N-Linked glycosylation is a common form of protein processing that can profoundly affect protein expression, structure, and function. N-Linked glycosylation generally occurs at the sequon Asn-X-Ser/Thr, where X is any amino acid except Pro. To assess the impact of the X amino acid on core glycosylation, rabies virus glycoprotein variants were generated by site-directed mutagenesis with each of the 20 common amino acids substituted at the X position of an Asn-X-Ser sequon. The efficiency of core

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... glycosylation at the sequon in each variant was quantified in a rabbit reticulocyte lysate cell-free translation system supplemented with canine pancreas microsomes. The presence of Pro at the X position completely blocked core glycosylation, whereas Trp, Asp, Glu, and Leu were associated with inefficient core glycosylation. The other variants were more efficiently glycosylated, and several were fully glycosylated. These findings demonstrate that the X amino acid is an important determinant of N-linked core-glycosylation efficiency. One of the most common types of protein modification is N-linked glycosylation, in which oligosaccharides are added to specific Asn residues (1, 2). N-Linked glycosylation plays a critical role in the expression of most cell-surface and secreted proteins and is often required for protein stability, antigenicity, and biological function (1, 3-6). The effects of N-linked glycosylation often depend on the number and position of N-linked oligosaccharides added to a protein chain (5, (7) (8) (9) (10) (11) . This is determined during core glycosylation, in which the oligosaccharide Glc 3 Man 9 GlcNAc 2 is transferred to a protein by the enzyme oligosaccharyltransferase (2, 12, 13). Oligosaccharyltransferase is integral to the endoplasmic reticulum membrane, and the active site of the enzyme resides near the endoplasmic reticulum membrane on the lumenal side (13-15). Core glycosylation usually occurs co-translationally as the glycosylation site on a nascent protein enters the endoplasmic reticulum lumen (14, 16 -18). Despite the importance of N-linked glycosylation, little is known about the protein signals that control the efficiency of oligosaccharide addition at specific Asn residues. N-Linked glycosylation generally occurs at the sequon Asn-X-Ser or Asn-X-Thr, where X is any amino acid except proline (Asn-X-Ser/ Thr) (15, 19, 20) . However, because many Asn-X-Ser/Thr se-1 The abbreviations used are: RGP, rabies virus glycoprotein; CGE, core-glycosylation efficiency.