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

Abstract

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.

Drosophila Rab2 controls endosome-lysosome fusion and LAMP delivery to late endosomes

“…In addition to showing that these processes clearly differ mechanistically, this fits well with the predominantly lysosomal localization of Drosophila Gie/Arl8 and its mammalian orthologs [25,71]. …”

“…The small GTPase ARL8B is required for LE-lysosome fusion as the lysosomal HOPS receptor in mammals [25]. Gie/Arl8, the single Drosophila ortholog for mammalian ARL8A/B, also localizes to lysosomes [71], but a phenotypic description of Gie/Arl8 loss-of-function in flies is lacking.…”

“…The current model for LE-lysosome fusion in mammals entails the ~ 30-nm long, seahorse-shaped HOPS complex bridging lysosomal and endosomal membranes by binding the RAB7A effector RILP in the LE membrane and ARL8B and its effector SKIP on the lysosome [22–25]. The binding is accomplished by the Vps39 and Vps41 subunits that are positioned at each end of the HOPS complex, based on electron tomography of the complex in yeast [20].…”

“…The HOPS complex then induces membrane fusion by the assembly of appropriate SNAREs into a trans-SNARE complex [15,21]. This model is largely believed to hold true also in metazoan systems, albeit with the RAB7A effector RILP together with the small lysosomal GTPase ARL8B and its effector PLEKHM2/SKIP playing the role of HOPS receptors in mammals [22–25]. Loss of HOPS subunits causes failure of lysosomal delivery of endocytosed and autophagic material in Drosophila and mammals [26–31].…”

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

“…Recent data has suggested that in metazoan cells, Arl8b directly binds to Vps41 and regulates assembly of the HOPS complex on lysosomal membranes. 23,25,46,47 Accordingly, Arl8b-depletion impaired lysosomal localization of Vps41 and other HOPS subunits. Furthermore, Arl8b-binding was required for Vps41 function in epidermal growth factor receptor (EGFR) trafficking to lysosomes and its lysosomal degradation.…”