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Mitochondrial localization of Arabidopsis thaliana Isu Fe–S scaffold proteins
FEBS Letters 579 (2005) 1930–1934
FEBS 29382
Mitochondrial localization of Arabidopsis thaliana Isu Fe–S scaffold proteins Se´bastien Le´on1, Brigitte Touraine, Jean-Franc¸ois Briat, Ste´phane Lobre´aux* Biochimie et Physiologie Mole´culaire des Plantes, Centre National de la Recherche Scientifique (Unite´ Mixte de Recherche 5004), Universite´ Montpellier-II, Institut National de la Recherche Agronomique et Ecole Nationale Supe´rieure dÕAgronomie, 2, place Viala, F-34060 Montpellier cedex 1, France Received 16 December 2004; revised 8 February 2005; accepted 8 February 2005 Available online 24 February 2005 Edited by Hans Eklund
Abstract Isu are scaffold proteins involved in iron–sulfur cluster biogenesis and playing a key role in yeast mitochondria and Escherichia coli. In this work, we have characterized the Arabidopsis thaliana Isu gene family. AtIsu1,2,3 genes encode polypeptides closely related to their bacterial and eukaryotic counterparts. AtIsu expression in a Saccharomyces cerevisiae Disu1Dnfu1 thermosensitive mutant led to the growth restoration of this strain at 37 C. Using Isu-GFP fusions expressed in leaf protoplasts and immunodetection in organelle extracts, we have shown that Arabidopsis Isu proteins are located only into mitochondria, supporting the existence of an Isu-independent Fe–S assembly machinery in plant plastids. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Plant; Iron–sulfur cluster; Mitochondria; Plastid
1. Introduction Isu proteins are scaffold proteins accepting sulfur and iron to build a transient Fe–S cluster, which is subsequently transferred to a target apoprotein [1]. Escherichia coli IscU is encoded by the isc operon together with other proteins like IscS, HscA and HscB [2]. IscU interacts with the IscS cysteine desulfurase [3], and a covalent complex is formed via disulfide bond, enabling sulfur transfer from IscS to IscU [4]. HscA and B are chaperones that are specifically devoted to Fe–S biogenesis. A binding of IscU to HscA has been reported, leading to the stimulation of HscA ATPase activity [5]. Saccharomyces cerevisiae Isu1p and Isu2p are homologues of bacterial IscU and are involved in a similar process [6]. Yeast Isu are essential proteins that are exclusively located into mitochondria [7,8]. This organelle is the site for Fe–S biogenesis in this eukaryotic cell [6]. Yeast cytosolic Fe–S protein maturation requires the export of some compounds from the mitochondria, and mitochondrial Isu proteins have been shown to be essential for this process [9]. Isu are therefore key components for Fe–S biogenesis in both E. coli and yeast. In plants, data support the exis* Corresponding author. Fax: +33 4 67 52 57 37. E-mail address: [email protected] (S. Lobre´aux). 1 Present address: Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 920930322, USA.
Abbreviations: Fe–S, iron–sulfur; GFP, green fluorescent protein; RTPCR, reverse-transcription PCR; Nt, NH2 terminal
tence of two distinct Fe–S assembly machineries: one in plastids and one in mitochondria [10–12]. In a recent paper, Tone et al. [13] described the characterization of Arabidopsis AtIsu1 which is targeted to mitochondria when expressed in yeast. In this paper, we have characterized the Arabidopsis AtIsu gene family, showing that the three AtIsu polypeptides are functional and targeted to mitochondria in planta.
2. Materials and methods 2.1. Plant materials Arabidopsis thaliana (ecotype Columbia) plants were grown as previously described [14]. Tobacco plants (Nicotiana tabacum cv petit havana SR1) were grown in vitro according to Vansuyt et al. [15]. 2.2. RT-PCR and cDNA cloning RNA samples described in [14] were used and RT-PCR was performed as described [14]. cDNAs were amplified using the following primers: 5 0 -TCCGTCTAGAAAAAGTTACTTCCGAAACCC-3 0 and 5 0 -TCCGGGTACCGGTTGTATGTATCATCCTCTTC-3 0 for Isu1, 5 0 -TCCGTCTAGACAAGCTCCATAGAGAGAAGCG-3 0 and 5 0 TCCGGGTACCTCTGTTTGGGTCATAAAACATC-3 0 for Isu2, 5 0 -TCCGTCTAGACTGCCCTTGATTCCGGCAAAGAG-3 0 and 5 0 for Isu3 TCCGGGTACCCTTGGGATCTGGGTCACATGC-3 0 (restriction sites are indicated as bold type). Digested PCR products were subcloned into the pYPGE15 vector at XbaI–KpnI sites [16] and sequenced. 2.3. Recombinant Isu1 expression in E. coli A fragment of Isu1 cDNA was amplified using primers 5 0 -GCTGACATATGCGAACCTACCATGAGAACGTC-3 0 and 5 0 -GCTAGCTCGAGAGCCTGTGTGGTTTCTCCTGC-3 0 , and subcloned into pET20 plasmid at NdeI–XhoI sites in frame with the His-Tag sequence. Plasmid constructs were introduced into E. coli BL21 strain and crude extract was prepared from a 3 h culture in LB medium at 37 C. Histagged recombinant Isu1 protein was purified using Ni-NTA agarose (Qiagen) according to the supplier instructions. Polyclonal antibodies were raised in rabbits against the recombinant protein (Elevage Scientifique des Dombes, France). 2.4. Immunodetection of Isu proteins Protein samples used have been described in [11]. Proteins were separated by SDS–PAGE and transferred to Hybond-P membrane (Amersham Biosciences, UK) according to [17]. Aurora kit (ICN) was used for chemiluminescent detection according to the manufacturer instructions.
