Vesicle-reconstituted Low Density Lipoprotein Receptor

Abstract

The low density lipoprotein (LDL) receptor is a key protein for maintaining cellular cholesterol homeostasis by binding cholesterol-rich lipoproteins through their apoB and apoE apoproteins. The LDL receptor is a transmembrane glycoprotein of M r ϳ115 kDa; based on its primary sequence, five distinct structural domains have been identified (Yamamoto, T., Davis, C. G., Brown, M. S., Schneider, W. J., Casey, M. L., Goldstein, J. L., and Russell, D. W. (1984) Cell 39, 27-38). As a first step toward providing a structural description of the intact LDL receptor, the receptor has been purified from bovine adrenal cortices, reconstituted into unilamellar egg yolk phosphatidylcholine vesicles, and imaged using cryoelectron microscopy (cryoEM). CryoEM has the advantage of providing images of the reconstituted LDL receptor in its frozen, fully hydrated state. LDL receptor molecules were visualized as elongated, stick-like projections from the vesicle surface with maximum dimensions ϳ120-Å length by ϳ45-Å width. In some of the images, a short arm (or arms) was visible at the distal end of the stick-like projections. The LDL receptor was labeled via accessible free cysteine residues, probably including that corresponding to Cys-431 of the known fulllength sequence of the human LDL receptor. The accessible cysteine was demonstrated using a maleimide-biotin⅐streptavidin conjugate and confirmed by labeling with monomaleimido-Nanogold. Images obtained by cryoEM showed that the extracellular stick-like domain of the reconstituted LDL receptor was labeled by Nanogold. This combined cryoEM-Nanogold labeling study has provided the first low resolution structural images of the reconstituted, full-length bovine LDL receptor. Low density lipoprotein (LDL)1 is the most abundant cholesterol-containing lipoprotein in human plasma (1). Since an elevated level of blood cholesterol is a major risk factor in developing atherosclerosis, the LDL receptor, which is responsible for the uptake of LDL into cells, has been studied extensively, notably by Brown and Goldstein (2). The LDL receptor is located in coated pit regions on the cell surface; it binds cholesterol-rich lipoproteins (LDL and ␤-VLDL) via receptor binding sub-domains of their apoB and apoE proteins, and the receptor-ligand complex is internalized by endocytosis. Acidification of the internal compartment of the endocytic vesicle leads to dissociation of the LDL receptor-LDL complex; the LDL receptor is recycled to the cell surface, and LDL is delivered to the lysosome. Release of cholesterol triggers a variety of intracellular metabolic and regulatory events, notably the regulation of cholesterol synthesis and expression of the LDL receptor and other genes (1-4). It should be stated that these studies of the LDL receptor were critical not only in providing a rationale for cholesterol homeostasis but also for laying the foundations for our understanding of intracellular trafficking mechanisms.The amino acid sequence of the LDL receptor has been reported for several species includin...

Localization of the N-terminal Domain of the Low Density Lipoprotein Receptor

“…Images of the reduced LDL receptor obtained by cryoEM showed more diffuse density at the end of the extracellular domain. Some images of the reduced LDL receptor still had visible stick-like or Y-shaped density as the previously reported images of the reconstituted receptor (31). The distal end of both the stick-like receptor and the Y-shaped receptor images showed additional diffuse density.…”

“…2B). Occasionally, images showed stick-like density linking the vesicle membrane to the bound LDL; this presumably represents part of the reconstituted LDL receptor (31). In the absence of calcium, the LDL receptors reconstituted into vesicles showed no interaction with LDL, even though the vesicles showed evidence of LDL receptor molecules extending from the bilayer surface (data not shown).…”

“…This characteristic of the LDL receptor has made it difficult to investigate its detailed structure by x-ray crystallography. Our cryoEM approach has provided low resolution images of the full-length bovine LDL receptor (31), and in this study we have used the gold labeling approach to determine the location of the cysteine-rich ligand binding domain. However, more detailed structural information must await improvements in specimen preparation for cryoEM and the crystallization of progressively larger extracellular domains of the LDL receptor (40,41).…”

“…Purification and Reconstitution-Purification of the LDL receptor from bovine adrenal cortices and reconstitution into egg yolk phosphatidylcholine (EYPC) vesicles followed the same procedures as reported previously (31).…”

“…Cryoelectron Microscopy-The specimens for cryoEM were prepared and visualized as described previously (31).…”