presence of a 10-fold excess of mitochondrial acceptor membrane and either SCP-2 (Fig. 5, top curve) or pro-SCP-2 (Fig. 5, middle curve), the DHE polarization increased linearly for a period of 10–15 min. Addition of SCP-2 or pro-SCP-2 accelerated the exchange of DHE in the order: SCP-2 \pro-SCP-2\ spontaneous. Through use of the appropriate standard curves (see earlier), as described in Section 2, the initial rates of DHE polarization change were converted to initial rates (pmol/min) of molecular sterol transfer (Table 1). This quantitative analysis of the data revealed that SCP-2 and pro-SCP-2 increased the initial rate of molecular sterol transfer from lysosomal donor membranes to mitochondrial membranes by 5.2- and 2.0-fold, respec-

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tively. These effects of SCP-2 and pro-SCP-2 on initial rates of molecular sterol transfer from lysosomal membranes to mitochondrial membranes were dependent on the dose of SCP-2 or pro-SCP2 (Fig. 6). With increasing concentration of SCP-2 the initial rate of molecular sterol transfer increased to a maximum near 1.8 pmol/min, or 5.7-fold that of spontaneous sterol transfer (Fig. 6, top curve). In contrast, over the same concentration range, pro-SCP-2 was considerably less effective and elicited a maximal initial rate of molecular sterol transfer of 0.73 pmol/min, or 2.3-fold that of spontaneous sterol transfer (Fig. 6, bottom curve). In summary, the 15 kDa proSCP-2 (contains a 20 amino acid N-terminal leader sequence as compared with the 13 kDa SCP-2) was 60% less effective, mole for mole, than SCP-2 in stimulating molecular sterol transfer from lysosomal membranes to mitochondrial membranes.

3.5. Spontaneous and SCP-2 -mediated sterol back-exchange between mitochondrial donor and lysosomal acceptor membranes The spontaneous exchange of fluorescent DHE involving mitochondrial donor membranes and lysosomal acceptor membranes is demonstrated in Fig. 7. Mitochondrial donor membrane DHE polarization was stable during the time of the assay in the absence of the lysosome acceptor membranes (not shown). However, addition of a 10fold excess of acceptor membranes resulted in an appreciable increase in polarization for the spontaneous DHE exchange (Fig. 7, bottom curve). Thus, the spontaneous transfer of sterol from mitochondria was about 1.5-fold faster than that observed for spontaneous sterol transfer to mitochondria. However, SCP-2 did not stimulate sterol exchange from mitochondrial membrane donors.

Fig. 3. (Panel A) Polarization as a function of dehydroergosterol concentration in media for lysosome donor membranes. For each sample, P was the average of 20 determinations. Individual values deviated less than 5% from the average. The computer-generated fit to the data as described in Section 2 was r 2 = 0.981. Higher values of dehydroergosterol (mg/ml) are not shown, since their polarizations were constant due to maximal self-quenching. (Panel B) Polarization as function of molar fraction of dehydroergosterol for lysosome donor membranes. Lipids were extracted and mole fraction dehydroergosterol was determined as described in Section 2.

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A.M. Gallegos et al. / Chemistry and Physics of Lipids 105 (2000) 9–29

Fig. 4.

A.M. Gallegos et al. / Chemistry and Physics of Lipids 105 (2000) 9–29

21

mal acceptor membranes and SCP-2 the initial phase of DHE polarization change remained unaltered (Fig. 7, middle curve). Similarly, addition of both acceptor membranes and pro-SCP-2 also did not appear to increase the initial phase of DHE polarization change (Fig. 7, top curve). However, the pro-SCP-2-mediated curve appeared to increase to higher polarization level at longer time (Fig. 7, top curve). These qualitative evaluations were confirmed by quantitative analysis of the initial rates of molecular sterol transfer (pmol/ min) which did not reveal statistical significance in initial rates between these curves (Table 1).

3.6. Mono-exponential 6ersus bi-exponential fit of the dehydroergosterol exchange between lysosomal donor membranes and mitochondrial acceptor membranes Fig. 5. Effect of SCP-2 and pro-SCP-2 on sterol exchange between lysosomal donor and mitochondrial acceptor membranes. The curves show the change in fluorescence polarization as a function of time after addition of a 10-fold excess of acceptor (dehydroergosterol-deficient) membranes to donor (dehydroergosterol-rich) membranes.

Addition of SCP-2 or pro-SCP-2 did not significantly alter mitochondrial donor membrane DHE polarization during the time of the assay (not shown). Furthermore, in the presence of lysoso-

In order to determine if DHE may be present in multiple kinetic pools in lysosomal membranes, the DHE polarization data in Fig. 7 were fit to one- and two-exponential equations. The exchange curves for lysosome donors and mitochondrial acceptors best fit to a second-order polynomial, i.e. P=y0 + ax+ bx 2 with an r 2 value of 0.9999. The resultant parameters y0 = 0.34039 0.0003, a= − 0.09559 0.0015 and b= − 0.09169 0.0015 were obtained and utilized to

Table 1 Initial rates for the different heterogeneous exchanges performed in this work Donor membrane (pmol/min)

Acceptor membrane

Proteina

Initial rateb

Lysosome

Mitochondria

Mitochondria

Lysosomes

None 13 kDa SCP-2 15 kDa SCP-2 None 13kDa SCP-2 15 kDa SCP-2

0.316 9 0.032 1.640 9 0.061** 0.635 9 0.026*† 0.540 90.012 0.650 9 0.027+ 0.730 9 0.011

Protein concentration was 1.5 mM SCP-2 or 1.5 mM pro-SCP-2. Values indicate mean9 standard deviation (n= 3 or 4). *, ** refer to PB 0.05 and PB0.01, respectively, as compared with no protein added in the presence of same donor acceptor pair; +, PB0.01 as compared with the opposite donor acceptor pair; †, PB0.01 versus 13 kDa protein in same donor–acceptor pair. a

b

Fig. 4. Standard curves of dehydroergosterol polarization in donor/acceptor pairs. (Panel A) Lysosomal membrane donor – mitochondrial membrane acceptor. (Panel B) Mitochondrial membrane donor – lysosomal membrane acceptor pairs. Polarization is a function of molar fraction of dehydroergosterol for these heterogeneous exchanges.

