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Chloroplast HCF101 is a scaffold protein for [4Fe-4S] cluster assembly.

Schwenkert S, Netz DJ, Frazzon J, Pierik AJ, Bill E, Gross J, Lill R, Meurer J - Biochem. J. (2009)

Bottom Line: Recently, the chloroplast protein HCF101 (high chlorophyll fluorescence 101) has been shown to be essential for the accumulation of the membrane complex Photosystem I and the soluble ferredoxin-thioredoxin reductases, both containing [4Fe-4S] clusters.We further demonstrate that the reconstituted cluster is transiently bound and can be transferred from HCF101 to a [4Fe-4S] apoprotein.Together, our findings suggest that HCF101 may serve as a chloroplast scaffold protein that specifically assembles [4Fe-4S] clusters and transfers them to the chloroplast membrane and soluble target proteins.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum der Ludwig-Maximilians-Universität München, Department Biologie I, Lehrstuhl für Botanik, Grosshaderner Strasse 2-4, 82152 Planegg-Martinsried, Germany.

ABSTRACT
Oxygen-evolving chloroplasts possess their own iron-sulfur cluster assembly proteins including members of the SUF (sulfur mobilization) and the NFU family. Recently, the chloroplast protein HCF101 (high chlorophyll fluorescence 101) has been shown to be essential for the accumulation of the membrane complex Photosystem I and the soluble ferredoxin-thioredoxin reductases, both containing [4Fe-4S] clusters. The protein belongs to the FSC-NTPase ([4Fe-4S]-cluster-containing P-loop NTPase) superfamily, several members of which play a crucial role in Fe/S cluster biosynthesis. Although the C-terminal ISC-binding site, conserved in other members of the FSC-NTPase family, is not present in chloroplast HCF101 homologues using Mössbauer and EPR spectroscopy, we provide evidence that HCF101 binds a [4Fe-4S] cluster. 55Fe incorporation studies of mitochondrially targeted HCF101 in Saccharomyces cerevisiae confirmed the assembly of an Fe/S cluster in HCF101 in an Nfs1-dependent manner. Site-directed mutagenesis identified three HCF101-specific cysteine residues required for assembly and/or stability of the cluster. We further demonstrate that the reconstituted cluster is transiently bound and can be transferred from HCF101 to a [4Fe-4S] apoprotein. Together, our findings suggest that HCF101 may serve as a chloroplast scaffold protein that specifically assembles [4Fe-4S] clusters and transfers them to the chloroplast membrane and soluble target proteins.

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Holo-HCF101-mediated transfer of the [4Fe-4S] cluster to the apo-Leu1 target proteinReconstituted and desalted holo-HCF101 (3.85 μM) was mixed with reduced apo-Leu1 (2.45 μM) at 23 °C in buffer A. Isopropylmalate isomerase activity was measured at the indicated time points (●). In a subsequent control experiment, activation of 2.45 μM apo-Leu1 with ferric ammonium citrate and Li2S at concentrations identical with those present in HCF101 (15.4 μM each) was assayed for isopropylmalate isomerase activity (▲).
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Figure 5: Holo-HCF101-mediated transfer of the [4Fe-4S] cluster to the apo-Leu1 target proteinReconstituted and desalted holo-HCF101 (3.85 μM) was mixed with reduced apo-Leu1 (2.45 μM) at 23 °C in buffer A. Isopropylmalate isomerase activity was measured at the indicated time points (●). In a subsequent control experiment, activation of 2.45 μM apo-Leu1 with ferric ammonium citrate and Li2S at concentrations identical with those present in HCF101 (15.4 μM each) was assayed for isopropylmalate isomerase activity (▲).

Mentions: A common feature of scaffold proteins is their capability to transfer the bound labile Fe/S cluster to the apo-form of acceptor proteins [3,10,42]. We tested the putative scaffold function of HCF101 in vitro. It is at present not feasible to use apo-PSI complexes of higher plants to test the transfer of Fe/S cluster from HCF101. We therefore chose yeast apo-Leu1 as a model acceptor protein. Upon binding of a [4Fe-4S] cluster Leu1 exhibits isopropylmalate isomerase activity [32]. Apo-HCF101 was chemically reconstituted to its holoform (typically containing 3.6±1.2 Fe and 3.0±0.8 S per monomer) and mixed with apo-Leu1. Upon incubation of the two proteins under anaerobic conditions isopropylmalate isomerase enzyme activity developed within 2 min, with more than 90% conversion of apo-Leu1 into the active enzyme (Figure 5). In contrast, upon chemical reconstitution of apo-Leu1 with similar amounts of iron and sulfide instead of a transferable Fe/S cluster, a comparatively slow and inefficient generation of the Leu1 enzyme activity was observed. Even after 30 min only approximately one-third of the activity was detected compared with using HCF101 as a donor after 2 min. These results show that the HCF101-bound Fe/S cluster is labile and can be efficiently transferred to acceptor proteins. Hence, HCF101 fulfils a critical requirement for a scaffold function, i.e. the capacity to transfer a labile Fe/S cluster to a target protein under conditions when chemical reconstitution is inefficient.


