. However, its importance appears to be species-dependent. In mammals, for example, the vast majority of proteins are targeted co-translationally, while in baker’s yeast and bacteria both translocation modes are equally important. Most post-translationally translocated proteins are small – less than 75 residues – and are equipped with a moderately hydrophobic signal sequence (7). Owing to this, these proteins escape recognition by the co-translational SRP system which preferentially interacts with hydrophobic targeting signals. Figure 2 (adapted from 2) is a schematic representation of post-translational translocation in yeast, which probably holds for all eukaryotes. Following completion of its synthesis, the polypeptide is released from the ribosome and kept in an unfolded state through association with cytosolic chaperones. These probably also deliver the polypeptide at the translocation site in the endoplasmic membrane, comprising the Sec61 channel and the associated auxiliary Sec62/63 complex. This latter complex is made up of four membrane proteins, namely: Sec62, Sec63, Sec71 and Sec72 (2). Sec72 contains multiple chaperone binding sites and it is therefore conceivable that Sec72 captures chaperone-bound substrate proteins. This represents the first step in the post-translational translocation cycle and during this step all chaperones are released from the polypeptide, which is subsequently inserted into the Sec61 channel as a hairpin. Once inside the channel, the polypeptide is bound by the luminal Hsp70 chaperone BiP that prevents its backsliding into the cytosol. This, therefore, creates an overall forward translocation movement. It has been established that BiP contains a nucleotide and substrate-binding domain (SBD). The latter can either be in an open or closed state depending on whether ATP or ADP is bound. ATP-bound BiP contains an open SBD and interaction with the J-domain of Sec63 triggers ATP hydrolysis and subsequent closure of the SBD around the polypeptide that emerges from the Sec61 channel (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 a