Figure 5. The signal-peptide cleavage site is buried in the membrane.

(A) Cartoon of the first 89 residues of gp160 attached to the P2 domain of protein leader peptidase (Lep) at the C-terminus. A first glycosylation site in Lep was used as translocation control. The second site was introduced at positions 39–47 to determine at which position the distance to the membrane is sufficient for glycosylation. Arrow indicates site of signal-peptide cleavage. (B) Gp160-Lep constructs with glycosylation sites N39, N40, N41, and N47, were in-vitro translated in presence (+) or absence (-) of dog pancreas microsomes (RM). Gp160-Lep received a single (<) or double (<<) glycan, or remained unglycosylated/untranslocated (o). (C) Band intensities of B were quantified and percentage of second glycosylation product was plotted against position of the engineered glycosylation site. The data in panels B and C are representative of multiple independent experiments (biological replicates).