2.5. Yeast complementation AtIsu cDNAs were subcloned into the pFL38 centromeric vector. Saccharomyces cerevisiae Disu1Dnfu1 strain was obtained previously [11]. Yeast were grown in yeast nitrogen base medium (2% glucose,
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.02.038
S. Le´on et al. / FEBS Letters 579 (2005) 1930–1934
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3. Results
conserved amino acids, three cysteines are strictly conserved in all Isu sequences (Fig. 1). These residues have been shown to be essential for the function of yeast Isu1p in vivo, as demonstrated by functional complementation of a yeast Dsod1Disu1 strain [7]. It has been established that IscU interact with HscA-like chaperones through an LPPVK motif [20]. This sequence is strictly conserved in AtIsu proteins suggesting a similar protein–protein interaction in plants. The existence of HscA homologous genes in Arabidopsis genome, whose product are predicted to be targeted to mitochondria, support such a hypothesis. Comparison of eukaryotic Isu and their bacterial counterparts revealed the presence of an Nt extension (Fig. 1). This additional sequence is known to be involved into the mitochondrial targeting of human IscU2 [21] and yeast Isu proteins [7,8]. Arabidopsis Isu primary sequences also include such a peptide, which could therefore play a role into the intracellular targeting of these proteins.
3.1. Identification of Arabidopsis Isu proteins Using E. coli IscU as a starting sequence, a search for genes encoding homologous proteins into the Arabidopsis genome database at TAIR website (http://arabidopsis.org) was performed. Three genes encoding putative proteins presenting significant sequence identity with EcIscU were identified (At4g22220, At3g01020, At4g04080). The corresponding cDNAs were cloned by an RT-PCR approach, enabling to correct inaccurate annotation of At3g01020 gene. AtIsu1,2,3 cDNAs correspond respectively to At4g22220, At3g01020, At4g04080 genes. Alignment of AtIsu primary sequences with Isu protein sequences from bacteria, human and yeast revealed a very high conservation of these polypeptides during evolution. AtIsu primary sequences share an average of 65% sequence identity with EcIscU, whereas Arabidopsis Isu1,2,3 share from 69% to 79% identity between them. Among the
3.2. Functional complementation of yeast Disu1Dnfu1 mutant by AtIsu Primary sequence analysis of AtIsu strongly support that these highly conserved proteins are functional members of the Isu Fe–S scaffold family of proteins. To investigate such a function in vivo, a yeast complementation assay was performed using a strain affected in Isu1 gene. Disu1Dnfu1 mutant strain was first described by Schilke et al. [8] who demonstrated its thermosensitive growth defect, while no phenotype was observed for single Isu1 or Nfu1 mutants. We have previously used Disu1Dnfu1 strain for the functional characterization of AtNfu proteins [11]. The thermosensitivity of the mutant strain was confirmed by its growth defect at 37 C when transformed with a pFL38 empty vector (Fig. 2). On the opposite, expression of AtIsu1,2 or 3 into the Disu1Dnfu1 yeast strain resulted in a growth restoration of the mutant at
0.17% YNB and 0.5% ammonium sulfate) containing the required amino acids. Disu1Dnfu1 strain was grown at 30 or 37 C as indicated. Yeast transformation was performed by the lithium acetate method [18]. 2.6. Subcellular localization of Isu::GFP fusion proteins A fragment of Isu cDNAs encoding the Nt region of the proteins (nt 1–171 for Isu1, nt 1–164 for Isu2, nt 1–167 for Isu3) was amplified using the upstream primers containing XbaI sites described above and the following primers: 5 0 -TCCGGGATCCAAGACCCAACGTTGCGGGGA-3 0 for Isu1, 5 0 -TCCGGGATCCATGATCCAACGTTACGGGG-3 0 for Isu2, 5 0 -TCCGGGATCCAGGAGCCAACGTTTCTAGGG-3 0 for Isu3. PCR fragments were digested and subcloned into XbaI–BamHI sites of pBi-GF vector [14], in frame with the GFP coding sequence. Tobacco leaf protoplasts were prepared from in vitro cultures according to Lukaszewicz et al. [19]. Protoplasts were transformed and observed using a confocal microscope as previously described [11].
