One important function of SSs is that protein translocation into the ER can be selectively attenuated in a signal sequence-dependent manner during ER stress to reduce the ER folding burden ( Kang et al., 2006). If the role of signal sequences is merely to mediate targeting and translocation into the ER, it is difficult to rationalize why signal sequences are evolved to use different accessory factors for their efficient translocation into the ER. These signal sequences require accessory factors including TRAM, TRAP, Sec63/Sec63, and luminal chaperones to improve their interaction with the Sec61 translocon and thereby promote the translocation of their mature domains into the ER ( Brodsky et al., 1995 Fons et al., 2003 Nguyen et al., 2018 Schorr et al., 2020b Voigt et al., 1996). Surprisingly, most SSs inefficiently interact with the Sec61 translon, leading to a nonlooped orientation that forces the mature domain of a nascent chain extruded into the cytosol ( Hegde and Kang, 2008). Hence, these SSs mediate efficient protein translocation through the Sec61 translocon. This results in a looped orientation in which the N-terminus of the SS is positioned at the cytosolic face of the translocon, and the following the hydrophobic portion of the SS becomes intercalated into the lateral gate of the Sec61 translocon ( Jungnickel and Rapoport, 1995 Kim et al., 2002 Voorhees and Hegde, 2016a). Surprisingly, only a minority of signal sequences, such as the signal sequence from prolactin, tightly engages with the Sec61 translocon. However, the interaction between the Sec61 translocon and SSs greatly varies. Earlier studies have shown that SRP can efficiently recognize a broad range of hydrophobic SSs of RNCs in the cytosol and target them to the ER of mammalian cells ( Hegde and Kang, 2008 Kim et al., 2002 Voorhees and Hegde, 2015). SSs are diverse and significantly differ in length, hydrophobicity, charge, and flanking mature domain, but all of them contain a core hydrophobic region of at least six non-hydrophilic residues. The SS is again recognized, and bound, this time, by the Sec61 translocon, allowing the translocation of the following nascent polypeptide across the ER membrane with concomitant cleavage of a signal peptide or integration of a TMD into the lipid bilayer. The SS is then transferred from the SRP to the heterotrimeric Sec61 translocon complex ( Rapoport, 2007).
The SRP-bound ribosome-associated nascent chains (RNCs) are then delivered to the ER membrane by an interaction with the SRP receptor ( Jiang et al., 2008). As SSs emerge from cytosolic ribosomes, they are co-translationally recognized and captured by the signal recognition particle (SRP) ( Shan and Walter, 2005). These nascent polypeptides typically contain either cleavable N-terminal signal sequences (SSs) or non-cleavable first transmembrane domains (TMDs). Thus, signal sequence-dependent protein folding explains why signal sequences are diverse and use multiple protein translocation pathways.Ībout 30% of newly synthesized proteins from the cytosolic ribosomes are delivered to the endoplasmic reticulum (ER) by the co-translational protein targeting pathway ( Juszkiewicz and Hegde, 2018). Increasing hydrophobicity of signal sequences bypasses Sec63/BiP-dependent protein translocation but translocated nascent chains misfold and aggregate under conditions of limited BiP availability in the ER. Surprisingly, BiP binding not only releases translocationally paused nascent chains into the ER lumen but also ensures protein folding. Using a substrate-trapping proteomic approach, we identify that nascent proteins with marginally hydrophobic signal sequences accumulate on the cytosolic side of the Sec61 translocon, which recruits BiP chaperone through Sec63 to bind onto nascent chains. We find that signal sequences cause a translocation pause at the Sec61 translocon until nascent chains engage with luminal chaperones for efficient translocation and folding in the ER.
It remains unclear how protein translocation coordinates with the chaperone availability to promote protein folding in the ER. One-third of newly synthesized proteins in mammals are translocated into the endoplasmic reticulum (ER) through the Sec61 translocon.