Translocon-Associated Protein Complex (TRAP) is Crucial for Co-Translational Translocation of Pre-Proinsulin

Many unanswered questions remain in understanding the biosynthesis of the peptide hormone insulin. Here we elucidate new aspects in the mechanism of co-translational translocation initiation of pre-proinsulin in the endoplasmic reticulum. We utilize a translational arrest peptide derived from the x-box-binding protein (Xbp1) to induce ribosomal stalling and generate translocation intermediates. We find that the insulin signal sequence is rather weakly gating and requires the assistance of auxiliary translocon components to initiate translocation. Probing the translational intermediates with chemical crosslinking, we identified an early interaction with the translocon-associated protein (TRAP) complex. The TRAPβ subunit interacts with pre-proinsulin before the peptide enters the Sec61 translocon channel in a signal sequence-dependent manner. We describe the substrate sequence determinants that are recognized by TRAP on the cytosolic site of the membrane to facilitate substrate-specific opening of the Sec61 translocon channel. Our findings support the hypothesis that the TRAP-dependence is in part determined by the content of glycine and proline residues mainly within the signal sequence.