Fig. 1. Comparison of Arabidopsis, yeast, human and bacterial Isu primary sequences. AtIsu1,2,3 protein sequences were aligned with Homo sapiens (Hs) Iscu1 and 2 (Accession Number: AAG37427 and AAG37428), Saccharomyces cerevisiae (Sc) Isu1 and 2 (NP_015190 and NP_014869), E. coli (Ec) IscU (BAB36818.1) and Azotobacter vinelandii(Av) IscU (T44282). Sequences were aligned using Clustal W [37]. Amino acid residues conserved in most sequences are boxed in black, and those which are partially conserved (identical or similar) are boxed in grey. Asterisks denote cysteine residues proposed to be involved in iron–sulfur cluster binding. The binding motif for HscA-like chaperones is overlined.
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S. Le´on et al. / FEBS Letters 579 (2005) 1930–1934
Fig. 3. Non-quantitative RT-PCR detection of Isu1,2,3 mRNA in various Arabidopsis tissues. Total RNA was extracted from flowers (Fl), Siliques (Si), floral stalk (St), leaves (L) and roots (R) of 35-dayold Arabidopsis plants. Specific primers were used to amplify each cDNA. Actin2 mRNA was detected as a control.
Fig. 2. Functional complementation of S. cerevisiae Disu1Dnfu1 mutant by AtIsu proteins. AtIsu1,2,3 cDNA were expressed in the yeast thermosensitive mutant. Strains were grown overnight in YNB medium and 5-fold serial dilutions were spotted on YNB-agar mediums. PFL38 line corresponds to the empty vector. Plates were incubated at the control permissive temperature 30 or 37 C for 72 h.
37 C (Fig. 2). Such an experiment argues in favour of the ability of the AtIsu proteins to act as Fe–S scaffold in yeast mitochondria. 3.3. AtIsu mRNA expression in planta AtIsu gene expression was investigated using non-quantitative RT-PCR in different organs of 35-day-old plants. Isu1
transcript appeared to be expressed in all tissues tested (Fig. 3), like the actin control, in agreement with data recently published by Tone et al. [13]. On the contrary, Isu2 and 3 mRNA showed distinct and specific patterns of expression. Both mRNA were detected in leaves and flowers RNA samples. Isu2 mRNA expression pattern was restricted to these two organs, whereas Isu3 was also present in roots. 3.4. Intracellular localization of Arabidopsis Isu proteins The presence of a putative targeting peptide within AtIsu protein sequences led us to investigate their intracellular localization. Yeast Isu proteins are located into mitochondria, the unique site for Fe–S biogenesis in this organism. However in plant cells, both plastids and mitochondria would be involved in Fe–S biogenesis. Tong et al. [21] have investigated the
Fig. 4. Subcellular localization of Isu-GFP fusion proteins. Constructs encoding the AtIsu Nt region fused to the GFP were transiently expressed in tobacco leaf protoplasts for 24 h. Transformed protoplasts were observed using a confocal laser scanning microscope (MRC1024, Bio-Rad) and a 60· Plan-Apo oil immersion objective. Light microscopy images of the cell, chlorophyll red auto-fluorescence used as a chloroplast marker and green fluorescent signals are shown. Cells were labeled with Mitotracker Orange as a mitochondrial fluorescent marker. Scale bar corresponds to 10 lm.
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4. Discussion
Fig. 5. Immunodetection of Isu proteins in Arabidopsis mitochondrial extract. 20 lg of protein samples were separated by SDS–PAGE and transferred to Hybond-P membrane. Protein extracts correspond to leaf crude extract (L), chloroplasts (Chl) and mitochondria enriched fraction (Mito). Chemiluminescent detection was performed using a-Isu1 antibody, a-RRF as chloroplast marker, and a-NAD9 as mitochondrial marker.
