Abstract
23 min readArticle Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract We follow the cotranslational biosynthesis of three multispanning Escherichia coli inner membrane proteins in vivo using high-resolution force profile analysis. The force profiles show that the nascent chain is subjected to rapidly varying pulling forces during translation and reveal unexpected complexities in the membrane integration process. We find that an N-terminal cytoplasmic domain can fold in the ribosome exit tunnel before membrane integration starts, that charged residues and membrane-interacting segments such as re-entrant loops and surface helices flanking a transmembrane helix (TMH) can advance or delay membrane integration, and that point mutations in an upstream TMH can affect the pulling forces generated by downstream TMHs in a highly position-dependent manner, suggestive of residue-specific interactions between TMHs during the integration process. Our results support the 'sliding' model of translocon-mediated membrane protein integration, in which hydrophobic segments are continually exposed to the lipid bilayer during their passage through the SecYEG translocon. Introduction Most integral membrane proteins are cotranslationally integrated into their target membrane with the help of translocons such as bacterial SecYEG and YidC, and the eukaryotic Sec61 and EMC complexes (Rapoport et al., 2017; Chitwood et al., 2018). While the energetics of translocon-mediated integration of a transmembrane α-helix (TMH) is reasonably well understood (Hessa et al., 2007), the actual integration process is not, other than in general terms. We have shown that force profile analysis (FPA) – a method in which a translational arrest peptide (AP) engineered into a target protein serves as a sensor to measure the force exerted on a nascent polypeptide chain during translation – can be used to follow the cotranslational folding of soluble proteins and the membrane integration of a model TMH (Ismail et al., 2012; Ismail et al., 2015; Farías-Rico et al., 2018). Here, we have applied FPA and coarse-grained molecular dynamics (CGMD) simulations to follow the cotranslational membrane integration of three multispanning Escherichia coli inner membrane proteins of increasing complexity (EmrE, GlpG, BtuC), providing the first residue-by-residue data on membrane protein integration in vivo. Results Force profile analysis FPA takes advantage of the ability of APs to bind in the upper parts of the ribosome exit tunnel and thereby pause translation when their last codon is in the ribosomal A-site (Ito and Chiba, 2013). The duration of an AP-induced pause is reduced in proportion to pulling forces exerted on the nascent chain (Goldman et al., 2015; Kemp et al., 2020), that is, APs can act as force sensors and can be tuned by mutation to react to different force levels (Cymer et al., 2015a). In an FPA experiment, a series of constructs is made in which a force-generating sequence element (e.g., a TMH) is placed an increasing number of residues away from an AP (reflected in N, the number of residues from the start of the protein to the end of the AP), which in turn is followed by a C-terminal tail (Figure 1a). In constructs where the TMH engages in an interaction that generates a strong enough pulling force F on the nascent chain at the point when the ribosome reaches the last codon of the AP, pausing will be prevented and mostly full-length protein will be produced during a short pulse with [35S]-Met (Figure 1b, middle). In contrast, in constructs where little force is exerted on the AP, pausing will be efficient and more of the arrested form of the protein will be produced (Figure 1b, left and right). The fraction full-length protein produced, fFL = IFL/(IFL+IA), where IFL and IA are the intensities of the bands representing the full-length (FL) and arrested (A) species on an SDS-PAGE gel (Figure 1c and Figure 1—figure supplement 1), can therefore be used as a proxy for F in a given construct (Kemp et al., 2020; Niesen et al., 2018; Leininger et al., 2019). A plot of fFL versus N – a force profile (FP) – thus can provide a detailed picture of the cotranslational process in question, as reflected in the