Nogo-B receptor is necessary for cellular dolichol biosynthesis and proteinN-glycosylation

Abstract

Dolichol monophosphate (Dol-P) functions as an obligate glycosyl carrier lipid in protein glycosylation reactions. Dol-P is synthesized by the successive condensation of isopentenyl diphosphate (IPP), with farnesyl diphosphate catalysed by a cis-isoprenyltransferase (cis-IPTase) activity. Despite the recognition of cis-IPTase activity 40 years ago and the molecular cloning of the human cDNA encoding the mammalian enzyme, the molecular machinery responsible for regulating this activity remains incompletely understood. Here, we identify Nogo-B receptor (NgBR) as an essential component of the Dol-P biosynthetic machinery. Loss of NgBR results in a robust deficit in cis-IPTase activity and Dol-P production, leading to diminished levels of dolichol-linked oligosaccharides and a broad reduction in protein N-glycosylation. NgBR interacts with the previously identified cis-IPTase hCIT, enhances hCIT protein stability, and promotes Dol-P production. Identification of NgBR as a component of the cis-IPTase machinery yields insights into the regulation of dolichol biosynthesis.

A Lettuce (Lactuca sativa) Homolog of Human Nogo-B Receptor Interacts with cis-Prenyltransferase and Is Necessary for Natural Rubber Biosynthesis

“…Upon careful examination, two lettuce unigenes were identified, which were annotated as homologs of Lew1 in Arabidopsis and Nogo-B receptor (NgBR) in human. Both genes have been implicated in dolichol metabolism (24,26).…”

“…Lacking all enzymatic activity, NgBR is able to interact with human CPT (24). Recently, NgBR and its homologs in mice and yeast (NUS1) were identified as necessary subunits for CPT activities in respective organisms (35).…”

“…CPT Enzyme Assay and TLC-Yeast microsomes (50 g of protein) containing recombinant CPT/CPTL was assayed as described (24). The 50-l reaction mixture contained: 50 M FPP (Echelon, Biosciences Inc., Salt Lake City, UT), 175 M IPP (Echelon, Biosciences Inc., Salt Lake City, UT), 25 M 14 C-IPP (1.1 ϫ 105 dpm; PerkinElmer Life Sciences), 50 mM HEPES, pH 7.5, 5 mM MgCl 2 , 2 mM DTT, 2 mM Na 3 VO 4 , and 2 mM NaF.…”

“…In humans, NgBR interacts with the Nogo-B peptide and Niemann-Pick type C2 (NPC2) protein to mediate signals for the proliferation of epithelial cells and to regulate intracellular cholesterol trafficking, respectively (23,25). Interestingly, it also interacts with the human CPT enzyme (24). In plant, its homolog in Arabidopsis (LEW1) was identified during genetic screening for a leaf wilting phenotype (26).…”

“…It was recently identified that the human Nogo-B receptor (NgBR) shares weak sequence homology to CPT (Ͻ15% at the amino acid level), and it lacks all five motifs conserved in all CPTs (23,24). In humans, NgBR interacts with the Nogo-B peptide and Niemann-Pick type C2 (NPC2) protein to mediate signals for the proliferation of epithelial cells and to regulate intracellular cholesterol trafficking, respectively (23,25).…”