expression of human IscU gene. This single gene produces two distinct mRNA by alternative splicing, differing only in their 5 0 region. Such a divergence lead to the expression of Iscu2 protein addressed to the mitochondria, and IscU1 remaining in the cytosol. We have performed 5 0 RACE experiments using mRNA extracted from the different tissues mentioned in Fig. 3, and we were able to detect only one mRNA species for each AtIsu (data not shown). Such data rule out the possibility of a differential targeting of Arabidopsis Isu by a similar mechanism as the one occurring in human. To determine the intracellular localization of AtIsu proteins, fusions were prepared between 5 0 part of the Isu cDNA, containing the putative targeting sequence, and the GFP coding region. The chimeric proteins were transiently expressed in tobacco leaf protoplasts under the control of the 35S promoter. GFP protein from the control vector was detected into the cytosol and the nucleus, whereas Isu::GFP fusions appeared to be targeted to an intracellular compartment (Fig. 4). Using Mitotracker as a fluorescent marker for mitochondria, AtIsu were located into mitochondria. No signal was detected in chloroplasts, visualized using the chlorophyll autofluorescence. To further confirm the intracellular localization of AtIsu polypeptides, recombinant Isu1 protein, deleted of its targeting sequence, was produced in E. coli and polyclonal antibodies were raised against this polypeptide. According to the very high conservation of Arabidopsis Isu primary sequences, we have chosen to produce antibodies against only one of the three AtIsu, assuming it will recognize the two other proteins. Fractionation of leaf material was performed to produce mitochondria and chloroplast enriched fractions. Purity of the fractions was assessed using antibodies raised against chloroplast ribosome recycling factor (RRF) or subunit 9 of mitochondrial NADH dehydrogenase (Fig. 5). Immunodetection of Isu proteins in these samples revealed a signal only into the mitochondrial fraction (Fig. 5), confirming therefore the results obtained using the GFP, and the absence of the Isu Fe–S scaffold protein in the chloroplasts.
In Saccharomyces cerevisiae, mitochondria represent the unique site for Fe–S biogenesis [6,22]. In this compartment, Isu are essential scaffold proteins and interact specifically with components of the Fe–S assembly machinery. It has been demonstrated in yeast that Isu proteins are the sulfide acceptor forming a transient complex with a Nfs1 cysteine desulfurase [3,4,23], a similar mechanism to the one described in E. coli. This Isu-Nfs1 complex is able to bind the frataxin (Yfh1p), which represents a potential iron donor for the Fe–S biogenesis process [24]. Furthermore, site specific binding of Isu and HscB-like chaperones have been demonstrated and shown to be required for Fe–S assembly in yeast mitochondria [25]. In Arabidopsis, the three Isu appeared to be located in mitochondria, as shown by Isu-GFP fusion localization and immunodetection (Figs. 4 and 5). The cysteine desulfurase AtNfs1 is the Arabidopsis homolog of yeast Nfs1p and has been characterized as a mitochondrial protein [12,26]. Recently, an Arabidopsis frataxin gene was identified, the encoded protein bearing a mitochondrial targeting sequence [27]. Several other genes encoding components of the yeast Fe–S assembly machinery have homologs in the Arabidopsis genome [22]. Such data are therefore in favour of a conserved Fe–S biogenesis process in plant mitochondria, in which Isu would act as an Fe–S scaffold protein. In order to gain further insight into AtIsu function into plant mitochondria, a biochemical characterization of these proteins will be required. In addition, other putative Fe–S scaffolds are present into this organelle. AtNfu4,5 are mitochondrial proteins. No function has been attributed so far to their yeast counterpart Nfu1p, whereas the plastid Nfu2 protein has been shown to act as an Fe–S scaffold [11,28]. IscA-like proteins are encoded by Arabidopsis genome and some are predicted to be located into mitochondria. An Fe–S scaffold function has been shown for IscA like proteins [29–31]. Therefore, the precise role of IscU, Nfu and IscA proteins into plant mitochondria will have to be defined. In plants, an additional organelle require Fe–S clusters for essential processes among which the photosynthesis: the plastid. Recent studies have reported the characterization of Arabidopsis genes encoding proteins involved in the plastid Fe–S assembly machinery [14,28,32,33] or affecting plastid 4Fe–4S protein maturation [34,35]. The absence of Isu proteins in plastids supports the hypothesis of an Isu-independent pathway for plastid Fe–S biogenesis. Indeed, plastid AtNfu2 protein has been shown to act in vitro as a scaffold protein, and its function in vivo has been investigated using T-DNA insertion Arabidopsis mutants interrupted in Nfu2 gene, revealing a role for this polypeptide in 4Fe–4S and ferredoxin Fe–S biogenesis into chloroplast [28,33]. Plastidial Nfu proteins share high sequence identity with Synechocystis NifU, which is supposed to act also as Fe–S scaffold protein in this cyanobacteria [36]. It has to be noticed that Synechocystis genome does not encode an Isu like protein. According to the endosymbiotic theory for the origin of mitochondria and plastids, this cyanobacteria represent the ancestor of plastids whereas mitochondria ancestor would be an aerobic bacteria. It appears therefore that an Isu-dependent process from the endosymbiotic bacteria would have been maintained into mitochondria, whereas plastids have conserved the Isu-independent mechanism derived from the cyanobacterial ancestor. These original Fe–S biogenesis processes have further evolved in the corresponding organelles
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as evidence by the proteins involved in yeast mitochondria Fe– S metabolism and not present in E. coli for example [22] or the plastid Nfu proteins having unique features when compared to their cyanobacterial counterpart [11]. Acknowledgements: We are grateful to Ge´rard Vansuyt for his help with tobacco culture. We are indebted to Dr. Geoffrey Duby for advices in tobacco protoplast preparation. Confocal microscopy was performed at the Centre Re´gional dÕImagerie Cellulaire (Montpellier, France) with the assistance of N. Lautre´dou.