variation in the force exerted on the nascent chain during translation. FPs can be recorded with up to single-residue resolution by increasing N in steps of one residue (corresponding to a lengthening of the nascent chain by ~3 Å). Figure 1 with 1 supplement see all Download asset Open asset The force profile assay. (a) Basic construct. Arrested (A) and full-length (FL) products are indicated. (b) At construct length N1, TMH2 has not yet entered the SecYEG channel and no pulling force F is generated. At N2, TMH2 is integrating into the membrane and F ≫0. At N3, TMH2 is already integrated and F ≈ 0. (c) SDS-PAGE gels showing A and FL products for [35S]-Met labeled and immunoprecipitated EmrE(Cout) (N = 105), GlpG (N = 196), and BtuC (N = 314). Control constructs AC and FLc have, respectively, a stop codon and an inactivating Ala codon replacing the last Pro codon in the arrest peptide (AP). The band just below the A band in the EmrE(Cout) (N = 105) lane most likely represents ribosomes stacked behind the AP-stalled ribosomes (Notari et al., 2018) and is not included in the calculation of fFL. See Figure 1—figure supplement 1 for additional gels. EmrE: 4 TMHs, 110 residues We chose EmrE as an example of a small, relatively simple 4-TMH protein. EmrE is a dual-topology protein, that is, the monomers integrate into the inner membrane in a 50–50 mixture of Nin-Cin and Nout-Cout topologies; two oppositely oriented monomers then assemble into an antiparallel dimer (Chen et al., 2007; Rapp et al., 2007). To avoid potential complications caused by the dual topology, we used EmrE(Cout), a mutant version that adopts the Nout-Cout topology (Rapp et al., 2007), and further used the relatively weak SecM(Ec) AP (Ismail et al., 2012) and included an HA tag for immunoprecipitation (Figure 2a). A series of EmrE(Cout)-AP constructs (see Supplementary file 1 for sequences) was used to obtain the FP shown in Figure 2b (orange curve), at 2–5 residues resolution. Also shown is an FP derived from a CGMD simulation (CGMD-FP, gray; Van Lehn et al., 2015); a hydrophobicity plot (HP) is included in Figure 2—figure supplement 1. Figure 2 with 1 supplement see all Download asset Open asset EmrE(Cout). (a) Construct design. EmrE(Cout) is shortened from the C-terminal end of the LepB-derived linker (dotted), as indicated by the arrow. Cytoplasmic (red) and periplasmic (blue) loops, and lengths of full-length EmrE(Cout), LepB-derived linker, HA tag + arrest peptide (AP), and C-terminal tail, are indicated. Since the 30-residue HA + AP segment is constant in all constructs, the force profile (FP) reflects nascent chain interactions occurring mainly outside the ribosome exit tunnel. (b) FPs for EmrE(Cout) (orange), EmrE(Cout,E14L) (green), EmrE(Cout) with SecM(Ec-sup1) AP (blue), EmrE(Cout, I37I38→NN) (magenta triangles), and coarse-grained molecular dynamics (CGMD-FP) calculated with a −100 mV membrane potential (gray). (c) Effects of mutations in E14 on fFL values for the N values are indicated by arrows in (b). p-values (two-sided t-test): *p < 0.05; **p < 0.01; ***p < 0.001. (d, e) Sequences corresponding to peaks I–IV aligned from their Nstart (d) and Nend (e) values. The + sign indicates 45 residues from the polypeptide transferase center (PTC). Hydrophobic transmembrane helix (TMH) segments are shown in orange and transmembrane α-helices underlined (PDB: 3B5D). Error bars in b and c indicate SEM values. We have previously shown that a model TMH composed of Ala and Leu residues generates a peak in an FP recorded with the SecM(Ec) AP that reaches half-maximal amplitude (Nstart) when the N-terminal end of the TMH is ~45 residues away from the polypeptide transferase center (PTC) (Ismail et al., 2012), and a recent real-time FRET study of cotranslational membrane integration found that the N-terminal end of the first TMH in a protein reaches the vicinity of the SecYEG translocon when it is 40–50 residues away from the PTC (Mercier et al., 2020). For EmrE(Cout) TMH1, this would correspond to constructs with N ≈ 50. However, the fFL values are hardly above background in this region of the FP. Due to the functionally important E14 residue, TMH1 is only marginally hydrophobic and does not become firmly embedded