Journal Article

An allosteric Sec61 inhibitor traps nascent transmembrane helices at the lateral gate

MacKinnon, Andrew L, Sharma, Ajay, Paavilainen, Ville O, Hegde, Ramanujan S, Taunton, Jack

2014

Overview

Membrane protein biogenesis requires the coordinated movement of hydrophobic transmembrane domains (TMD) from the cytosolic vestibule of the Sec61 channel into the lipid bilayer. Molecular insight into TMD integration has been hampered by the difficulty of characterizing intermediates during this intrinsically dynamic process. In this study, we show that cotransin, a substrate-selective Sec61 inhibitor, traps nascent TMDs in the cytosolic vestibule, permitting detailed interrogation of an early pre-integration intermediate. Site-specific crosslinking revealed the pre-integrated TMD docked to Sec61 near the cytosolic tip of the lateral gate. Escape from cotransin-arrest depends not only on cotransin concentration, but also on the biophysical properties of the TMD. Genetic selection of cotransin-resistant cancer cells uncovered multiple mutations clustered near the lumenal plug of Sec61α, thus revealing cotransin’s likely site of action. Our results suggest that TMD/lateral gate interactions facilitate TMD transfer into the membrane, a process that is allosterically modulated by cotransin binding to the plug. Cells are surrounded by a plasma membrane that acts like a barrier around the cell—keeping the cell’s boundaries distinct from surrounding cells and helping to regulate the contents of the cell. This plasma membrane is made up mostly of two layers of fatty molecules, and is also studded with proteins. Some of these membrane proteins act as channels that allow nutrients and other chemicals to enter and leave the cell, while others allow the cell to communicate with other cells and the outside environment. Like all proteins, membrane proteins are chains of amino acids that are linked together by a molecular machine called a ribosome. The ribosomes that make membrane proteins are located on the outside of a membrane-enclosed compartment within the cell called the endoplasmic reticulum. To eventually become embedded within a membrane, a new protein must—at the same time as it is being built—enter a channel within the membrane of the endoplasmic reticulum. The newly synthesized protein chain enters this channel, called Sec61, via an entrance near the ribosome and then threads its way toward the inside of the endoplasmic reticulum. However, there is also a ‘side-gate’ in Sec61 that allows specific segments the new protein to escape the channel and become embedded within the membrane. From here, the membrane protein can be trafficked to other destinations within the cell, including the plasma membrane. However, how the newly forming protein chain passes through the side-gate of Sec61 is not well understood. Now MacKinnon, Paavilainen et al. have used a small molecule called cotransin—which is known to interfere with the passage of proteins through Sec61—to observe the interactions between the Sec61 channel and the new protein. Cotransin appears to trap the new protein chain within the Sec61 channel by essentially ‘locking’ the side-gate. MacKinnon, Paavilainen et al. observed that the trapped protein interacts with the inside of the channel at the end closest to the ribosome—which is the likely location of the side-gate. In contrast, cotransin likely binds at the other end of the channel, to a piece of Sec61 that serves to plug the exit into the endoplasmic reticulum; and this plug is directly connected to the side-gate. By preventing the plug from moving out of the way, cotransin can somehow stop the new protein from passing through the side-gate. However, MacKinnon, Paavilainen et al. did find that some membrane proteins with certain physical and chemical properties could get through the gate, despite the presence of cotransin. The next challenge is to resolve exactly how interactions between cotransin and the Sec61 plug can block the escape of new proteins into the membrane.

• Publisher: eLife Sciences Publications Ltd,eLife Sciences Publications, Ltd
• Subject: Allosteric properties / Animals / Binding Sites / Biochemistry / Cancer / Cell Membrane - drug effects / Cell Membrane - metabolism / cotransin / Cross-Linking Reagents - pharmacology / Dose-Response Relationship, Drug / endoplasmic reticulum / Evolution / HCT116 Cells / HEK293 Cells / Humans / Hydrophobicity / Intermediates / Lipids / Membrane proteins / Membrane Proteins - antagonists & inhibitors / Membrane Proteins - chemistry / Membrane Proteins - genetics / Membrane Proteins - metabolism / Molecular Docking Simulation / Mutation / Peptides, Cyclic - metabolism / Peptides, Cyclic - pharmacology / Polypeptides / Protein Binding / Protein Structure, Secondary / Protein Structure, Tertiary / Protein Transport / Proteins / SEC Translocation Channels / Structure-Activity Relationship / Transfection / transmembrane domain / Transmembrane domains / Tumor Necrosis Factor-alpha - metabolism