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FEBS 29382
Mitochondrial localization of Arabidopsis thaliana Isu Fe–S scaffold proteins Se´bastien Le´on1, Brigitte Touraine, Jean-Franc¸ois Briat, Ste´phane Lobre´aux* Biochimie et Physiologie Mole´culaire des Plantes, Centre National de la Recherche Scientifique (Unite´ Mixte de Recherche 5004), Universite´ Montpellier-II, Institut National de la Recherche Agronomique et Ecole Nationale Supe´rieure dÕAgronomie, 2, place Viala, F-34060 Montpellier cedex 1, France Received 16 December 2004; revised 8 February 2005; accepted 8 February 2005 Available online 24 February 2005 Edited by Hans Eklund
Abstract Isu are scaffold proteins involved in iron–sulfur cluster biogenesis and playing a key role in yeast mitochondria and Escherichia coli. In this work, we have characterized the Arabidopsis thaliana Isu gene family. AtIsu1,2,3 genes encode polypeptides closely related to their bacterial and eukaryotic counterparts. AtIsu expression in a Saccharomyces cerevisiae Disu1Dnfu1 thermosensitive mutant led to the growth restoration of this strain at 37 C. Using Isu-GFP fusions expressed in leaf protoplasts and immunodetection in organelle extracts, we have shown that Arabidopsis Isu proteins are located only into mitochondria, supporting the existence of an Isu-independent Fe–S assembly machinery in plant plastids. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Plant; Iron–sulfur cluster; Mitochondria; Plastid
1. Introduction Isu proteins are scaffold proteins accepting sulfur and iron to build a transient Fe–S cluster, which is subsequently transferred to a target apoprotein [1]. Escherichia coli IscU is encoded by the isc operon together with other proteins like IscS, HscA and HscB [2]. IscU interacts with the IscS cysteine desulfurase [3], and a covalent complex is formed via disulfide bond, enabling sulfur transfer from IscS to IscU [4]. HscA and B are chaperones that are specifically devoted to Fe–S biogenesis. A binding of IscU to HscA has been reported, leading to the stimulation of HscA ATPase activity [5]. Saccharomyces cerevisiae Isu1p and Isu2p are homologues of bacterial IscU and are involved in a similar process [6]. Yeast Isu are essential proteins that are exclusively located into mitochondria [7,8]. This organelle is the site for Fe–S biogenesis in this eukaryotic cell [6]. Yeast cytosolic Fe–S protein maturation requires the export of some compounds from the mitochondria, and mitochondrial Isu proteins have been shown to be essential for this process [9]. Isu are therefore key components for Fe–S biogenesis in both E. coli and yeast. In plants, data support the exis* Corresponding author. Fax: +33 4 67 52 57 37. E-mail address: [email protected] (S. Lobre´aux). 1 Present address: Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 920930322, USA.
Abbreviations: Fe–S, iron–sulfur; GFP, green fluorescent protein; RTPCR, reverse-transcription PCR; Nt, NH2 terminal
tence of two distinct Fe–S assembly machineries: one in plastids and one in mitochondria [10–12]. In a recent paper, Tone et al. [13] described the characterization of Arabidopsis AtIsu1 which is targeted to mitochondria when expressed in yeast. In this paper, we have characterized the Arabidopsis AtIsu gene family, showing that the three AtIsu polypeptides are functional and targeted to mitochondria in planta.
2. Materials and methods 2.1. Plant materials Arabidopsis thaliana (ecotype Columbia) plants were grown as previously described [14]. Tobacco plants (Nicotiana tabacum cv petit havana SR1) were grown in vitro according to Vansuyt et al. [15]. 2.2. RT-PCR and cDNA cloning RNA samples described in [14] were used and RT-PCR was performed as described [14]. cDNAs were amplified using the following primers: 5 0 -TCCGTCTAGAAAAAGTTACTTCCGAAACCC-3 0 and 5 0 -TCCGGGTACCGGTTGTATGTATCATCCTCTTC-3 0 for Isu1, 5 0 -TCCGTCTAGACAAGCTCCATAGAGAGAAGCG-3 0 and 5 0 TCCGGGTACCTCTGTTTGGGTCATAAAACATC-3 0 for Isu2, 5 0 -TCCGTCTAGACTGCCCTTGATTCCGGCAAAGAG-3 0 and 5 0 for Isu3 TCCGGGTACCCTTGGGATCTGGGTCACATGC-3 0 (restriction sites are indicated as bold type). Digested PCR products were subcloned into the pYPGE15 vector at XbaI–KpnI sites [16] and sequenced. 2.3. Recombinant Isu1 expression in E. coli A fragment of Isu1 cDNA was amplified using primers 5 0 -GCTGACATATGCGAACCTACCATGAGAACGTC-3 0 and 5 0 -GCTAGCTCGAGAGCCTGTGTGGTTTCTCCTGC-3 0 , and subcloned into pET20 plasmid at NdeI–XhoI sites in frame with the His-Tag sequence. Plasmid constructs were introduced into E. coli BL21 strain and crude extract was prepared from a 3 h culture in LB medium at 37 C. Histagged recombinant Isu1 protein was purified using Ni-NTA agarose (Qiagen) according to the supplier instructions. Polyclonal antibodies were raised in rabbits against the recombinant protein (Elevage Scientifique des Dombes, France). 2.4. Immunodetection of Isu proteins Protein samples used have been described in [11]. Proteins were separated by SDS–PAGE and transferred to Hybond-P membrane (Amersham Biosciences, UK) according to [17]. Aurora kit (ICN) was used for chemiluminescent detection according to the manufacturer instructions.
