UC Berkeley

The endoplasmic reticulum (ER) is a major site for protein biosynthesis, required for production of about a third of all proteins in eukaryotic cells. The heterotrimeric Sec61 complex is a protein-conducting channel which mediates the transport or membrane integration of a majority of the ER-targeted proteins, such as secretory proteins and membrane proteins. Using a sophisticated gating mechanism, Sec61 transports soluble amino acid segments through its vertical water filled pore, and integrates hydrophobic transmembrane helices laterally into the membrane. The Sec61 channel translocates client polypeptides either co- or post-translationally. The posttranslational mode requires the formation of a larger complex with the membrane proteins Sec62 and Sec63. Substrate translocation further requires the essential ER resident Hsp70, BiP. The post translational complex in fungal species is additionally associated with the non-essential proteins Sec71 and Sec72. The structure and mechanism by which these proteins activate Sec61 for transport of client proteins has been poorly understood.

Using cryo-electron microscopy (cryo-EM), we determined the structures of several variants of the Sec61–Sec62–Sec63–Sec71–Sec72 complex (shortly, the Sec complex) from Saccharomyces cerevisiae and Thermomyces lanuginosus and showed that Sec62 and Sec63 induce opening of the Sec61 channel. The structures reveal that Sec63 is positioned in the back of Sec61 and interacts with Sec61 and Sec71-Sec72, while Sec62 is flexibly associated with the complex in front of the lateral gate. The J-domain of Sec63 is optimally positioned under the channel pore to bind BiP and enable efficient polypeptide translocation. Sec63 associates with Sec61 tightly through interactions in cytosolic, transmembrane, and ER-luminal domains. These extensive interactions pry open Sec61’s lateral gate. Association of Sec62 further opens the luminal end of the lateral gate causing dislocation of the small helix blocking the pore called the plug domain. A simultaneous disruption of the cytosolic and luminal interactions of Sec63 with Sec61 completely closes the channel and abolishes its binding with Sec62. The structures and molecular dynamics simulations suggest that Sec62 may also prevent lipids from invading the channel through the open lateral gate. Our studies show how Sec63 and Sec62 work together in a hierarchical manner to activate Sec61 for post-translational protein translocation.