Fig. 3.

Sequential integration of Shaker S3–S4 into the membrane. (A) Constructs for the translocation intermediates. The native glycosylation acceptor site, N263 (red square), was preserved in the S1–S2 loop to monitor the translocation of S1–S2. The G loop (red circle) was introduced into the S3–S4 loop to monitor the translocation of S3–S4. In the S1–Q348 protein, the glycosylation acceptor site in the S3–S4 loop was positioned inside the ribosome as a control. The position of the ribosome (ellipse) is shown. The distance between ribosomes and the translocon is estimated to be ≈30–40 residues based on the estimation reported by Voss et al. (38). (B) (Top and Middle) Assessment of the cotranslational membrane integration of the S3–S4 integration intermediates. Double dots, diglycosylated product. (Bottom) Efficiency of membrane integration of S3–S4 in truncated peptides. Efficiency (%) = diglycosylated form × 100/(monoglycosylated form + diglycosylated form). n = 3 and P < 0.05 (S1–S392 vs. S1–F402, A417, and K427).