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A.M. Gallegos et al. / Chemistry and Physics of Lipids 105 (2000) 9–29

Fig. 6. Initial rates (pmol/min) for the lysosome donor–mitochondria acceptor exchanges as a function of concentration of SCP-2 (single exponential fit, r 2 = 0.999) and pro-SCP-2 (single exponential fit, r 2 =0.974).

resolve the kinetic parameters of the exchange (Table 2). Spontaneous transfer of sterol between lysosomal donor membranes and mitochondrial acceptor membranes displayed two kinetic pools of cholesterol, a slowly exchangeable sterol pool with a t1/2 of 202 9 19 min, comprising 11.1% of the total sterol, and an essentially nonexchangeable sterol pool (t1/2 was too slow to be measured), comprising 89% of the total sterol (Table 2). The exchange curves for SCP-2-mediated transfer of sterol from lysosomal donor membranes to mitochondrial acceptor membranes were also examined for one- and two-component exponential fits. Attempts at fitting the SCP-2-mediated sterol transfer to a one-exponential equation, y =

A exp(− Bt)+ constant, resulted in a low r 2 value of 0.17. However, the same curves (n= 4) yielded an excellent fit to a two-exponential equation as indicated by an r 2 value of 0.97. In order to determine whether these fitting parameters depended on the length of the exchange, additional exchanges up to 96 h were performed as follows. The 4 h exchanges were performed either under continuous illumination or for 30 min initially, followed by closing the shutters and opening them periodically for 5 min intervals. No differences were noted for the 4 h data fittings done by these methods. For times longer than 4 h, data were taken continuously for the first 30 min, and thereafter the shutters were closed in order to avoid photobleaching effects of extended light exposure

A.M. Gallegos et al. / Chemistry and Physics of Lipids 105 (2000) 9–29

Fig. 7. Effect of SCP-2 and pro-SCP-2 on sterol exchange between mitochondrial donor and lysosomal acceptor membranes. The curves show the change in fluorescence polarization as function of time after addition of a 10-fold excess of acceptor (dehydroergosterol-deficient) membranes to donor (dehydroergosterol-rich) membranes.

23

cantly different from those obtained with 24, 48, or 96 h exchanges. Addition of SCP-2 increased the size of the slowly exchangeable sterol pool 3.5-fold from 11.1 to 38.6% (Table 2). Equally important, SCP-2 induced the formation of a very rapidly exchangeable sterol pool (t1/2 of 4.190.6 min), comprising 11.4% of the total sterol (Table 2). These data showed that SCP-2 increased the sum total size of the exchangeable sterol pools nearly 5-fold. In contrast, the pro-SCP-2-mediated sterol exchange curve best fit a single exponential equation, y= A exp(− Bt)+ constant. Again, increasing the exchange time from 4 to 24, 48, or 96 h and acquiring data as earlier did not significantly affect the fitting parameters (Table 2). Addition of pro-SCP-2 did not induce formation of a rapidly exchangeable sterol pool (Table 2). However, pro-SCP-2 did increase the size of the slowly exchangeable sterol pool 3.2-fold, from 11.1 to 35.7%.

4. Discussion (which did occur if continuous illumination was used). Shutters were opened every few hours, at which time data were taken for 5 min intervals. Under these circumstances, the total light exposure was limited to B 4 h and photobleaching was avoided. The data in Table 2 showed that for a 4 h exchange, the fitting parameters were not signifi-

Although the majority of cholesterol utilized for steroidogenesis is derived from lipoproteins via the cell surface receptor linked endocytic mechanisms and lysosomes, relatively little is known how this cholesterol is transported intracellularly to mitochondria for oxidation (reviewed

Table 2 The resultant parameters obtained from fit of lysosome–mitochondria exchanges to one- and two-exponential equationsa Protein

Time (h)

Half time 1

None SCP-2

Pro-SCP-2

a

4b 4c 24c 48c 96c 4b 24b 48b 96b

t1/2 (min)

– 4.19 0.6 5.19 1.1 4.690.9 5.09 1.2 – – – –

Fraction 2

f1

f2

f3

2029 19 578 9 35 590 940 589 9 38 599945 267 921 270 930 2729 31 2759 29

– 0.114 90.009 0.115 90.010 0.114 90.008 0.115 9 0.008 – – – –

0.111 90.009 0.386 90.015 0.385 9 0.010 0.383 90.012 0.383 90.016 0.386 90.015 0.360 90.016 0.359 90.023 0.359 9 0.025

0.889 901 0.500 9 0.008 0.500 9 0.011 0.503 9 0.012 0.502 9 0.015 0.643 9 0.021 0.640 9 0.020 0.641 9 0.023 0.641 90.025

t1/2 (min)

The half times of the runs are in minutes. For each type of curve, a total of n =3 runs were used. One exponential best fit. c Two exponential best fit. b