in the membrane until the protein dimerizes (Seurig et al., 2019). To ascertain whether the lack of a peak in the FP corresponding to the membrane integration of TMH1 is because of its low hydrophobicity, we mutated E14 to L. Indeed, in the FP obtained for EmrE(Cout,E14L) (Figure 2b, green curve), a clear peak appears at the expected chain length Nstart ≈ 50 residues. Mutation E14A yields an fFL value intermediate between EmrE(Cout,E14L) and EmrE(Cout) at N = 55 (Figure 2c), while fFL for the mutants EmrE(Cout,E14D) and EmrE(Cout,E14Q) is the same as for EmrE(Cout). Peak II has Nstart ≈ 76, corresponding to a situation where the N-terminal end of TMH2 is ~45 residues from the PTC (Figure 2d). The double mutation I37I38→NN in TMH2 reduces fFL at N = 80 and 85 (magenta triangles), as expected. Unexpectedly, however, the E14L, E14A, and E14Q (but not the E14D) mutations in TMH1 increase fFL at N = 85 (Figure 2c), showing that a negatively charged residue (D or E) in position 14 in TMH1 specifically reduces the pulling force generated by TMH2 at N = 85, that is, when about one-half of TMH2 has integrated into the membrane. Likewise, fFL values at N = 115 and 130 (but not at N = 105, included as a negative control) are specifically affected by mutations in E14: at N = 115 (one-half of TMH3 integrated), all four mutations in position 14 increase fFL relative to E14, while at N = 130 (beginning of TMH4 integration) the E14A and E14L mutations decrease fFL (Figure 2c). FPA thus reveals long-range effects of mutations in E14 on three specific steps in the membrane integration of the downstream TMHs. This implies that TMH1 remains in the vicinity of the translocon and that E14 makes specific interactions with residues in the TMH2–TMH4 region during the membrane integration process. Further studies will be required to pinpoint these interactions and understand the role played by the slow dynamics of TMH1 integration (Seurig et al., 2019). Peak III has Nstart ≈ 102 residues, with the N-terminal end of TMH3 ~45 residues from the PTC (Figure 2d). Peak IV is difficult to locate precisely in the FP because fFL values are high throughout the TMH3–TMH4 region, but is seen at Nstart ≈ 132 residues when the strong SecM(Ec-sup1) AP (Yap and Bernstein, 2009) is used (blue curve), again with the N-terminal end of TMH4 ~45 residues from the PTC (Figure 2d). As shown in Figure 2e, the TMHs cease generating a pulling force when their C-terminal ends are ~45 residues away from the PTC, indicating that they are fully integrated at this point. GlpG: 6 TMHs, 276 residues We next studied GlpG, a medium-sized monomeric 6-TMH rhomboid protease with an ~60 residue cytoplasmic N-terminal domain (NTD) (Sherratt et al., 2012; Wang et al., 2006) (Figure 3a), a protein that allows us to follow the cotranslational folding of a soluble domain and integration of a membrane domain in the same experiment. Figure 3 with 1 supplement see all Download asset Open asset GplG. (a) Construct design, c.f., Figure 2a. The N-terminal LepB fusion is indicated. (b) Force profiles (FPs) for GlpG and LepB-GlpG (N = 131–224) (orange), NTD(F16E) (green), in vitro translated N-terminal domain (NTD) (magenta), and NTD(F16E) (black), LepB-GlpG with SecM(Ec-Sup1) AP (blue), and coarse-grained molecular dynamics (CGMD)-FP calculated with a −100 mV membrane potential (gray). Error bars indicate SEM values. Note that the LepB-GlpG constructs are two residues shorter than the corresponding GlpG constructs but are plotted with the same N values as the latter to facilitate comparison. (c) NTD (PDB ID: 2LEP), with F16 in spacefill. (d) Enlarged FPs for LepB-GlpG with SecM(Ec) AP (orange), SecM(Ec-Ms) AP (green), SecM(Ec-sup1) AP (blue), and GlpG(Y138F139L143→NNN) with SecM(Ec-Ms) AP (magenta). CGMD-FP in gray. (e) Structure of GlpG with the periplasmic surface helix in blue, TMH2 in red, the membrane-associated cytoplasmic segment in cyan, and TMH5 in yellow. Y138F139L143 and G222I223Y224L225 are shown as sticks. (f) LepB-GlpG peak III-a and III-c sequences aligned, respectively, from their Nstart and Nmax values, and the mutant LepB-GlpG(Y138F139L143→NNN) peak III-c sequence aligned from its Nmax value. Hydrophobic transmembrane helix (TMH) segments are shown in orange and transmembrane α-helices (PDB: 2IC8)underlined. The periplasmic surface helix is italicized. AP: arrest peptide; PTC: polypeptide transferase center. The FP is shown in Figure 3b (orange curve). It was obtained at 5-residue resolution, except for the portion N = 168–224, which we measured with single-residue resolution. For unknown reasons, constructs with N ≈ 140–190 residues gave rise to a slowly migrating band on the gel that was difficult to interpret (Figure 1—figure supplement 1j,k); this problem did not arise when the NTD (GlpG residues 1–60) was replaced by residues 1–58 of the LepB protein (Figure 3a), and the corresponding fFL values are shown in the FP (N = 131–224). The LepB part contains an N-terminal, Nout-Cin-oriented TMH (Wolfe et al., 1983; von Heijne, 1989), that interacts with the signal recognition particle Ffh (Schibich et al., 2016) and hence targets the LepB-GlpG constructs to the SecYEG translocon before GlpG TMH1 is translated. This could in principle affect the FP; however, because the C-terminal end of the LepB part is ≥70 residues away from the C-terminal end of the SecM AP in these constructs, LepB is far outside the ribosome exit tunnel and therefore unlikely to exert a strong effect. Indeed, fFL values for GlpG (calculated either including or excluding the slowly migrating band in IFL) and LepB-GlpG are very similar in the peak III region (N = 166–231) of the FP (Figure 3—figure supplement 1a). Nstart and Nend values for peaks II–VII are indicated in Figure 3—figure supplement 1c,d. Peak I, at Nstart ≈ 84 residues, is conspicuously close to what would be expected for the folding of the NTD from previous studies of cotranslational folding of small globular domains in the ribosome exit tunnel (Farías-Rico et al., 2018). To verify that the peak indeed represents folding of the NTD, we recorded an FP for the NTD by in vitro transcription-translation in the PURE system (Shimizu et al., 2005) and further made a destabilizing point mutation (F16E) in the core of the NTD (Figure 3c). The FP obtained in vitro (magenta) overlaps peak I in the in vivo FP, and the mutation strongly reduces fFL values for peak I both in vivo (green) and in vitro (black). Given that the NTD has a relative contact order of 15% and is predicted to fold on the ms time scale (Plaxco et al., 1998) while the elongation cycle on the ribosome takes ~100 ms/codon (Young and Bremer, 1976), the NTD has ample time to equilibrate between the unfolded and accessible folded states at each elongation step (Kemp et al., 2019). We conclude that the ~60 residue NTD folds inside the ribosome exit tunnel when its C-terminal end is 25–30 residues from the PTC, well before synthesis of the membrane domain has commenced. Peaks II–VII in the FP correspond reasonably well to the CGMD-FP (gray) and HP (Figure 3—figure supplement 1b). The unexpectedly low Nstart value for peak III seems to be caused by an upstream periplasmic surface helix (Figure 3f) (see below). Likewise, peak VI-a likely reflects the membrane integration of a hydrophobic, membrane-associated cytoplasmic segment located just upstream of TMH5 (Figure 3—figure supplement 1c). In contrast, the unexpectedly high Nstart value for peak IV indicates that integration of TMH3 commences only when its N-terminal end is ~52 residues away from the PTC, possibly because of the tight spacing between TMH2 and TMH3. As peak III saturates at fFL ≈ 0.9 over a rather wide range, we sought a more detailed view by using the strong SecM(Ec-Sup1) AP (Yap and Bernstein, 2009) (Figure 3b,d, blue) and the medium-strong SecM(Ec-Ms) AP (Farías-Rico et al., 2017) (Figure 3d, green). The SecM(Ec-Sup1) FP allows a precise determination of Nmax = 200, at which point the middle of TMH2 (L155) is located 45 residues from the PTC (Figure 3f). The SecM(Ec-Ms) FP reveals additional detail: peak III is now seen to be composed of three subpeaks III-a, III-b, and III-c. III-a has Nstart = 182, coinciding with the N-terminal end of the periplasmic surface helix reaching 45 resides away from the PTC. For III-b, Nstart ≈ 190, with the N-terminal end of TMH2 ~45 residues from the PTC. The major subpeak III-c at N ≈ 197–204 finally corresponds