2.5. Yeast complementation AtIsu cDNAs were subcloned into the pFL38 centromeric vector. Saccharomyces cerevisiae Disu1Dnfu1 strain was obtained previously [11]. Yeast were grown in yeast nitrogen base medium (2% glucose,
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.02.038
S. Le´on et al. / FEBS Letters 579 (2005) 1930–1934
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3. Results
conserved amino acids, three cysteines are strictly conserved in all Isu sequences (Fig. 1). These residues have been shown to be essential for the function of yeast Isu1p in vivo, as demonstrated by functional complementation of a yeast Dsod1Disu1 strain [7]. It has been established that IscU interact with HscA-like chaperones through an LPPVK motif [20]. This sequence is strictly conserved in AtIsu proteins suggesting a similar protein–protein interaction in plants. The existence of HscA homologous genes in Arabidopsis genome, whose product are predicted to be targeted to mitochondria, support such a hypothesis. Comparison of eukaryotic Isu and their bacterial counterparts revealed the presence of an Nt extension (Fig. 1). This additional sequence is known to be involved into the mitochondrial targeting of human IscU2 [21] and yeast Isu proteins [7,8]. Arabidopsis Isu primary sequences also include such a peptide, which could therefore play a role into the intracellular targeting of these proteins.
3.1. Identification of Arabidopsis Isu proteins Using E. coli IscU as a starting sequence, a search for genes encoding homologous proteins into the Arabidopsis genome database at TAIR website (http://arabidopsis.org) was performed. Three genes encoding putative proteins presenting significant sequence identity with EcIscU were identified (At4g22220, At3g01020, At4g04080). The corresponding cDNAs were cloned by an RT-PCR approach, enabling to correct inaccurate annotation of At3g01020 gene. AtIsu1,2,3 cDNAs correspond respectively to At4g22220, At3g01020, At4g04080 genes. Alignment of AtIsu primary sequences with Isu protein sequences from bacteria, human and yeast revealed a very high conservation of these polypeptides during evolution. AtIsu primary sequences share an average of 65% sequence identity with EcIscU, whereas Arabidopsis Isu1,2,3 share from 69% to 79% identity between them. Among the
3.2. Functional complementation of yeast Disu1Dnfu1 mutant by AtIsu Primary sequence analysis of AtIsu strongly support that these highly conserved proteins are functional members of the Isu Fe–S scaffold family of proteins. To investigate such a function in vivo, a yeast complementation assay was performed using a strain affected in Isu1 gene. Disu1Dnfu1 mutant strain was first described by Schilke et al. [8] who demonstrated its thermosensitive growth defect, while no phenotype was observed for single Isu1 or Nfu1 mutants. We have previously used Disu1Dnfu1 strain for the functional characterization of AtNfu proteins [11]. The thermosensitivity of the mutant strain was confirmed by its growth defect at 37 C when transformed with a pFL38 empty vector (Fig. 2). On the opposite, expression of AtIsu1,2 or 3 into the Disu1Dnfu1 yeast strain resulted in a growth restoration of the mutant at
0.17% YNB and 0.5% ammonium sulfate) containing the required amino acids. Disu1Dnfu1 strain was grown at 30 or 37 C as indicated. Yeast transformation was performed by the lithium acetate method [18]. 2.6. Subcellular localization of Isu::GFP fusion proteins A fragment of Isu cDNAs encoding the Nt region of the proteins (nt 1–171 for Isu1, nt 1–164 for Isu2, nt 1–167 for Isu3) was amplified using the upstream primers containing XbaI sites described above and the following primers: 5 0 -TCCGGGATCCAAGACCCAACGTTGCGGGGA-3 0 for Isu1, 5 0 -TCCGGGATCCATGATCCAACGTTACGGGG-3 0 for Isu2, 5 0 -TCCGGGATCCAGGAGCCAACGTTTCTAGGG-3 0 for Isu3. PCR fragments were digested and subcloned into XbaI–BamHI sites of pBi-GF vector [14], in frame with the GFP coding sequence. Tobacco leaf protoplasts were prepared from in vitro cultures according to Lukaszewicz et al. [19]. Protoplasts were transformed and observed using a confocal microscope as previously described [11].