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A.M. Gallegos et al. / Chemistry and Physics of Lipids 105 (2000) 9–29

in (Hechtor et al., 1953; Thomson, 1998). As indicated in Section 1, SCP-2 is believed to play a role in this process by an as yet unresolved mechanism(s). To our knowledge, to date there have been no reports with intact cells or animals examining the possibility that (i) the lysosome could be a source of mitochondrial cholesterol, or that (ii) SCP-2 might play a role in this transfer. The difficulty in using intact cells or tissues has been the presence of multiple competing pathways for intracellular sterol transfer from lysosomes to plasma membranes (Brasaemle and Attie, 1990; Liscum and Dahl, 1992; Liscum and Underwood, 1995; Lange et al., 1998), endoplasmic reticulum (Underwood et al., 1998) and, possibly golgi (reviewed in Liscum and Dahl, 1992; Neufeld et al., 1996). To our knowledge, there are no known specific inhibitors of lysosomal sterol transfer to mitochondria that do not simultaneously block the other pathways. Therefore, the present investigation addressed these questions with isolated lysosomal and mitochondrial membranes in an in vitro sterol exchange assay. Although we recognize the fact that results in vitro may not necessarily equate to function in vivo, the data presented herein and by other laboratories contribute to identifying potential mechanism(s) whereby SCP-2 may target cholesterol trafficking to mitochondria. First, among the various candidate proteins thought to be involved in cholesterol transfer to mitochondria for oxidation, only SCP-2 has been shown to bind cholesterol. Data from this and other laboratories show that SCP-2 binds cholesterol at a single binding site with reported Kd values as low as 6 nM (Chanderbhan et al., 1982; Sams et al., 1990; Schroeder et al., 1990, 1998; Colles et al., 1995; Stolowich et al., 1999). Furthermore, an intact sterol binding site appears necessary for intermembrane sterol transfer activity (Woodford et al., 1995). Second, SCP-2 interacts with membranes (Woodford et al., 1995; Huang et al., 1999) and such interaction appears to be required for maximal intermembrane sterol transfer activity (Woodford et al., 1995). These and other studies (reviewed in Schroeder et al., 1996, 1998) showed that acidic phospholipids were required for this activity.

Third, the gene for SCP-2 encodes a 15 kDa pro-SCP-2, which has both a peroxisomal C-terminal targeting sequence and a 20 amino acid N-terminal leader sequence that was suggested over a decade ago to be a mitochondrial targeting sequence (reviewed in Pastuszyn et al., 1987). Thus, it might be expected that SCP-2 may target to both organelles, the relative proportion being dependent on the rapidity and location of posttranslational proteolytic cleavage of the 20 amino acid leader sequence, which in almost all cells and tissues studied is rarely detected on Western blotting (Matsuura et al., 1993). Immunogold electron microscopy (Keller et al., 1989; MendisHandagama et al., 1990, 1995; van Haren et al., 1992a; Reinhart et al., 1993; Ossendorp et al., 1996; Baum et al., 1997), immunofluorescence imaging (Schroeder et al., 1998; Atshaves et al., 1999), and Western blotting (van Haren et al., 1992a) all revealed that SCP-2 is present in highest concentration in peroxisomes. However, peroxisomes represent less than 1% of cellular protein and significant SCP-2 was extraperoxisomal. As much as one-half of the immunoreactivity was present in other subcellular organelles as shown by immunogold electron microscopy (Keller et al., 1989; Mendis-Handagama et al., 1990; Baum et al., 1997), immunofluorescence imaging (Schroeder et al., 1998; Atshaves et al., 1999), and Western blotting of isolated subcellular organelles (Tsuneoka et al., 1988; van Heusden et al., 1990; van Haren et al., 1992a; Seedorf et al., 1994). In fact, subcellular fractionation of rat tissues revealed that SCP-2 did not cofractionate with peroxisomes, while another SCP-2 gene product, 58 kDa SCP-x did (van Heusden et al., 1990). In short, while the earlier investigations all agree that peroxisomes contain the highest concentration of SCP-2, it is essential to note these reports also indicate that a significant proportion of total SCP-2 immunoreactivity is extraperoxisomal. For the purposes of the present investigation of SCP-2-mediated transfer of cholesterol between lysosomes and mitochondria to be physiologically significant, it is essential to know whether SCP-2 is associated with mitochondria, lysosomes, and/ or cytosol. The fluorescence immunofluorescence

A.M. Gallegos et al. / Chemistry and Physics of Lipids 105 (2000) 9–29

imaging data presented herein with transfected L-cell fibroblasts overexpressing SCP-2 show, for the first time, that transfected cells in culture have significant SCP-2 colocalized with mitochondria. This observation is supported by findings from a variety of animal tissues including liver, intestine, steroidogenic tissue, etc. using immunogold electron microscopy (Keller et al., 1989; MendisHandagama et al., 1990, 1992, 1995; Reinhart et al., 1993; Ossendorp et al., 1996), and Western blotting (Tsuneoka et al., 1988; van Heusden et al., 1990; van Haren et al., 1992b) to show that mitochondria were the intracellular organelle containing the second highest concentration of immunoreactive SCP-2, with the total amount of mitochondrial SCP-2 ranging from nearly the same as in peroxisomes to 10-fold lower than peroxisomes. SCP-2 is also associated with lysosomes and cytoplasm. Western blotting of isolated lysosomes obtained upon subfractionation of rat liver, testis, lung, intestine, and tumor Leydig cells all reveals significant immunoreactive SCP-2 (van Heusden et al., 1990; van Haren et al., 1992a). Likewise immunogold electron microscopy (Van der Krift et al., 1985; Tsuneoka et al., 1988; Keller et al., 1989; Ossendorp et al., 1996) and Western blotting (Tsuneoka et al., 1988) showed that cytosol (obtained upon subfractionation of rat liver, lung, and intestine) had a 20-fold lower concentration of SCP-2 than peroxisomes, but the total SCP-2 immunoreactivity in cytosol was nearly the same as in peroxisomes (Tsuneoka et al., 1988). The presence of SCP-2 in highest concentration in peroxisomes, lower levels in mitochondria, and significant association with lysosomes and cytoplasm is consistent with SCP-2 being localized to transfer cholesterol between intracellular membranes such as lysosomes and mitochondria. A variety of observations with transfected cells, antisense DNA treatment, and functional observations also support this view (reviewed in Schroeder et al., 1998). Several reports suggest multiple sources of extramitochondrial cholesterol destined for oxidation including: plasma membranes (Freeman, 1989) and lipid droplets (Chanderbhan et al., 1982; Bisgaier et al., 1985; Mendis-Handagama et al., 1995). However, the possibility of cholesterol

