Splitting the Functions of a Mitochondrial Iron/Pyrimidine Carrier

The biosynthesis of the iron-cofactors heme and iron-sulfur [Fe-S] clusters takes place in the interior of the mitochondria, but the mechanism of delivery of iron across the impermeable inner mitochondrion membrane is still unclear. In Saccharomyces cerevisiae the mitochondrial carrier proteins Mrs3 and Mrs4, are involved in iron transport, as is the human homolog mitoferrin 1, but yeast lacking these genes still have normal mitochondrial iron content, suggesting involvement of other proteins. A synthetic lethal screen identified a requirement of Rim2 in the absence of Mrs3 and Mrs4. Rim2 of S. cerevisiae, and the human homologs SLC25A33 and SLC25A36, import and export pyrimidine nucleotides in and out of mitochondria. Rim2 also delivers iron into intact mitochondria, allowing conversion of a fluorescent porphyrin precursor into non-fluorescent heme inside the organelle. Measurement of [Fe-S] assembly, using ³⁵S-cysteine, and loading into the aconitase, demonstrated that Rim2 mediates mitochondrial iron uptake for [Fe-S] cluster biogenesis.

Point mutations were generated in the predicted substrate binding site of Rim2. K299 on helix 5 and E248 on helix 4 showed asymmetry and were predicted to be substrate binding residues, and these were mutated respectively to alanine. In intact mitochondria, incorporation of radiolabeled iron (⁵⁵Fe) into heme, was significantly decreased in the Rim2-E248A mutant. Additionally protein levels of aconitase in the Rim2-E248 mutant were much lower, suggestive of a destabilization of aconitase in the absence of [Fe-S] cluster incorporation. Interestingly, TTP (pyrimidine) homoexchange was entirely preserved in this mutant. The TTP/TMP heteroexchange was compromised, perhaps because the E248 residue was required for proton coupled transport. Conversely for the Rim2-K299A mutant, all pyrimidine exchange functions were severely compromised almost to the point of the Rim2 null. By contrast, iron dependent functions, such as ⁵⁵Fe transport and incorporation into heme, were maintained at wild-type levels in the K299A mutant.

In summary the lysine at position 299 of Rim2 was critical for pyrimidine transport/exchange where as the glutamic acid at position 248 was required for iron transport/use and proton coupled TTP/TMP heteroexchange across the mitochondrial inner membrane. Iron transport by Rim2 is likely to be proton coupled and pyrimidine independent.