More than 30% of human genes encode secretory or membrane proteins. Most secretory proteins are targeted to the Endoplasmic reticulum (ER) membrane via cleavable N-terminal signal sequences either in a co- or post-translational manner. They enter or cross the membrane using a protein translocating channel (translocon). Although the core of the translocon, formed by the Sec61 complex, was identified some time ago, the details of how signal sequences can facilitate channel opening and initiate protein translocation still remain unclear. Interestingly, the signal sequences of different proteins do not share any sequence homology—only general motifs have been described—but the precise sequence has been found to substantially affect the efficiency of translocation initiation. Many proteins require auxiliary components in order to enter the ER lumen. During ER stress conditions, these weakly gating proteins are prevented from entering, reducing the load of unfolded protein within the ER and protecting the cell. Consequently, it is tempting to hypothesize that the “inefficiencies” of signal sequences may actually provide a different message that works as a protective mechanism during ER stress conditions.

Here, we employed a translational arrest peptide, which pauses the ribosome until a force—such as the interaction of the signal sequence with the translocon—acts on the nascent chain.  We analyzed the different forces that are experienced by efficient and inefficient signal sequences during their biosynthesis in vitro. Our data shows that the efficient signal sequence of prolactin (Prl) experiences a strong biphasic pulling force while less efficient sequences, such as the ones from the Prion protein (PrP) or insulin, are pulled to a much lesser extent, indicating different modes of engagement with the translocon. The Prl signal sequence interacts first with a hydrophobic patch within the channel (the first pulling event), next it is inverted and intercalates into the lateral gate of the translocon, facilitating channel opening both laterally and axially. In the case of PrP or insulin, the initiation of translocation is delayed, suggesting that the opening of the channel might require auxiliary components. In order to explore this, we made use of semi-permeabilized cells (SPCs) prepared after siRNA knockdown of components of the translocation machinery and studied the effect on the observed pulling events and translocation efficiency. We found that the translocon-associated protein (TRAP) complex enhanced translocation of client proteins bearing weakly gating signal sequences that contained more glycine and proline residues. Additionally, we showed that TRAP plays a role in the translocation of intrinsically disordered domains with a high content of proline and glycine residues, and other regions of the mature protein enriched in positively charged amino acids. Chemical crosslinking revealed that TRAP contacts the insulin nascent chain before it enters the translocon channel suggesting that TRAP scans along the translocating protein and provides sequence-dependent assistance in facilitating channel opening and as a ratchet for challenging regions of the mature protein. Taken together, all of this data expands our understanding of the interplay between the signal sequence and the mature protein during translocation and protein folding and how the cell may take advantage of this to regulate translocation during ER stress.