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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 controls recruitment of p150Glued to the Rab7–RILP receptor. (A) ORP1L domain structure and constructs. Five domains are predicted in ORP1L. Numbers indicate amino acid residue positions. Constructs were N-terminally tagged with mRFP. The ΔORDPHDPHD chimera had ORD exchanged for a tandem PH domain derived from ORP1L. (B) Effect of ORP1L deletion or chimeric constructs on RILP-mediated p150Glued recruitment. (left) Mel JuSo cells transfected with GFP-RILP and mRFP-ORP1L constructs and stained with anti-p150Glued antibodies before CLSM. For pixel analyses of the images, see Fig. S2 E. n > 200 for each condition. (right) Mel JuSo cells transfected with the indicated mRFP-ORP1L constructs and whole cell lysates analyzed by immunoblotting with anti-mRFP antibodies (WB: α-mRFP). Molecular standards are indicated. WB, Western blot. (C) Effect of ORP1L constructs on RILP recruitment of the p150Glued-associated dynein motor adapter DIC. Mel JuSo cells were transfected with GFP-RILP and the mRFP-ORP1L constructs indicated and stained with anti-DIC antibodies. n > 100 for each condition. Bars, 10 µm.
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fig2: ORP1L controls recruitment of p150Glued to the Rab7–RILP receptor. (A) ORP1L domain structure and constructs. Five domains are predicted in ORP1L. Numbers indicate amino acid residue positions. Constructs were N-terminally tagged with mRFP. The ΔORDPHDPHD chimera had ORD exchanged for a tandem PH domain derived from ORP1L. (B) Effect of ORP1L deletion or chimeric constructs on RILP-mediated p150Glued recruitment. (left) Mel JuSo cells transfected with GFP-RILP and mRFP-ORP1L constructs and stained with anti-p150Glued antibodies before CLSM. For pixel analyses of the images, see Fig. S2 E. n > 200 for each condition. (right) Mel JuSo cells transfected with the indicated mRFP-ORP1L constructs and whole cell lysates analyzed by immunoblotting with anti-mRFP antibodies (WB: α-mRFP). Molecular standards are indicated. WB, Western blot. (C) Effect of ORP1L constructs on RILP recruitment of the p150Glued-associated dynein motor adapter DIC. Mel JuSo cells were transfected with GFP-RILP and the mRFP-ORP1L constructs indicated and stained with anti-DIC antibodies. n > 100 for each condition. Bars, 10 µm.

Mentions: The N-terminal ankyrin repeats of ORP1L bind to Rab7 and are involved in dynein motor transport of LEs (Johansson et al., 2005, 2007). To resolve the ORP1L domains involved in the control of LE transport, a series of monomeric RFP (mRFP)–tagged C-terminal truncations of ORP1L constructs (Fig. 2 A and Fig. S2 A) were coexpressed with GFP-RILP in MelJuSo cells. qPCR data revealed that the ORP1L constructs and RILP were expressed 4- to 10-fold over endogenous expression levels (unpublished data). RILP expression was required to visualize recruitment of endogenous p150Glued and dynein motor binding, but the many motors present on a vesicle (Jordens et al., 2001) rendered localization insensitive to cholesterol manipulation (Fig. S1 C). Vesicles labeled by the mRFP-ORP1L constructs were characterized by immunostaining for LE labeling for CD63, LAMP-1, Rab7, and cholesterol, as detected by filipin (Fig. S2, B and C). Markers for the ER, Golgi, or early endosomes were absent on these vesicles (Fig. S2 D).


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 controls recruitment of p150Glued to the Rab7–RILP receptor. (A) ORP1L domain structure and constructs. Five domains are predicted in ORP1L. Numbers indicate amino acid residue positions. Constructs were N-terminally tagged with mRFP. The ΔORDPHDPHD chimera had ORD exchanged for a tandem PH domain derived from ORP1L. (B) Effect of ORP1L deletion or chimeric constructs on RILP-mediated p150Glued recruitment. (left) Mel JuSo cells transfected with GFP-RILP and mRFP-ORP1L constructs and stained with anti-p150Glued antibodies before CLSM. For pixel analyses of the images, see Fig. S2 E. n > 200 for each condition. (right) Mel JuSo cells transfected with the indicated mRFP-ORP1L constructs and whole cell lysates analyzed by immunoblotting with anti-mRFP antibodies (WB: α-mRFP). Molecular standards are indicated. WB, Western blot. (C) Effect of ORP1L constructs on RILP recruitment of the p150Glued-associated dynein motor adapter DIC. Mel JuSo cells were transfected with GFP-RILP and the mRFP-ORP1L constructs indicated and stained with anti-DIC antibodies. n > 100 for each condition. Bars, 10 µm.
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fig2: ORP1L controls recruitment of p150Glued to the Rab7–RILP receptor. (A) ORP1L domain structure and constructs. Five domains are predicted in ORP1L. Numbers indicate amino acid residue positions. Constructs were N-terminally tagged with mRFP. The ΔORDPHDPHD chimera had ORD exchanged for a tandem PH domain derived from ORP1L. (B) Effect of ORP1L deletion or chimeric constructs on RILP-mediated p150Glued recruitment. (left) Mel JuSo cells transfected with GFP-RILP and mRFP-ORP1L constructs and stained with anti-p150Glued antibodies before CLSM. For pixel analyses of the images, see Fig. S2 E. n > 200 for each condition. (right) Mel JuSo cells transfected with the indicated mRFP-ORP1L constructs and whole cell lysates analyzed by immunoblotting with anti-mRFP antibodies (WB: α-mRFP). Molecular standards are indicated. WB, Western blot. (C) Effect of ORP1L constructs on RILP recruitment of the p150Glued-associated dynein motor adapter DIC. Mel JuSo cells were transfected with GFP-RILP and the mRFP-ORP1L constructs indicated and stained with anti-DIC antibodies. n > 100 for each condition. Bars, 10 µm.
Mentions: The N-terminal ankyrin repeats of ORP1L bind to Rab7 and are involved in dynein motor transport of LEs (Johansson et al., 2005, 2007). To resolve the ORP1L domains involved in the control of LE transport, a series of monomeric RFP (mRFP)–tagged C-terminal truncations of ORP1L constructs (Fig. 2 A and Fig. S2 A) were coexpressed with GFP-RILP in MelJuSo cells. qPCR data revealed that the ORP1L constructs and RILP were expressed 4- to 10-fold over endogenous expression levels (unpublished data). RILP expression was required to visualize recruitment of endogenous p150Glued and dynein motor binding, but the many motors present on a vesicle (Jordens et al., 2001) rendered localization insensitive to cholesterol manipulation (Fig. S1 C). Vesicles labeled by the mRFP-ORP1L constructs were characterized by immunostaining for LE labeling for CD63, LAMP-1, Rab7, and cholesterol, as detected by filipin (Fig. S2, B and C). Markers for the ER, Golgi, or early endosomes were absent on these vesicles (Fig. S2 D).

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