Bip Binding Affects Integration of Transmembrane Domains

The Sec61 translocon mediates translocation of proteins into the endoplasmic reticulum (ER) and enables integration of hydrophobic (H) segments into the lipid bilayer via its lateral gate. The luminal chaperone BiP (Kar2 in yeast) has been shown to act as a molecular ratchet, recruited to the emerging polypeptide by the J-domain of Sec63, and to be involved in post- and cotranslational translocation. In this study, we address the question whether BiP binding to the nascent chain affects the process of membrane integration vs. translocation, i.e. whether integration is governed by thermodynamic equilibrium between pore and membrane.To determine the hydrophobicity threshold of membrane integration, a series of model proteins based on the sequence of dipeptidylaminopeptidase B (DPAPB) with oligo-alanine H-segments containing increasing numbers of leucines. The translocated loop preceding the H-segments was replaced by a sequence of similar length containing multiple copies of segments known to bind BiP with high affinity or not to interact with BiP. While H-segments preceded by the DPAPB sequence required ∼3.7 Leu for 50% integration, this threshold was reduced to ∼2.1 Leu with the BiP-binding sequence and increased to ∼5.2 Leu with the non-binding sequence. These results show that membrane integration of an H-segment is influenced by the already translocated sequence preceding it and suggest that BiP binding promotes membrane integration. We observed changes of the apparent free energy in integration between 1 to 3 kcal/mol. We propose a model in which Brownian motion of the translocated loop with bound chaperones preferentially promotes release of the H-segment into the lipid bilayer. The work was supported by grant W0125 of the Austrian science fund (FWF).