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

Abstract

Environmental factors such as extracellular pH (pHe) and nutritional status influence lysosomal localization and autophagy. However, the mechanisms by which pHe, intracellular pH (pHi), and Ca2+ 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 pHi and Ca2+ levels. Specifically, we demonstrate that Ca2+-activated apoptosis-linked gene 2 (ALG-2) promotes retrograde lysosomal transport while simultaneously increasing pHi and decreasing lysosomal pH (pHlys). Elevated pHi deprotonates RNF13 at His332, enabling its interaction with Ca2+-bound ALG-2. This interaction activates RNF13, which inhibits ARL8B-mediated anterograde lysosomal transport. Furthermore, we show that alkaline pHe elevates pHlys, activating the lysosomal Ca2+ channel TRPML3. This activation enhances RNF13 activity, driving lysosomes to adopt a perinuclear localization. Thus, conditions such as starvation or alkaline pHe, which induce ALG-2 activation and pHi elevation, facilitate RNF13- mediated ARL8B degradation. In contrast, under acidic pHi 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 Ca2+ release and dynamic changes in cytoplasmic and lysosomal pH.