his, the ribosome-nascent-chain-SRP complex is delivered at SRP receptor at the translocation channel and translation is resumed. The elongating polypeptide chain moves subsequently into the translocation channel and into the lumen of the ER. Hydrolysis of GTP triggers disassembly of the SRP-receptor complex, thereby initiating another targeting cycle (2). The SRP has typically a strong affinity for hydrophobic targeting sequences and moderately hydrophobic signal sequences are therefore overlooked by it. Hence, secretory proteins equipped with these signal sequences escape recognition by the SRP but need to remain unfolded after release from the ribosome (2). This is achieved through association with molecular chaperones of the Hsp40 class that probably also target the unfolded polypeptide to the translocation channel (4). While the Sec61 machinery is required for protein translocation in both the co and post-translational targeting pathways, both processes depend on a structurally different Sec61 complex. Specifically, during post-translational translocation in yeast the Sec61 channel associates with another membrane protein complex, known as the Sec62/Sec63 complex. This represents a tetrameric complex comprising Sec62, Sec63, Sec71 and Sec72. Additionally, the Sec61 channel partners with Bip, a luminal Hsp70 chaperone, that binds to the polypeptide during translocation, and thereby prevents its backsliding through the channel (2). In contrast to co-translational translocation, the molecular details of post-translational translocation are not well understood. However, recently determined high resolution structures of the post-translational translocation machinery from yeast have provided the first mechanistic insights into this process (5,6). Here, I will discuss these novel structures as well as our current understanding of post-translational translocation in eukaryotes.
Overview of post-translational translocation
Accumulating evidence indicates that post-translational translocation is found in all organisms. 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 