Scanning N-glycosylation mutagenesis of membrane proteins.
N-Glycosylation of eukaryotic membrane proteins is a co-translational event that occurs in the lumen of the endoplasmic reticulum (ER). This process is catalyzed by a membrane-associated oligosaccharyl transferase (OST) complex that transfers a preformed oligosaccharide (Glc(3)Man(9)GlcNAc(2)-) to an asparagine (Asn) side-chain acceptor located within the sequon (-Asn-X-Ser/Thr-). Scanning N-glycosylation mutagenesis experiments, where novel acceptor sites are introduced at unique sites within membrane proteins, have shown that the acceptor sites must be located a minimum distance (12-14 amino acids) away from the luminal membrane surface of the ER in order to be efficiently N-glycosylated. Scanning N-glycosylation mutagenesis can therefore be used to determine membrane protein topology and it can also serve as a molecular ruler to define the ends of transmembrane (TM) segments. Furthermore, since N-glycosylation is a co-translational event, N-glycosylation mutagenesis can be used to identify folding intermediates in membrane proteins that may expose segments to the ER lumen transiently during biosynthesis.
Cheung
Joanne C
JC
Department of Biochemistry, University of Toronto, Room 5216, Medical Sciences Building, Toronto, Ont., Canada M5S 1A8.
Reithmeier
Reinhart A F
RA
D000595:Amino Acid Sequence; D002474:Cell-Free System; D004798:Enzymes; D006031:Glycosylation; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009844:Oligosaccharides / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D004798:Enzymes; D008565:Membrane Proteins; D009844:Oligosaccharides