Mutations in the phospho-FFAT motif of VPS13D but not in conventional FFAT motifs disrupt VAP binding. (A) Cartoon depicting full-length VPS13D and the constructs used for C and D, displaying the localization of the predicted three conventional FFAT motifs and the single phospho-FFAT motif and the mutations that were introduced to disrupt them. (B) Sequence of each of the predicted conventional FFAT and phospho-FFAT motifs. *, Conventional FFAT motif score was calculated using a previously described algorithm; scores 3.5 and 4 are considered weak FFAT motifs (Slee and Levine, 2019). (C) Left: Coexpression of an EGFP-tagged N-terminal fragment of VPS13D (amino acid 1–1576) with the ER protein VAP-B shows robust recruitment of the fragment to the ER. Right: The combined disruption of the three best predicted conventional FFAT motifs as indicated in A did not affect ER recruitment of this VPS13D fragment by VAP-B. (D) The point mutation T770D alone (without the additional mutation of the adjacent proline to alanine; see Fig. 5 E and Di Mattia et al., 2020) abolishes the VAP-dependent recruitment of the N-terminal fragment of VPS13D to the ER. Scale bars, 10 µm.