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The rare autosomal dominant Charcot-Marie-Tooth type 2B (CMT2B) is associated with mutations in the RAB7A gene, involved in the late endocytic pathway. CMT2B is characterized by predominant sensory loss, ulceromutilating features, with lesser-to-absent motor deficits. We characterized clinically and genetically a family harboring a novel pathogenic RAB7A variant and performed structural and functional analysis of the mutant protein. A 39-year-old woman presented with early-onset walking difficulties, progressive distal muscle wasting and weakness in lower limbs and only mild sensory signs. Electrophysiology demonstrated an axonal sensorimotor neuropathy. Nerve biopsy showed a chronic axonal neuropathy with moderate loss of all caliber myelinated fibers. Next-generation sequencing (NGS) technology revealed in the proband and in her similarly affected father the novel c.377A&gt, G (p.K126R) heterozygous variant predicted to be deleterious. The mutation affects the biochemical properties of RAB7 GTPase, causes altered interaction with peripherin, and inhibition of neurite outgrowth, as for previously reported CMT2B mutants. However, it also shows differences, particularly in the epidermal growth factor receptor degradation process. Altogether, our findings indicate that this RAB7A variant is pathogenic and widens the phenotypic spectrum of CMT2B to include predominantly motor CMT2. Alteration of the receptor degradation process might explain the different clinical presentations in this family.

The main organelles of the secretory and endocytic pathways--the endoplasmic reticulum (ER) and endosomes, respectively--are connected through contact sites whose numbers increase as endosomes mature. One function of such sites is to enable dephosphorylation of the cytosolic tails of endosomal signalling receptors by an ER-associated phosphatase, whereas others serve to negatively control the association of endosomes with the minus-end-directed microtubule motor dynein or mediate endosome fission. Cholesterol transfer and Ca(2+) exchange have been proposed as additional functions of such sites. However, the compositions, activities and regulations of ER-endosome contact sites remain incompletely understood. Here we show in human and rat cell lines that protrudin, an ER protein that promotes protrusion and neurite outgrowth, forms contact sites with late endosomes (LEs) via coincident detection of the small GTPase RAB7 and phosphatidylinositol 3-phosphate (PtdIns(3)P). These contact sites mediate transfer of the microtubule motor kinesin 1 from protrudin to the motor adaptor FYCO1 on LEs. Repeated LE-ER contacts promote microtubule-dependent translocation of LEs to the cell periphery and subsequent synaptotagmin-VII-dependent fusion with the plasma membrane. Such fusion induces outgrowth of protrusions and neurites, which requires the abilities of protrudin and FYCO1 to interact with LEs and kinesin 1. Thus, protrudin-containing ER-LE contact sites are platforms for kinesin-1 loading onto LEs, and kinesin-1-mediated translocation of LEs to the plasma membrane, fuelled by repeated ER contacts, promotes protrusion and neurite outgrowth.

Rab-interacting lysosomal protein (RILP) is a downstream effector of the Rab7 GTPase. GTP-bound Rab7 recruits RILP to endosomal membranes and, together, they control late endocytic traffic, phagosome and autophagosome maturation and are responsible for signaling receptor degradation. We have identified, using different approaches, the V1G1 (officially known as ATP6V1G1) subunit of the vacuolar ATPase (V-ATPase) as a RILP-interacting protein. V1G1 is a component of the peripheral stalk and is fundamental for correct V-ATPase assembly. We show here that RILP regulates the recruitment of V1G1 to late endosomal and lysosomal membranes but also controls V1G1 stability. Indeed, we demonstrate that V1G1 can be ubiquitylated and that RILP is responsible for proteasomal degradation of V1G1. Furthermore, we demonstrate that alterations in V1G1 expression levels impair V-ATPase activity. Thus, our data demonstrate for the first time that RILP regulates the activity of the V-ATPase through its interaction with V1G1. Given the importance of V-ATPase in several cellular processes and human diseases, these data suggest that modulation of RILP activity could be used to control V-ATPase function.

Rab-interacting lysosomal protein (RILP) is a downstream effector of the Rab7 GTPase. GTP-bound Rab7 recruits RILP to endosomal membranes and, together, they control late endocytic traffic, phagosome and autophagosome maturation and are responsible for signaling receptor degradation. We have identified, using different approaches, the V1G1 (officially known as ATP6V1G1) subunit of the vacuolar ATPase (V-ATPase) as a RILP-interacting protein. V1G1 is a component of the peripheral stalk and is fundamental for correct V-ATPase assembly. We show here that RILP regulates the recruitment of V1G1 to late endosomal and lysosomal membranes but also controls V1G1 stability. Indeed, we demonstrate that V1G1 can be ubiquitylated and that RILP is responsible for proteasomal degradation of V1G1. Furthermore, we demonstrate that alterations in V1G1 expression levels impair V-ATPase activity. Thus, our data demonstrate for the first time that RILP regulates the activity of the V-ATPase through its interaction with V1G1. Given the importance of V-ATPase in several cellular processes and human diseases, these data suggest that modulation of RILP activity could be used to control V-ATPase function.