Fig. 1. Comparison of Arabidopsis, yeast, human and bacterial Isu primary sequences. AtIsu1,2,3 protein sequences were aligned with Homo sapiens (Hs) Iscu1 and 2 (Accession Number: AAG37427 and AAG37428), Saccharomyces cerevisiae (Sc) Isu1 and 2 (NP_015190 and NP_014869), E. coli (Ec) IscU (BAB36818.1) and Azotobacter vinelandii(Av) IscU (T44282). Sequences were aligned using Clustal W [37]. Amino acid residues conserved in most sequences are boxed in black, and those which are partially conserved (identical or similar) are boxed in grey. Asterisks denote cysteine residues proposed to be involved in iron–sulfur cluster binding. The binding motif for HscA-like chaperones is overlined.
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Fig. 3. Non-quantitative RT-PCR detection of Isu1,2,3 mRNA in various Arabidopsis tissues. Total RNA was extracted from flowers (Fl), Siliques (Si), floral stalk (St), leaves (L) and roots (R) of 35-dayold Arabidopsis plants. Specific primers were used to amplify each cDNA. Actin2 mRNA was detected as a control.
Fig. 2. Functional complementation of S. cerevisiae Disu1Dnfu1 mutant by AtIsu proteins. AtIsu1,2,3 cDNA were expressed in the yeast thermosensitive mutant. Strains were grown overnight in YNB medium and 5-fold serial dilutions were spotted on YNB-agar mediums. PFL38 line corresponds to the empty vector. Plates were incubated at the control permissive temperature 30 or 37 C for 72 h.
37 C (Fig. 2). Such an experiment argues in favour of the ability of the AtIsu proteins to act as Fe–S scaffold in yeast mitochondria. 3.3. AtIsu mRNA expression in planta AtIsu gene expression was investigated using non-quantitative RT-PCR in different organs of 35-day-old plants. Isu1
transcript appeared to be expressed in all tissues tested (Fig. 3), like the actin control, in agreement with data recently published by Tone et al. [13]. On the contrary, Isu2 and 3 mRNA showed distinct and specific patterns of expression. Both mRNA were detected in leaves and flowers RNA samples. Isu2 mRNA expression pattern was restricted to these two organs, whereas Isu3 was also present in roots. 3.4. Intracellular localization of Arabidopsis Isu proteins The presence of a putative targeting peptide within AtIsu protein sequences led us to investigate their intracellular localization. Yeast Isu proteins are located into mitochondria, the unique site for Fe–S biogenesis in this organism. However in plant cells, both plastids and mitochondria would be involved in Fe–S biogenesis. Tong et al. [21] have investigated the
Fig. 4. Subcellular localization of Isu-GFP fusion proteins. Constructs encoding the AtIsu Nt region fused to the GFP were transiently expressed in tobacco leaf protoplasts for 24 h. Transformed protoplasts were observed using a confocal laser scanning microscope (MRC1024, Bio-Rad) and a 60· Plan-Apo oil immersion objective. Light microscopy images of the cell, chlorophyll red auto-fluorescence used as a chloroplast marker and green fluorescent signals are shown. Cells were labeled with Mitotracker Orange as a mitochondrial fluorescent marker. Scale bar corresponds to 10 lm.
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4. Discussion
Fig. 5. Immunodetection of Isu proteins in Arabidopsis mitochondrial extract. 20 lg of protein samples were separated by SDS–PAGE and transferred to Hybond-P membrane. Protein extracts correspond to leaf crude extract (L), chloroplasts (Chl) and mitochondria enriched fraction (Mito). Chemiluminescent detection was performed using a-Isu1 antibody, a-RRF as chloroplast marker, and a-NAD9 as mitochondrial marker.
expression of human IscU gene. This single gene produces two distinct mRNA by alternative splicing, differing only in their 5 0 region. Such a divergence lead to the expression of Iscu2 protein addressed to the mitochondria, and IscU1 remaining in the cytosol. We have performed 5 0 RACE experiments using mRNA extracted from the different tissues mentioned in Fig. 3, and we were able to detect only one mRNA species for each AtIsu (data not shown). Such data rule out the possibility of a differential targeting of Arabidopsis Isu by a similar mechanism as the one occurring in human. To determine the intracellular localization of AtIsu proteins, fusions were prepared between 5 0 part of the Isu cDNA, containing the putative targeting sequence, and the GFP coding region. The chimeric proteins were transiently expressed in tobacco leaf protoplasts under the control of the 35S promoter. GFP protein from the control vector was detected into the cytosol and the nucleus, whereas Isu::GFP fusions appeared to be targeted to an intracellular compartment (Fig. 4). Using Mitotracker as a fluorescent marker for mitochondria, AtIsu were located into mitochondria. No signal was detected in chloroplasts, visualized using the chlorophyll autofluorescence. To further confirm the intracellular localization of AtIsu polypeptides, recombinant Isu1 protein, deleted of its targeting sequence, was produced in E. coli and polyclonal antibodies were raised against this polypeptide. According to the very high conservation of Arabidopsis Isu primary sequences, we have chosen to produce antibodies against only one of the three AtIsu, assuming it will recognize the two other proteins. Fractionation of leaf material was performed to produce mitochondria and chloroplast enriched fractions. Purity of the fractions was assessed using antibodies raised against chloroplast ribosome recycling factor (RRF) or subunit 9 of mitochondrial NADH dehydrogenase (Fig. 5). Immunodetection of Isu proteins in these samples revealed a signal only into the mitochondrial fraction (Fig. 5), confirming therefore the results obtained using the GFP, and the absence of the Isu Fe–S scaffold protein in the chloroplasts.
