[19] Receptor-mediated endocytosis of low-density lipoprotein in cultured cells

doi:10.1016/0076-6879(83)98152-1

Methods in Enzymology

Volume 98, 1983, Pages 241-260

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Studies of the cell surface binding, internalization, and metabolism of low-density lipoprotein (LDL) in cultured cells have provided useful information regarding the general aspects of receptor-mediated endocytosis. The study of the LDL receptor has been facilitated by analysis of mutant fibroblasts obtained from human subjects with disorders of cholesterol metabolism. The most informative cells, obtained from patients with familial hypercholesterolemia (FH), have defects in the gene encoding the LDL receptor. The existence of three classes of mutant alleles at the LDL receptor locus has been deduced on the basis of genetic and kinetic data. One of these alleles specifies a receptor that is unable to bind LDL. The second type of allele specifies a receptor that can bind small amounts of LDL; and the third type of allele specifies a receptor that can bind LDL but cannot be incorporated into coated pits and hence cannot carry the LDL into the cell. The first two alleles are common among FH patients, whereas the third allele is extremely rare.

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The enzymatic pathway of cholesterol biosynthesis has been well characterized. However, there remain several potential interacting proteins that may play ancillary roles in the regulation of cholesterol production. Here, we identified ERG28 (chromosome 14 open reading frame 1 [C14orf1]), a homologue of the yeast protein Erg28p, as a player in mammalian cholesterol synthesis. ERG28 is conserved from yeast to humans but has been largely overlooked in mammals. Using quantitative RT-PCR, luciferase assays, and publicly available chromatin immunoprecipitation sequencing data, we found that transcription of this gene is driven by the transcription factor SREBP-2, akin to most cholesterol synthesis enzymes, as well as identifying sterol-responsive elements and cofactor binding sites in its proximal promoter. Based on a split luciferase system, ERG28 interacted with itself and two enzymes of cholesterol synthesis (NSDHL and SC4MOL). Huh7 ERG28-KO cell lines were generated, revealing reduced total cholesterol levels in sterol-depleted environments. In addition, radiolabeled metabolic flux assays showed a 60–75% reduction in the rate of cholesterol synthesis in the KO versus wild-type cells, which could be rescued by expression of ectopic ERG28. Unexpectedly, KO of ERG28 also impaired the activation of SREBP-2 under sterol-replete conditions, by a yet-to-be defined mechanism. These results indicate that ERG28 is clearly involved in cholesterol synthesis, although the precise role this noncatalytic protein plays in this complex metabolic pathway remains to be fully elucidated. A deeper understanding of ERG28, and other ancillary proteins of cholesterol synthesis, may help inform therapeutic strategies for diseases associated with aberrant cholesterol metabolism.
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[19] Receptor-mediated endocytosis of low-density lipoprotein in cultured cells

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J. Biol. Chem.

Cell

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Cell

Cell

Cell

Biochem. Biophys. Acta

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Cell

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Nature (London)