Unraveling the mechanism of [4Fe‐4S] cluster assembly on the N‐terminal cluster binding site of NUBP1

Bargagna, Beatrice; Matteucci, Sara; Ciofi‐Baffoni, Simone; Camponeschi, Francesca; Banci, Lucia

doi:10.1002/pro.4625

Cited by 5 publications

(6 citation statements)

References 37 publications

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“…We next focused on the proposed [2Fe-2S] cluster-trafficking function of cGrxs to the scaffold components Cfd1 and Nbp35. As noted above, such transfer has been observed in vitro from human GLRX3 to NBP35 ( 10 , 26 ). cGrx-depleted yeast cells expressing TAP-tagged Cfd1 and Nbp35 either alone or together were radiolabeled with 55 Fe.…”

Section: Resultsmentioning

confidence: 52%

“…S1 ) ( 4 , 7 – 9 ). The CIA system initiates the synthesis of a [4Fe-4S] cluster on the cytosolic scaffold complex Cfd1-Nbp35, a reaction depending on the electron transport chain NADPH-Tah18-Dre2 ( 1 , 10 ). The [4Fe-4S] cluster is then presumably passed on via Nar1 to the CIA targeting complex (CTC) composed of Cia1-Cia2-Mms19 that mediate the maturation of target [4Fe-4S] proteins like DNA maintenance enzymes ( 11 – 14 ).…”

mentioning

confidence: 99%

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Requirements for the biogenesis of [2Fe-2S] proteins in the human and yeast cytosol

Braymer,

Stehling,

Stümpfig

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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The biogenesis of iron–sulfur (Fe/S) proteins entails the synthesis and trafficking of Fe/S clusters, followed by their insertion into target apoproteins. In eukaryotes, the multiple steps of biogenesis are accomplished by complex protein machineries in both mitochondria and cytosol. The underlying biochemical pathways have been elucidated over the past decades, yet the mechanisms of cytosolic [2Fe-2S] protein assembly have remained ill-defined. Similarly, the precise site of glutathione (GSH) requirement in cytosolic and nuclear Fe/S protein biogenesis is unclear, as is the molecular role of the GSH-dependent cytosolic monothiol glutaredoxins (cGrxs). Here, we investigated these questions in human and yeast cells by various in vivo approaches. [2Fe-2S] cluster assembly of cytosolic target apoproteins required the mitochondrial ISC machinery, the mitochondrial transporter Atm1/ABCB7 and GSH, yet occurred independently of both the CIA system and cGrxs. This mechanism was strikingly different from the ISC-, Atm1/ABCB7-, GSH-, and CIA-dependent assembly of cytosolic–nuclear [4Fe-4S] proteins. One notable exception to this cytosolic [2Fe-2S] protein maturation pathway defined here was yeast Apd1 which used the CIA system via binding to the CIA targeting complex through its C-terminal tryptophan. cGrxs, although attributed as [2Fe-2S] cluster chaperones or trafficking proteins, were not essential in vivo for delivering [2Fe-2S] clusters to either CIA components or target apoproteins. Finally, the most critical GSH requirement was assigned to Atm1-dependent export, i.e. a step before GSH-dependent cGrxs function. Our findings extend the general model of eukaryotic Fe/S protein biogenesis by adding the molecular requirements for cytosolic [2Fe-2S] protein maturation.

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Section: Resultsmentioning

confidence: 52%

mentioning

confidence: 99%

Requirements for the biogenesis of [2Fe-2S] proteins in the human and yeast cytosol

Braymer,

Stehling,

Stümpfig

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…We next focused on the proposed [2Fe-2S] cluster-trafficking function of cGrxs to the scaffold components Cfd1 and Nbp35. As noted above, such transfer has been observed in vitro from human GLRX3 to NBP35 (10,26). cGrx-depleted yeast cells expressing TAP-tagged Cfd1 and Nbp35 either alone or together were radiolabeled with 55 Fe.…”

Section: Yeast Cgrxs Impact But Are Not Essential For the Cia Pathwaymentioning

confidence: 69%

“…The CIA system initiates the synthesis of a [4Fe-4S] cluster on the cytosolic scaffold complex Cfd1-Nbp35, a reaction depending on the electron transport chain NADPH-Tah18-Dre2 (1, 10). The [4Fe-4S] cluster is then presumably passed on via Nar1 to the CIA targeting complex (CTC) composed of Cia1-Cia2-Mms19 that mediate the maturation of target [4Fe-4S] proteins like DNA maintenance enzymes (11–14).…”

