Fig. 8: Sequence analysis reveals a consensus C-terminal Retriever-binding motif.

a Sequence alignment of the CT tail of indicated proteins across representative species. Gold, light gold, and white shading denote sequences identical to, similar to, and not conserved with the human SNX17 CT sequence, respectively. The identified 6-residue acidic sequences are highlighted by the black box, with each position denoted by a colored dot corresponding to the residues mutated in b. The conserved NxxF motifs are marked in pink, with the assignment of QDVF in the Choanoflagellate sequence based on AlphaFold predictions. b Definition of the consensus RICT motif and Coomassie blue-stained SDS PAGE gels showing in vitro pull-down of Retriever by GST-SNX17 CT tails containing indicated point mutations. Binding signals of VPS35L bands are quantified and normalized to the WT CT and shown beneath corresponding mutations. Each dot represents a repeat (n = 2 or 3). c Sequence alignment of the CT tail of indicated proteins in humans or pathogens containing a RICT motif at the C-termini of unstructured tails. Gold, light gold, and white shading denote sequences identical to, similar to, and not conserved with the human SNX17 CT sequence, respectively. Black and gray arrowheads indicate strong and weak binding in the pull-down assay shown in d, respectively. White arrowheads indicate no detectable interaction. Proteins without an arrowhead were not tested. d Coomassie blue-stained SDS PAGE gels showing in vitro pull-down of Retriever WT vs. the DM mutant by GST-tagged CT of the indicated proteins. e Sequences and Coomassie blue-stained SDS PAGE gel showing in vitro pull-down of Retriever by indicated chimera CT tails. Representative results from two independent experiments are shown for RICT motif screening and chimera CT tail pull-down.