Figure 3.

Downstream of the LA8-segment is exposed to the cytoplasmic side. (A) Cysteine scanning model protein to assess membrane topology. Cys-less model proteins included one glycosylation site (N⁶⁷) and either the LA8-segment or LA11-segment. A single Cys residue and the 6K-cluster were included at the indicated positions. (B) Position dependency of PEGmal reaction. The models were translated in the presence of RM and then subjected to PEGmal treatment. Where indicated, RM was not included in the reaction (w/o RM). The PEGylated form (←), glycosylated form (▶), and nonglycosylated form (○) are indicated. The experiments were repeated two times with each model and the mean PEGylation efficiency was plotted. (C) Reactivity to PEGmal and sensitivity to ProK digestion demonstrate that the LA8-segment spanned the membrane as the LA11-segment.