Determination of the distance between the oligosaccharyltransferase active site and the endoplasmic reticulum membrane.

By in vitro transcription/translation of model proteins in the presence of dog pancreas microsomes, we have measured the minimum distance of an acceptor site from the lumenal end of a transmembrane segment required for N-linked glycosylation, both when the acceptor site is placed N-and C-terminally to the membrane anchor.We observe a sharp threshold at a distance of 12-14 residues, suggesting that the oligosaccharyltransferase active site is 30-40 A above the membrane and is oriented roughly parallel to the membrane surface.