Iron-sulfur cluster biogenesis and trafficking in mitochondria

Abstract

The biogenesis of iron-sulfur (Fe/S) proteins in eukaryotes is a multistage, multicompartment process that is essential for a broad range of cellular functions, including genome maintenance, protein translation, energy conversion, and the antiviral response. Genetic and cell biological studies over almost 2 decades have revealed some 30 proteins involved in the synthesis of cellular [2Fe-2S] and [4Fe-4S] clusters and their incorporation into numerous apoproteins. Mechanistic aspects of Fe/S protein biogenesis continue to be elucidated by biochemical and ultrastructural investigations. Here, we review recent developments in the pursuit of constructing a comprehensive model of Fe/S protein assembly in the mitochondrion.

Keywords: acyl carrier protein (ACP); chaperone; cysteine desulfurase; fatty acid metabolism; ferredoxin; frataxin; glutaredoxin; lipoic acid; metal biology; mitochondrial disease.

Figure 1.

Simplified overview of the assembly of eukaryotic Fe/S proteins and their cellular functions. The process begins in the mitochondrion with components of the early ISC machinery synthesizing a [2Fe-2S] cluster on a scaffold protein. This requires iron, cysteine, and electrons as the basic components. Following the de novo assembly reaction, the cluster is released from the scaffold and bound to a transfer protein, from where the transiently associated [2Fe-2S] cluster is trafficked to [2Fe-2S] targets or the late ISC machinery for [4Fe-4S] cluster synthesis. Additionally, an unknown sulfur-containing component, X-S, is generated (shown exported from the mitochondrion) and delivered to the CIA system for maturation of cytosolic and nuclear Fe/S proteins. Mitochondrial [4Fe-4S] clusters are finally trafficked to apoproteins by ISC targeting factors. Prominent examples of eukaryotic Fe/S proteins are listed with their corresponding cellular roles.

Figure 2.

Cartoon model of the mitochondrial Fe/S protein assembly process. A cascade of ISC proteins is required for the de novo synthesis of [2Fe-2S] and [4Fe-4S] clusters and their proper trafficking to target apoproteins in mitochondria. Initially, a [2Fe-2S] cluster is synthesized by the early ISC machinery, composed of the Isu1 scaffold protein requiring sulfide from the cysteine desulfurase complex Nfs1-Isd11-Acp1, electrons from the transfer chain NADPH-Arh1 and the ferredoxin Yah1, and the regulator and/or iron donor Yfh1. The Isu1-bound [2Fe-2S] cluster is then delivered to the monothiol glutaredoxin Grx5, a reaction accomplished by the Hsp70 chaperone Ssq1 with the help of the J-type co-chaperone Jac1. This reaction is dependent on ATP hydrolysis by Ssq1. The exchange factor Mge1 facilitates the exchange of ADP for ATP. The resulting bridging [2Fe-2S] cluster on a Grx5 dimer is inserted directly into [2Fe-2S] recipient apoproteins or trafficked to the late ISC machinery for [4Fe-4S] cluster biogenesis. The early ISC machinery, including the chaperones and Grx5, is also responsible for generating the component X-S for transport of sulfur out of the mitochondria to the CIA machinery for cytosolic-nuclear Fe/S protein biogenesis. The late ISC machinery consists of the yet structurally and functionally uncharacterized Isa1-Isa2-Iba57 complex and is needed for the generation of [4Fe-4S] clusters. Trafficking and insertion of the [4Fe-4S] clusters into target Fe/S proteins are facilitated by specific ISC targeting factors, such as Nfu1, the complex I-specific Ind1, and the Bol proteins. Dashed arrows indicate steps that remain poorly elucidated on the biochemical level.

Figure 3.

Structural insights of the early ISC machinery for [2Fe-2S] cluster synthesis. Structural and functional studies of the early ISC machinery have suggested a six-membered multimeric protein complex composed of two copies of each of the following proteins. The cysteine desulfurase Nfs1 (modeled from IscS-IscU PDB code 4EB7) (100), which binds its partner proteins Isd11 and Acp1 at an unknown location; the scaffold Isu1 (modeled from IscS-IscU PDB code 3LVL); the ferredoxin Yah1 (PDB code 2MJE), and Yfh1 (PDB code 3FQL). All three proteins are expected to bind close to the Cys loop (red) of Nfs1. Note that the second set of Yah1, Yfh1, and Isd11-Acp1 components are not shown for clarity. Paramount to the synthesis of a [2Fe-2S] cluster is the scaffold protein Isu1 containing a possible cluster-binding site of three cysteines and a histidine (lower inset). The exact coordination of the [2Fe-2S] cluster is unclear throughout the biosynthetic process (designated by X). Nfs1 delivers two persulfides (–SSH) from the PLP active site (PLP represented as spheres) via the Cys loop (lower inset, red) to the active site of Isu1. Electrons are shuttled via the ferredoxin Yah1. Interaction of reduced Yah1 with Isu1 is proposed to be facilitated by α-helix 3 (red). How 2 eq of ferrous iron gain access to the active site of Fe/S cluster synthesis remains unclear. Allosteric regulation of sulfur transfer from Nfs1 to Isu1 is conferred by Yfh1. Isd11-Acp1 stabilizes the 200-kDa complex with Nfs1, yet their precise function is unknown. The Isd11-Acp1 structure may be similar to that of an LYR protein-Acp1 dimer in respiratory complex I (upper inset). The subunit B14 from Bos taurus containing an LYR motif (red) interacts with SDAP-α (Acp1), which covalently binds a 4′-phosphopantetheine moiety via a conserved serine (orange) (PDB code 5LDW) (41, 43).

Figure 4.

ISC transfer proteins and targeting factors assisting [2Fe-2S] and [4Fe-4S] cluster trafficking. A, crystal structure of the human Grx5 homologue GLRX5 (PDB code 2WUL) as a homodimer. The different protomers are different shades of gray with the GSH carbon atoms colored in magenta (blue, nitrogen; red, oxygen; orange, iron; yellow, sulfur). B, [2Fe-2S] cluster coordination in the Grx5 homodimer involves a cysteine from each protomer and each GSH molecule, with coordination bonds shown by dotted lines. C–E, solution structures of human ISC targeting factors. C, C-terminal domain of NFU1 (PDB code 2M5O); D, BOLA1 (PDB code 5LCI); and E, BOLA3 (PDB code 2NCL). Potential Fe/S cluster coordinating residues are shown in orange as sticks (yellow, sulfur; blue, nitrogen).