Monty Krieger

Murine SR-BI, a High Density Lipoprotein Receptor That Mediates Selective Lipid Uptake, Is N-Glycosylated and Fatty Acylated and Colocalizes with Plasma Membrane Caveolae

The class B, type I scavenger receptor, SR-BI, was the first molecularly well defined cell surface high density lipoprotein (HDL) receptor to be described. It mediates transfer of lipid from HDL to cells via selective lipid uptake, a mechanism distinct from receptor-mediated endocytosis via clathrin-coated pits and vesicles. SR-BI is expressed most abundantly in steroidogenic tissues (adrenal gland, ovary), where trophic hormones coordinately regulate its expression with steroidogenesis, and in the liver, where it may participate in reverse cholesterol transport. Here we have used immunochemical methods to study the structure and subcellular localization of murine SR-BI (mSR-BI) expressed either in transfected Chinese hamster ovary cells or in murine adrenocortical Y1-BS1 cells. mSR-BI, an ∼82-kDa glycoprotein, was initially synthesized with multiple high mannose N-linked oligosaccharide chains, and some, but not all, of these were processed to complex forms during maturation of the protein in the Golgi apparatus. Metabolic labeling with [3H]palmitate and [3H]myristate demonstrated that mSR-BI was fatty acylated, a property shared with CD36, another class B scavenger receptor, and other proteins that concentrate in specialized, cholesterol- and glycolipid-rich plasma membrane microdomains called caveolae. OptiPrep density gradient fractionation of plasma membranes established that mSR-BI copurified with caveolin-1, a constituent of caveolae; and immunofluorescence microscopy demonstrated that mSR-BI colocalized with caveolin-1 in punctate microdomains across the surface of cells and on the edges of cells. Thus, mSR-BI colocalizes with caveolae, and this raises the possibility that the unique properties of these specialized cell surface domains may play a critical role in SR-BI-mediated transfer of lipids between lipoproteins and cells.

Isolation of Chinese hamster cell mutants defective in the receptor-mediated endocytosis of low density lipoprotein☆

This paper describes a procedure for the isolation of mutant cells with defects in receptor-mediated endocytosis. The procedure takes advantage of the unique structure of low density lipoprotein, a plasma cholesterol transport protein that enters cells by receptor-mediated endocytosis. LDL contains a core of cholesteryl ester that can be extracted and reconstituted with hydrophobic molecules that convert the LDL into a toxic or fluorescent particle. Mutagenized Chinese hamster ovary cells were incubated with reconstituted LDL containing toxic 25-hydroxycholesteryl oleate. Wild-type cells take up this lipoprotein via the LDL receptor, liberate the 25-hydroxycholesterol in lysosomes, and die. To identify colonies of receptor-deficient cells from among the few survivors of the first selection step, we incubated the cells with LDL reconstituted with a fluorescent cholesteryl ester and picked colonies that failed to accumulate fluorescence. The two-step isolation procedure yielded receptor-deficient cells at a frequency of 1 in 105. The mutant cells grew in the presence of LDL reconstituted with 25-hydroxycholesteryl oleate at concentrations 100-fold higher than those that killed parental cells. The altered phenotypes have remained stable for more than 200 population doublings under non-selective conditions. Inasmuch as LDL can be coupled to ligands that bind to receptors other than the LDL receptor, the above method may have general utility in isolating cells with mutations affecting other receptor systems.