Spatial Regulation of Lysosomal Vesicle Acidification Along the Axon via mRAVE-Dependent v-ATPase Assembly

Lysosomal acidification is essential for neuronal homeostasis, supporting degradative clearance and metabolic signaling in all neuronal domains. Yet, how lysosomal acidification is spatially regulated within neurons remains unclear. Here, we show that assembly of the membrane-embedded V₀ and cytosolic V₁ domains of the vacuolar H⁺-ATPase (v-ATPase) - the proton pump that drives lysosomal acidification - governs spatial and functional lysosome diversity. In non-neuronal cells, V₁-V₀ association is higher in perinuclear lysosomes, correlating with increased acidity of this population. In neurons, axonal V₀-positive vesicles move bidirectionally, whereas V₁-V₀-positive vesicles move almost exclusively in the retrograde direction, consistent with the higher acidity of retrograde lysosomal vesicles. Depletion of DMXL2, a subunit of the mRAVE complex that promotes V₁-V₀ assembly, reduces V₁ association, acidification, transport, and proteolytic activity of retrograde lysosomal vesicles in the axon. Together, these findings reveal a spatially regulated mechanism for the acidification of axonal lysosomal vesicles and identify mRAVE-dependent v-ATPase assembly as a key determinant of this process.