25

transfer from lysosomal membranes to mitochondria has previously not been examined either in vitro or in vivo. While the data presented herein have confirmed the former possibility, demonstration of function in vivo awaits the development of new techniques, inhibitors, mutant cells, etc., wherein the other competing pathways of intracellular sterol transfer are blocked. Fourth, SCP-2-mediated sterol transfer between lysosomes and mitochondria was vectorial. SCP-2 stimulated sterol transfer from lysosomes to mitochondria, but not in the reverse direction. As already indicated, immunogold electron microscopy, fluorescence imaging, and/or Western blotting all showed that SCP-2 was associated in part with both the donor (lysosomes) and acceptor (mitochondria) membranes as well as the intervening fluid (cytosolic). While this was functionally consistent with the kinetic results of SCP-2-stimulated transfer of sterol from lysosomes to mitochondria, it did not explain the lack of SCP-2-mediated sterol transfer from mitochondria to lysosomes. This was probably due to the fact that these membranes differ markedly in sterol content as evidenced by sterol/phospholipid ratios of 0.6 and 0.02–0.1, respectively (reviewed in Schroeder et al., 1996). The six- to 30-fold higher sterol content of lysosomes, therefore, would be expected to favor SCP-2-accelerated sterol transfer down a sterol concentration gradient (i.e. from lysosomal to mitochondrial membrane), but in the opposite direction it would not favor SCP-2-mediated sterol transfer up a sterol concentration gradient (i.e. from mitochondrial to lysosomal membranes). This type of vectorial SCP-2-mediated sterol transfer between lysosomes and mitochondria has precedent in earlier studies with other types of membranes by this and other laboratories. SCP-2 preferentially stimulates sterol transfer between plasma membrane and endoplasmic reticulum (27-fold) or plasma membranes to mitochondria (12-fold) in vitro (Frolov et al., 1996a). SCP-2 expression in transfected cells also stimulated sterol transfer from plasma membranes to endoplasmic reticulum (Murphy and Schroeder, 1997). Finally, SCP-2 enhanced cholesterol transfer from other intracellular cholesterol sources to mitochondria. For example, the rate-

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A.M. Gallegos et al. / Chemistry and Physics of Lipids 105 (2000) 9–29

limiting step in mitochondrial cholesterol oxidation is the transfer of cholesterol to the inner mitochondrial membrane. SCP-2 expression in transfected cells stimulated mitochondrial cholesterol oxidation (Yamamoto et al., 1991). SCP-2 enhanced cholesterol transfer from adrenal lipid droplets or other intracellular sources to mitochondria in vitro (Chanderbhan et al., 1986, 1998). Finally, the lack of SCP-2 in SCP-2-gene ablated mice resulted in 4-fold upregulation of liver 7a-hydroxylase, the rate-limiting enzyme in liver cholesterol oxidation (Seedorf et al., 1998). Fifth, the data presented herein show that SCP2 converted nonexchangeable sterol to exchangeable sterol (nearly 6-fold total increase). The significance of these findings may be seen by comparison with previously reported (Frolov et al., 1996a) initial rates of spontaneous sterol transfer (nmol/min) from other organelles to mitochondria in vitro, which reveals the following order: lysosomal membranes – mitochondria (0.31 9 0.03)\plasma membranes – mitochondria (0.15 90.03)\microsome – mitochondria (0.10 9 0.02). Thus, the spontaneous rate of sterol transfer from lysosomal membranes to mitochondria was 2-fold faster than from other cholesterol donors (plasma membranes and microsomes). Comparison of the SCP-2-stimulated rates reveals: lysosomal membranes – mitochondria (1.64 90.06)=plasma membranes – mitochondria (1.7590.27)\ \ microsome – mitochondria (0.25 90.04). Thus, the initial rate of SCP-2-mediated sterol transfer from lysosomal membranes to mitochondria was 7-fold faster than from microsomes and similar to that from plasma membranes. Taken together, these data suggest that lysosomal membranes may be at least as good a source of mitochondrial cholesterol as plasma membranes (thought to be the primary route for cholesterol transfer out of lysosomes; reviewed in Schroeder et al., 1998) or even more so than microsomes (site of endogenous sterol synthesis, and a major source of biliary cholesterol) (reviewed in Paz Marzolo et al., 1990). In summary, the present results show for the first time that SCP-2 may stimulate sterol transfer from lysosomal membranes to mitochondria. The effect of SCP-2 was not just to accelerate the rate

