Organelle-centered ISG15 biology: distinguishing covalent ISGylation from interferon-associated responses

ISGylation is an interferon-inducible ubiquitin-like post-translational modification mediated by the interferon-stimulated gene 15 conjugation system. Initially characterized as an antiviral effector pathway, ISGylation is now increasingly recognized as a regulator of organelle homeostasis and cellular stress responses. This review summarizes emerging evidence linking ISG15-related mechanisms and covalent ISGylation to major organelle systems, including mitochondria, the endoplasmic reticulum–Golgi axis, endolysosomal compartments, ribosome-associated translation, and lipid droplets. We distinguish covalent ISGylation from free ISG15 signaling and ubiquitin specific protease 18-mediated interferon regulation, and further classify existing findings into evidence-based levels ranging from substrate-validated modification to correlative interferon signatures. At the mitochondrial level, direct and pathway-level evidence implicates ISG15 biology in DRP1-mediated fission, MFN1/2-associated mitophagy, oxidative metabolism, and redox regulation; direct effects of MFN1/2 ISGylation on mitochondrial fusion remain unproven. Along the ER–Golgi axis, ISG15-related pathways intersect with unfolded protein response signaling, endoplasmic reticulum-associated degradation, and stimulator of interferon genes-mediated innate immune activation. In the endolysosomal system, ISGylation and ISG15-associated pathways modulate autophagic flux, multivesicular body fate, and exosome secretion in a context-dependent manner. Ribosome-associated co-translational ISGylation links nascent protein surveillance with antiviral defense, whereas lipid droplet-associated ISG15/ISGylation pathways are linked to lipid metabolism and immune signaling. Current evidence supports an organelle-centered view of ISG15 biology but indicates that validated covalent mechanisms remain confined to selected substrates and contexts. Clinical translation will require organelle-resolved ISGylome mapping, substrate-level validation, and standardized biomarker assays before context-selective targeting can be considered.