well to the peak seen in the SecM(Ec-Sup1) and the CGMD FPs, and therefore represents the membrane insertion of the most hydrophobic part of TMH2. Taken together, subpeaks III-b and III-c are reminiscent of the biphasic pulling force pattern previously recorded for a model hydrophobic transmembrane segment using the medium-strong SecM(Ms) AP (Ismail et al., 2012), which is closely related to the SecM(Ec-Ms) AP used here. We further recorded a SecM(Ec-Ms) FP (magenta) for the triple mutation Y138F139L143→NNN (Figure 3e) that renders the periplasmic surface helix less hydrophobic: the mutation strongly reduces the amplitude of peak III-a, has only a small effect on peak III-b, and both reduces the amplitude and shifts Nstart and Nmax for peak III-c by approximately four residues (Figure 3d,f). Thus, the periplasmic surface helix engages in hydrophobic interactions already during its passage through the translocon, presumably by sliding along a partly open lateral gate (Cymer et al., 2015b). It also adds to the force generated by the membrane integration of TMH2, possibly by partitioning into the periplasmic leaflet of the inner membrane at approximately the same time that TMH2 enters the translocon. BtuC: 10 TMHs, 326 residues Finally, we studied BtuC, a vitamin B12 transporter with 10 TMHs, as an example of a large, multispanning protein with a complex fold (Hvorup et al., 2007). In order to improve expression, we added the N-terminal part of LepB to the BtuC constructs (Figure 4a) and used a LepB antiserum for immunoprecipitation. The Nout-Cin orientation of LepB TMH1 ensures that the Nin-Cin topology of BtuC will be maintained, and constructs that we could measure without the LepB fusion gave similar fFL values as those seen for the LepB fusions (Figure 4—figure supplement 1b). Figure 4 with 3 supplements see all Download asset Open asset BtuC. (a) Construct design, cf. Figure 2a. The N-terminal LepB fusion is indicated. N values are calculated from the N-terminus of BtuC. For constructs with N ≥ 298, the C-terminal tail is 75 residues long. Circles indicate constructs for which mutations were made in the corresponding transmembrane helix (TMH) (see Figure 4—figure supplement 2. (b) Force profiles (FPs) for BtuC (orange), BtuC-TMH2 (green), BtuC(R47R56R59→QQQ) (black), BtuC-TMH6 (dark blue), BtuC-TMH8 (blue), BtuC-TMH10 (pink), and CGMD-FP calculated with a −100 mV membrane potential (gray). Error bars indicate SEM values. Note that the BtuC-TMH2, BtuC-TMH6, BtuC-TMH8, and BtuC-TMH10 constructs are plotted with the same N values as the corresponding BtuC constructs to facilitate comparison (i.e., the number of residues between the TMH in question and the last residue of the AP is the same in both types of constructs, see Supplementary file 1). (c) Sequences corresponding to peaks I–XI aligned from their Nstart values. Hydrophobic TMH segments are shown in orange and membrane-embedded α-helices according to the OPM database (Lomize et al., 2012) underlined. Re-entrant loops and surface helices discussed in the text are italicized. (d) Construct design for obtaining FPs of isolated Nout-oriented BtuC TMHs. Dashed segments are derived from LepB. (e) Enlarged FPs for BtuC (orange) and (R47R56R59→QQQ) (black), together with coarse-grained molecular dynamics (CGMD)-FPs calculated with (gray) and without (dashed gray) a −100 mV potential. (f) BtuC TMH9-TMH10, with hydrophobic flanking residues in stick representation (PDB ID: 2QI9). (g) Enlarged FPs for BtuC (orange), isolated TMH6 (residues 187–206; blue), and isolated TMH5-6 (residues 138–206; green). In the latter construct, LepB TMH2 was not included in order to maintain the correct membrane topology of the BtuC TMH5-TMH6 part. The CGMD-FP is in gray. (h) Structure of TMH6 including the upstream periplasmic re-entrant helix and the downstream cytoplasmic surface helix, with hydrophobic flanking residues in stick representation. AP: arrest peptide; PTC: polypeptide transferase center. We identified 11 peaks in the FP (Figure 4b, orange), one more than could be accounted for by the 10 TMHs. Since it was not possible to provide an unequivocal match between the BtuC FP and the CGMD-FP (or HP, Figure 4—figure supplement 