Section: Introductionmentioning

confidence: 99%

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Requirements for the Biogenesis of [2Fe-2S] Proteins in the Human and Yeast Cytosol

Braymer,

Stehling,

Stümpfig

et al. 2024

Preprint

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The biogenesis of iron-sulfur (Fe/S) proteins entails the synthesis and trafficking of Fe/S clusters, followed by their insertion into target apoproteins. In eukaryotes, the multiple steps of biogenesis are accomplished by complex protein machineries in both mitochondria (ISC) and cytosol (CIA). The underlying biochemical pathways have been elucidated over the past decades, yet the mechanisms of cytosolic [2Fe-2S] protein assembly have remained ill-defined. Similarly, the precise site of glutathione (GSH) requirement in cytosolic and nuclear Fe/S protein biogenesis is unclear, as is the molecular role of the GSH-dependent cytosolic monothiol glutaredoxins (cGrxs). Here, we investigated these questions in human and yeast cells by various in vivo approaches. [2Fe-2S] cluster assembly of cytosolic target apoproteins required the mitochondrial ISC machinery, the ABC transporter Atm1/ABCB7 and GSH, yet occurred independently of both the CIA system and cGrxs. This mechanism was strikingly different from the ISC-, Atm1/ABCB7-, GSH-, and CIA-dependent assembly of cytosolic-nuclear [4Fe-4S] proteins. One notable exception to this newly defined cytosolic [2Fe-2S] protein maturation pathway was the yeast protein Apd1 which used the CIA system via binding to the CIA targeting complex through its C-terminal tryptophan. cGrxs, although attributed as [2Fe-2S] cluster chaperones or trafficking proteins, were not essential in vivo for delivering [2Fe-2S] clusters to either CIA components or apoproteins. Finally, GSH function was assigned to Atm1-dependent export, i.e. a step before GSH-dependent cGrxs function. Our findings extend the general model of eukaryotic Fe/S protein biogenesis by adding the molecular requirements for cytosolic [2Fe-2S] protein maturation.

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Unraveling the mechanism of [4Fe‐4S] cluster assembly on the N‐terminal cluster binding site of NUBP1

et al. 2023

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[4Fe‐4S]2+ cluster assembly in human cytosol requires both a [2Fe‐2S] cluster chaperone being able to donate two [2Fe‐2S]2+ clusters and an electron donor providing two electrons to reductively couple the two [2Fe‐2S]2+ clusters into a [4Fe‐4S]2+ cluster. The mechanism through which the cytosolic [4Fe‐4S]2+ cluster assembly works is still not defined. Here, we show that a hetero‐tetrameric complex formed by two molecules of cluster‐reduced [2Fe‐2S]+2‐anamorsin and one molecule of dimeric cluster‐oxidized [2Fe‐2S]2+2‐GLRX32 orchestrates the assembly of a [4Fe‐4S]2+ cluster on the N‐terminal cluster binding site of the cytosolic protein NUBP1. We demonstrate that the hetero‐tetrameric complex is able to synergically provide two [2Fe‐2S]2+ clusters from GLRX3 and two electrons from anamorsin for the assembly of the [4Fe‐4S]2+ cluster on the N‐terminal cluster binding site of NUBP1. We also showed that only one of the two [2Fe‐2S] clusters bound to anamorsin, that is, that bound to the CX8CX2CXC motif, provides the electrons required to form the [4Fe‐4S]2+ cluster. Our study contributes to the molecular understanding of the mechanism of [4Fe‐4S] protein biogenesis in the cytosol.

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Unraveling the mechanism of [4Fe‐4S] cluster assembly on the N‐terminal cluster binding site of NUBP1

Cited by 5 publications

References 37 publications

Requirements for the biogenesis of [2Fe-2S] proteins in the human and yeast cytosol

Requirements for the biogenesis of [2Fe-2S] proteins in the human and yeast cytosol

Requirements for the Biogenesis of [2Fe-2S] Proteins in the Human and Yeast Cytosol

Unraveling the mechanism of [4Fe‐4S] cluster assembly on the N‐terminal cluster binding site of NUBP1

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