Figure 3

Determination of stop-transfer activity of different full-length constructs. (A) Model segments (white) lacking stop-transfer activity are translocated into the ER lumen and both glycosylation acceptor sites become modified (left). For model segments with stop-transfer function, only the upstream site will become modified (right; Y denotes a glycosylated site, Φ a non-glycosylated site). The C-terminal glycosylation site is 6 residues away from the C-terminus of the protein (see Materials and Methods). (B) In vitro translation in the absence (-) and presence (+) of rough dog pancreas microsomes (RM) of constructs with the indicated model segments. Non-glycosylated and mono- and di-glycosylated molecules are indicated by a black dot, a white dot, and two white dots, respectively. (C) Proteinase K (PK) treatment of microsomes carrying in vitro translated wild type Lep (left panel) or the full-length V¹⁸ construct (right panel). The detergent Triton X-100 was included to dissolve the microsomal membrane in lanes 4 and 8. Non-glycosylated and glycosylated molecules are indicated by a black dot and a white dot, respectively. Protease-protected fragments are indicated by a white square.