Signal sequences encode information for protein folding in the endoplasmic reticulum.

One-third of newly synthesized proteins in mammals are translocated into the endoplasmic reticulum (ER) through the Sec61 translocon. How protein translocation coordinates with chaperone availability in the ER to promote protein folding remains unclear. We find that marginally hydrophobic signal sequences and transmembrane domains cause transient retention at the Sec61 translocon and require the luminal BiP chaperone for efficient protein translocation. Using a substrate-trapping proteomic approach, we identify that nascent proteins bearing marginally hydrophobic signal sequences accumulate on the cytosolic side of the Sec61 translocon. Sec63 is co-translationally recruited to the translocation site and mediates BiP binding to incoming polypeptides. BiP binding not only releases translocationally paused nascent chains but also ensures protein folding in the ER. Increasing hydrophobicity of signal sequences bypasses Sec63/BiP-dependent translocation, but translocated proteins are prone to misfold and aggregate in the ER under limited BiP availability. Thus, the signal sequence-guided protein folding may explain why signal sequences are diverse and use multiple protein translocation pathways.