Sequential triage of transmembrane segments by Sec61α during biogenesis of a native multispanning membrane protein

During polytopic protein biogenesis, the Sec61 translocon must rapidly orient and integrate multiple transmembrane segments (TMs) into the endoplasmic reticulum membrane. To understand this process, we examined interactions between Sec61 alpha and all six TMs of the aquaporin-4 (AQP4) water channel at defined stages of synthesis using incorporated photo-cross-linking probes. Each TM interacted with and moved through the translocon in a highly ordered and sequential fashion. Strong asymmetric Sec61 alpha cross-linking was observed for only one helix at a time, suggesting the presence of a single primary binding site. However, up to four TMs simultaneously contacted Sec61 alpha from different molecular environments. Thus, AQP4 integration by Sec61 alpha involves sequential triage of TMs from their initial portal of entry into multiple secondary sites within the translocon. This mechanism provides a means to facilitate early folding events before release into the lipid bilayer.