Assembly of mitochondrial iron-sulfur (Fe/S) proteins is a key process of cells, and defects cause many rare diseases. In the first phase of this pathway, ten Fe/S cluster (ISC) assembly components synthesize and insert [2Fe-2S] clusters. The second phase is dedicated to the assembly of [4Fe-4S] proteins, yet this part is poorly understood. Here, we characterize the BOLA family proteins Bol1 and Bol3 as specific mitochondrial ISC assembly factors that facilitate [4Fe-4S] cluster insertion into a subset of mitochondrial proteins such as lipoate synthase and succinate dehydrogenase. Bol1-Bol3 perform largely overlapping functions, yet cannot replace the ISC protein Nfu1 that also participates in this phase of Fe/S protein biogenesis. Bol1 and Bol3 form dimeric complexes with both monothiol glutaredoxin Grx5 and Nfu1. Complex formation differentially influences the stability of the Grx5-Bol-shared Fe/S clusters. Our findings provide the biochemical basis for explaining the pathological phenotypes of patients with mutations in BOLA3.DOI: http://dx.doi.org/10.7554/eLife.16673.001

The biogenesis of iron-sulfur (Fe-S) proteins in humans is a multistage process occurring in different cellular compartments. The mitochondrial iron-sulfur cluster (ISC) assembly machinery composed of at least 17 proteins assembles mitochondrial Fe-S proteins. A cytosolic iron-sulfur assembly (CIA) machinery composed of at least 13 proteins has been more recently identified and shown to be responsible for the Fe-S cluster incorporation into cytosolic and nuclear Fe-S proteins. Cytosolic and nuclear Fe-S protein maturation requires not only the CIA machinery, but also the components of the mitochondrial ISC assembly machinery. An ISC export machinery, composed of a protein transporter located in the mitochondrial inner membrane, has been proposed to act in mediating the export process of a still unknown component that is required for the CIA machinery. Several functional and molecular aspects of the protein networks operative in the three machineries are still largely obscure. This Review focuses on the Fe-S protein maturation processes in humans with the specific aim of providing a molecular picture of the currently known protein-protein interaction networks. The human ISC and CIA machineries are presented, and the ISC export machinery is discussed with respect to possible molecules being the substrates of the mitochondrial protein transporter.

Members of the monothiol glutaredoxin family and members of the BolA-like protein family have recently emerged as specific interacting partners involved in iron-sulfur protein maturation and redox regulation pathways. It is known that human mitochondrial BOLA1 and BOLA3 form [2Fe-2S] cluster-bridged dimeric heterocomplexes with the monothiol glutaredoxin GRX5. The structure and cluster coordination of the two [2Fe-2S] heterocomplexes as well as their molecular function are, however, not defined yet. Experimentally-driven structural models of the two [2Fe-2S] cluster-bridged dimeric heterocomplexes, the relative stability of the two complexes and the redox properties of the [2Fe-2S] cluster bound to these complexes are here presented on the basis of UV/vis, CD, EPR and NMR spectroscopies and computational protein-protein docking. While the BOLA1-GRX5 complex coordinates a reduced, Rieske-type [2Fe-2S] cluster, an oxidized, ferredoxin-like [2Fe-2S] cluster is present in the BOLA3-GRX5 complex. The [2Fe-2S] BOLA1-GRX5 complex is preferentially formed over the [2Fe-2S] BOLA3-GRX5 complex, as a result of a higher cluster binding affinity. All these observed differences provide the first indications discriminating the molecular function of the two [2Fe-2S] heterocomplexes.