How does Sec63 affect the conformation of Sec61 in yeast?

The Sec complex catalyzes the translocation of proteins of the secretory pathway into the endoplasmic reticulum and the integration of membrane proteins into the endoplasmic reticulum membrane. Some substrate peptides require the presence and involvement of accessory proteins such as Sec63. Recently, a structure of the Sec complex from Saccharomyces cerevisiae, consisting of the Sec61 channel and the Sec62, Sec63, Sec71 and Sec72 proteins was determined by cryo-electron microscopy (cryo-EM). Here, we show by co-precipitation that the accessory membrane protein Sec62 is not required for formation of stable Sec63-Sec61 contacts. Molecular dynamics simulations started from the cryo-EM conformation of Sec61 bound to Sec63 and of unbound Sec61 revealed how Sec63 affects the conformation of Sec61 lateral gate, plug, pore region and pore ring diameter via three intermolecular contact regions. Molecular docking of SRP-dependent vs. SRP-independent peptide chains into the Sec61 channel showed that the pore regions affected by presence/absence of Sec63 play a crucial role in positioning the signal anchors of SRP-dependent substrates nearby the lateral gate. Author summary The ribosome particles of a cell constantly synthesize fresh proteins. Some of these will be either integrated into organellar membranes or will be secreted from the cell. To this aim, they need to enter a channel protein complex termed translocon in the membrane of the endoplasmatic reticulum. The translocon needs to determine based on certain sequence features whether the peptide should be translocated into the endoplasmatic reticulum lumen or whether opening of a lateral gate will enable that its transmembrane domains slide laterally into the membrane. Whereas it is known that some peptides cannot be translocated without the involvement of accessory membrane proteins such as the Sec63 protein, the mechanistic details how Sec63 affects the conformation and/or dynamics of the translocon are so far not known. Here, we used molecular simulations, molecular docking and co-precipitation experiments to characterize protein contact residues and conformational shifts in the channel pore. Our simulations reveal that the plug moiety adopts different conformations in the channel in the presence, respectively the absence of Sec63, and we describe the influence of Sec63 on the conformation of the lateral gate. This study contributes to our understanding of the functionality of translocon and accessory proteins, and may eventually aid in characterizing and overcoming the effects of mutations related to protein translocation defects.

Automated summary

The Sec complex mediates protein translocation into the endoplasmic reticulum and membrane integration. Cryo-EM determined the structure of the Sec complex, showing that Sec62 is not necessary for stable Sec63-Sec61 contacts. Molecular simulations revealed the influence of Sec63 on the conformation of Sec61, particularly in the pore region. The study helps understand the functionality of the translocon and accessory proteins, potentially aiding in addressing protein translocation defects caused by mutations.