of transfer, but, equally important, SCP-2 induced formation of a rapidly exchangeable sterol pool with t1/2 less than 1 min. These data obtained in vitro are consistent with the suggestion that SCP2 may play a role in the rapid replenishment of cholesterol to sterol-depleted mitochondria after the action of acute phase steroidogenic regulatory proteins such as StAR (reviewed in Chanderbhan et al., 1998). Furthermore, SCP-2 may possibly act in part in the acute phase response through induction of the rapidly exchangeable sterol pool, both in steroidogenic tissues expressing StAR and, equally important, in tissues in which StAR is not present. This action of SCP-2 may be through alteration of SCP-2 content (Vahouny et al., 1983; Kawata et al., 1991; Puglielli et al., 1996; Fuchs et al., 1997) and/or through a redistribution of SCP-2 within the cell. Consistent with the latter possibility, recent immunogold electron microscopic results showed that in rat luteal cells stimulated with luteinizing hormone, the SCP-2 became increasingly colocalized with lipid droplets and with mitochondria (Wanders et al., 1992).

Acknowledgements This work was supported in part by a grant from the USPHS National Institutes of Health (GM31651).

References Arakane, F., Kallen, C.B., Watari, H., Foster, J.A., Sepuri, N.B.V., Pain, D., Stayrook, S.E., Lewis, M., Gerton, G.L., Strauss, J.F.I., 1998. The mechanism of action of steroidogenic acute regulatory protein (StAR). J. Biol. Chem. 273, 16339 – 16345. Atshaves, B.P., Foxworth, W.B., Frolov, A.A., Roths, J.B., Kier, A.B., Oetama, B.K., Piedrahita, J.A., Schroeder, F., 1998. Cellular differentiation and I-FABP protein expression modulate fatty acid uptake and diffusion. Am. J. Physiol. 274, C633 – C644. Atshaves, B.P., Petrescu, A., Starodub, O., Roths, J., Kier, A.B., Schroeder, F., 1999. Expression andintracellular processing of the 58 kDa sterol carrier protein 2/3-oxoacylCoA thiolase in transfected mouse L-cell fibroblasts. J. Lipid Res. 40, 610 – 622.

A.M. Gallegos et al. / Chemistry and Physics of Lipids 105 (2000) 9–29 Baum, C.L., Reschly, E.J., Gayen, A.K., Groh, M.E., Schadick, K., 1997. Sterol carrier protein-2 overexpression enhances sterol cycling and inhibits cholesterol ester synthesis and high density lipoprotein cholesterol secretion. J. Biol. Chem. 272, 6490–6498. Bisgaier, C.L., Chanderbhan, R., Hinds, R.W., Vahouny, G.V., 1985. Adrenal cholesterol esters as substrate source for steroidogenesis. J. Steroid Biochem. 23, 967–974. Brasaemle, D.L., Attie, A.D., 1990. Rapid intracellular transport of LDL-derived cholesterol to the plasma membrane in cultured fibroblasts. J. Lipid Res. 31, 103–112. Brown, M.S., Goldstein, J.L., 1986. A receptor-mediated pathway for cholesterol homeostasis. Science 232, 34–47 (Review). Butko, P., Hapala, I., Nemecz, G., Schroeder, F., 1992. Sterol domains in phospholipid membranes: dehydroergosterol polarization measures molecular sterol transfer. J. Biochem. Biophys. Methods 24, 15–37. Chanderbhan, R., Noland, B.J., Scallen, T.J., Vahouny, G.V., 1982. Sterol carrier protein2. Delivery of cholesterol from adrenal lipid droplets to mitochondria for pregnenolone synthesis. J. Biol. Chem. 257, 8928–8934. Chanderbhan, R., Kharroubi, A., Noland, B.J., Scallen, T.J., Vahouny, G.V., 1986. Sterol carrier protein 2: further evidence for its role in adrenol steroidogenesis. Endocrine Res. 12, 351 – 370. Chanderbhan, R.F., Kharoubi, A., Pastuszyn, A., Gallo, L.L., Scallen, T., 1998. Direct evidence for sterol carrier protein2 (SCP-2) participation in ACTH stimulated steroidogenesis in isolated adrenal cells. In: Chang, T.Y., Freeman, D. (Eds.), Intracellular Cholesterol Trafficking. Kluwer, Boston, pp. 197 – 212. Choi, Y.-S., Freeman, D.A., 1998. The movement of plasma membrane cholesterol through the cell. In: Chang, T.Y., Freeman, D.A. (Eds.), Intracellular Cholesterol Trafficking. Kluwer, Boston, pp. 109–121. Clark, B.J., Soo, S.C., Caron, K.M., Ikeda, Y., Parker, K.L., Stocco, D.M., 1995. Hormonal and developmental regulation of the steroidogenic acute regulatory protein. Mol. Endocrinol. 9, 1346 – 1355. Colbeau, A., Nachbaur, J., Vignais, P.M., 1971. Enzymic characterization and lipid composition of rat liver subcellular membranes. Biochim. Biophys. Acta 249, 462–492. Colles, S.M., Woodford, J.K., Moncecchi, D., Myers-Payne, S.C., McLean, L.R., Billheimer, J.T., Schroeder, F., 1995. Cholesterol interactions with recombinant human sterol carrier protein-2. Lipids 30, 795–804. Cooper, A.D., 1997. Editorial on bile acid synthesis. Gastroenterology 113, 2005 – 2008. Daum, G., Vance, J.E., 1997. Import of lipids into mitochondria. Prog. Lipid Res 36, 103–130. Edwards, P.A., Davis, R., 1996. Isoprenoids, sterols and bile acids. In: Vance, D.E., Vance, J.E. (Eds.), Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier Science BV, Amsterdam, pp. 341 –362. Fielding, C.J., Fielding, P.E., 1997. Intracellular cholesterol transport. J. Lipid Res. 38, 1503–1521.

