Abstract
SummaryProtein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation channel formed by the Sec61 complex. Unrelated small-molecule natural products and synthetic compounds inhibit Sec61 with differential effects for different substrates or for Sec61 from different organisms, making this a promising target for therapeutic intervention. To understand the mode of inhibition and provide insight into the molecular mechanism of this dynamic translocon, we determined the structure of mammalian Sec61 inhibited by the Mycobacterium ulcerans exotoxin mycolactone via electron cryo-microscopy. Unexpectedly, the conformation of inhibited Sec61 is optimal for substrate engagement, with mycolactone wedging open the cytosolic side of the lateral gate. The inability of mycolactone-inhibited Sec61 to effectively transport substrate proteins implies that signal peptides and transmembrane domains pass through the site occupied by mycolactone. This provides a foundation for understanding the molecular mechanism of Sec61 inhibitors and reveals novel features of translocon function and dynamics.
Highlights
Biogenesis of eukaryotic secretory or transmembrane proteins often occurs at the surface of the endoplasmic reticulum through the process of co-translational translocation
We purified ribosome-translocon complexes (RTCs) from canine microsomal membranes that had been incubated with mycolactone at a concentration that completely prevented prepro-a factor translocation (Figure S1)
To ensure that the observed changes were due to the presence of mycolactone rather than due to details of our preparation or imaging protocols, we prepared a control sample of ribosome-translocon complexes in the absence of inhibitor
Summary
Biogenesis of eukaryotic secretory or transmembrane proteins often occurs at the surface of the endoplasmic reticulum through the process of co-translational translocation. Proteins targeted to the membrane by a signal sequence are translocated during translation through a membrane-spanning protein conduit formed by the Sec61abg complex. The structure of the heterotrimeric Sec complex is conserved throughout evolution and contains the core channel-forming subunit Sec61a/SecY together with the smaller subunits Sec61b/SecG and Sec61g/SecE (Van den Berg et al, 2004; Voorhees et al, 2014). The translocon forms a gated channel that maintains membrane integrity while selectively opening to allow passage of the unfolded polypeptide chain. The translocon must open laterally to allow the transmembrane helices of membrane proteins to pass into the membrane environment as they are translated
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