| dc.description.abstract | Precise regulation of nutrient availability is crucial for cellular function and survival. Iron, in particular, is tightly regulated as it serves as an essential cofactor for numerous enzymes but can catalyze the formation of toxic radicals at elevated levels. To maintain the necessary cytoplasmic iron concentration, cells store excess iron in large proteinaceous cages called ferritin and, when available iron levels fall, they degrade these cages, liberating the stored iron for use. This thesis focuses on the molecular mechanisms underlying cellular iron sensing, as well as the molecular interactions supporting regulated ferritin degradation and subsequent iron release. Specifically, this work interrogates the protein interactions involved in ferritinophagy, a form of selective autophagy that leads to the lysosomal degradation of ferritin. Extending prior work that identified key components supporting ferritinophagy, including the selective autophagy receptor protein NCOA4 and its cognate autophagosomal receptor GATE16, experiments described here uncover the molecular contacts between these proteins. I found that NCOA4 bears two short linear motifs that each bind to GATE16 with weak affinity. However, these binding motifs are highly avid and, in concert, support high-affinity binding of NCOA4 to oligomerized GATE16. I further describe that ferritin degradation in cultured human cells relies on the contacts I identified biochemically. Moreover, I found that iron decreases NCOA4’s affinity for GATE16, providing a plausible mechanism for irondependent regulation of ferritinophagy. Taken together, this work suggests a general mechanism by which selective autophagy receptors can distinguish between inactive monomeric GATE16 and the active oligomerized forms that primarily drive autophagy. In related studies, I have biochemically probed the NCOA4•ferritin interface, with these experiments suggesting a novel function of NCOA4 in modulating ferritin cage structure – either through cage dismantling or through the formation of higher order structures. Taken together, these studies further define the molecular mechanisms by which NCOA4 aids cells in maintaining iron homeostasis, and they provide the requisite reagents for future work aimed at building a unified model for how mammalian cells regulate this vital but toxic metal. | |
