RNF13 mediates pH- and Ca2+-dependent regulation of lysosomal positioning

Summary Environmental factors such as extracellular pH (pH e ) and nutritional status influence lysosomal localization and autophagy. However, the mechanisms by which pH e , intracellular pH (pH i ), and Ca 2+ levels coordinate the bidirectional transport of lysosomes remain poorly understood. In this study, we identify RNF13 as a critical regulator of lysosomal positioning through its ubiquitin- dependent degradation of ARL8B. RNF13 activity is modulated by both pH i and Ca 2+ levels. Specifically, we demonstrate that Ca 2+ -activated apoptosis-linked gene 2 (ALG-2) promotes retrograde lysosomal transport while simultaneously increasing pH i and decreasing lysosomal pH (pH lys ). Elevated pH i deprotonates RNF13 at His332, enabling its interaction with Ca 2+ -bound ALG-2. This interaction activates RNF13, which inhibits ARL8B-mediated anterograde lysosomal transport. Furthermore, we show that alkaline pH e elevates pH lys , activating the lysosomal Ca 2+ channel TRPML3. This activation enhances RNF13 activity, driving lysosomes to adopt a perinuclear localization. Thus, conditions such as starvation or alkaline pH e , which induce ALG-2 activation and pH i elevation, facilitate RNF13- mediated ARL8B degradation. In contrast, under acidic pH i conditions, RNF13 activity remains suppressed regardless of ALG-2 activation, leading to increased ARL8B levels. Additionally, we provide evidence linking the loss of RNF13 activity to developmental and epileptic encephalopathy-73, a neurological disorder characterized by severe developmental symptoms. These findings deepen our understanding of lysosomal positioning mechanisms, highlighting the interplay between lysosomal Ca 2+ release and dynamic changes in cytoplasmic and lysosomal pH.