A.
In the ER, individual sugars are not added one-by-one to the protein to create an oligosaccharide side chain. Instead, a preformed, branched oligosaccharide containing a total of 14 sugars is attached en bloc to all proteins that carry the appropriate site for glycosylation.
B.
The oligosaccharide is originally attached to a specialized lipid, called dolichol, in the ER membrane; it is then transferred to the amino (NH 2 ) group of an asparagine side chain on the protein, immediately after a target asparagine emerges in the ER lumen during protein translocation. Asparagines that are glycosylated are always present in the tripeptide sequences asparagine-X-serine or asparagine-X-threonine, where X can be almost any amino acid.
C.
The addition takes place in a single enzymatic step that is catalyzed by a membrane-bound enzyme (an oligosaccharyl transferase) that has its active site exposed on the lumenal side of the ER membrane— which explains why cytosolic proteins are not glycosylated in this way.
D.
A simple sequence of three amino acids, of which the target asparagine is one, defines which sites in a protein receive the oligosaccharide. Oligosaccharide side chains linked to an asparagine NH 2 group in a protein are said to be N-linked and this is by far the most common type of linkage found on glycoproteins.