Our results show that the signal sequence binds directly to a site at which it contacts both Sec61p and Sec62p/71p. Cross-links to these components occur with the same kinetics during the incubation of ppαF with the Sec complex, and the signal sequence contacts these components simultaneously. These results argue against the possibility that the signal sequence stably interacts with Sec62p/71p before being transferred into the channel. Rather, the signal sequence seems to bind in a single step to a site that is formed by both Sec61p and Sec62p/71p. However, we cannot exclude that there is a preceding, fast recognition step that went undetected in our kinetic experiments. Sec62/71p seems to be localized at a defined site relative to TM2 and TM7 of Sec61p, presumably the site where the signal sequence laterally exits into the lipid phase. It is unlikely that Sec62p/71p is directly involved in signal sequence recognition, simply because it is not required in the cotranslational mode of translocation in mammals or in the posttranslational mode in bacteria. Its role still remains to be clarified. Previously, a consecutive interaction of the signal sequence with Sec62/63p complex components and Sec61p was proposed on the basis of experiments with a bifunctional, amino group cross-linking reagent (Lyman and Schekman, 1997
). An initial interaction of ppαF with Sec62p, Sec71p, and Sec72p was seen, whereas cross-links to Sec61p required a subsequent Kar2p- and ATP-dependent step. However, these experiments did not actually address interactions of the signal sequence because all cross-linkable lysines of ppαF are in the C-terminal portion of the polypeptide chain. A plausible alternative interpretation of these results is that in the initial binding stage, this portion is localized in the cytosol and cross-linkable to cytoplasmic domains of Sec62p, Sec71p, and Sec72p, whereas upon addition of Kar2p and ATP, it moves into the channel and is cross-linkable to Sec61p.
Our mapping experiments show that the translocation pore is lined by several regions of Sec61p. Single photoreactive probes in a translocating polypeptide chain that were located inside the channel gave cross-links to regions in the Sec61p molecule that are far apart in the primary sequence. For example, in experiments with proteoliposomes containing the purified Sec complex, ~20–30% of all molecules cross-linked to the N-terminal two TM segments, about the same percentage to the C-terminal two TM segments, and a significant fraction cross-linked to internal regions. Surprisingly, proteoliposomes containing purified Sec complex and native microsomes gave reproducibly somewhat different results. One possibility is that the mutant Sec complexes containing factor Xa cleavage sites may be more stable in native membranes than after purification and reconstitution into membranes, and the results in particular for internal regions of Sec61p may therefore differ. Another possibility is that the cross-linking yields may not be completely additive when the results of different cleavage mutants are compared. Furthermore, we have used different sec61 mutants for the experiments with native microsomes and purified Sec complex. Despite the quantitative differences, the major conclusion, i.e., that different regions are involved in forming the translocation pore, is true for experiments with both types of membranes. The strong contribution by TM segment 7 suggests that it may play a dual role in forming both the signal sequence binding site and the translocation pore, and supports the idea that they are in proximity of one another.
In contrast to the results with probes in the signal sequence, different positions in a polypeptide segment located inside the actual channel pore gave identical cross-linking patterns. Obviously, the pore must be designed to allow the passage of polypeptides with widely different amino acid sequences, and our results indicate that there are indeed no specific contacts with the translocating polypeptide chain. The signal sequence, on the other hand, must be recognized by Sec61p and must therefore make specific interactions with certain TM segments.
Our experiments with two simultaneous photoreactive probes incorporated into ppαF did not give any evidence for ppαF-Sec61p-Sec61p double-cross-links. With cross-linking probes at opposite sides of a helix formed by the hydrophobic core of the signal sequence (e.g., positions 9 and 14 or 10 and 15; ), one may have expected such a double-cross-link, if TM2 and TM7 belonged to different Sec61p molecules. Double-cross-links to Sec61p and Sec62p were observed with some combinations of photoreactive probes, and quantification indicates that the double-cross-links to two Sec61p molecules should have been even stronger if they occurred. Although of negative nature, these data therefore suggest that the signal sequence is not at the interface of two different Sec61p molecules, but rather intercalated into TM segments of a single molecule. Because the pore of the channel must be adjacent to the signal sequence binding site, it may also be generated by only one Sec61p molecule.