In Saccharomyces cerevisiae, mitochondria represent the unique site for Fe–S biogenesis [6,22]. In this compartment, Isu are essential scaffold proteins and interact specifically with components of the Fe–S assembly machinery. It has been demonstrated in yeast that Isu proteins are the sulfide acceptor forming a transient complex with a Nfs1 cysteine desulfurase [3,4,23], a similar mechanism to the one described in E. coli. This Isu-Nfs1 complex is able to bind the frataxin (Yfh1p), which represents a potential iron donor for the Fe–S biogenesis process [24]. Furthermore, site specific binding of Isu and HscB-like chaperones have been demonstrated and shown to be required for Fe–S assembly in yeast mitochondria [25]. In Arabidopsis, the three Isu appeared to be located in mitochondria, as shown by Isu-GFP fusion localization and immunodetection (Figs. 4 and 5). The cysteine desulfurase AtNfs1 is the Arabidopsis homolog of yeast Nfs1p and has been characterized as a mitochondrial protein [12,26]. Recently, an Arabidopsis frataxin gene was identified, the encoded protein bearing a mitochondrial targeting sequence [27]. Several other genes encoding components of the yeast Fe–S assembly machinery have homologs in the Arabidopsis genome [22]. Such data are therefore in favour of a conserved Fe–S biogenesis process in plant mitochondria, in which Isu would act as an Fe–S scaffold protein. In order to gain further insight into AtIsu function into plant mitochondria, a biochemical characterization of these proteins will be required. In addition, other putative Fe–S scaffolds are present into this organelle. AtNfu4,5 are mitochondrial proteins. No function has been attributed so far to their yeast counterpart Nfu1p, whereas the plastid Nfu2 protein has been shown to act as an Fe–S scaffold [11,28]. IscA-like proteins are encoded by Arabidopsis genome and some are predicted to be located into mitochondria. An Fe–S scaffold function has been shown for IscA like proteins [29–31]. Therefore, the precise role of IscU, Nfu and IscA proteins into plant mitochondria will have to be defined. In plants, an additional organelle require Fe–S clusters for essential processes among which the photosynthesis: the plastid. Recent studies have reported the characterization of Arabidopsis genes encoding proteins involved in the plastid Fe–S assembly machinery [14,28,32,33] or affecting plastid 4Fe–4S protein maturation [34,35]. The absence of Isu proteins in plastids supports the hypothesis of an Isu-independent pathway for plastid Fe–S biogenesis. Indeed, plastid AtNfu2 protein has been shown to act in vitro as a scaffold protein, and its function in vivo has been investigated using T-DNA insertion Arabidopsis mutants interrupted in Nfu2 gene, revealing a role for this polypeptide in 4Fe–4S and ferredoxin Fe–S biogenesis into chloroplast [28,33]. Plastidial Nfu proteins share high sequence identity with Synechocystis NifU, which is supposed to act also as Fe–S scaffold protein in this cyanobacteria [36]. It has to be noticed that Synechocystis genome does not encode an Isu like protein. According to the endosymbiotic theory for the origin of mitochondria and plastids, this cyanobacteria represent the ancestor of plastids whereas mitochondria ancestor would be an aerobic bacteria. It appears therefore that an Isu-dependent process from the endosymbiotic bacteria would have been maintained into mitochondria, whereas plastids have conserved the Isu-independent mechanism derived from the cyanobacterial ancestor. These original Fe–S biogenesis processes have further evolved in the corresponding organelles
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as evidence by the proteins involved in yeast mitochondria Fe– S metabolism and not present in E. coli for example [22] or the plastid Nfu proteins having unique features when compared to their cyanobacterial counterpart [11]. Acknowledgements: We are grateful to Ge´rard Vansuyt for his help with tobacco culture. We are indebted to Dr. Geoffrey Duby for advices in tobacco protoplast preparation. Confocal microscopy was performed at the Centre Re´gional dÕImagerie Cellulaire (Montpellier, France) with the assistance of N. Lautre´dou.
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