1a), we did two sets of controls. First, we chose constructs at or near peaks in the FP and CGMD-FP and mutated multiple hydrophobic residues (Leu, Ile, Val, Met) located 40–50 residues from the PTC to less hydrophobic Ala residues (Figure 4—figure supplement 2). The mutations caused significant drops in fFL (p < 0.01, two-sided t-test), except for construct N = 191 that is mutated at the extreme N-terminus of TMH5. The mutation data allowed us to identify the membrane integration of TMHs 1, 2, 3, 4, 5, 7, 8, 9, and 10 with peaks I, II, III, IV, V, VIII, IX, X, and XI, respectively; the overlapping peaks VIII and IX appear to represent the concerted integration of the closely spaced TMH7 and TMH8. However, peak II (corresponding to TMH2) is shifted to unexpectedly high, and peaks V (corresponding to TMH5), X (corresponding to TMH9), and XI (corresponding to TMH10) to unexpectedly low, Nstart values (Figure 4c). To confirm these assignments, we obtained FPs for the isolated TMH2 (dashed green), TMH8 (dashed light blue), and TMH10 (dashed pink) sequences (Figure 4b) by introducing them into the periplasmic domain of LepB such that they maintained their natural Nout-Cin orientation (Figure 4d); the FPs for the individual TMHs overlap the corresponding peaks II, IX, and XI in the full FP. Likewise, an FP obtained for a construct lacking TMH1-TMH4 overlaps the full FP, except that peak V is shifted to a higher Nstart value (Figure 4—figure supplement 3), more in line with the peak seen in the CGMD-FP. The low Nstart value for the Nin-Cout-oriented TMH5 in full-length BtuC may result from an early interaction between a positively charged patch (RFARRHLSTSR) just upstream of TMH5 and negatively charged lipid headgroups (note that only two of the four Arg residues are present in the ΔTMH1-TMH4 construct; Figure 4—figure supplement 3), while the low Nstart values for peaks X and XI are likely caused by the short upstream hydrophobic segments LCGL and LAAALEL (Figure 4c,f), similar to peak III in GlpG. Remarkably, the N-terminal end of the isolated TMH2 is ~45 residues away from the PTC at Nstart, suggesting that upstream sequence elements present in full-length BtuC delay the integration of TMH2 by ~10 residues (compare II* and II in Figure 4c). The most conspicuous feature in the upstream region of TMH2 is the presence of three positively charged Arg residues, an uncommon occurrence in a periplasmic loop (Heijne, 1986). Indeed, when these residues are replaced by uncharged Gln residues in LepB-BtuC, peak II (dashed black in Figure 4b,e) becomes almost identical to the FP for the isolated TMH2; a similar behavior is seen when the CGMD-FP simulation is run without an electrical membrane potential (Figure 4e). Upstream positively charged residues thus delay the membrane integration of the Nout-oriented TMH2, possibly because of the energetic cost of translocating them against the membrane potential (Ismail et al., 2015), or because they are temporarily retained in the negatively charged exit tunnel (Mercier et al., 2020). Neither peak VI nor VII seems to represent the integration of TMH6, but instead flanks the location expected from the CGMD-FP and HP and apparently corresponds, respectively, to the membrane insertion of a short periplasmic re-entrant helix and of a short cytoplasmic surface helix (Figure 4c,h). Mutation of three hydrophobic residues to Ala in the latter significantly reduces the amplitude of peak VII (Figure 4—figure supplement 2, construct N = 259). Further, the FP for the isolated TMH6 (Figure 4b,g, dashed dark blue) peaks in the location expected from the CGMD-FP, between peaks VI and VII, and the FP for the isolated TMH5-6 part that includes the re-entrant helix but lacks the downstream surface helix is intermediate between the LepB-BtuC and the TMH6 FPs (Figure 4g, dashed green). Thus, the membrane interactions of the periplasmic re-entrant helix and the cytoplasmic surface helix exert a strong effect on the membrane integration of the intervening TMH6. Discussion A detailed view of the cotranslational integration of three multispanning membrane proteins provided here shows that translocating nascent chains experience a distinct transition to a more hydrophobic