RUFY1 binds Arl8b and mediates endosome-to-TGN CI-M6PR retrieval for cargo sorting to lysosomes.

Arl8b, an Arf-like GTP-binding protein, regulates cargo trafficking and positioning of lysosomes. However, it is unknown whether Arl8b regulates lysosomal cargo sorting. Here, we report that Arl8b binds to the Rab4 and Rab14 interaction partner, RUN and FYVE domain-containing protein (RUFY) 1, a known regulator of cargo sorting from recycling endosomes. Arl8b determines RUFY1 endosomal localization through regulating its interaction with Rab14. RUFY1 depletion led to a delay in CI-M6PR retrieval from endosomes to the TGN, resulting in impaired delivery of newly synthesized hydrolases to lysosomes. We identified the dynein-dynactin complex as an RUFY1 interaction partner, and similar to a subset of activating dynein adaptors, the coiled-coil region of RUFY1 was required for interaction with dynein and the ability to mediate dynein-dependent organelle clustering. Our findings suggest that Arl8b and RUFY1 play a novel role on recycling endosomes, from where this machinery regulates endosomes to TGN retrieval of CI-M6PR and, consequently, lysosomal cargo sorting.

The small GTPase Arl8b regulates assembly of the mammalian HOPS complex on lysosomes.

The homotypic fusion and protein sorting (HOPS) complex is a multi-subunit complex conserved from yeast to mammals that regulates late endosome and lysosome fusion. However, little is known about how the HOPS complex is recruited to lysosomes in mammalian cells. Here, we report that the small GTPase Arl8b, but not Rab7 (also known as RAB7A), is essential for membrane localization of the human (h)Vps41 subunit of the HOPS complex. Assembly of the core HOPS subunits to Arl8b- and hVps41-positive lysosomes is guided by their subunit-subunit interactions. RNA interference (RNAi)-mediated depletion of hVps41 resulted in the impaired degradation of EGFR that was rescued upon expression of wild-type but not an Arl8b-binding-defective mutant of hVps41, suggesting that Arl8b-dependent lysosomal localization of hVps41 is required for its endocytic function. Furthermore, we have also identified that the Arl8b effector SKIP (also known as PLEKHM2) interacts with and recruits HOPS subunits to Arl8b and kinesin-positive peripheral lysosomes. Accordingly, RNAi-mediated depletion of SKIP impaired lysosomal trafficking and degradation of EGFR. These findings reveal that Arl8b regulates the association of the human HOPS complex with lysosomal membranes, which is crucial for the function of this tethering complex in endocytic degradation.

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

Lysosomes are dynamic organelles that not only mediate degradation of cellular substrates but also play critical roles in processes such as cholesterol homeostasis, plasma membrane repair, antigen presentation, and cell migration. The small GTPase Arl8, a member of Arf-like (Arl) family of proteins, has recently emerged as a crucial regulator of lysosome positioning and membrane trafficking toward lysosomes. Through interaction with its effector SKIP, the human Arl8 paralog (Arl8b) mediates kinesin-1 dependent motility of lysosomes on microtubule tracks toward the cell periphery. Arl8b-mediated kinesin-driven motility is also implicated in regulating lytic granule polarization in NK cells, lysosome tubulation in macrophages, cell spreading, and migration. Moreover, Arl8b regulates membrane traffic toward lysosomes by recruiting subunits of the HOPS complex, a multi-subunit tethering complex that mediates endo-lysosome fusion. Here we provide a brief review on this recently characterized lysosomal GTPase and summarize the studies focusing on its known functions in regulating lysosomal motility and delivery of endocytic cargo to the lysosomes. We also explore the role of human Arl8b and its orthologs upon infection by intracellular pathogens.