Fig. 6: Lateral tunnel as a general substrate-recognition site.

a In-vivo UV photocrosslinking experiment testing a direct interaction between the CL1 degron and lateral tunnel of Doa10. p-Benzoyl-L-phenylalanine (Bpa) was incorporated into either amino acid position 734 (tunnel interior) or 926 (cytosolic surface) of Doa10. Deg1-Ura3-2xStrep was used as a positive control. IP, immunoprecipitation. b As in (a), but testing mScarlet-Sbh2 and mScarlet-Pex15Δ30 as substrates. Note that these substrates also contain 2xStrep-tag immediately after mScarlet for immunodetection. c mScarlet fluorescence intensity measurements on yeast cells expressing mScarlet-Sbh2 (under the TEF1 promoter) and Doa10 variants (under the RET2 promoter). Where indicated, the substrate or Doa10 or both were omitted in constructing strains. Intensities were normalized with respect to the WT Doa10-expressing strain. Mean ± s.e.m. of 6 independent experiments. d As in (c), but testing mScarlet-Pex15Δ30 as a substrate. Mean ± s.e.m. of 7 independent experiments. e, f Cycloheximide chase experiments using mScarlet-Sbh2 (e) or mScarlet-Pex15Δ30 (f) and indicated Doa10 variants (strains identical to those in c and d). Pgk1 was used as a loading control. Data in (a, b, e, f) are representative of three independent experiments. Statistical significance was accessed by unpaired t-test (two-tailed). *p = 0.0091; ***p < 0.0001. Source data are provided as a Source Data file.