Abstract
Signal peptide cleavage and N-glycosylation of proteins are co-translational processes, but little is known about their interplay if they compete for adjacent sites. Here we report two unique findings for processing of glycoprotein 3 of equine arteritis virus. Glycoprotein 3 (Gp3) contains an N-terminal signal peptide, which is not removed, although bioinformatics predicts cleavage with high probability. There is an overlapping sequon, NNTT, adjacent to the signal peptide that we show to be glycosylated at both asparagines. Exchanging the overlapping sequon and blocking glycosylation allows signal peptide cleavage, indicating that carbohydrate attachment inhibits processing of a potentially cleavable signal peptide. Bioinformatics analyses suggest that a similar processing scheme may exist for some cellular proteins. Membrane fractionation and secretion experiments revealed that the signal peptide of Gp3 does not act as a membrane anchor, indicating that it is completely translocated into the lumen of the endoplasmic reticulum. Membrane attachment is caused by the hydrophobic C terminus of Gp3, which, however, does not span the membrane but rather attaches the protein peripherally to endoplasmic reticulum membranes.
Highlights
N-Glycosylation and signal peptide cleavage are co-translational modifications of membrane proteins
We hypothesized that attachment of carbohydrates to the overlapping sequon (NNTT) located just two amino acids downstream of the predicted signal peptide cleavage site prevents cleavage (Fig. 1A)
When Glycoprotein 3 (Gp3)-YFP was expressed in CHO cells, it ran as two bands upon labeling with [35S]methionine/cysteine in SDS-PAGE
Summary
N-Glycosylation and signal peptide cleavage are co-translational modifications of membrane proteins. Results: Attachment of carbohydrates adjacent to a potentially cleavable signal peptide inhibits its processing in a glycoprotein from an arterivirus. Signal peptide cleavage and N-glycosylation of proteins are co-translational processes, but little is known about their interplay if they compete for adjacent sites. Glycoprotein 3 (Gp3) contains an N-terminal signal peptide, which is not removed, bioinformatics predicts cleavage with high probability. There is an overlapping sequon, NNTT, adjacent to the signal peptide that we show to be glycosylated at both asparagines. Exchanging the overlapping sequon and blocking glycosylation allows signal peptide cleavage, indicating that carbohydrate attachment inhibits processing of a potentially cleavable signal peptide. Membrane fractionation and secretion experiments revealed that the signal peptide of Gp3 does not act as a membrane anchor, indicating that it is completely translocated into the lumen of the endoplasmic reticulum. Membrane attachment is caused by the hydrophobic C terminus of Gp3, which, does not span the membrane but rather attaches the protein peripherally to endoplasmic reticulum membranes
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have