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.

Hook proteins are evolutionarily conserved dynein adaptors that promote assembly of highly processive dynein–dynactin motor complexes. Mammals express three Hook paralogs, namely Hook1, Hook2, and Hook3, that have distinct subcellular localizations and expectedly, distinct cellular functions. Here we demonstrate that Hook2 binds to and promotes dynein–dynactin assembly specifically during mitosis. During the late G2 phase, Hook2 mediates dynein–dynactin localization at the nuclear envelope (NE), which is required for centrosome anchoring to the NE. Independent of its binding to dynein, Hook2 regulates microtubule nucleation at the centrosome; accordingly, Hook2-depleted cells have reduced astral microtubules and spindle positioning defects. Besides the centrosome, Hook2 localizes to and recruits dynactin and dynein to the central spindle. Dynactin-dependent targeting of centralspindlin complex to the midzone is abrogated upon Hook2 depletion; accordingly, Hook2 depletion results in cytokinesis failure. We find that the zebrafish Hook2 homologue promotes dynein–dynactin association and was essential for zebrafish early development. Together, these results suggest that Hook2 mediates assembly of the dynein–dynactin complex and regulates mitotic progression and cytokinesis.

Cytoplasmic dynein is a retrograde microtubule-based motor transporting cellular cargo, including organelles, vesicular intermediates, RNA granules, and proteins, thus regulating their subcellular distribution and function. Mammalian dynein associates with dynactin, a multisubunit protein complex that is necessary for the processive motility of dynein along the microtubule tracks. Recent studies have shown that the interaction between dynein and dynactin is enhanced in the presence of a coiled-coil activating adaptor protein, which performs dual functions of recruiting dynein and dynactin to their cargoes and inducing the superprocessive motility of the motor complex. One such family of coiled-coil activating adaptor proteins is the Hook family of proteins that are conserved across evolution with three paralogs in the case of mammals, namely, HOOK1− HOOK3. This Perspective aims to provide an overview of the Hook protein structure and the cellular functions of Hook proteins, with an emphasis on the recent developments in understanding their role as activating dynein adaptors.

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Members of Rab and ADP-ribosylation factor (Arf) family of small GTP-binding (G) proteins regulate several aspects of intracellular transport and cytoskeleton organization. The phylogenetic analysis, combined with database mining approaches has led to the identification of Arf-like (Arl) G proteins as a sub-group of the Arf family with approximately 20 members in mammals. Arls are similar in structure to the Arfs, but exhibit immense diversity pertaining to their mechanisms of membrane recruitment, subcellular distribution and cellular functions. Only a few members of this sub-group are currently characterized, while information on the majority of Arl proteins remains scanty or is not known. In this review, we will cover our current understanding of the functions performed by Arl sub-family members, described under three broad categories: Arls involved in primary cilia formation and function, Arls engaged in secretory and endocytic transport, and Arls regulating microtubule and actin organization.