JCB_201607085 1051..1070

Late endosome (LE) and lysosome motility and their fusion with other compartments are regulated by action of two small GTPases, Rab7 and Arl8b, and their numerous effectors, including adaptors, tethering factors, and microtubule-based motor-binding proteins (Wang et al., 2011; Khatter et al., 2015b). As with other members of the Rab and Arf-like (Arl) family, Rab7 and Arl8 cycle between inactive (GDP-bound) cytosolic and active (GTP-bound) membrane-bound conformations, recruiting their effectors to lysosomes in their GTP-bound state to mediate downstream functions. Rab7, the better characterized of the two small GTPases, is primarily enriched on the LE/lysosome pool present in the perinuclear region of the cell near the microtubule organizing center (Wang et al., 2011). Herein, Rab7 recruits its effectors, RILP and PLE KHM1, to promote dynein-driven retrograde transport of LEs/lysosome and their fusion with endocytic, phagocytic, and autophagic vesicles (Jordens et al., 2001; McEwan et al., 2015a,b). RILP and PLE KHM1 interact with and recruit the multisubunit tethering factor HOPS complex to Rab7-positive LE/autophagosome–lysosome contact sites (van der Kant et al., 2013; Lin et al., 2014; McEwan et al., 2015a; Wijdeven et al., 2016). HOPS complex facilitates tethering of LEs/autophagosomes to lysosomes and binds with SNA RE proteins to mediate membrane fusion (Balderhaar and Ungermann, 2013; Jiang et al., 2014). ORP1L, another Rab7 effector, induces formation of ER–LE membrane contact sites that inhibit recruitment of the PLE KHM1–HOPS complex to Rab7 (Rocha et al., 2009; Wijdeven et al., 2016). Finally, the Rab7 effector FYCO1 plays an opposing role to RILP by recruiting the motor protein kinesin-1 to promote anterograde movement of LEs/ lysosomes (Pankiv et al., 2010). Unlike Rab7, Arl8b is enriched on the peripheral lysosomes, which are less acidic and have reduced density of Rab7-RILP proteins on their surface (Hofmann and Munro, 2006; Johnson et al., 2016). Arl8b mediates anterograde lysosomal motility by recruiting SKIP (also known as PLE KHM2), which in turn recruits the motor protein kinesin-1 on lysosomes (Rosa-Ferreira and Munro, 2011). Recent studies have established that Arl8b-mediated positioning of lysosomes and lysosome-related organelles is important for nutrient sensing, cell migration, cancer cell metastasis, natural killer cell–mediated cytotoxicity, antigen presentation, and the formation of tubular lysosomes in macrophages (Korolchuk et al., 2011; Mrakovic et al., 2012; Tuli et al., 2013; Schiefermeier et al., 2014; Michelet et al., 2015; Dykes et al., 2016; Pu et al., 2016). Arl8b also regulates cargo trafficking to lysosomes by directly binding to the HOPS subunit Vps41, resulting in functional assembly of the HOPS complex on lysosomal membranes (Garg et al., 2011; Khatter et al., 2015a). Although Rab7 and Arl8b have an overlapping distribution and function, it is not known if they coordinate their Endocytic, autophagic, and phagocytic vesicles move on microtubule tracks to fuse with lysosomes. Small GTPases, such as Rab7 and Arl8b, recruit their downstream effectors to mediate this transport and fusion. However, the potential cross talk between these two GTPases is unclear. Here, we show that the Rab7 effector PLE KHM1 simultaneously binds Rab7 and Arl8b, bringing about clustering and fusion of late endosomes and lysosomes. We show that the N-terminal RUN domain of PLE KHM1 is necessary and sufficient for interaction with Arl8b and its subsequent localization to lysosomes. Notably, we also demonstrate that Arl8b mediates recruitment of HOPS complex to PLE KHM1-positive vesicle contact sites. Consequently, Arl8b binding to PLE KHM1 is required for its function in delivery and, therefore, degradation of endocytic and autophagic cargo in lysosomes. Finally, we also show that PLE KHM1 competes with SKIP for Arl8b binding, which dictates lysosome positioning. These findings suggest that Arl8b, along with its effectors, orchestrates lysosomal transport and fusion. The Rab7 effector PLE KHM1 binds Arl8b to promote cargo traffic to lysosomes