Structure of Monomeric Yeast and Mammalian Sec61 Complexes Interacting with the Translating Ribosome

Abstract

The trimeric Sec61/SecY complex is a protein-conducting channel (PCC) for secretory and membrane proteins. Although Sec complexes can form oligomers, it has been suggested that a single copy may serve as an active PCC. We determined sub-nanometer resolution cryo-electron microscopy structures of eukaryotic ribosome-Sec61 complexes. In combination with biochemical data we found that in both idle and active states, the Sec complex is not oligomeric and interacts mainly via two cytoplasmic loops with the universal ribosomal adaptor site. In the active state the ribosomal tunnel and a central pore of the monomeric PCC were occupied by the nascent chain contacting loop 6 of the Sec complex. This provides a structural basis for the activity of a solitary Sec complex in cotranslational protein translocation.The protein-conducting channel (PCC) of the canonical secretory pathway is formed in all cells by the Sec61/SecY complex. It engages in the post-and co-translational translocation of secretory proteins across, and the insertion of integral membrane proteins into the membrane of the endoplasmic reticulum (ER) in eukaryotes and the plasma membrane of bacteria (1,2).# To whom correspondence should be addressed. beckmann@lmb.uni-muenchen.de; elisabet.mandon@umassmed.edu. In the co-translational translocation mode the ribosome with an emerging signal sequence is targeted to the membrane by the signal recognition particle (SRP) and its receptor (3). Here, the Sec complex acts as a receptor for the ribosome via its cytosolic loops (4). The alignment of the ribosomal tunnel with a central pore of the PCC allows direct movement of the nascent chain from the ribosomal tunnel exit across or into the membrane (5,6).The PCC-forming heterotrimeric Sec complex consists of one large subunit (Sec61α in Mammalia, Sec61p/Ssh1p in yeast, SecY in Bacteria) and two small subunits (Sec61β, γ in eukaryotes and SecE, G in Bacteria). Conflicting models have been presented as to how many of these heterotrimers are necessary to build an active PCC and what the actual path of the polypeptide chain is. The Escherichia coli SecYEG complex forms back-to-back dimers in two-dimensional crystals (7), and low resolution single particle electron microscopic (EM) data revealed a pentagonal ringlike morphology of the PCC interpreted as oligomers (5,6,(8)(9)(10)(11)(12). The monomeric crystal structure of an archaeal SecYEß complex (13), in combination with chemical cross-linking data (14), led to the interpretation that a single copy of the Sec complex is sufficient to serve as an active PCC, even when assembled into a dimer for posttranslational translocation (15) Cryo-EM and 3D reconstructionFor structure determination by cryo-EM we used digitonin-solubilized purified Ssh1 complex (Sec sixty-one homologue 1 from the yeast Saccharomyces cerevisiae) containing Ssh1p, Sbh2p and Sss1p (19). This complex is active in the co-translational translocation mode only, i.e. when ribosome-bound (20,21).We reconstituted the Ssh1 complex with in vitro progra...

Structure of the mammalian oligosaccharyl-transferase complex in the native ER protein translocon

“…13) 15 . Notably, the position and overall arrangement of the Sec61 complex in the presence of TRAP and OST remain indistinguishable from the canonical position observed previously for the ribosome-bound Sec61 or SecYEG complexes alone 17,18 . Superposition of the subtomogram average and the single-particle reconstruction showed a spatial coincidence of the translocon densities (Fig.…”

Cryo-EM structure and rRNA model of a translating eukaryotic 80S ribosome at 5.5-Å resolution

“…Similarly, we have previously reported a cryo-EM structure of a translating Saccharomyces cerevisiae 80S ribosome at 6.1-Å resolution (Fig. 1B) (27). For both reconstructions, ribosome-nascent chain complexes in the posttranslocational state were utilized (27,28), after in silico sorting (see Experimental Procedures) to increase conformational homogeneity.…”

“…1B) (27). For both reconstructions, ribosome-nascent chain complexes in the posttranslocational state were utilized (27,28), after in silico sorting (see Experimental Procedures) to increase conformational homogeneity. The final reconstruction of the T. aestivum 80S ribosome was derived from 1,362,920 particles sorted for the presence of peptidyl-tRNA in the P site (Fig.…”

Free-energy cost for translocon-assisted insertion of membrane proteins

“…Insertion into the bilayer does not happen directly, but rather occurs via a highly conserved protein-conducting channel in the membrane, the Sec translocon (2)(3)(4). At an early stage of synthesis, the ribosome docks to the channel, forming a tightly bound complex (5,6). The polypeptide is then inserted into the translocon prior to entering the membrane, the former step requiring a driving force, such as nucleotide hydrolysis, a membrane potential gradient, or the pressure exerted by the growing nascent chain (2).…”