27

Fischer, R.T., Stephenson, F.A., Shafiee, A., Schroeder, F., 1984. Delta 5,7,9(11)-Cholestatrien-3 beta-ol: a fluorescent cholesterol analogue. Chem. Phys. Lipids 36, 1 – 14. Fischer, R.T., Stephenson, F.A., Shafiee, A., Schroeder, F., 1985. Structure and dynamic properties of dehydroergosterol, delta 5,7,9(11),22-ergostatetraen-3 beta-ol. J. Biol. Phys. 13, 13 – 24. Freeman, D.A., 1989. Plasma membrane cholesterol: removal and insertion into the membrane and utilization as substrate for steroidogenesis. Endocrinology 124, 2527 – 2530. Frolov, A., Woodford, J.K., Murphy, E.J., Billheimer, J.T., Schroeder, F., 1996a. Spontaneous and protein-mediated sterol transfer between intracellular membranes. J. Biol. Chem. 271, 16075 – 16083. Frolov, A.A., Woodford, J.K., Murphy, E.J., Billheimer, J.T., Schroeder, F., 1996b. Fibroblast membrane sterol kinetic domains: modulation by sterol carrier protein 2 and liver fatty acid binding protein. J. Lipid Res. 37, 1862 – 1874. Fuchs, M., Lammert, F., Wang, D.Q.H., Paigen, B., Carey, M.C., Cohen, D.E., 1998. Sterol carrier protein-2 participates in hypersecretion of biliary cholesterol during cholesterol gallstone formation in genetically gallstone susceptible mice. Biochem. J. 336, 33 – 37. Gossett, R.E., Edmondson, R.D., Jolly, C.A., Cho, T.H., Russell, D.H., Knudsen, J., Kier, A.B., Schroeder, F., 1998. Structure and function of normal and transformed murine acyl CoA binding proteins. Arch. Biochem. Biophys. 350, 201 – 213. Harmala, A.-S., Porn, M.I., Mattjus, P., Slotte, J.P., 1994. Flow cholesterol transport from plasma membranes to intracellular membranes is inhibited by 3 beta-[2-(diethylamino)ethoxy]androst-5-ene-17-one. Biochim. Biophys. Acta 1211, 317 – 325. Hechtor, O., Solomon, M.M., Zafforoni, A., Pincus, G., 1953. Transformation of cholesterol and acetate to adrenal cortical hormones. Arch. Biochem. Biophys 46, 201 – 214. Huang, H., Schoer, J., Ball, J.M., Knittel, J., Myers-Payne, S., Billheimer, J.T., Schroeder, F., 1999. The sterol carrier protein-2 amino terminus: role in membrane interaction and sterol transfer. Biochemistry 38, 13231 – 13243. Kavecansky, J., Joiner, C.H., Schroeder, F., 1994. Erythrocyte membrane lateral sterol domains: a dehydroergosterol fluorescence polarization study. Biochemistry 33, 2880 – 2890. Kawata, S., Imai, Y., Inada, M., Inui, M., Kakimoto, H., Fukuda, K., Maeda, Y., Tarui, S., 1991. Modulation of cholesterol 7-a hydroxylase activity by nsLTP in human liver — possibly alters regulation of cytosolic level in patients with gallstones. Clin. Chim. Acta 197, 201 – 208. Keller, G.A., Scallen, T.J., Clarke, D., Maher, P.A., Krisans, S.K., Singer, S.J., 1989. Subcellular localization of sterol carrier protein-2 in rat hepatocytes: its primary localization to peroxisomes. J. Cell Biol. 108, 1353 – 1361. Lange, Y., Ye, J., Steck, T.L., 1998. Circulation of cholesterol between lysosomes and the plasma membrane. J. Biol. Chem. 272, 18915 – 18922.

