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. 2021 Feb 8;10:e64302. doi: 10.7554/eLife.64302

Figure 1. 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.

Figure 1.

Figure 1—figure supplement 1. Gel gallery with selected EmrE (a–e), GlpG (f–l), and BtuC (m–r) constructs.

Figure 1—figure supplement 1.

Full-length (FL) and arrested (A) products are indicated by black and white circles, respectively. Repeat experiments are indicated by single and double lines above the lanes for EmrE (a–e), by short lines beneath the lanes for GlpG (f–i), and are on neighboring gels for BtuC. For BtuC, some full-length (FLc) and arrest (Ac) controls are included (c.f., Figure 1c); these are for the construct immediately to the right of the control construct lanes. GlpG (j, k) show GlpG constructs (indicated by –) and the corresponding LepB-GlpG constructs (indicated by +) for N = 136–196. For N = 141–186, GlpG constructs have the expected full-length band (lower gray circles) that runs slightly faster than the corresponding LepB-GlpG construct (black circles), plus an extra band of unknown provenance that runs more slowly than the full-length LepB-GlpG construct (upper gray circles). The GlpG fFL values shown in Figure 3—figure supplement 1a were calculated by assigning only the lower (dashed magenta curve), or both (dashed green curve), of the bands indicated by gray circles as full-length product. Panel l shows a repeat experiment for the LepB-GlpG constructs included in panels j and k.