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Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150 Glued and late endosome positioning.

Rocha N, Kuijl C, van der Kant R, Janssen L, Houben D, Janssen H, Zwart W, Neefjes J - J. Cell Biol. (2009)

Bottom Line: Motor proteins associated to the dynactin subunit p150(Glued) bind to LEs via the Rab7 effector Rab7-interacting lysosomal protein (RILP) in association with the oxysterol-binding protein ORP1L.At these sites, the ER protein VAP (VAMP [vesicle-associated membrane protein]-associated ER protein) can interact in trans with the Rab7-RILP complex to remove p150(Glued) and associated motors.Under high cholesterol conditions, as in Niemann-Pick type C disease, this process is prevented, and LEs accumulate at the microtubule minus end as the result of dynein motor activity.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, The Netherlands Cancer Institute, 1066CX Amsterdam, Netherlands.

ABSTRACT
Late endosomes (LEs) have characteristic intracellular distributions determined by their interactions with various motor proteins. Motor proteins associated to the dynactin subunit p150(Glued) bind to LEs via the Rab7 effector Rab7-interacting lysosomal protein (RILP) in association with the oxysterol-binding protein ORP1L. We found that cholesterol levels in LEs are sensed by ORP1L and are lower in peripheral vesicles. Under low cholesterol conditions, ORP1L conformation induces the formation of endoplasmic reticulum (ER)-LE membrane contact sites. At these sites, the ER protein VAP (VAMP [vesicle-associated membrane protein]-associated ER protein) can interact in trans with the Rab7-RILP complex to remove p150(Glued) and associated motors. LEs then move to the microtubule plus end. Under high cholesterol conditions, as in Niemann-Pick type C disease, this process is prevented, and LEs accumulate at the microtubule minus end as the result of dynein motor activity. These data explain how the ER and cholesterol control the association of LEs with motor proteins and their positioning in cells.

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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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fig6: 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.

Mentions: ORP1L allows RILP-induced clustering of LEs. ΔORD relocates LEs to the cell periphery, suggesting the exclusion of p150Glued from RILP (Fig. 2 B). Removal of the 20-kD region preceding the ORD restored RILP-induced clustering and p150Glued binding to RILP (Fig. 2 B). This region contains a predicted coiled-coil region (aa 430–463; Johansson et al., 2007) followed by a FFAT motif (aa 472–482; sequence, SEDEFYDALSD; Fig. 2 A; Loewen et al., 2003) and should contain the information for removal of p150Glued from RILP. To test whether the FFAT motif controlled p150Glued binding to RILP, an inactivating point mutation (D478A) in the FFAT motif (Loewen et al., 2003) was introduced in mRFP-ΔORD. This mutant was coexpressed with GFP-RILP before staining for p150Glued (Fig. 6 A). Whereas ΔORD excludes RILP-mediated recruitment of p150Glued (resulting in LE scattering), the ΔORDFFAT(D478A) point mutant permitted p150Glued binding to RILP, resulting in LE clustering around the MTOC.


Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150 Glued and late endosome positioning.

Rocha N, Kuijl C, van der Kant R, Janssen L, Houben D, Janssen H, Zwart W, Neefjes J - J. Cell Biol. (2009)

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.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2712958&req=5

fig6: 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.
Mentions: ORP1L allows RILP-induced clustering of LEs. ΔORD relocates LEs to the cell periphery, suggesting the exclusion of p150Glued from RILP (Fig. 2 B). Removal of the 20-kD region preceding the ORD restored RILP-induced clustering and p150Glued binding to RILP (Fig. 2 B). This region contains a predicted coiled-coil region (aa 430–463; Johansson et al., 2007) followed by a FFAT motif (aa 472–482; sequence, SEDEFYDALSD; Fig. 2 A; Loewen et al., 2003) and should contain the information for removal of p150Glued from RILP. To test whether the FFAT motif controlled p150Glued binding to RILP, an inactivating point mutation (D478A) in the FFAT motif (Loewen et al., 2003) was introduced in mRFP-ΔORD. This mutant was coexpressed with GFP-RILP before staining for p150Glued (Fig. 6 A). Whereas ΔORD excludes RILP-mediated recruitment of p150Glued (resulting in LE scattering), the ΔORDFFAT(D478A) point mutant permitted p150Glued binding to RILP, resulting in LE clustering around the MTOC.

Bottom Line: Motor proteins associated to the dynactin subunit p150(Glued) bind to LEs via the Rab7 effector Rab7-interacting lysosomal protein (RILP) in association with the oxysterol-binding protein ORP1L.At these sites, the ER protein VAP (VAMP [vesicle-associated membrane protein]-associated ER protein) can interact in trans with the Rab7-RILP complex to remove p150(Glued) and associated motors.Under high cholesterol conditions, as in Niemann-Pick type C disease, this process is prevented, and LEs accumulate at the microtubule minus end as the result of dynein motor activity.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, The Netherlands Cancer Institute, 1066CX Amsterdam, Netherlands.

ABSTRACT
Late endosomes (LEs) have characteristic intracellular distributions determined by their interactions with various motor proteins. Motor proteins associated to the dynactin subunit p150(Glued) bind to LEs via the Rab7 effector Rab7-interacting lysosomal protein (RILP) in association with the oxysterol-binding protein ORP1L. We found that cholesterol levels in LEs are sensed by ORP1L and are lower in peripheral vesicles. Under low cholesterol conditions, ORP1L conformation induces the formation of endoplasmic reticulum (ER)-LE membrane contact sites. At these sites, the ER protein VAP (VAMP [vesicle-associated membrane protein]-associated ER protein) can interact in trans with the Rab7-RILP complex to remove p150(Glued) and associated motors. LEs then move to the microtubule plus end. Under high cholesterol conditions, as in Niemann-Pick type C disease, this process is prevented, and LEs accumulate at the microtubule minus end as the result of dynein motor activity. These data explain how the ER and cholesterol control the association of LEs with motor proteins and their positioning in cells.

Show MeSH
Related in: MedlinePlus