Most, if not all, proteins destined for the organelles and membranes of the endomembrane system - the Golgi complex, lysosomes, endosomes, the nuclear and plasma membrane – are in eukaryotic cells initially translocated across the endoplasmic reticulum membrane (1). These proteins are known as secretory proteins and their transport can be accomplished through mechanistically distinct translocation modes (figure 1 adapted from 1). For example, in co-translational targeting proteins are directed to the endoplasmic membrane while they are being synthesized on ribosomes, whereas some proteins are transported after their translation, thus, post-translationally (2). Pioneering experiments by Günter Blobel and coworkers established that signal sequences are responsible for targeting secretory proteins to the endoplasmic membrane (3). A signal sequence comprises an N-terminal extension of about 20 to 30 residues that specifies membrane translocation of the adjacent mature domain and is typically cleaved following transport. Likewise, membrane proteins are also synthesized with an N-terminal targeting signal, which usually comprises the first transmembrane domain (TMD), but this is not proteolytically removed and is therefore known as a signal anchor sequence (1). Hydrophobicity of the targeting signal is an important factor in determining whether a protein is funneled into the co-translational or post-translational pathway. It has been shown that secretory proteins with a hydrophobic signal sequence as well as membrane proteins are in general translocated co-translationally, whereas proteins with a less hydrophobic signal sequence are transported in a post-translational fashion (2). Both pathways converge at the Sec61 translocation channel made up of an α, β and γ subunit embedded into the endoplasmic membrane (figure 1). In co-translational targeting, the signal sequence is recognized by the signal recognition particle (SRP) as soon as it emerges from the ribosomal exit tunnel, resulting in translational arrest. After this, 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