Pathways of Protein Secretion in Prokaryotes and Eukaryotes: Molecular Mechanisms, Biological Functions, and Therapeutic Opportunities

Protein secretion is a fundamental biological process essential for cellular communication, matrix remodeling, and overall homeostasis across all domains of life. While the classical endoplasmic reticulum (ER)‐to‐Golgi pathway has long been recognized as the primary route for eukaryotic protein export, decades of research reveal that numerous cytosolic and membrane proteins bypass this canonical route. This alternative paradigm, termed unconventional protein secretion (UcPS), encompasses direct transmembrane translocation, vesicle‐mediated release, and Golgi‐bypass mechanisms. Simultaneously, prokaryotes utilize highly specialized secretion machineries to deliver effector proteins. Despite these established frameworks, the precise molecular regulation, cargo sorting mechanisms, and dynamic crosstalk between these diverse pathways remain incompletely understood. Here, we comprehensively review the molecular mechanisms of UcPS and prokaryotic secretion systems, synthesizing their evolutionary adaptations and operational frameworks. By mapping nonvesicular pores, extracellular vesicles intermediates, and complex bacterial nanomachine assemblies, we delineate how cells rapidly mobilize proteins under stress. Furthermore, we highlight the dual role of these pathways in driving physiological adaptation versus fueling pathological dissemination. Abnormalities in these secretory nodes are now recognized as primary drivers of neurodegeneration, inflammatory disorders, and cancer metastasis. Consequently, manipulating UcPS mechanisms offers promising, multidimensional therapeutic opportunities for future medicine.