environment at a distance of ~45 residues from the PTC, generating an oscillating force on the nascent chain that is ultimately transmitted to the PTC and varies in step with the appearance of each TMH in the vicinity of the SecYEG translocon channel. It seems likely that such oscillations can have multiple effects on the translation of membrane proteins, as recently demonstrated for ribosomal frameshifting (Harrington et al., 2020), and may affect protein quality control (Lakshminarayan et al., 2020). Notably, TMHs also stop generating a force on the nascent chain when their C-terminal end reaches ~45 residues from the PTC, irrespective of whether their orientation is Nout-Cin or Nin-Cout. This is in agreement with the 'sliding' model of TMH integration (Cymer et al., 2015b), which posits that Nout-Cin TMHs have continuous lipid contact as they slide across the membrane along the open lateral gate in the SecYEG translocon, while Nin-Cout TMHs first partition into the cytoplasmic interface region of the membrane as they exit the ribosome (and therefore generate less pulling force than Nout-Cin TMHs (Cymer et al., 2014) and only insert across the membrane as their polar C-terminal flanking region translocates through the central translocon channel. In both cases, the TMHs are embedded in the membrane (albeit in perpendicular orientations) when their C-terminal end is ~45 residues from the PTC. In the sliding model, the translocon channel serves as a conduit for polar nascent chain segments while hydrophobic segments are always in contact with surrounding lipid, similar to what has been proposed for the YidC/Oxa1 translocon family (He et al., 2020). The lateral gate region in the SecYEG translocon thus in a certain sense mimics the water–bilayer interface environment (Marx and Fleming, 2021). We also find that the cytoplasmic NTD in GlpG folds already in the ribosome exit tunnel, before the first TMH has been synthesized. Further, the FPs for EmrE, GlpG, and BtuC to a first approximation match those predicted by CGMD calculations, but uncover a much richer picture of the membrane integration process where charged residues and membrane-interacting segments such as re-entrant loops and surface helices flanking a TMH show prominent interactions with the translocon and surrounding lipid. Finally, point mutations in EmrE TMH1 affect the pulling force generated by downstream TMHs in a highly position-dependent manner, suggestive of residue-specific interactions between TMHs during the membrane integration process. Complementing in vitro unfolding/folding studies (Yu et al., 2017; Choi et al., 2019), real-time FRET analyses (Mercier et al., 2020), chemical crosslinking (He et al., 2020), structure determination (Kater et al., 2019), and computational modeling (Lu et al., 2018), high-resolution in vivo FPA can thus help identify the molecular interactions underlying cotranslational membrane protein biogenesis with up to single-residue precision. Materials and methods Key resources table Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional informationStrain, strain background (Escherichia coli)BL21(DE3)Sigma-AldrichCMC0016Electrocompetent cellsStrain, strain background (Escherichia coli)MC1061J Biol Chem. 261:13844–9. PMID:3531212NAElectrocompetent cellsOtherProtein-G-agaroseRoche11243233001Resin used for immunoprecipitationAntibodyAnti-HA.11 epitope tag antibody (mouse monoclonal) IgGBioLegendCat# 901533Used for immunoprecipitation (1 μl of 1 mg/ml, diluted 1:820)AntibodyLepB antibody (rabbit polyclonal) IgGGenerated in-houseNAUsed for immunoprecipitation (dilution 1:820)Recombinant DNA reagentpET Duet-1 (plasmid)NovagenCat# 71146Expression plasmidRecombinant DNA reagentpING1 (plasmid)Gene 34:137–45. PMID:4007491NAExpression plasmidCommercial assay, kitGeneJET Plasmid miniprep kitThermo Fisher Scientific RRID: SCR_008452Cat# 0502Used to purify plasmidsCommercial assay, kitGeneJET PCR Purification KitThermo Fisher ScientificCat# K0701Used to purify linear fragments for in vitro expressionCommercial assay, kitPURExpressNew England BiolabsCat# E6800LUsed for in vitro expressionChemical compound, drug35S methioninePerkinElm
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