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1 Stepwise Gating of the Sec61 Protein-Conducting Channel by Sec63 And bioRxiv preprint doi: https://doi.org/10.1101/2020.12.14.422728; this version posted December 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Stepwise gating of the Sec61 protein-conducting channel by Sec63 and Sec62 2 Samuel Itskanov1 and Eunyong Park2,* 1Biophysics Graduate Program, University of California, Berkeley, Berkeley, CA 94720, USA. 2Department of Molecular and Cell Biology and California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA. *Corresponding author. Email: [email protected] 3 4 Summary 5 The universally conserved Sec61/SecY channel mediates transport of many newly synthesized 6 polypeptides across membranes, an essential step in protein secretion and membrane protein integration1- 7 5. The channel has two gating mechanisms—a lipid-facing lateral gate, through which hydrophobic signal 8 sequences or transmembrane helices (TMs) are released into the membrane, and a vertical gate, called the 9 plug, which regulates the water-filled pore required for translocation of hydrophilic polypeptide 10 segments6. Currently, how these gates are controlled and how they regulate the translocation process 11 remain poorly understood. Here, by analyzing cryo-electron microscopy (cryo-EM) structures of several 12 variants of the eukaryotic post-translational translocation complex Sec61-Sec62-Sec63, we reveal discrete 13 gating steps of Sec61 and the mechanism by which Sec62 and Sec63 induce these gating events. We show 14 that Sec62 forms a V-shaped structure in front of the lateral gate to fully open both gates of Sec61. 15 Without Sec62, the lateral gate opening narrows, and the vertical pore becomes closed by the plug, 16 rendering the channel inactive. We further show that the lateral gate is opened first by interactions 17 between Sec61 and Sec63 in both cytosolic and luminal domains, a simultaneous disruption of which 18 fully closes the channel. Our study defines the function of Sec62 and illuminates how Sec63 and Sec62 19 work together in a hierarchical manner to activate the Sec61 channel for post-translational translocation. 20 21 Main text 22 In all organisms, about one third of proteins are transported across or integrated into a membrane upon 23 synthesis by the ribosome. This translocation process mainly occurs in the endoplasmic reticulum (ER) 24 membrane of eukaryotes or the plasma membrane of prokaryotes, mediated by the conserved 25 heterotrimeric protein-conducting channel called the Sec61 (SecY in prokaryotes) complex1-5. The main α 26 subunit of the channel, comprised of ten TMs, forms an hourglass-shaped cavity, through which 27 polypeptide chains are transported, whereas the small β and γ subunits peripherally associate with the α 28 subunit in the membrane6. Previous structures of Sec61/SecY showed that in the idle state the pore is 29 blocked in the ER luminal (or extracellular) funnel by the plug domain—a structure formed by a segment 30 immediately following TM1 of the α subunit6-9, whereas in translocating states the plug moves away10-12. 31 Sec61/SecY can also release polypeptides to the lipid phase through a lateral gate formed between TM2 32 and TM7 of the α subunit, a process required for recognition of hydrophobic targeting signals of soluble 33 secretory proteins and integration of membrane proteins. 34 The Sec61/SecY channel alone is inactive and thus must associate with partners to enable translocation. 35 In the co-translational mode, common in both prokaryotes and eukaryotes, The channel directly docks 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.14.422728; this version posted December 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 36 with the ribosome-nascent-chain complex11,13,14. Many secretory proteins are targeted to the channel post- 37 translationally after their release from the ribosome15-20. In eukaryotes, post-translational translocation is 38 enabled by association between the Sec61 complex and the two essential integral membrane proteins 39 Sec62 and Sec63, forming a machinery called the Sec complex21-24. In fungal species, the Sec complex 40 also contains the additional nonessential proteins Sec71 and Sec72, which are bound to Sec63 in the 41 cytosol. Sec63 recruits the ER luminal Hsp70 ATPase BiP to the complex to power translocation25,26. In 42 bacteria, a single cytosolic ATPase called SecA binds to the SecY complex to drive post-translational 43 translocation10,12,19,20,27. 