Figure 6.

Mutating residues NGGLQP between TM7a and TM7b shift chains toward conventional topology. PK digestion and carbonate extraction of polypeptides containing native (lanes 1–3) or ΔNGGLQP (lanes 4–10) from plasmids TM7a/b.P (A) or TM7a/b-8.P (B). Downward arrows indicate full-length chains, whereas upward arrows indicate protease-protected, P-reactive fragments. Topology of chains relative to the ER membrane is indicated. In B, downward arrows (lanes 1 and 4) indicate full-length glycosylated polypeptides. Additional bands in lane 1 represent unglycosylated polypeptides and cleavage products. The small 15-kDa band (lanes 2 and 5) represents <10% of total chains and is also partially resistant to digestion in the presence of detergent and therefore likely represents a residual fragment of the P reporter.