Membrane Protein TM Segments Are Retained at the Translocon during Integration until the Nascent Chain Cues FRET-Detected Release into Bulk Lipid  Bo Hou, Pen-Jen Lin, Arthur E. Johnson  Molecular Cell  Volume 48, Issue 3, Pages 398-408 (November 2012) DOI: 10.1016/j.molcel.2012.08.023 Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 When Does a TMS Enter the Bulk Lipid? (A) The translocon (yellow) is viewed from above the plane of the membrane. A nascent chain TMS (black) is depicted as having entered the aqueous pore (white), from which the TMS moves directly (red arrow) into the bulk lipid of the ER membrane (gray) (Martoglio et al., 1995; Heinrich et al., 2000). (B) A TMS helix remains adjacent to translocon proteins even though more than 400 Å of nascent chain have emerged from the ribosome (blue) (Do et al., 1996) and are exposed to the cytosol (Liao et al., 1997). Ion movement through the pore is inhibited by the combined actions of, among others, BiP (green) and a J domain-containing ER membrane protein (magenta) (Haigh and Johnson, 2002; Alder et al., 2005). (C) Acceptor-labeled PLs (BOP-PE; red) are distributed by diffusion within the bulk lipid. After release from the translocon, donor-acceptor proximity is maximal because the TMS labeled with a donor dye (green) is completely surrounded by BOP-PE. (D) A TMS donor adjacent to or inside the translocon has a reduced proximity to acceptors because BOP-PE is excluded from area occupied by the translocon and associated proteins. Distances, sizes, and concentrations are not to scale. Molecular Cell 2012 48, 398-408DOI: (10.1016/j.molcel.2012.08.023) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 SSMP TMS Proximity to BOP-PE (A) Topogenic sequences in SSMP 111p: VSVG TMS (green); SS, preprolactin signal sequence (orange). NBD (red) is attached at residue 75. (B) Average NBD-to-BOP E values (±SD; n = 3) are shown for 111p232 that has been terminated normally (green), has been released from tRNA by puromycin (yellow), or is still attached to the tRNA (magenta). (C) Nascent, normally terminated (FL), or puromycin-reacted 111p232 with an εANB-Lys probe at 75 were photolyzed, immunoprecipitated with antibodies to Sec61α (α), Sec61β (β), or TRAM (T), and analyzed by SDS-PAGE. Photoadducts containing Sec61α (●), Sec61β (▴), and TRAM (♦) are indicated. The arrow indicates unreacted 111p. See also Figure S1 and Table S1. Molecular Cell 2012 48, 398-408DOI: (10.1016/j.molcel.2012.08.023) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 TMS1 and TMS2 Are Retained at the Translocon (A) Topogenic sequences for PMPs with an opsin 2 TMS (OP2; yellow) are identified as in Figure 2A. NBD (red) is positioned in either TMS1 (K1) or TMS2 (K2). (B) 2TM topology before (i) and after (ii) termination. (C) E values (±SD; n = 3–4) of each NBD-TMS to BOP-PE FRET before and after PMP release from tRNA. (D) Photocrosslinking of TMS1 and TMS2 to translocon components before and after termination. Symbols are defined in Figure 2C. (E) A longer TMS1-TMS2 loop had little effect on E (±SD; n = 4–5). (F) Inverting TMS1 and TMS2 in the PMP did not alter protein topology (Lin et al., 2011a) and had little effect on E (±SD; n = 3). See also Table S1. Molecular Cell 2012 48, 398-408DOI: (10.1016/j.molcel.2012.08.023) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 TMS3 Stays at Translocon, but Triggers TMS1 and TMS2 Release into Bulk Lipid (A) Topogenic sequences for PMPs with an opsin 3 TMS (OP3; magenta) are identified as in Figure 3A. NBD (red) is positioned in TMS1 (K1), TMS2 (K2), or TMS3 (K3). (B) 3TM topology before (i) and after (ii) termination. (C) E values (±SD; n = 3) of each NBD-TMS to BOP-PE FRET before and after PMP release from tRNA. (D) A longer TMS2-TMS3 loop had little effect on E (±SD; n = 4). (E) Photocrosslinking of TMS1, TMS2, and TMS3 to translocon components before and after termination of PMP. (F) Nascent chain length dependence of TMS2 and TMS3 photocrosslinking to Sec61α. Symbols are defined in Figure 2C. See also Table S1. Molecular Cell 2012 48, 398-408DOI: (10.1016/j.molcel.2012.08.023) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 SA Proximity to Bulk Lipid and Translocon Proteins (A) Location of Lep SA sequence (blue) and NBD dye (red) in Lep1 derivative (McCormick et al., 2003) that lacked the second TMS. (B) Nascent chain length dependence of NBD-SA to BOP-PE FRET E (±SD; n = 3–4) before and after termination. (C) Sec61α proximity to four different helical surfaces of SA was assessed by photoadduct formation (●) before and after termination. (D) Nascent chain length dependence of Sec61α photocrosslinking from four different SA probe locations. Symbols are defined in Figure 2C. See also Table S1. Molecular Cell 2012 48, 398-408DOI: (10.1016/j.molcel.2012.08.023) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 TMS1-TMS2 Separation (A) Donor (green) and acceptor (red) dyes were incorporated into TMS2 and TMS1, respectively. (B) E (±SD; n = 3) was determined before and after termination. Molecular Cell 2012 48, 398-408DOI: (10.1016/j.molcel.2012.08.023) Copyright © 2012 Elsevier Inc. Terms and Conditions