Late endosomal transport and tethering are coupled processes controlled by RILP and the cholesterol sensor ORP1L

Abstract

SummaryLate endosomes and lysosomes are dynamic organelles that constantly move and fuse to acquire cargo from early endosomes, phagosomes and autophagosome. Defects in lysosomal dynamics cause severe neurodegenerative and developmental diseases, such as Niemann-Pick type C disease and ARC syndrome, yet little is known about the regulation of late endosomal fusion in a mammalian system. Mammalian endosomes destined for fusion need to be transported over very long distances before they tether to initiate contact. Here, we describe that lysosomal tethering and transport are combined processes co-regulated by one multi-protein complex: RAB7-RILP-ORP1L. We show that RILP directly and concomitantly binds the tethering HOPS complex and the p150Glued subunit of the dynein motor. ORP1L then functions as a cholesterol-sensing switch controlling RILP-HOPS-p150Glued interactions. We show that RILP and ORP1L control Ebola virus infection, a process dependent on late endosomal fusion. By combining recruitment and regulation of both the dynein motor and HOPS complex into a single multiprotein complex, the RAB7-RILP-ORP1L complex efficiently couples and regulates the timing of microtubule minus-end transport and fusion, two major events in endosomal biology.

Characterization of the Mammalian CORVET and HOPS Complexes and Their Modular Restructuring for Endosome Specificity

“…We previously showed that expression levels of these tagged proteins in our systems are low (comparable to endogenous) and that the tagged subunits incorporate into functional complexes (30).…”

“…Constructs-RAB7 and RILP (29) and full-length VPS constructs and GFP-VPS33b L30P (30)have been described previously. GFP-VPS16 and GFP-VPS18 were gifts from Chengyu Liang (Department of Molecular Microbiology and Immunology, University of Southern California, Los Angeles).…”

“…Correlation coefficient for colocalization assays were calculated from plot profiles measuring intensity for the indicated fluorescence channels over a vector through the cells (as in van der Kant et al (30)). Correlation coefficients for the two plots were calculated in Excel using the function,…”

Arf-like GTPase Arl8: Moving from the periphery to the center of lysosomal biology

“…47 Interestingly, 2 recent studies have shown that late endosomal Rab7 effector Rab-interacting lysosomal protein (RILP) that binds to the dynein adaptor, dynactin, also interacts with subunits of the HOPS complex. 48,49 These findings suggest a model whereby Rab7-RILP and Arl8b-SKIP both interact with HOPS complex and play opposing roles in regulating positioning of HOPS endosomes by mediating their association with motor proteins (Fig. 4).…”

In vitro reconstitution of a highly processive recombinant human dynein complex

“…Our current study reveals that a strong candidate to mediate this stimulation is the single fly BICD protein, which is known to be one of a small number of proteins recruited to the RNA element (Dix et al , 2013). It will be important to determine in the future whether other BICD family members such as BICDR proteins (Schlager et al , 2010) and unrelated cargo adaptors for dynein (Engelender et al , 1997; Horgan et al , 2010; van der Kant et al , 2013; van Spronsen et al , 2013) also regulate motor processivity by promoting the interaction with dynactin.…”