The yeast mitochondrial carrier proteins Mrs3p/Mrs4p mediate iron transport across the inner mitochondrial membrane

The yeast proteins Mrs3p and Mrs4p are two closely related members of the mitochondrial carrier family (MCF), which had previously been implicated in mitochondrial Fe²⁺ homeostasis. A vertebrate Mrs3/4 homologue named mitoferrin was shown to be essential for erythroid iron utilization and proposed to function as an essential mitochondrial iron importer. Indirect reporter assays in isolated yeast mitochondria indicated that the Mrs3/4 proteins are involved in mitochondrial Fe²⁺ utilization or transport under iron-limiting conditions. To have a more direct test for Mrs3/4p mediated iron uptake into mitochondria we studied iron (II) transport across yeast inner mitochondrial membrane vesicles (SMPs) using the iron-sensitive fluorophore PhenGreen SK (PGSK). Wild-type SMPs showed rapid uptake of Fe²⁺ which was driven by the external Fe²⁺ concentration and stimulated by acidic pH. SMPs from the double deletion strain mrs3/4Δ failed to show this rapid Fe²⁺ uptake, while SMPs from cells overproducing Mrs3/4p exhibited increased Fe²⁺ uptake rates. Cu²⁺ was transported at similar rates as Fe²⁺, while other divalent cations, such as Zn²⁺ and Cd²⁺ apparently did not serve as substrates for the Mrs3/4p transporters. We conclude that the carrier proteins Mrs3p and Mrs4p transport Fe²⁺ across the inner mitochondrial membrane. Their activity is dependent on the pH gradient and it is stimulated by iron shortage.