Spatiotemporal regulation of endocytic membrane trafficking by voltage-sensing phosphatase (VSP) in zebrafish enterocytes

In developing vertebrates, nutrient uptake by specialized epithelial cells is primarily mediated by endocytosis, a process driven by dynamic phosphoinositide remodeling that regulates vesicle formation and endosomal maturation. Voltage-sensing phosphatase (VSP), a unique membrane protein that couples changes in membrane potential to phosphoinositide hydrolysis, is expressed in zebrafish lysosome-rich enterocytes (LREs), which mediate endocytosis-dependent nutrient absorption during development. However, the molecular mechanisms by which zebrafish VSP (Dr-VSP) regulates endocytic membrane trafficking remain unclear. Here, we elucidate by confocal imaging that Dr-VSP localizes to subapical endomembranes and dynamically redistributes to the apical plasma membrane during nutrient uptake, where it promotes early vesicle formation and maintains proper endolysosomal organization. Loss of Dr-VSP reduces early endocytic vesicles and disrupts downstream recycling and lysosomal compartments, leading to defective nutrient absorption. Electrophysiological analyses showed that extracellular or luminal acidic pH suppresses Dr-VSP voltage sensing, consistent with its activity being confined to the apical plasma membrane where voltage, pH, and phosphoinositide conditions are favorable for activation. These findings indicate that Dr-VSP acts as a voltage- and pH-regulated phosphoinositide phosphatase during the early phase of endocytosis at the plasma membrane, preceding lysosomal digestion. This work defines a functional role for VSPs in epithelial nutrient uptake in vertebrate enterocytes and points to a novel electrochemical mechanism underlying membrane trafficking in vertebrates. • Voltage-sensing phosphatase (VSP) regulates epithelial endocytic trafficking. • Dr-VSP remodels phosphoinositides to support nutrient absorption in enterocytes. • Apical recruitment positions Dr-VSP at sites of early vesicle formation. • Extracellular or luminal acidic pH suppresses Dr-VSP activation, defining compartmental specificity.