44 Recently, two cryo-EM studies reported structures of the Sec complex from Saccharomyces cerevisiae at 45 ~4 Å resolution28,29, which suggested a putative role of Sec63 in activating the Sec61 channel for 46 translocation. However, in both structures, Sec62 was barely visible, and thus its function remains elusive 47 despite its essentiality. Furthermore, the two structures displayed noticeable conformational differences in 48 Sec61, despite essentially identical specimen compositions. Most notably, in one structure the pore is 49 blocked by the plug29, whereas in the other structure the plug is displaced leaving the pore open28. 50 Although deemed important given the role of the plug in channel gating, the cause of this difference 51 remains a puzzle. Finally, although Sec63 has been suggested to open the lateral gate of Sec6128,29, the 52 mechanism of opening remains speculative without structures of mutants and other conformations. Thus, 53 whether and how Sec62 and Sec63 regulate gating of Sec61 are poorly understood. Addressing these 54 issues is essential for our understanding of eukaryotic post-translational translocation and the mechanism 55 of the Sec61/SecY channel in general. Mutations in Sec61 and Sec63 have been implicated in 56 hyperuricemic nephropathy30, diabetes31, and polycystic liver disease32. 57 Cryo-EM analysis of two fungal Sec complexes 58 To determine how the gates are regulated in the Sec complex, we first analyzed a large cryo-EM dataset 59 of the wildtype (WT) Sec complex from S. cerevisiae (ScSec), which yielded three structures at 3.1–3.2-Å 60 resolutions with distinct conformations (Fig. 1a,b, Table 1, and Extended Data Fig. 1). While 61 reconstruction from approximately 1 million particles yielded a 3.0-Å-resolution consensus map 62 (Extended Data Fig. 1b–d), we found that the particle set contained subpopulations lacking Sec62 or 63 Sec71-Sec72, despite apparent sample homogeneity (Extended Data Fig. 1a). We therefore performed 64 additional three-dimensional (3D) classifications to separate particles with and without Sec62 (referred to 65 as Sec62+ and Sec62−) (Extended Data Fig. 1b,e). Furthermore, the Sec62+ class could be further 66 separated into two distinct subclasses (referred to as C1 and C2), which show notable conformational 67 differences in Sec62, the lateral gate, and the plug (Fig 1b and Extended Data Fig. 1f,g; see below). 68 Although an atomic model for Sec62 could not be built due to insufficient local resolution, the 69 classification significantly improved Sec62 features, enabling unambiguous assignment of individual 70 domains (Fig. 1d). 71 To gain insights into structural and mechanistic conservation across species, we also determined 72 structures of the Sec complex from the thermophilic fungus Thermomyces lanuginosus (TlSec) at overall 73 resolution of 3.6 to 3.9 Å (Fig. 1c, Table 1, and Extended Data Fig. 2). TlSec classes with and without 74 Sec62 closely resemble the ScSec [C2] and [Sec62−] structures, respectively (brackets denote classes). 75 We could not isolate a C1-equivalent class from the TlSec dataset perhaps because the specimen freezing 76 condition (4°C) might have biased the conformation distribution of this thermophilic complex towards 77 C2. The structure of TlSec[Sec62−] is essentially identical to a separately determined structure of a 78 mutant complex completely lacking Sec62 (ΔSec62 TlSec) (Extended Data Fig. 2g–i). Importantly, the 79 domain arrangement of TlSec62 is the same as that of ScSec62 despite ~30% overall sequence identity 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.14.422728; this version posted December 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 80 (Fig. 1c and Extended Data Fig. 3). Compared to ScSec62, TlSec62 is better resolved such that we could 81 register amino acids to its TM1. 82 Sec62 forms a V-shaped structure 83 Sec62 consists of a cytosolic, globular N-terminal domain (NTD), two TMs (TM1 and TM2) connected 84 by a short ER luminal loop (L1/2), and a cytosolic C-terminal segment (Fig. 1d). Functionally essential 85 regions have previously been mapped to the two TMs and a segment of ~30 amino acids immediately 86 following TM2 (ref. 33). The TMs of Sec62 are arranged as a V shape in front of the lateral gate with L1/2 87 directed to the lateral gate opening (Fig. 1a–d). The contact with the channel is mainly formed by an 88 interaction of Sec62-TM1 with the TM3 and N-terminal segment of Sec61α. 89 Following TM2, Sec62 contains an oval-shaped structure lying flat on the membrane interface (Fig. 1a–d, 90 and Extended Data Fig 4a–b). This amphipathic structure, which we termed the anchor domain, is most 91 likely formed by an ~20-residue-long conserved segment within the abovementioned 30 amino acids, and 92 is rich in hydrophobic amino acids (Extended Data Fig.
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