28

A.M. Gallegos et al. / Chemistry and Physics of Lipids 105 (2000) 9–29

Lin, D., Sugawara, T., Strauss, J.F.I., Clark, B.J., Stocco, D.M., Saenger, P., Rogol, A., Miller, W.L., 1995. Role of steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis. Science 267, 1828–1831. Liscum, L., Dahl, N.K., 1992. Intracellular cholesterol transport. J. Lipid Res. 33, 1239–1254. Liscum, L., Underwood, K.W., 1995. Intracellular cholesterol transport and compartmentation. J. Biol. Chem. 270, 15443 – 15446. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 264–275. Madden, E.A., Storrie, B., 1987. The preparation isolation of mitochondria from Chinese hamster ovary cells. Anal. Biochem. 163, 350 – 357. Madden, E.A., Wirt, J.B., Storrie, B., 1987. Purification and characterization of lysosomes from Chinese hamster ovary cells. Arch. Biochem. Biophys. 257, 27–38. Magalhaes, M.M., Magalhaes, M.C., 1997. Peroxisomes in adrenal steroidogenesis. Microsc. Res. Technol. 36, 493– 502. Matsuura, J.E., George, H.J., Ramachandran, N., Alvarez, J.G., Strauss, J.F.I., Billheimer, J.T., 1993. Expression of the mature and the pro-form of human sterol carrier protein 2 in Escherichia coli alters bacterial lipids. Biochemistry 32, 567 – 572. Mendis-Handagama, S.M., Watkins, P.A., Gelber, S.J., Scallen, T.J., Zirkin, B.R., Ewing, L.L., 1990. Luteinizing hormone causes rapid and transient changes in rat Leydig cell peroxisome volume and intraperoxisomal sterol carrier protein-2 content. Endocrinology 127, 2947–2954. Mendis-Handagama, S.M., Watkins, P.A., Gelber, S.J., Scallen, T.J., 1992. Leydig cell peroxisomes and sterol carrier protein-2 in luteinizing hormone-deprived rats. Endocrinology 131, 2839–2845. Mendis-Handagama, S.M., Aten, R.F., Watkins, P.A., Scallen, T.J., Berhman, H.R., 1995. Peroxisomes and sterol carrier protein-2 in luteal cell steroidogenesis: a possible role in cholesterol transport from lipid droplets to mitochondria. Tissue Cell 27, 483–490. Miller, W.L., 1988. Molecular biology of steroid hormone synthesis. Endocrinol. Rev. 9, 295–318. Miller, W.L., 1995. Mitochondrial specificity of the early steps in steroidogenesis. J. Steroid Biochem. Mol. Biol. 55, 607– 616. Moncecchi, D.M., Murphy, E.J., Prows, D.R., Schroeder, F., 1996. Sterol carrier protein-2 expression in mouse L-cell fibroblasts alters cholesterol uptake. Biochim. Biophys. Acta 1302, 110 – 116. Murphy, E.J., Schroeder, F., 1997. Sterol carrier protein-2 mediated cholesterol uptake and transfer in transfected L-cell fibroblasts. Biochim. Biophys. Acta 1345, 283–292. Neufeld, E.B., Cooney, A.M., Pitha, J., Dawidowicz, E.A., Dwyer, N.K., Pentchev, P.G., Blanchette-Mackie, E.J., 1996. Intracellular trafficking of cholesterol monitored with a cyclodextrin. J. Biol. Chem. 271, 21604–21613.

Ossendorp, B.C., Voorhout, W.F., Van Amerongen, A., Brunink, F., Batenburg, J.J., Wirtz, K.W.A., 1996. Tissuespecific distribution of a peroxisomal 46-kDa protein related to the 58-kDa protein (sterol carrier protein X; sterol carrier protein 2/3-oxoacyl-CoA thiolase). Arch. Biochem. Biophys. 334, 251 – 260. Pastuszyn, A., Noland, B.J., Bazan, F., Fletterick, R.J., Scallen, T.J., 1987. Primary sequence and structural analysis of sterol protein-2 from rat liver: homology with immunoglobins. J. Biol. Chem. 262, 13219 – 13227. Paz Marzolo, M., Rigotti, A., Nervi, F., 1990. Secretion of biliary lipids from the hepatocyte. Hepatology 12, 134S – 142S. Pfeifer, S.M., Furth, E.E., Ohba, T., Chang, Y.J., Rennert, H., Sakuragi, N., Billheimer, J.T., Strauss, J.F.I., 1993. Sterol carrier protein 2: a role in steroid hormone synthesis? J. Steroid Biochem. Mol. Biol. 47, 167 – 172. Pu, L., Foxworth, W.B., Kier, A.B., Annan, R.S., Carr, S.A., Edmondson, R., Russell, D., Wood, W.G., Schroeder, F., 1998. Isolation and characterization of 26- and 30-kDa Rat liver proteins immunoreactive to anti-sterol carrier protein2 antibodies. Protein Expression Purif. 13, 337 – 348. Puglielli, L., Rigotti, A., Amigo, L., Nunez, L., Greco, A.V., Santos, M.J., Nervi, F., 1996. Modulation on introhepatic cholesterol trafficking: evidence by in 6i6o antisense treatment for the involvement of sterol carrier protein-2 in newly synthesized cholesterol transfer into bile. Biochem. J. 317, 681 – 687. Reinhart, M.P., Avart, S.J., Dobson, T.O., Foglia, T.A., 1993. The presence and subcellular distribution of sterol carrier protein-2 in embyonic chick tissue. Biochem. J. 295, 787 – 792. Sams, G.H., Hargis, B.M., Hargis, P.S., 1990. Isolation and characterization of a fatty acid binding protein in adipose tissue of Gallus domesticus. Comp. Biochem. Phys-B: Comp. Biochem. 96, 585 – 590. Schroeder, F., Butko, P., Nemecz, G., Scallen, T.J., 1990. Interaction of fluorescent delta 5,7,9(11),22-ergostatetraen3b-ol with sterol carrier protein-2. J. Biol. Chem. 265, 151 – 157. Schroeder, F., Frolov, A.A., Murphy, E.J., Atshaves, B.P., Jefferson, J.R., Pu, L., Wood, W.G., Foxworth, W.B., Kier, A.B., 1996. Recent advances in membrane cholesterol domain dynamics and intracellular cholesterol trafficking. Proc. Soc. Exp. Biol. Med. 213, 150 – 177. Schroeder, F., Frolov, A., Schoer, J., Gallegos, A., Atshaves, B.P., Stolowich, N.J., Scott, A.I., Kier, A.B., 1998. Intracellular cholesterol binding proteins, cholesterol transport and membrane domains. In: Freeman, D., Chang, T.Y. (Eds.), Intracellular Cholesterol Trafficking. Kluwer Academic Publishers, Boston, pp. 213 – 234. Seedorf, U., Brysch, P., Engel, T., Schrage, K., Assmann, G., 1994. Sterol carrier protein X is peroxisomal 3-oxoacyl coenzyme A thiolase with intrinsic sterol carrier and lipid transfer activity. J. Biol. Chem. 269, 21277 – 21283. Seedorf, U., Raabe, M., Ellinghaus, P., Kannenberg, F., Fobker, M., Engel, T., Denis, S., Wouters, F., Wirtz,

