Fig. 4: Mutational analysis of the lateral tunnel of Doa10.

a Schematic diagram of Doa10-dependent yeast growth inhibition assay using Deg1-Ura3. b Structure of TMs 5 to 7 (TD ___domain). The view is similar to Fig. 3b. Positions of amino acid residues tested for the effects on Deg1-Ura3 degradation are shown with sticks and spheres (magenta: defects observed; cyan/blue: no substantial defects observed; see c and d). c, d Yeast growth inhibition assay with indicated Doa10 mutants (GFP-tagged Doa10 was expressed at a relatively low level from the DOA10 promoter in a single-copy plasmid). e As in (c) and (d), but testing different mutants of Doa10 (ALFA-tagged). Note that a higher expression level of Doa10-ALFA compared to Doa10-GFP produces stronger growth inhibition. f Cycloheximide chase experiments on Deg1-Ura3 using indicated Doa10 mutants (‘EV/SV’ = E633V/S738V, ‘GS’ = Δ710-718::GS, ‘GS+3Val’=Δ710-718::GS+3Val, and ‘EV/DV’ = E713V/D714V). Mean ± s.e.m. of three independent experiments. g–i Example snapshots of MD simulations for WT and indicated Doa10 mutants illustrating the collapse of the L6/7 loop. L6/7 are rendered in blue licorice, along with the sidechains for positions 710–718. The residues forming the hydrophobic wedge of the tunnel (F637, M686, F689, and Y742) are rendered in both gray semitransparent surface and licorice, with the center of mass (COM) of the loop residues 712–715 and the COM of the wedge residues rendered as orange spheres. Their separation is indicated with a black line. In these snapshots, the separation distances are 15.9 Å, 11.4 Å, and 14.0 Å for WT, E633V/S738V, and E713V/D714V, respectively. The mutated valine residues are rendered in yellow. See Supplementary Fig. 8 for GS and GS+3Val mutants. j–l The distance distribution was computed between the center of mass of the loop residues (712–715) and the hydrophobic wedge residues (F637, M686, F689, and Y742) for the wild type and mutant systems. The first 150 ns of each trajectory were removed to allow loop relaxation and the 2 replicas were combined. Data in (c–e) are representative of three independent experiments. Data in (f) are mean ± s.e.m. from three independent experiments. Source data are provided as a Source Data file.