Examining whether Multicopper Oxidase 4 (Mco4) acts as a high affinity iron importer in Drosophila melanogaster

Abstract

Iron is a crucial micronutrient for nearly all living organisms, playing a vital role in various biological processes. In humans, it is essential for the production of hemoglobin, which is necessary for oxygen transport in red blood cells. Beyond oxygen transport, iron is a key component of heme and iron-sulfur clusters, which are involved in numerous cellular functions, including DNA repair and hormone synthesis. The model organism Drosophila melanogaster has proven to be a valuable tool for studying iron metabolism due to its genetic similarities to humans and its high iron demand for steroid hormone synthesis. Multicopper oxidase 4 (Mco4) was first discovered as a gene upregulated in response to genetic or dietary disruption of iron homeostasis. Mco4 is expressed on the cell membrane and has a yeast ortholog, Fet3p, which acts as a component of a high-affinity iron import system. This thesis investigates whether Mco4 also plays a role in high-affinity iron import in Drosophila, which would represent the first such system identified in animals. In this study, I performed the phenotypic analysis of an Mco4 knockout line, which showed that loss of Mco4 function causes sensitivity to iron starvation. Specifically, multigenerational iron depletion showed that Mco4 null mutant flies exhibit decreased survival rates when reared on iron-deprived food due to lower body iron content compared to wild-type flies. I also constructed a Mco4-3xFlag knock-in line to analyze the expression pattern of Mco4. Immunofluorescence experiments revealed that Mco4 is predominantly expressed in the proventriculus. In other tissues, primarily the gut, Mco4 is only detectable under low dietary iron level, indicating a role during iron starvation. To further understand its function, I explored the effects of Mco4 overexpression and found that whole-body overexpression of Mco4 leads to increased iron content in the gut and strongly enhanced resistance to iron deprivation. Remarkably, Mco4 overexpression allows animals to survive extreme iron deprivation, which is not seen in controls. Additionally, Mco4 overexpression in S2 cells demonstrated ferroxidase activity comparable to that of human Hephaestin, a known ferroxidase. Using the TurboID proximity labeling technique, I identified interactions between Mco4 and ferritin heavy and light chains. Together, these findings indicate that Mco4 plays a critical role in the cellular response to iron deprivation and likely functions as a high-affinity iron importer, thus facilitating the import of iron when it is scarce.