Fig. 6: The key residues of RPN10 for SAP05 binding.

a Sequence alignment of RPN10 homologs between plants and animals. AtRPN10, Arabidopsis thaliana RPN10 (Uniprot ID: P55034); SlRPN10, Solanum lycopersicum RPN10 (Uniprot ID: A0A3Q7F6N7); OsRPN10, Oryza sativa RPN10 (Uniprot ID: O82143); ZmRPN10, Zea mays RPN10 (Uniprot ID: B6TK61); HsRPN10, Homo sapiens RPN10 (Uniprot ID: Q5VWC4); MqRPN10, Macrosteles quadrilineatus RPN10 (GenBanK: XP_054291051.1); FcRPN10, Folsomia candida (Uniprot ID: A0A226E266); HvRPN10, Hydra vulgaris (Uniprot ID: T2MF29). The residues involved in SAP05 binding are marked with black triangles. b Structural analysis of steric hindrance caused by His38 of human RPN10 (PDB: 6MSE). c Structural analysis of steric hindrance caused by Asn68 of FcRPN10 (corresponding to Gly70 in AtRPN10). The structure of FcRPN10 is predicted by Alphafold2, and superimposed with AtRPN10 in the SAP05-AtRPN10 complex. d GST pull-down assay using wild-type and mutant AtRPN10 to pull down OY-M SAP05. Source data are provided as a Source Data file. Representative images, n = 3. e Structural analysis of the absence of hydrophobic interaction caused by Thr68 in human RPN10 instead of Leu69 in AtRPN10. f Structural analysis of steric hindrance caused by Arg42 of human RPN10. g GST pull-down assay using OY-M SAP05 to pull down wild-type and mutant human RPN10. h, i Western blot analysis of OY-M SAP05-mediated degradation of SPL5 in the presence of wild-type and mutant human vWA domain (HS38-39GA, R42Q, T68L) instead of AtRPN10 vWA domain in purified human 26 S proteasomes. Source data are provided as a Source Data file. Representative images, n = 3. j Quantification of the percentage of retained GST-SPL5 in the degradation assay, corresponding to h, i and Fig. 4a (Mean ± S.E.M.; n = 3 independent experiments).