Figure 5.
VPS13D binds VAP on the ER via a phospho-FFAT in its N-terminal region.(A) Left: Confocal image of a COS7 cell coexpressing Halo–VAP-B and VPS13D^EGFP showing weak recruitment of VPS13D^EGFP to the ER. Scale bar, 10 µm. Right: High-magnification view of Halo–VAP-B and VPS13D^EGFP fluorescence of the field enclosed by a rectangle. (B) VAP-dependent binding of the N-terminal portion of VPS13D to the ER. Left: Confocal images of a COS7 cell coexpressing the N-terminal portion of VPS13D fused to EGFP and mCherry-VAP-B. Right: Confocal images of a COS7 cell coexpressing the N-terminal portion of VPS13D fused to EGFP and Sec61β-RFP, but not VAP. Scale bar, 3 µm. (C) Cartoons showing VPS13D constructs used for the experiments shown in E, indicating the position of the predicted phospho-FFAT motif. (D) Comparison of the conventional FFAT motif with phospho-FFAT motifs, including the one found in VPS13D (Di Mattia et al., 2020). The aromatic residue indicated in green, present in both conventional and phospho-FFAT motifs, is essential for binding to VAP. Acidic amino acid residues in the region contribute to the binding in conventional FFAT motifs, but based on a previous study (Di Mattia et al., 2020), they can be replaced by phosphorylatable residues in phospho-FFAT motifs. (E) Evidence for a phospho-FFAT motif-dependent binding of the N-terminal region of VPS13D to VAP. First column: The N-terminal construct (1–1576) of VPS13D is recruited to the ER upon VAP overexpression. Second column: Two mutations in the MSP domain of VAP that disrupt the FFAT motif binding pocket also disrupt the recruitment of the VPS13D construct. Third column: Mutation to alanine of the aromatic residue of the phospho-FFAT motif disrupts binding. Fourth and fifth columns: No binding occurs when the threonine that corresponds to an aspartate in the conventional FFAT motif is replaced by a nonphosphorylatable alanine, but binding is restored when the threonine is replaced by aspartate, as long as the adjacent proline is also mutated (Di Mattia et al., 2020). Scale bar, 10 µm. (F) Alignment of the region of VPS13D orthologues from different species centered on the amino acid region required for Miro binding in human VPS13D. The alignment shows a high degree of conservation of the key residues of the phospho-FFAT motif among several chordates and also observed in flies. The phylogenetic tree was generated by maximum likelihood.

VPS13D binds VAP on the ER via a phospho-FFAT in its N-terminal region. (A) Left: Confocal image of a COS7 cell coexpressing Halo–VAP-B and VPS13D^EGFP showing weak recruitment of VPS13D^EGFP to the ER. Scale bar, 10 µm. Right: High-magnification view of Halo–VAP-B and VPS13D^EGFP fluorescence of the field enclosed by a rectangle. (B) VAP-dependent binding of the N-terminal portion of VPS13D to the ER. Left: Confocal images of a COS7 cell coexpressing the N-terminal portion of VPS13D fused to EGFP and mCherry-VAP-B. Right: Confocal images of a COS7 cell coexpressing the N-terminal portion of VPS13D fused to EGFP and Sec61β-RFP, but not VAP. Scale bar, 3 µm. (C) Cartoons showing VPS13D constructs used for the experiments shown in E, indicating the position of the predicted phospho-FFAT motif. (D) Comparison of the conventional FFAT motif with phospho-FFAT motifs, including the one found in VPS13D (Di Mattia et al., 2020). The aromatic residue indicated in green, present in both conventional and phospho-FFAT motifs, is essential for binding to VAP. Acidic amino acid residues in the region contribute to the binding in conventional FFAT motifs, but based on a previous study (Di Mattia et al., 2020), they can be replaced by phosphorylatable residues in phospho-FFAT motifs. (E) Evidence for a phospho-FFAT motif-dependent binding of the N-terminal region of VPS13D to VAP. First column: The N-terminal construct (1–1576) of VPS13D is recruited to the ER upon VAP overexpression. Second column: Two mutations in the MSP domain of VAP that disrupt the FFAT motif binding pocket also disrupt the recruitment of the VPS13D construct. Third column: Mutation to alanine of the aromatic residue of the phospho-FFAT motif disrupts binding. Fourth and fifth columns: No binding occurs when the threonine that corresponds to an aspartate in the conventional FFAT motif is replaced by a nonphosphorylatable alanine, but binding is restored when the threonine is replaced by aspartate, as long as the adjacent proline is also mutated (Di Mattia et al., 2020). Scale bar, 10 µm. (F) Alignment of the region of VPS13D orthologues from different species centered on the amino acid region required for Miro binding in human VPS13D. The alignment shows a high degree of conservation of the key residues of the phospho-FFAT motif among several chordates and also observed in flies. The phylogenetic tree was generated by maximum likelihood.

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