Stepwise insertion and inversion of a type II signal anchor sequence in the ribosome-Sec61 translocon complex

Abstract

In eukaryotic cells, the ribosome-Sec61 translocon complex (RTC) establishes membrane protein topology by cotranslationally partitioning nascent polypeptides into the cytosol, ER lumen, and lipid bilayer. Using photocrosslinking, collisional quenching, cysteine accessibility, and protease protection, we show that a canonical type II signal anchor (SA) acquires its topology through four tightly coupled and mechanistically distinct steps: (1) head-first insertion into Sec61α, (2) nascent chain accumulation within the RTC, (3) inversion from type I to type II topology, and (4) stable translocation of C-terminal flanking residues. Progression through each stage is induced by incremental increases in chain length and involves abrupt changes in the molecular environment of the SA. Importantly, type II SA inversion deviates from a type I SA at an unstable intermediate whose topology is controlled by dynamic interactions between the ribosome and translocon. Thus, the RTC coordinates SA topogenesis within a protected environment via sequential energetic transitions of the TM segment.

Figure 1. Nascent chain targeting to Sec61α

(A) Autoradiogram of truncated in vitro-translated AQP4-TM1.P showing total products (T), supernatant (S) and membrane pellet (P) fractions analyzed by SDS-PAGE. Peptidyl-tRNA bands (asterisk) and prematurely released nascent chains lacking tRNA (double asterisk) are indicated. (B) Fraction of nascent chains (as in panel A) that remained ER associated ± NaCl treatment as indicated. (C) Translation products containing a photoactive crosslinker (εANB-Lys) at residue 28 (and WT constructs lacking UAG codon) were UV irradiated and pelleted. Translation products were quantified by phosphorimaging (see also Fig. S1). Equal amounts were immunoprecipitated with Sec61α antisera and subjected to SDS-PAGE. (D) Quantification of photocrosslinks (as in panel C) after correcting for WT signal. (E, F) Autoradiogram showing photoadducts to residues 2, 28, 44 and 65 immunoprecipitated with Sec61α (E) or TRAM (F). Graphs show mean (n ≥ 3 ± SEM).

Figure 2. TM1 inserts head first into the translocon

(A) Schematic diagram of RNC and RTC showing relative location of residues 2 and 44. (B,C) Stern Volmer plots obtained for RNCs and RTCs truncated at codon 71 and containing εNBD-Lys at residue 2 (panel B) or 44 (panel C) (see also Fig. S2). K_sv was determined before and after microsome permeabilization with melittin. Results show mean (n≥ 3 ± SEM).

Figure 3. Initiation of TM1 inversion from a type I to a type II topology

Ksv values were determined as in Figure 2 for probes located at residue 44 (A) or2 (B) in RTCs before (grey bars) and after (black bars) membrane permeabilization. Data show average K_sv values obtained for indicated chain lengths (n ≥ 3 ± SEM). (C) Schematic of RTC showing probable location of fluorescent probes (circles).

Figure 4. Nascent chain shielding by the RTC

(A,B) Transcripts containing L44C were translated in the absence (A) or presence (B) of ER microsomes. Pelleted RNCs and RTCs were pelleted and incubated with PEG-Mal ± SDS as indicated. Pegylated (P) and unpegylated (UP) peptidyl-tRNA bands are indicated (see also Fig. S3). (C) Fraction of pegylated nascent chains (as in panels A&B) were quantified and plotted against chain length. Fraction of pegylated RTCs containing Cys44 (D) or Cys34, Cys49, Cys65 (E) following permeabilization with digitonin or melittin. (F) Pegylation of Cys34, Cys44 and Cys49 in RTCs solubilized with TX-100. (G) Pegylation efficiency of residue Cys9 in intact and digitonin solubilized RTCs (see also Fig. S4). Graphs show mean (n=3 ± SEM).

Figure 5. Ribosome shielding is salt sensitive, reversible, and required for TM1 inversion

(A) Schematic RTC showing potential effect of NaCl before and after TM1 inversion. (B–E) RTCs containing a Cys residue at position 34 (B), 44 (C), 49 (D), or 65 (E) were pegylated ± addition of 0.5 M NaCl. Fraction of pegylated peptidyl tRNA bands was determined as in Figure 4. (F) RTCs containing L44C (truncated at residue 98 or 110aa) were incubated with (lanes 3–4) or without (lanes 1–2) 0.5 M NaCl and pegylated directly (lanes 1–4) or repelleted and pegylated in the presence (lanes 7 & 10) and absence (lanes 6 & 9) of 0. 5M NaCl. (G) Quantification of experiments (as in panel F) showing no preincubation (white bars), preincubation without NaCl (grey bars), or preincubation with NaCl (black bars). Mean values (n=3, ± SEM). (H, I) Pegylation efficiency of Cys9 in RTCs digested with RNase and treated as indicated (see also Fig S5). Graphs show mean (n=3, ± SEM)

Figure 6. Proteolytic susceptibility of the ribosome-translocon junction

(A) Autoradiogram showing truncated peptidyl-tRNA bands ± PK digestion (downward arrows show protected bands). (B) PK digestion of nascent chains labeled with [¹⁴C]Lys at indicated UAG stop codons. 5–7 kDa N-terminal fragments (bracket) contain residues 2 and 44 while C-terminal peptidyl-tRNA fragments (horizontal arrow) contain residue 65. For truncation 110, intensity of latter bands reflect partial removal by PK. Double asterisks indicate prematurely released nascent chains. (C) RTCs were subject to PK digestion as in panel A, but in the presence of 0.5M NaCl and/or digitonin as indicated. PK protected Peptidyl tRNA bands in the presence of NaCl (double arrow).

Figure 7. Type I signal anchor insertion

(A) Schematic showing engineered N-linked glycosylation site (N-glc) and mutations used to reverse TM1 topology. (B) PK protection of (membrane targeted) type I and type II AQP4-TM1.P ± N-glc. Asterisk indicates full-length protein (see also Fig. S6). Double asterisk shows a minor population cleaved at a cryptic signal peptidase site (describe previously (Foster et al., 2000)). Cleavage is observed only for truncations >133 aa (not shown). Downward arrow indicates glycosylated band (See also Figure S6). Graph shows percent of chains with translocated C-terminus (type II topology, mean of 2 experiments). (C, D) Mean pegylation efficiency of Cys46 for type I construct (n=3, ± SEM). Truncations are at same sites as type II constructs, but numbering reflects addition of Arg residues (E41R3). (E) Protease protection of type I RTCs at indicated truncations. Graph shows mean C-terminus translocation efficiency for Type I and type II nascent chains (n=3, ± SEM (type II) or average of 2 experiments (type I)).