Figure 6.
Figure 6. ORP1L requires VAP-A to remove p150Glued from Rab7–RILP. (A) Effect of the FFAT motif in ΔORD on RILP-mediated p150Glued recruitment. (left) Mel JuSo cells were transfected with GFP-RILP and mRFP-ΔORD containing a point mutation in its FFAT motif, ΔORDFFAT(D478A). Fixed cells were stained with anti-p150Glued antibodies. n > 200. (right) Mel JuSo cells were transfected with the mRFP-ΔORD or mRFP-ΔORDFFAT(D478A) constructs, and whole cell lysates were analyzed by immunoblotting with anti-mRFP antibodies (WB: α-mRFP). (B) p150Glued exclusion by ΔORD and VAP-A. VAP-A was silenced by siRNA (siVAP-A) in cells expressing GFP-RILP and mRFP-ΔORD. (left) Cells stained for p150Glued. Pixel analyses are shown in Fig. S2 E. n > 100 for each condition. (right) Western blot analysis of cells transfected with transfection reagent (mock), control (siCTRL), or VAP-A siRNA (siVAP-A) and probed for α-tubulin (as loading control) and anti–VAP-A antibodies. (C) VAP-A removes p150Glued from Rab7–RILP. GTP-locked His-Rab7(Q67L) was GTP loaded and coupled to Talon beads before adding purified RILP, ORP1L, and p150Glued(C25) fragments in equimolar amounts to form a preassembled ORP1L–Rab7–RILP–p150Glued(C25) complex. Subsequently, the complex was incubated in the presence or absence of purified VAP-A. After washing, the bead-bound proteins were analyzed by SDS-PAGE and Western blotting. (D) ORP1L requirements for p150Glued removal by VAP-A. GTP-loaded His-Rab7(Q67L) was coupled to Talon Co2+ beads before adding the isolated proteins indicated in equimolar amounts to form a preassembled complex. After washing, these complexes were exposed to isolated VAP-A or the p150Glued(C25) fragment, as indicated, and the effects on the preformed complex were assessed by SDS-PAGE and Western blot analyses with antibodies, as indicated. (E) VAP-A interacts with p150Glued. GST or GST–VAP-A was coupled to beads before exposure to the p150Glued(C25) fragment. After washing, the complexes were analyzed by SDS-PAGE and Western blotting. (A–E) Molecular masses are indicated. WB, Western blot. Bars, 10 µm.

ORP1L requires VAP-A to remove p150Glued from Rab7–RILP. (A) Effect of the FFAT motif in ΔORD on RILP-mediated p150Glued recruitment. (left) Mel JuSo cells were transfected with GFP-RILP and mRFP-ΔORD containing a point mutation in its FFAT motif, ΔORDFFAT(D478A). Fixed cells were stained with anti-p150Glued antibodies. n > 200. (right) Mel JuSo cells were transfected with the mRFP-ΔORD or mRFP-ΔORDFFAT(D478A) constructs, and whole cell lysates were analyzed by immunoblotting with anti-mRFP antibodies (WB: α-mRFP). (B) p150Glued exclusion by ΔORD and VAP-A. VAP-A was silenced by siRNA (siVAP-A) in cells expressing GFP-RILP and mRFP-ΔORD. (left) Cells stained for p150Glued. Pixel analyses are shown in Fig. S2 E. n > 100 for each condition. (right) Western blot analysis of cells transfected with transfection reagent (mock), control (siCTRL), or VAP-A siRNA (siVAP-A) and probed for α-tubulin (as loading control) and anti–VAP-A antibodies. (C) VAP-A removes p150Glued from Rab7–RILP. GTP-locked His-Rab7(Q67L) was GTP loaded and coupled to Talon beads before adding purified RILP, ORP1L, and p150Glued(C25) fragments in equimolar amounts to form a preassembled ORP1L–Rab7–RILP–p150Glued(C25) complex. Subsequently, the complex was incubated in the presence or absence of purified VAP-A. After washing, the bead-bound proteins were analyzed by SDS-PAGE and Western blotting. (D) ORP1L requirements for p150Glued removal by VAP-A. GTP-loaded His-Rab7(Q67L) was coupled to Talon Co2+ beads before adding the isolated proteins indicated in equimolar amounts to form a preassembled complex. After washing, these complexes were exposed to isolated VAP-A or the p150Glued(C25) fragment, as indicated, and the effects on the preformed complex were assessed by SDS-PAGE and Western blot analyses with antibodies, as indicated. (E) VAP-A interacts with p150Glued. GST or GST–VAP-A was coupled to beads before exposure to the p150Glued(C25) fragment. After washing, the complexes were analyzed by SDS-PAGE and Western blotting. (A–E) Molecular masses are indicated. WB, Western blot. Bars, 10 µm.

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