Sec61: A static framework for membrane-protein insertion

Abstract

During their synthesis at the ribosome, many proteins have to be either translocated across or inserted into the endoplasmic reticulum (ER) membrane by the translocon, a multi-subunit complex located in the ER membrane. The universally conserved protein-conducting channel Sec61 forms the functional core of the translocon. Accessory translocon components, most notably the stoichiometric translocon associated protein complex (TRAP) and the near-stoichiometric oligosaccharyl-transferase (OST) complex, complement Sec61 and assist in protein transport and membrane protein integration or facilitate maturation of nascent chains by covalent modifications. Early characterization of Sec61 by conductance measurements indicated that it adopts at least 2 distinct conformational states to enable protein translocation and membrane insertion while preventing extensive ion flux: a moreconductive state when bound to the ribosome and a less-conductive state upon ribosome release. Sec61 is a hetero-trimeric complex, consisting of the central Sec61a subunit and 2 much smaller peripheral subunits, Sec61b and Sec61g. X-ray crystallographic analyses of prokaryotic Sec61 homologs revealed that Sec61a consists of 2 pseudo-symmetrical Nand C-terminal halves, each comprising 5 transmembrane helices, which form the translocation channel. The two domains are connected by a short ‘hinge’ helix allowing a jaw-like motion of the Nand C-terminal halves with respect to each other. Consistent with the early characterization of the protein-conducting channel, Sec61 was found to adopt 2 functionally different conformations: a state with a lateral opening between the 2 Sec61a halves, which allows hydrophobic helices to partition into the lipid bilayer (termed the lateral gate), as well as a laterally closed state (Fig. 1). Recent mechanistic models for the interplay of the ribosome and Sec61 were derived from single particle cryo-EM structures of ribosome-bound, detergent-solubilized Sec61 in distinct functional states. They suggested that ribosome-bound Sec61 is mostly present in a closed state and opens only transiently for integration of a nascent transmembrane helix into the membrane. However, these models were inconsistent with the earlier conductance measurements, which indicated that ribosome binding alone induces conformational changes of the native protein-conducting channel toward a more conductive state. This discrepancy illustrates the need for visualizing the conformation of ribosome-bound Sec61 in a lipid environment and in presence of all other translocon components. Cryo-electron tomography (CET) in combination with subtomogram analysis is an excellent method for studying the structures of large macromolecules in their natural environment. It is particularly attractive for studying membrane-embedded and –associated complexes, because detergent solubilization is not required, avoiding destabilization of the complex during purification. Developments in direct detector technology, automated tomography