Reconstitution of the lipoprotein cholesteryl ester transfer process using isolated rat ovary plasma membranes

Journal of Biological Chemistry

Tsai

Azhar

1996

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126

132

The current study utilizes human, apoE-free high density lipoprotein reconstituted with a highly specific fluorescent-cholesteryl ester probe to define the initial steps and regulatory sites associated with the "selective" uptake and intracellular itinerary of lipoproteinderived cholesteryl esters. Bt 2 cAMP-stimulated ovarian granulosa cells were used as the experimental model, and both morphological and biochemical fluorescence data were obtained. The data show that cholesteryl ester provided through the selective pathway is a process which begins with a temperature-independent transfer of cholesteryl ester to the cell's plasma membrane. Thereafter transfer of the lipid proceeds rapidly and accumulates prominently in a perinuclear region (presumed to be the Golgi/membrane sorting compartment) and in lipid storage droplets of the cells. The data suggest that lipid transfer proteins (or other small soluble proteins) are not required for the intracellular transport of the cholesteryl esters, nor is an intact Golgi complex or an intact cell cytoskeleton (although the transfer is less efficient in the presence of certain microtubule-disrupting agents). The intracellular transfer of the cholesteryl esters is also somewhat dependent on an energy source in that a glucose-deficient culture medium or a combination of metabolic inhibitors reduces the efficiency of the transfer. A protein-mediated event may be required for cholesteryl ester internalization from the plasma membrane, in that N-ethylmaleimide dramatically blocks the internalization phase of the selective uptake process. Taken together these data suggest that the selective pathway is a factor-dependent, energy-requiring cholesteryl ester transport system, in which lipoprotein-donated cholesteryl esters probably flow through vesicles or intracellular membrane sheets and their connections, rather than through the cell cytosol.In the intact rat ovary and adrenal, blood-borne cholesteryl ester-rich high density lipoproteins (HDL) 1 supply the major share of the cholesterol used for progesterone and corticosterone production (1-4). The circulating lipoproteins are trapped in an intricate microvillar channel compartment present on the surface of the steroidogenic cells (2, 4, 5-9), and with time, cholesteryl esters (CEs) are extracted from the lipoproteins and interiorized by the cells, leaving behind the remaining lipoprotein components (2,4,(5)(6)(7)(8). This nonendocytic delivery of cholesteryl esters to cells has been termed the "selective" cholesteryl ester pathway (10

Section: Discussionsupporting

confidence: 88%

Intracellular Events in the “Selective” Transport of Lipoprotein-derived Cholesteryl Esters

Journal of Biological Chemistry

Tsai

Azhar

1996

Self Cite

126

132

“…During in situ perfusions of adrenal and luteinized ovarian tissue, the perfused lipoproteins are trapped in such channels and selective uptake of lipoprotein CEs is believed to begin at these sites (1-3, 6, 26). Enriched membrane preparations isolated from the luteinized ovary retain many such microvillar channels in vitro, which are morphologically intact, functionally able to trap lipoproteins, and able to carry out selective HDL-CE uptake (4,27). Recent studies have shown that in all these situations, the doublemembraned channels strongly express the HDL receptor protein, SR-BI.…”

Section: Discussionmentioning

confidence: 99%

Expression of scavenger receptor class B type 1 (SR-BI) promotes microvillar channel formation and selective cholesteryl ester transport in a heterologous reconstituted system

Proc. Natl. Acad. Sci. U.S.A.

Leers-Sucheta²,

Nomoto³

et al. 2001

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In the ''selective'' cholesteryl ester (CE) uptake process, surfaceassociated lipoproteins [high density lipoprotein (HDL) and low density lipoprotein] are trapped in the space formed between closely apposed surface microvilli (microvillar channels) in hormone-stimulated steroidogenic cells. This is the same location where an HDL receptor (SR-BI) is found. In the current study, we sought to understand the relationship between SR-BI and selective CE uptake in a heterologous insect cell system. Sf9 (Spodoptera frugiperda) cells overexpressing recombinant SR-BI were examined for (i) SR-BI protein by Western blot analysis and light or electron immunomicroscopy, and (ii) selective lipoprotein CE uptake by the use of radiolabeled or fluorescent (BODIPY-CE)-labeled HDL. Noninfected or infected control Sf9 cells do not express SR-BI, show microvillar channels, or internalize CEs. An unexpected finding was the induction of a complex channel system in Sf9 cells expressing SR-BI. SR-BI-expressing cells showed many cell surface doublemembraned channels, immunogold SR-BI, apolipoprotein (HDL) labeling of the channels, and high levels of selective HDL-CE uptake. Thus, double-membraned channels can be induced by expression of recombinant SR-BI in a heterologous system, and these specialized structures facilitate both the binding of HDL and selective HDL-CE uptake.

“…Light and heavy plasma membrane fractions were isolated by a slight modification of the procedure described previously (16,(30)(31)(32). In brief, pooled ovaries from a group of four superovulated luteinized rats [day 7 post-hCG] or desensitized rats [luteinized ovaries treated with a second (24 h) dose of hCG (25 IU) on day 6] were freed of adhering fat and connective tissue, minced with scissors, and homogenized with 10 strokes in 10 ml of ice-cold STE medium (0.3 M sucrose, 1 M EDTA, and 10 mM Tris-HCl, pH 7.4) at 4 Њ C. After filtration through cheesecloth, the homogenates were subjected to differential centrifugation, and 800 g (10 min) and 20,000 g (20 min) pellets were prepared and used for the isolation of light and heavy plasma membranes, respectively.…”

Section: Isolation Of Light and Heavy Plasma Membrane Fractions From mentioning

confidence: 99%

Dimerization of the scavenger receptor class B type I

Journal of Lipid Research

Cortez

Leers-Sucheta

et al. 2004

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This study has examined the dimeric/oligomeric forms of scavenger receptor class B type I (SR-BI) and its alternatively spliced form, SR-BII, in a diverse group of cells and tissues: i.e., normal and hormonally altered tissues of mice and rats as well as tissues of transgenic animals and genetically altered steroidogenic and nonsteroidogenic cells overexpressing the SR-B proteins. Using both biochemical and morphological techniques, we have seen that these dimeric and higher order oligomeric forms of SR-BI expression are strongly associated with both functional and morphological expression of the selective HDL cholesteryl ester uptake pathway. Rats and mice show some species differences in expression of SR-BII dimeric forms; this difference does not extend to the use of SR-B cDNA types for transfection purposes. In a separate study, cotransfection of HEK293 cells with cMyc and V5 epitope-tagged SR-BI permitted coprecipitation and quantitative coimmunocytochemical measurements at the electron microscope level, suggesting that much of the newly expressed SR-BI protein in stimulated cells dimerizes and that the SR-BI dimers are localized to the cell surface and specifically to microvillar or double membraned intracellular channels.These combined data suggest that SR-BI self-association represents an integral step in the selective cholesteryl ester uptake process.