Figure 2.
Figure 2. FAPP1 interacts with VAPs and Sac1 and promotes the in-trans 4-phosphatase activity of Sac1 in vitro. (A) HeLa cells transfected with FAPP1-BioID2 or Sac1-BioID2 constructs were treated with 50 µM biotin. Biotinylated proteins were used in a PD assay with streptavidin-conjugated beads, resolved by SDS-PAGE, and immunoblotted with the indicated antibodies. (B) In vitro PD of GST-FAPP1 proteins (FL, residues 1–100, or residues 97–300) with His-tagged forms of Sac1 and VAPA showing direct binding to Sac1 (residues 1–522 and 1–188) and to VAPA. (C) Top: In vitro PD of His-Sac1 (residues 1–188) with GST-tagged VAPA and VAPB proteins. Bottom: GST-FAPP1 PD assay shows simultaneous binding with Sac1 (1–522) and VAPA. The asterisk indicates cross-reaction of the anti-His-antibody with FL-FAPP1. (D) SIM–super resolution images of endogenous FAPP1 localizing at ERTGoCS. Right panels: Magnification of boxed area; arrowheads indicate interposition of FAPP1 (green) between the TGN (TGN46 in blue) and the ER (Cb5 in red), as highlighted in the orthogonal projection (bottom). Bars, 1 µm. (E) Immuno-EM image showing FAPP1 localizing at the ERTGoCS. FAPP1 was enriched at the TGN as compared with earlier Golgi compartments, as previously described (Godi et al., 2004). (F) In vitro 4-phosphatase activity of liposome-bound His6-Sac1 (1–522) in the presence of GST-FAPP1 (+FAPP1) or GST alone (without FAPP1) in the cis (solid line) or trans (dotted line) conformation (schematized above panel). The reaction was performed in 100 µl in the presence of 100 nM Sac1; 12.5 µl was taken at the indicated time points to measure phosphate release (see Materials and methods). Means ± SD, three technical replicates of a representative experiment, n = 3. (G) Effects of GST-FAPP1 or GST alone (w/o FAPP1) on liposome-bound His6-Sac1 (1–522) activity toward soluble diC8-PI4P. Means ± SD, three technical replicates of a representative experiment, n = 3. (H) Dose response of phosphate release in the trans conformation in the presence of increasing concentrations of GST-FAPP1. Means ± SD, three technical replicates of a representative experiment, n = 3 (see Materials and methods for details). (I) OSBP1 but not FAPP1 can extract PI4P from membranes. 3 µM GST-OSBP1-FL (red line), GST-FAPP1-FL (green line) or GST (blue line) was added to liposomes containing TopFluor-PI4P and DilC18 (see Materials and methods).

FAPP1 interacts with VAPs and Sac1 and promotes the in-trans 4-phosphatase activity of Sac1 in vitro. (A) HeLa cells transfected with FAPP1-BioID2 or Sac1-BioID2 constructs were treated with 50 µM biotin. Biotinylated proteins were used in a PD assay with streptavidin-conjugated beads, resolved by SDS-PAGE, and immunoblotted with the indicated antibodies. (B) In vitro PD of GST-FAPP1 proteins (FL, residues 1–100, or residues 97–300) with His-tagged forms of Sac1 and VAPA showing direct binding to Sac1 (residues 1–522 and 1–188) and to VAPA. (C) Top: In vitro PD of His-Sac1 (residues 1–188) with GST-tagged VAPA and VAPB proteins. Bottom: GST-FAPP1 PD assay shows simultaneous binding with Sac1 (1–522) and VAPA. The asterisk indicates cross-reaction of the anti-His-antibody with FL-FAPP1. (D) SIM–super resolution images of endogenous FAPP1 localizing at ERTGoCS. Right panels: Magnification of boxed area; arrowheads indicate interposition of FAPP1 (green) between the TGN (TGN46 in blue) and the ER (Cb5 in red), as highlighted in the orthogonal projection (bottom). Bars, 1 µm. (E) Immuno-EM image showing FAPP1 localizing at the ERTGoCS. FAPP1 was enriched at the TGN as compared with earlier Golgi compartments, as previously described (Godi et al., 2004). (F) In vitro 4-phosphatase activity of liposome-bound His6-Sac1 (1–522) in the presence of GST-FAPP1 (+FAPP1) or GST alone (without FAPP1) in the cis (solid line) or trans (dotted line) conformation (schematized above panel). The reaction was performed in 100 µl in the presence of 100 nM Sac1; 12.5 µl was taken at the indicated time points to measure phosphate release (see Materials and methods). Means ± SD, three technical replicates of a representative experiment, n = 3. (G) Effects of GST-FAPP1 or GST alone (w/o FAPP1) on liposome-bound His6-Sac1 (1–522) activity toward soluble diC8-PI4P. Means ± SD, three technical replicates of a representative experiment, n = 3. (H) Dose response of phosphate release in the trans conformation in the presence of increasing concentrations of GST-FAPP1. Means ± SD, three technical replicates of a representative experiment, n = 3 (see Materials and methods for details). (I) OSBP1 but not FAPP1 can extract PI4P from membranes. 3 µM GST-OSBP1-FL (red line), GST-FAPP1-FL (green line) or GST (blue line) was added to liposomes containing TopFluor-PI4P and DilC18 (see Materials and methods).

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