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Originally published In Press as doi:10.1074/jbc.M002583200 on July 10, 2000

J. Biol. Chem., Vol. 275, Issue 39, 30458-30464, September 29, 2000
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Vesicle-reconstituted Low Density Lipoprotein Receptor
VISUALIZATION BY CRYOELECTRON MICROSCOPY*

Hyesung JeonDagger and G. Graham Shipley§

From the Departments of Biophysics and Biochemistry, Center for Advanced Biomedical Research, Boston University School of Medicine, Boston, Massachusetts 02118

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 Mr ~115 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 ~120-Å length by ~45-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 full-length sequence of the human LDL receptor. The accessible cysteine was demonstrated using a maleimide-biotin·streptavidin 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.

* This work was supported by National Institutes of Health Research Grants HL 57405 and HL 26335.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Dept. of Pathology, Harvard Medical School, Boston, MA 02115.

§ To whom correspondence should be addressed: Biophysics Dept., Center for Advanced Biomedical Research, Boston University School of Medicine, 715 Albany St., Boston, MA 02118. Tel.: 617-638-4009; Fax: 617-638-4041; E-mail: shipley@med-biophd.bu.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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