Recombinant Mouse Cytosolic Fe-S cluster assembly factor NARFL (Narfl)

Introduction to Recombinant Mouse Cytosolic Fe-S Cluster Assembly Factor NARFL (Narfl)

Recombinant Mouse Cytosolic Fe-S cluster assembly factor NARFL (Narfl), also known as cytosolic iron-sulfur assembly component 3 (CIAO3), plays a crucial role in the cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway . This pathway is essential for delivering Fe-S clusters to various nuclear and cytosolic Fe-S proteins that are involved in critical cellular functions . Fe-S clusters are cofactors composed of iron and inorganic sulfur, which are generally attached to cysteine residues in proteins and facilitate numerous reactions .

Function and Importance of Fe-S Clusters

Fe-S clusters are integral components in various biological processes, facilitating a wide range of reactions within proteins . These clusters are most commonly found in a cubane form, containing four iron and four inorganic sulfur atoms . Their chemical versatility arises from the ability of iron and sulfur to readily donate or accept multiple electrons, enabling fine-tuning of electron affinity across a broad electrochemical range by the surrounding protein residues .

Fe-S clusters perform several critical functions:

• Electron Transfer: They facilitate electron movement, such as in mitochondrial complex I, where seven Fe-S clusters with increasing reduction potentials form a wire-like pathway for electron transfer .

• Substrate Binding: They directly facilitate chemical reactions by binding to an Fe-S protein’s substrate, as seen in the enzyme aconitase .

• Sensors: They function as sensors, exemplified by bacterial FNR and IscR proteins, and mammalian IRP1, which regulates cytosolic iron metabolism .

The Cytosolic Iron-Sulfur Assembly (CIA) Pathway

The CIA pathway is essential for the maturation of cytosolic Fe-S proteins, a tightly regulated multistep process . In human cells, bioavailable iron is delivered by poly(rC)-binding protein 1 (PCBP1) to a chaperone consisting of BolA-like protein 2 (BOLA2) and glutaredoxin-3 (GLRX3) for [2Fe-2S] cluster biogenesis . The [4Fe-4S] clusters are first assembled on the CIA scaffold complex, composed of nucleotide-binding protein 1 (NUBP1) and nucleotide-binding protein 2 (NUBP2) . This step requires a sulfur-containing compound produced by the mitochondrial Fe-S cluster biogenesis (ISC) machinery and transported to the cytosol via the mitochondrial inner membrane protein ABCB7 .

NARFL/CIAO3 in Fe-S Cluster Assembly

Cytosolic iron-sulfur assembly component 3 (CIAO3/NARFL) is crucial for bridging early and late steps in the CIA pathway . NARFL contains two Fe-S cluster binding sites, one at the N terminus and the other at the C terminus . The interaction of CIAO3 may be regulated by its cluster incorporation status, given the sensitivity of Fe-S clusters to the cellular environment, which can affect the stability and function of Fe-S proteins .

NARFL Interactions and Complex Formation

NARFL interacts with several components of the CIA machinery, including the CIA scaffold complex (NUBP1/NUBP2) and CIA substrates . These interactions suggest the potential assembly of a higher-order protein complex that facilitates Fe-S cluster transfer into substrates .

Key observations regarding NARFL interactions:

• CIA Scaffold Complex: NARFL associates with NUBP1 and NUBP2, components of the CIA scaffold complex .

• CIA Targeting Complex: NARFL also associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B), indicating the existence of a higher-order complex .

• CIA Substrates: NARFL interacts with CIA substrates like ABCE1 and CDKAL1, and this association is strongly enhanced when the C-terminal Fe-S cluster binding site of CIAO3 is mutated .

• ERCC2 Interaction: NARFL binding by ERCC2 requires the CIA targeting complex binding region of ERCC2, supporting the model that the CIA scaffold complex, CIAO3, the CIA targeting complex, and CIA substrates form higher-order complexes facilitating Fe-S protein maturation .

Regulation of NARFL Interactions by Fe-S Cluster Incorporation

The incorporation of Fe-S clusters into CIAO3 controls its interactions and incorporation into the CIA metabolon . Studies involving mutations at cysteine residues (C71S, C190S/C395S, and C71S/C190S/C395S) that impair cluster incorporation reveal that the association of substrates like ABCE1 and CDKAL1 with CIAO3 increases when the CIAO3 C-terminal Fe-S cluster binding site is mutated . This suggests that CIAO3 can associate with CIA substrates independently of the CIA targeting complex .

NARFL and Cellular Defense Against Oxidative Stress

NARFL is a crucial element in the cellular defense against oxidative stress . The precise mechanisms through which NARFL provides this protection are still being explored, but it is clear that its role in Fe-S cluster assembly is vital for maintaining cellular health under oxidative stress conditions .