The Asp1 pyrophosphatase from S. pombe hosts a [2Fe-2S]2+ cluster in vivo

Rosenbach, Hannah; Walla, Eva; Cutsail, George E.; Birrell, James A.; Pascual-Ortiz, Marina; DeBeer, Serena; Fleig, Ursula; Span, Ingrid

doi:10.1007/s00775-020-01840-w

Cited by 5 publications

(3 citation statements)

References 50 publications

(81 reference statements)

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“…These strategies are not limited to proteins containing one [4Fe-4S] cluster. We have previously shown that the same strategies can be applied for the maturation of a [2Fe-2S] protein [83]. In addition, we have demonstrated that this approach can be also used for radical SAM enzymes.…”

Section: Discussionmentioning

confidence: 90%

Maturation strategy influences expression levels and cofactor occupancy in Fe–S proteins

et al. 2022

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Iron–sulfur clusters are ubiquitous cofactors required for fundamental biological processes. Structural and spectroscopic analysis of Fe–S proteins is often limited by low cluster occupancy in recombinantly produced proteins. In this work, we report a systematic comparison of different maturation strategies for three well-established [4Fe–4S] proteins. Aconitase B, HMBPP reductase (IspH), and quinolinate synthase (NadA) were used as model proteins as they have previously been characterized. The protein production strategies include expression of the gene of interest in BL21(DE3) cells, maturation of the apo protein using chemical or semi-enzymatic reconstitution, co-expression with two different plasmids containing the iron–sulfur cluster (isc) or sulfur formation (suf) operon, a cell strain lacking IscR, the transcriptional regulator of the ISC machinery, and an engineered “SufFeScient” derivative of BL21(DE3). Our results show that co-expression of a Fe–S biogenesis pathway influences the protein yield and the cluster content of the proteins. The presence of the Fe–S cluster is contributing to correct folding and structural stability of the proteins. In vivo maturation reduces the formation of Fe–S aggregates, which occur frequently when performing chemical reconstitution. Furthermore, we show that the in vivo strategies can be extended to the radical SAM protein ThnB, which was previously only maturated by chemical reconstitution. Our results shed light on the differences of in vitro and in vivo Fe–S cluster maturation and points out the pitfalls of chemical reconstitution. Graphical abstract

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Section: Discussionmentioning

confidence: 90%

Maturation strategy influences expression levels and cofactor occupancy in Fe–S proteins

et al. 2022

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“…Relatively few cytosolic [2Fe-2S] proteins per species are known to date (Fig. S1) (4,19,(29)(30)(31)(32)(33). In addition to true target proteins, also the cGrxs and the early CIA components Dre2, Cfd1, and Nbp35 have been reported to (transiently) bind [2Fe-2S] clusters in vitro (and in cellulo for Dre2) suggesting that also these proteins are acceptors of binuclear clusters (26,(34)(35)(36)(37).…”

Section: Introductionmentioning

confidence: 99%

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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“…S1 ). Relatively few cytosolic [2Fe-2S] proteins per species are known to date ( 4 , 19 , 29 – 33 ). In addition to true target proteins, also the cGrxs and the early CIA components Dre2, Cfd1, and Nbp35 have been reported to (transiently) bind [2Fe-2S] clusters in vitro (and in cellulo for Dre2) suggesting that also these proteins are acceptors of binuclear clusters ( 26 , 34 – 37 ).…”

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

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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The Asp1 pyrophosphatase from S. pombe hosts a [2Fe-2S]2+ cluster in vivo

Cited by 5 publications

References 50 publications

Maturation strategy influences expression levels and cofactor occupancy in Fe–S proteins

Maturation strategy influences expression levels and cofactor occupancy in Fe–S proteins

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

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