A.M. Gallegos et al. / Chemistry and Physics of Lipids 105 (2000) 9–29 K.W.A., Wanders, R.J.A., Maeda, N., Assmann, G., 1998. Defective peroxisomal catabolism of branched fatty acyl coenzyme A in mice lacking the sterol carrier protein-2/ sterol carrier protein-x gene function. Genes Dev. 12, 1189 – 1201. Smythe, E., 1996. In: Lloyd, J.B., Mason, R.W. (Eds.), Endocytosis in Biology of the Lysosome. Plenum Press, New York, pp. 51 – 92. Stocco, D.M., 1996. Acute regulation of Leydig cell steroidogenesis. In: Payne, A.H., Hardy, M.P., Russell, L.D. (Eds.), The Leydig Cell. Cache River Press, Vienna, IL, pp. 241 – 258. Stolowich, N.J., Frolov, A., Petrescu, A., Scott, A.I., Billheimer, J.T., Schroeder, F., 1999. 13C-NMR Elucidation of the sterol carrier protein-2 cholesterol and fatty acid binding sites. J. Biol. Chem. 274, 35425–35433. Sugawara, T., Holt, J.A., Driscoll, D., Strauss, J.F.I., Lin, D., Miller, W.L., Patterson, D., Clancy, K.P., Hart, I.M., Clark, B.J., 1995a. Human steroidogenic acute regulatory protein: functional activity in COS-1 cells, tissue-specific expression, and mapping of the structural gene to 8p11.2 and a pseudogene to chromosome 13. Proc. Natl. Acad. Sci. USA 92, 4778 – 4782. Sugawara, T., Lin, D., Holt, J.A., Martin, K.O., Javitt, N.B., Miller, W.L., Strauss, J.F.3., 1995b. Structure of the human steroidogenic acute regulatory protein (StAR) gene: StAR stimulates mitochondrial cholesterol 27-hydroxylase activity. Biochemistry 34, 12506–12512. Thomson, M., 1998. Molecular and cellular mechanisms used in the acute phase of stimulated steroidogenesis. Horm. Metab. Res. 30, 16 – 28. Tsuneoka, M., Yamamoto, A., Fujiki, V., Tashiro, Y., 1988. Nonspecific lipid transfer protein (sterol carrier protein2) is located in rat liver peroxisomes. J. Biochem. 104, 560–564. Underwood, K.W., Jacobs, N.L., Howley, A., Liscum, L., 1998. Evidence for a cholesterol transport pathway from lysosomes to endoplasmic reticulum that is independent of the plasma membrane. J. Biol. Chem. 273, 4266–4274. Vahouny, G.V., Chanderbhan, R., Noland, B., Irwin, D., Dennis, P., Lambeth, J.D., Scallen, T.J., 1983. Sterol carrier protein. J. Biol. Chem. 258, 11731–11737.

.

29

Van der Krift, T.P., Leunissen, J., Teerlink, T., van Heusden, G.P.H., Verklij, A.J., Wirtz, K.W.A., 1985. Ultrastructural localization of a peroxisomal protein in rat liver using the specific antibody against the nonspecific lipid transfer protein (sterol carrier protein-2). Biochim. Biophys. Acta 812, 387 – 392. van Haren, L., Teerds, K.J., Ossendorp, B.C., van Heusden, G.P.H., Orly, J., Stocco, D.M., Wirtz, K.W.A., Rommerts, F.F.G., 1992a. Sterol carrier protein 2 (non-specific lipid transfer protein) is localized in membranous fractions of Leydig cells and Sertoli cells but not in germ cells. Biochim. Biophys.Acta 1124, 288 – 296. van Haren, L., Teerds, K.J., Ossendorp, B.C., VanHeusden, G.P.H., Orly, J., Stocco, D.M., Wirtz, K.W.A., Rommerts, F.F.G., 1992b. Sterol carrier protein-2 (nonspecific lipid transfer protein) is localized in membranes fractions of Leydig cells and Sertoli cells but not in germ cells. Biochim. Biophys. Acta 1124, 288 – 296. van Heusden, G.P.H., Bos, K., Wirtz, K.W.A., 1990. The occurrence of soluble and membrane-bound non-specific lipid transfer protein (sterol carrier protein 2) in rat tissues. Biochim. Biophys. Acta 1046, 315 – 321. Wanders, R.J., van Roermund, C.W., Lageweg, W., Jakobs, B.S., Schutgens, R.B., Nijenhuis, A.A., Tager, J.M., 1992. X-linked adrenoleukodystrophy: biochemical diagnosis and enzyme defect. J. Inher. Metab. Dis. 15, 634 – 644. Weber, G., 1954. Dependence of the polarization of the fluorescence on the concentration. Trans. Faraday Soc. 50, 552 – 555. Woodford, J.K., Colles, S.M., Myers-Payne, S., Billheimer, J.T., Schroeder, F., 1995. Sterol carrier protein-2 stimulates intermembrane sterol transfer by direct membrane interaction. Chem. Phys. Lipids 76, 73 – 84. Xu, G., Salen, G., Shefer, S., Tint, G.S., Kren, B.T., Nguyen, L.B., Steer, C.J., Chen, T.S., Salen, L., Greenblatt, D., 1997. Increased bile acid pool inhibits cholesterol 7 alphahydroxylase in cholesterol-fed rabbits. Gastroenterology 113, 1958 – 1965. Yamamoto, R., Kallen, C.B., Babalola, G.O., Rennert, H., Billheimer, J.T., Strauss, J.F.I., 1991. Cloning and expression of a cDNA encoding human sterol carrier protein 2. Proc. Natl. Acad. Sci. USA 88, 463 – 467.