Spontaneous and Protein-mediated Sterol Transfer between Intracellular Membranes

Andrey Frolov,Judith K Woodford,Eric J Murphy,Jeffrey T Billheimer,Friedhelm Schroeder

doi:10.1074/jbc.271.27.16075

Andrey Frolov, Judith K Woodford + Show 3 more

Open Access

https://doi.org/10.1074/jbc.271.27.16075

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Abstract

Relatively little is known regarding intracellular cholesterol trafficking pathways. To resolve some of these potential pathways, spontaneous and protein-mediated sterol transfer was examined between different donor-acceptor membrane pairs in vitro using L-cell fibroblast plasma membrane (PM) and microsomal (MICRO) and mitochondrial (MITO) membranes. Several new exciting insights were provided. First, the initial rate of spontaneous molecular sterol transfer was more dependent on the type of acceptor than donor membrane, i.e. spontaneous intracellular sterol trafficking was vectorial. Therefore, the rate of sterol desorption from the donor membrane was not necessarily the rate-limiting step in molecular sterol transfer. Second, the rate of molecular sterol transfer was not obligatorily correlated with the direction of the cholesterol gradient. For example, although PM had a 3.2-fold higher cholesterol/phospholipid ratio than MITO, spontaneous sterol transfer was 4-5-fold faster up (MITO to PM) rather than down (PM to MITO) the concentration gradient. Third, sterol carrier protein-2 differentially stimulated the initial rate of sterol transfer for all donor-acceptor combinations, being most effective with PM donors: PM-MICRO, 27-fold; and PM-MITO, 12-fold. Sterol carrier protein-2 was less effective in enhancing sterol transfer in the reverse direction, i.e. MICRO-PM and MITO-PM (5- and 4-fold, respectively). Fourth, liver fatty acid-binding protein was limited in stimulating the initial rate of sterol transfer from PM to PM (1.5-fold), from PM to MITO (3-fold), and from MICRO to MITO (3-fold). In summary, these observations present important insights into potential sterol trafficking pathways between the major membrane components of the cell.

Highlights

Terol biosynthesis and synthesis of DNA, RNA, and phospholipids (9, 10)
Intermembrane Sterol Exchange: Plasma Membranes as Donors—Plasma membrane (PM) donor DHE fluorescence polarization in the absence of acceptor membranes was stable during the time of assay (Fig. 1A, curve 1)
Spontaneous sterol transfer occurs most rapidly between the cholesterol-rich plasma membranes as compared with relatively cholesterol-poor mitochondria. This surprising finding is complemented by data showing for the first time the existence and vectorial nature, at least in vitro, of the spontaneous intracellular sterol trafficking process

Summary

Introduction

Terol biosynthesis and synthesis of DNA, RNA, and phospholipids (9, 10). In addition, cholesterol biosynthetic intermediates such as isoprenoids are required for glycoprotein synthesis and the synthesis of ubiquinones (11, 12). Extensive sterol transfer and targeting must occur between these intracellular membranes, either against or with the cholesterol gradient, respectively (4, 16 –18). Despite their importance to sterol homeostasis in the cell, the major pathways of intracellular sterol trafficking are still unclear. The second objective was to investigate the role of SCP-21 and L-FABP, cytosolic lipid transfer proteins, in targeting potential pathways of intracellular sterol trafficking. These specialized cytosolic proteins modulate specific reactions of sterol metabolism in vitro (23–25) and enhance sterol transfer between model membranes (25–29) and cell plasma membranes in vitro (4, 22).

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