Figure 3. Kinetics of EmrE topogenesis.
(A) Fraction of CG trajectories in which all TMDs are fully integrated in a multispanning topology, plotted as a function of time for several mutants. (B) Fraction of CG trajectories in which each TMD is integrated, plotted as a function of time for the cotranslationally-biased (CB) mutant (top) and EmrE (bottom). The snapshots show an example of a simulation in which TMD4 of EmrE does not integrate during translation. In both panels, 0 s corresponds to the end of translation and negative values of time correspond to the period that precedes the end of ribosomal translation.
Figure 3—figure supplement 1. Pathways for the cotranslational integration of TMDs into the membrane.
At left, three distinct integration pathways are illustrated; the definition of each pathway is described in the Cotranslational integration pathways section of the ‘Materials and methods’. In the ‘channel-sliding’ pathway, the TMD partially enters the channel, then crosses the lateral gate (LG), then fully integrates into the membrane. In the ‘interface-sliding’ pathway, the TMD enters the cytoplasm through the gap between the translocon and ribosome, prior to undergoing membrane integration. In the ‘in-out’ pathway, the TMD fully spans the channel prior to membrane integration. At right, the fraction of cotranslational TMD integration events that exhibit each of these three pathways is presented for all four TMDs and for both the EmrE and nEmrE mutants. The dominant cotranslational integration pathway is the ‘channel-sliding’ pathway for all TMDs in both mutants.
Figure 3—figure supplement 2. Simulation time necessary for 50%, 90%, and 95% of the CG trajectories to reach fully integrated topologies for each mutant.
Figure 3—figure supplement 3. Effect of loop length on integration trajectories.
(A) Schematic illustrations of a new EmrE mutant with elongated, 10-bead loops, and a new CB mutant with shortened, 5-bead loops. (B) Fraction of CG trajectories that reach fully integrated topologies as a function of time for the EmrE and CB mutants with both loop lengths. The integration kinetics for the EmrE mutants clearly show that longer loops increase the timescale of post-translational annealing, due to the reduction of the loop-flipping frequency for the longer soluble loops. The corresponding effect is less clear for the two CB mutants, since the timescale for the loop-flipping frequency is dominated by the large positive charges on these loops, masking the effect of changing the loop length. (C) Average loop positions in the end-of-translation (EOT) ensemble for both the EmrE and CB mutants, presented in terms of the fraction of configurations for which each loop occupies the cytoplasm. For the EmrE mutants, changing the loop length leads to relatively small shifts in the EOT ensemble; however, for the CB mutants, the effect of loop length on the EOT ensemble is much larger, with the new short-loop CB mutant exhibiting a much weaker bias towards the Ncyto/Ccyto topology in the EOT ensemble.