Fig. 2.

Topogenic functions of Shaker S3 or S4. (A) The model proteins used to assess the topogenic function of S3 or S4 (SA-I, SA-II, translocation-reinitiating function, and stop-transfer function). H1 and S indicate, respectively, the H1 segment and the signal peptide. (B) Experimental results and the deduced topologies. a_g, glycosylated product; a_ng, nonglycosylated product; b, proteinase K-resistant product; c, proteinase K-resistant product with a cleaved signal peptide. The dots indicate monoglycosylated products. (C) Summary of the topogenic functions of Shaker S3 or S4 (∗). The results on the stop-transfer function of S3 and the translocation-reinitiating function of S4 are given in SI Text (∗∗). The data for KAT1 are from Sato et al. (26). The efficiency of topogenic function was calculated using the following formulas: SA-I efficiency = a_g × 100/(a_g + a_ng); SA-II efficiency = b × k⁻¹ × 100/(a_g + a_ng); translocation-reinitiating efficiency = b × k⁻¹ × 100/a_g; and stop-transfer efficiency = c × 100/(b + c), where k is the proteinase K protection efficiency of the translocated PL estimated under our experimental conditions using the formula k = (PL after proteinase K treatment)/(PL before proteinase K treatment). The number of [³⁵S]methionines in each digested form was taken into consideration. (D) (Upper) The model E. coli leader peptidase protein has two N-terminal TM segments (H1 and H2) and a large C-terminal domain. S4 from Shaker or KAT1 was inserted into the C-terminal domain at a position where it was flanked by two glycosylation acceptor sites (G1 and G2) (31). (Lower) S4 sequences of KvAP, Shaker, and KAT1. GGPG-GPGG flanking sequences were added at the ends of S4 in the model proteins. (E) Membrane integration of Shaker and KAT1 S4. (Left) Plasmids encoding the constructs were transcribed and translated in vitro in the absence (−) and presence (+) of RMs. White circle, nonglycosylated product; single black circle, monoglycosylated product; two black circles, diglycosylated product. (Right) If S4 is integrated into the membrane, only G1 will be glycosylated (see left side); if not, both G1 and G2 will be glycosylated (see right side). (F) ΔG_app values deduced from the data in E. The apparent free energy (ΔG_app) of S4 insertion is defined as ΔG_app = −RT ln(f_1g/f_2g), where R is the gas constant, T the absolute temperature, and f_1g and f_2g are the fractions of monoglycosylated and diglycosylated molecules, respectively. The ΔG_app value for KvAP (∗∗∗) is from Hessa et al. (31).