Figure 1.

The signal sequence binds to Sec61p and Sec62p/71p with the same kinetics. (A) ppαF mutant containing a single photoreactive lysine derivative at position 10 (ppαF K10) was incubated with proteoliposomes containing purified Sec complex. At different time points, the binding reaction was stopped, and samples were irradiated with UV light as indicated. After solubilization with digitonin, the Sec complex was immunoprecipitated together with bound and cross-linked ppαF. The samples were analyzed by SDS-PAGE and autoradiography. Slow and fast mobility Sec61p cross-linked products are indicated by closed and open arrows, respectively; cross-linked products containing Sec62/71p by open squares; and lipid cross-links by open circles. (B) As in A, with a photoreactive lysine derivative at position 11 (ppαF K11). (C) As in A, with a photoreactive lysine derivative at position 12 (ppαF K12). (D) As in A, with a photoreactive lysine derivative at position 13 (ppαF K13). (E) As in A, with a photoreactive lysine derivative at position 14 (ppαF K14). (F) Quantification of the Sec61p cross-linked products (closed squares) and Sec62p/71p cross-linked products (stars) observed in (A-E) was performed with a PhosphorImager. Yields of cross-links are expressed relative to the amount of ppαF coimmunoprecipitated with the Sec complex. The Sec61p cross-links are the sum of all products containing Sec61p. Note that the outlier with position 11 at 0.5-min incubation is likely due to errors in the quantitation of the particularly weak Sec61p cross-links seen with this position.