A lysosomal targeting defect of V0a1 suppresses V‐ATPase activity elevating lysosomal pH in Ppt1‐/‐ mice: amelioration by NtBuHA

Abstract

Lysosomal pH regulation is impaired in virtually all lysosomal storage disorders (LSDs). However, the mechanism of this defect remains unexplained. The vacuolar H+‐transporting ATPase (v‐ATPase), a multi‐subunit protein‐complex, regulates endosomal/lysosomal pH. The reversible assembly of its membrane‐bound V0 sector and cytosolic V1 sector regulates acidification of the lysosomes. Infantile Neuronal Ceroid Lipofuscinoses (INCL) is a neurodegenerative LSD caused by mutations in the CLN1 gene encoding palmitoyl‐protein thioesterase‐1(PPT1), a depalmitoylating enzyme. Dynamic palmitoylation (palmitoylation‐depalmitoylation) is critical for regulating the functions of many proteins, which requires palmitoyl‐acyltransferases (PATs) for palmitoylation and palmitoyl‐protein thioesterases (PPTs) for depalmitoylation.Using Ppt1‐/‐ mice that mimic INCL, we uncovered that subunit a1 of V0‐ATPase (V0a1) requires palmitoylation on Cys‐25 for localization to endosomal/lysosomal membrane. V0a1 is transported via clathrin/AP2 pathway from plasma membrane to early endosomes where PPT1‐deficiency suppressed depalmitoylation impairing clathrin/AP2/V0a1 dissociation. This defect prevented interaction of V0a1 with AP3, essential for trafficking to lysosomes and clathrin/AP2/V0a1 was recycled back to the plasma membrane. Consequently, disruption of V0/V1 assembly on lysosomal membrane suppressed V‐ATPase activity, essential for pH regulation. Importantly, a PPT1‐mimetic, NtBuHA, ameliorated these defects. Our findings uncover a novel mechanism underlying increased lysosomal pH in INCL and suggest that varying factors impairing V‐ATPase activity may disrupt lysosomal pH regulation in other LSDs.