A proteomic atlas of organelle remodeling identifies lysosomal SNX3 as a regulator of Notch signaling in epidermal differentiation

Differentiation of epidermal keratinocytes is accompanied by profound reorganization of intracellular architecture, but how organelle remodeling interfaces with cell fate control is not well understood. Here, we generate a compartment-resolved proteomic map of keratinocyte differentiation and identify extensive remodeling of lysosomes, mitochondria, autophagic vesicles, plasma membrane, and nucleus. Differentiating keratinocytes display coordinated enrichment of lysosomal degradative machinery, vesicular trafficking factors, and mitochondrial metabolic proteins, revealing organelle remodeling as a prominent feature of epidermal differentiation. From the lysosomal proteome, we identify Sorting Nexin 3 (SNX3) as a critical regulator of epidermal homeostasis. SNX3 increasingly localizes to LAMP1-positive vesicles during differentiation, and its loss impairs epidermal differentiation, suppresses Notch signaling, and promotes proliferative gene expression. In vivo, SNX3-deficient skin grafts fail to maintain normal epidermal architecture and instead develop into squamous cell carcinoma. Mechanistically, SNX3 mediates efficient Notch receptor activation, as SNX3 loss reduces nuclear Notch1 and NICD production, whereas NICD re-expression can rescue the differentiation defect. Our study defines a proteomic framework for organelle remodeling during epidermal differentiation and identifies lysosome-associated SNX3 as a key link between endolysosomal trafficking, Notch signaling, and epidermal tissue homeostasis.