Lipid-anchored melanotransferrin mediates transferrin-independent iron uptake and ferritin storage in mammals.

Non-transferrin-bound iron (NTBI) transport constitutes a critical pathway for cellular iron uptake in the kingdom Animalia that remains mechanistically unresolved. Its physiological importance is underscored by atransferrinemia, a rare disorder in which individuals lacking plasma transferrin nonetheless retain the capacity to distribute dietary iron to essential organs, implying the presence of compensatory iron transport routes. Melanotransferrin (MFI2; also designated p97 or CD228) is an evolutionarily conserved iron-binding protein that exists in both a secreted form and a glycosylphosphatidylinositol (GPI)-anchored membrane-bound form, suggesting a fundamental role in iron homeostasis. In mammals, the secreted isoform mediates iron transport across the blood-brain barrier, whereas GPI-anchored MFI2 is expressed by microglia in proximity to β-amyloid plaques in Alzheimer's disease, implicating it in neuroinflammatory processes. Moreover, it is also recognized as a tumor-associated antigen in melanoma, indicating a potential role in tumor progression. In the present study, we delineate a previously uncharacterized NTBI internalization pathway mediated by GPI-MFI2. Using human melanoma cells, we demonstrate that GPI-MFI2, together with its bound iron, undergoes caveolae-dependent internalization followed by trafficking through a Rab5-mediated endosomal pathway. The internalized iron is subsequently trafficked to ferritin, underscoring its functional importance in maintaining intracellular iron stores. These findings establish the first molecularly defined pathway for transferrin-independent iron uptake in mammalian cells, providing a framework to interrogate MFI2's role in iron mobilization and dysregulation in neurodegeneration and cancer.