The protein ferritin consists of two kinds of subunits, H and L, that assemble to a 24-subunit heteropolymer complex that can store up to 4500 iron atoms. Mammalian ferritin is an actively secreted protein even though it lacks a signal peptide sequence for classical ER-Golgi secretion. However, little is known about the mechanisms underlying ferritin secretion and reuptake in mammals. This research focused on deciphering the mechanisms controlling ferritin secretion and its regulation. We used cell line models of wild-type and knockout HeLa cells for both ferritin subunits (HeLaFtKO cells) and transfected them with different ferritin constructs. We investigated the role of a 13 amino-acid long motif in ferritin secretion. This motif was identified on both ferritin subunits, which lack the classical signal peptide sequence. Additionally, we examined the role of iron in intracellular distribution and secretion of ferritin. Our preliminary results showed that the ferritin motif led to enhance ferritin secretion. However, as the research on the ferritin motif continued, we realized that the mutations on the ferritin motif inhibit its internalization via TfR1. Hence, the elevation of motif-mutated ferritin in the medium was due to impaired ferritin uptake rather than increased secretion. When we compared the secretion of mutated and wild-type ferritin, while blocking the reabsorption of the wild-type ferritin via TfR1, we saw that the H-subunit motif does not play a role in the regulated secretion of ferritin. In contrast, our results show that the ferritin-iron core is an important factor in the regulation of ferritin secretion, whereas the cytosolic iron levels do not affect the secretion of ferritin. Our results also demonstrate that the secretion and uptake of cellular ferritin is an active cycle, regulated by iron at the level of secretion and the level of uptake. In summary, these results enhance our understanding on the mechanism controlling ferritin secretion, which is an important piece in the puzzle of tissue iron homeostasis.
