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Published online
doi:10.1083/jcb.200606077
The Journal of Cell Biology, Vol. 176, No. 4, 459-471
The Rockefeller University Press, 0021-9525 $30.00
© Johansson et al.
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Article

Activation of endosomal dynein motors by stepwise assembly of Rab7–RILP–p150Glued, ORP1L, and the receptor ßlll spectrin



Marie Johansson1, Nuno Rocha2, Wilbert Zwart2, Ingrid Jordens2, Lennert Janssen2, Coenraad Kuijl2, Vesa M. Olkkonen1, and Jacques Neefjes2

1 Department of Molecular Medicine, National Public Health Institute, Biomedicum, FI-00251 Helsinki, Finland
2 Division of Tumor Biology, The Netherlands Cancer Institute, 1066CX Amsterdam, Netherlands

Correspondence to Jacques Neefjes: j.neefjes{at}nki.nl

The small GTPase Rab7 controls late endocytic transport by the minus end–directed motor protein complex dynein–dynactin, but how it does this is unclear. Rab7-interacting lysosomal protein (RILP) and oxysterol-binding protein–related protein 1L (ORP1L) are two effectors of Rab7. We show that GTP-bound Rab7 simultaneously binds RILP and ORP1L to form a RILP–Rab7–ORP1L complex. RILP interacts directly with the C-terminal 25-kD region of the dynactin projecting arm p150Glued, which is required for dynein motor recruitment to late endocytic compartments (LEs). Still, p150Glued recruitment by Rab7–RILP does not suffice to induce dynein-driven minus-end transport of LEs. ORP1L, as well as ßIII spectrin, which is the general receptor for dynactin on vesicles, are essential for dynein motor activity. Our results illustrate that the assembly of microtubule motors on endosomes involves a cascade of linked events. First, Rab7 recruits two effectors, RILP and ORP1L, to form a tripartite complex. Next, RILP directly binds to the p150Glued dynactin subunit to recruit the dynein motor. Finally, the specific dynein motor receptor Rab7–RILP is transferred by ORP1L to ßIII spectrin. Dynein will initiate translocation of late endosomes to microtubule minus ends only after interacting with ßIII spectrin, which requires the activities of Rab7–RILP and ORP1L.

M. Johansson and N. Rocha contributed equally to this paper.

Abbreviations used in this paper: Arp, actin-related protein; FLIM, fluorescence lifetime imaging microscopy; FRET, fluorescence resonance energy transfer; LE, late endocytic compartment; MIIC, major histocompatibility complex class II–containing compartment; MBP, maltose-binding protein; mRFP, monomeric red fluorescent protein; MTOC, microtubule organizing center; ORD, oxysterol-binding protein–related domain; ORP, oxysterol-binding protein–related protein; RILP, Rab7-interacting lysosomal protein; shRNA, short hairpin RNA.


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