(2). Ongoing translocation allows binding of other BiP molecules, which continues until translocation, is completed. After this, ADP is exchanged for ATP which triggers opening of the SBD and BiP is released from the substrate.
Despite its prevalence in yeast and bacteria, post-translational translocation in prokaryotes is mechanistically different from its eukaryotic counterpart. In bacteria, the SecY channel cooperates with SecA during post-translational translocation. This is an ATPase that associates with the SecY complex and uses the energy of ATP hydrolysis to push the polypeptide through the channel (8). Post-translational translocation is initiated by binding of SecB, a cytosolic chaperone, to the newly synthesized polypeptide chain. Subsequently, SecB delivers the polypeptide chain at the translocation site and interacts with SecA after which the polypeptide is handed over to SecA. Following insertion into the channel, the substrate protein is translocated by SecA using a pushing mechanism.
Biochemical and structural features of the post-translational translocation machinery
Co-translational targeting by the SRP was the first pathway that was discovered for protein transport across the endoplasmic membrane in eukaryotes (9). However, it was subsequently found that the model protein prepro-α factor is post-translationally translocated into yeast microsomes (10). Genetic and biochemical experiments identified several components that are required for protein transport in yeast. These include the Sec61 channel, made up of an α,β and γ subunit, as well as another complex comprising Sec62, Sec63, Sec71 and Sec72 (11). Of these, Sec62 and Sec63 are essential for cell viability unlike Sec71 and Sec72. It was, moreover, next found that Sec63, Sec71 and Sec72 copurify with BiP, a luminal Hsp70 chaperone (12). Using an in vitro translocation system, post-translational transport was reconstituted using proteoliposomes containing the purified Sec61 channel and the Sec62/63 complex from yeast, while BiP and ATP were also 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 f