Immunocytochemical localization of mutant low density lipoprotein receptors that fail to reach the Golgi complex.

Abstract

Abstract. In the low density lipoprotein (LDL) receptor system, blocks in intracellular movement of a cell surface receptor result from naturally occurring mutations. These mutations occur in patients with familial hypercholesterolemia. One class of mutant LDL receptor genes (class 2 mutations) produces a receptor that is synthesized and glycosylated in the endoplasmic reticulum (ER) but does not reach the cell surface. These receptors contain serine/threonine-linked (O-linked) carbohydrate chains with core N-acetylgalactosamine residues and asparagine-linked (N-linked) carbohydrate chains of the high mannose type that are only partially trimmed. To determine the site of blockage in transport, we used electron microscope immunohistochemistry to compare the intracellular location of LDL receptors in normal human fibroblasts with their location in class 2 mutant fibroblasts. In normal cells, LDL receptors were located in coated pits, coated vesicles, endosomes, multivesicular bodies, and portions of the Golgi complex, In contrast, the mutant receptors in class 2 cells were almost entirely confined to rough ER and irregular extensions of the rough ER. Metabolic labeling studies with [3H]glucosamine confirmed that these mutant receptors contain core O-linked sugars, suggesting that the enzymes that attach these residues are located in the rough ER or the transitional zone of the ER. These studies establish that naturally occurring mutations in cell surface receptors can cause the receptors to remain trapped in the ER, thereby preventing their normal function and producing a genetic disease.

Intracellular transport of rubella virus structural proteins expressed from cloned cDNA

“…poorly (Baenziger & Fiete, 1979). The initial sugar of Olinked glycans is added in the ER (Pathak et al, 1988;Tooze et al, 1988). It has recently been shown that E2, but not E 1, contains O-linked glycans (Lundstr6m et al, 1991 ;; in agreement with this, we found that E2, but not E 1, bound to the O-glycan-specific lectin Jacalin (not shown).…”

A Highly Conserved Motif at the COOH Terminus Dictates Endoplasmic Reticulum Exit and Cell Surface Expression of NKCC2

“…The heterologous expression system in X. laevis oocytes has been shown to be an excellent tool for a robust expression and reproducible expression of NKCC2 proteins. However, it has also been shown for several mutated proteins that, whereas they are completely retained in the ER when expressed in mammalian cells (41)(42)(43), such ER-retained proteins are often transported to the plasma membrane of oocytes to some extent. For example, the ER-retained cystic fibrosis transmembrane conductance regulator lacking Phe-508 (CFTR-⌬F508) confers chloride conductance in oocytes, but it does not exit the ER in mammalian cells (44 -46).…”

Subdomains of the rough endoplasmic reticulum in colon goblet cells of the rat: lectin-cytochemical characterization.

“…Enzymes of initial 0-glycosylation have been found highly enriched in membranes derived from the Golgi apparatus (1,7,8,23), and the sugar nucleotides UDP-GalNAc, UDP-Gal, CMP-NeuNAc, and GDP-Fuc have been shown to be translocated predominantly into Golgi vesicles (26,43). On the other hand, 0-glycosylation of glycoprotein produced by gastric surface cells has been found to be initiated cotranslationally (50); studies with mutant LDL receptor (38). with proglucagon (39, and with El protein of mouse hepatitis virus A-59 (53) indicate that the synthesis of GalNAc-serinelthreonine linkages is an early event and occurs in a pre-Golgi compartment.…”