lso required for efficient translocation (13). Together, these results show that the Sec61 channel not only mediates co-translational translocation but is also required for post-translational protein transport. In the co-translational pathway the Sec61 channel associates with the ribosome, while the Sec62/63 complex is the major channel partner during post-translational translocation.
However, due to the lack of structural information on the posttranslocational Sec complex the mechanistic details of post-translational protein transport are poorly defined. With regards to the latter it should be noted that recently detailed structures of this complex were determined by cryo-EM (5,6). Figure 3 shows the structure of the post-translational Sec complex from yeast at 4.1 Å (5). This was obtained by single particle cryo-EM of the affinity purified complex from detergent solubilized yeast membranes and reveals that the post-translational Sec complex is made up of the Sec61 channel, Sec63 (in green), Sec71 (in magenta) and Sec72 (in grey). In turn, the Sec61 channel comprises Sec61(in blue), Sss1 (in pink) and Sbh1 (in yellow). No clear density for Sec62 was observed in the final electron density map, preventing inclusion of its atomic model in the final structure. The overall structure of the complex is shown as a surface representation (upper left panel with subunits indicated) or as a ribbon diagram (upper right panel with subunits indicated). The latter panel reveals that Sec61 contains 10 TMDs and is tightly associated with Sss1 and Sbh1, two smaller proteins that span the membrane one time. Sec63 has two TMDs, but its J domain that interacts with BiP is not observed in the structure possibly due to its flexible nature. Sec71 is a single-spanning membrane protein that anchors Sec72 to the ER membrane. Importantly, Sec71 and 72 are not found in mammals. A front (left) and top view (center) of Sec61 is provided in the bottom panels with the plug in red, TMD2 in orange and TMD7 in grey. All other subunits are omitted for clarity. Sec61 contains 10 TMDs that divide the protein into N- and C-terminal halves with TMD 1-5 and 6-10. Sec61 is open on one side (the front) that probably functions as a lateral gate (between TMD 2 and 7), enabling the channel to open toward the lipid bilayer. A structure of the homologous bacterial SecY channel in the closed state is provided in the bottom right panel with TMD7 in pink and TMD2 in green. One striking feature of Sec61 structure is a fully open lateral gate, while the plug is located in the center of the channel suggesting that it is closed. Opening of the lateral gate is caused by major conformational changes in TMD2, 3 and 4. Thus, opening of the gate and channel are probably separate events. Channel opening is caused by insertion of a polypeptide and subsequent displacement of the plug, whereas opening of the lateral gate is triggered by binding of a translocation partner, such as the ribosome, SecA, or Sec62/63. In the case of co-translational translocation binding of the ribosome opens the lateral gate partially, while in post-translational translocation, binding of Sec62/63 triggers wide opening of the lateral gate. This allows moderately hydrophobic signal sequences to exit into the bulk lipid and explains why these signal sequences are functional in post-translational translocation. Sec63 is associated with Sec61 via cytosolic loops 6/7 and 8/9, resulting in similar interactions as binding of the ribosome. This forces Sec61 in a gate-opened conformation. Binding of Sec63 not only controls opening of the lateral gate but also prevents ribosome binding to Sec61.
Model for protein post-translational translocation
Based on the structural data discussed above, a model for post-translational translocation has been proposed p(figure 4 adapted from 5). In the resting state (A) the channel and lateral gate are closed. The plug domain that blocks the channel is indicated by an asterisks. Binding of the Sec62/63 complex (B) primes the channel for translocation. The lateral gate is fully open