Post-translational translocation of secretory proteins at the endoplasmic reticulum

In eukaryotes, many secretory protein precursors are thought to translocate across the membrane of the endoplasmic reticulum (ER) co-translationally, in a process that is also dependent on their targeting via the signal recognition particle (SRP). However, a previously unsuspected number of metazoan secretory proteins have been shown to utilise a distinct, post-translational, mechanism(s) for their delivery to and translocation across the ER. Such proteins are unusually short; therefore SRP does not have sufficient opportunity to engage with their N-terminal signal sequence before polypeptide synthesis is complete. In this study, I examine the properties of secretory proteins that are known or potential candidates for post-translational translocation, and identify cytosolic components that mediate this process (Chapters 2.1 to 2.3). In vitro analysis using chimeric proteins and inhibitors of ER translocation suggested that both the signal sequence and mature domains of secretory proteins might influence their capacity for post-translational translocation (Chapter 2.1). These effects are most likely exerted by the Sec61 translocon of the ER membrane. The ER delivery of this class of proteins bears a striking resemblance to that of tail-anchored membrane proteins, with clear evidence for multiple overlapping pathways. Furthermore, a known tail-anchor protein delivery factor, TRC40, was found to mediate one of at least two distinct pathways that target short secretory proteins to the ER where they utilise the Sec61 complex for translocation (Chapter 2.2). During an in vitro screen of other cytosolic components potentially influencing post-translational translocation, Pex19 was observed to affect this process. Preliminary findings suggest that Pex19 appears to interact with proteins delivered to the ER post-translationally, and these observations merit further investigation (Chapter 2.3). Based on these results and current knowledge in the wider field, a working model describing the molecular mechanisms that underlie the post-translational translocation of short secretory proteins is presented, and I speculate on the delivery pathways and specific properties of these unique polypeptide substrates.