Chloroplast HCF101 is a scaffold protein for [4Fe-4S] cluster assembly.

Schwenkert S, Netz DJ, Frazzon J, Pierik AJ, Bill E, Gross J, Lill R, Meurer J - Biochem. J. (2009)

Holo-HCF101-mediated transfer of the [4Fe-4S] cluster to the apo-Leu1 target proteinReconstituted and desalted holo-HCF101 (3.85 μM) was mixed with reduced apo-Leu1 (2.45 μM) at 23 °C in buffer A. Isopropylmalate isomerase activity was measured at the indicated time points (●). In a subsequent control experiment, activation of 2.45 μM apo-Leu1 with ferric ammonium citrate and Li2S at concentrations identical with those present in HCF101 (15.4 μM each) was assayed for isopropylmalate isomerase activity (▲).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2805927&req=5

Figure 5: Holo-HCF101-mediated transfer of the [4Fe-4S] cluster to the apo-Leu1 target proteinReconstituted and desalted holo-HCF101 (3.85 μM) was mixed with reduced apo-Leu1 (2.45 μM) at 23 °C in buffer A. Isopropylmalate isomerase activity was measured at the indicated time points (●). In a subsequent control experiment, activation of 2.45 μM apo-Leu1 with ferric ammonium citrate and Li2S at concentrations identical with those present in HCF101 (15.4 μM each) was assayed for isopropylmalate isomerase activity (▲).
Mentions: A common feature of scaffold proteins is their capability to transfer the bound labile Fe/S cluster to the apo-form of acceptor proteins [3,10,42]. We tested the putative scaffold function of HCF101 in vitro. It is at present not feasible to use apo-PSI complexes of higher plants to test the transfer of Fe/S cluster from HCF101. We therefore chose yeast apo-Leu1 as a model acceptor protein. Upon binding of a [4Fe-4S] cluster Leu1 exhibits isopropylmalate isomerase activity [32]. Apo-HCF101 was chemically reconstituted to its holoform (typically containing 3.6±1.2 Fe and 3.0±0.8 S per monomer) and mixed with apo-Leu1. Upon incubation of the two proteins under anaerobic conditions isopropylmalate isomerase enzyme activity developed within 2 min, with more than 90% conversion of apo-Leu1 into the active enzyme (Figure 5). In contrast, upon chemical reconstitution of apo-Leu1 with similar amounts of iron and sulfide instead of a transferable Fe/S cluster, a comparatively slow and inefficient generation of the Leu1 enzyme activity was observed. Even after 30 min only approximately one-third of the activity was detected compared with using HCF101 as a donor after 2 min. These results show that the HCF101-bound Fe/S cluster is labile and can be efficiently transferred to acceptor proteins. Hence, HCF101 fulfils a critical requirement for a scaffold function, i.e. the capacity to transfer a labile Fe/S cluster to a target protein under conditions when chemical reconstitution is inefficient.

Bottom Line: Recently, the chloroplast protein HCF101 (high chlorophyll fluorescence 101) has been shown to be essential for the accumulation of the membrane complex Photosystem I and the soluble ferredoxin-thioredoxin reductases, both containing [4Fe-4S] clusters.We further demonstrate that the reconstituted cluster is transiently bound and can be transferred from HCF101 to a [4Fe-4S] apoprotein.Together, our findings suggest that HCF101 may serve as a chloroplast scaffold protein that specifically assembles [4Fe-4S] clusters and transfers them to the chloroplast membrane and soluble target proteins.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum der Ludwig-Maximilians-Universität München, Department Biologie I, Lehrstuhl für Botanik, Grosshaderner Strasse 2-4, 82152 Planegg-Martinsried, Germany.

ABSTRACT
Oxygen-evolving chloroplasts possess their own iron-sulfur cluster assembly proteins including members of the SUF (sulfur mobilization) and the NFU family. Recently, the chloroplast protein HCF101 (high chlorophyll fluorescence 101) has been shown to be essential for the accumulation of the membrane complex Photosystem I and the soluble ferredoxin-thioredoxin reductases, both containing [4Fe-4S] clusters. The protein belongs to the FSC-NTPase ([4Fe-4S]-cluster-containing P-loop NTPase) superfamily, several members of which play a crucial role in Fe/S cluster biosynthesis. Although the C-terminal ISC-binding site, conserved in other members of the FSC-NTPase family, is not present in chloroplast HCF101 homologues using Mössbauer and EPR spectroscopy, we provide evidence that HCF101 binds a [4Fe-4S] cluster. 55Fe incorporation studies of mitochondrially targeted HCF101 in Saccharomyces cerevisiae confirmed the assembly of an Fe/S cluster in HCF101 in an Nfs1-dependent manner. Site-directed mutagenesis identified three HCF101-specific cysteine residues required for assembly and/or stability of the cluster. We further demonstrate that the reconstituted cluster is transiently bound and can be transferred from HCF101 to a [4Fe-4S] apoprotein. Together, our findings suggest that HCF101 may serve as a chloroplast scaffold protein that specifically assembles [4Fe-4S] clusters and transfers them to the chloroplast membrane and soluble target proteins.

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