SufU Is an Essential Iron-Sulfur Cluster Scaffold Protein in Bacillus subtilis

Bacteria use three distinct systems for iron-sulfur (Fe/S) cluster biogenesis: the ISC, SUF, and NIF machineries. The ISC and SUF systems are widely distributed, and many bacteria possess both of them. In Escherichia coli, ISC is the major and constitutive system, whereas SUF is induced under iron starvation and/or oxidative stress. Genomic analysis of the Fe/S cluster biosynthesis genes in Bacillus subtilis suggests that this bacterium''s genome encodes only a SUF system consisting of a sufCDSUB gene cluster and a distant sufA gene. Mutant analysis of the putative Fe/S scaffold genes sufU and sufA revealed that sufU is essential for growth under minimal standard conditions, but not sufA. The drastic growth retardation of a conditional mutant depleted of SufU was coupled with a severe reduction of aconitase and succinate dehydrogenase activities in total-cell lysates, suggesting a crucial function of SufU in Fe/S protein biogenesis. Recombinant SufU was devoid of Fe/S clusters after aerobic purification. Upon in vitro reconstitution, SufU bound an Fe/S cluster with up to ∼1.5 Fe and S per monomer. The assembled Fe/S cluster could be transferred from SufU to the apo form of isopropylmalate isomerase Leu1, rapidly forming catalytically active [4Fe-4S]-containing holo-enzyme. In contrast to native SufU, its D43A variant carried a Fe/S cluster after aerobic purification, indicating that the cluster is stabilized by this mutation. Further, we show that apo-SufU is an activator of the cysteine desulfurase SufS by enhancing its activity about 40-fold in vitro. SufS-dependent formation of holo-SufU suggests that SufU functions as an Fe/S cluster scaffold protein tightly cooperating with the SufS cysteine desulfurase.Iron-sulfur (Fe/S) clusters are one of the most ubiquitous and versatile cofactors employed by nature for catalyzing a variety of redox reactions or for serving as redox sensors in a broad range of regulatory processes (10). Iron and sulfide are toxic for the cells in concentrations needed for spontaneous chemical Fe/S protein maturation. Hence, cells have developed complex biosynthesis machineries which are essential in vivo to assemble Fe/S proteins. Three phylogenetically distinct biosynthesis systems have been found in bacteria: ISC (iron-sulfur cluster), SUF (sulfur mobilization), and NIF (nitrogen fixation) (9, 11, 24). The ISC machinery is the most widely distributed bacterial Fe/S cluster biogenesis system and is also present in eukaryotes (31). In Escherichia coli a second system for Fe/S cluster assembly, SUF, is induced under conditions of iron limitation and/or oxidative stress, thus replacing the housekeeping ISC system for assembly of Fe/S proteins. In contrast, SUF was found as the exclusive Fe/S biogenesis system in mycobacteria (22) and Enterococcus faecalis (39) and hence may also serve as a constitutive system. Furthermore, SUF is present in plastids of green plants, resembling the situation found in their cyanobacterial ancestors (25, 53). The NIF system is responsible for the dedicated maturation of the complex Fe/S protein nitrogenase involved in nitrogen fixation, e.g., in Azotobacter vinelandii. Some NIF genes are associated with anaerobic or microaerobic growth in Helicobacter pylori and Entamoeba histolytica (24).Common principles for Fe/S protein assembly in each system have been defined (30). The de novo assembly of an Fe/S cluster occurs on scaffold proteins which transiently bind the Fe/S cluster before transfer to target apoproteins. Cysteine desulfurases such as IscS and SufS serve as sulfur donors, which acquire sulfur from free l-cysteine by pyridoxal-5′-phosphate-dependent desulfuration. The sulfur is transiently bound in the form of a persulfide to an active-site cysteine of the desulfurase and is subsequently transferred to the scaffold protein. Several SUF systems contain SufE, which specifically forms a complex with the cysteine desulfurase SufS (36, 42). SufE enhances SufS activity significantly and assists the sulfur transfer to scaffold proteins. In this case the persulfide is transiently bound to SufE and not to the desulfurase. Recent studies show that the E. coli cysteine desulfurase CsdA is able to complement the SUF system and interacts with SufE if SufS is inactivated (50). A general iron donor involved in Fe/S cluster assembly is not known yet; however, frataxin homologs in prokaryotes and eukaryotes are postulated to deliver iron to the scaffold protein IscU in the ISC system (5, 9, 31, 34).Several components have been suggested to act as scaffold proteins. U-type scaffold proteins such as bacterial NifU, IscU, and eukaryotic Isu1 preferentially bind [2Fe-2S] clusters. However, the assembly of [4Fe-4S] clusters was described to proceed by reductive coupling of two [2Fe-2S] clusters that bind successively to an IscU dimer (1). A-type scaffolds like bacterial SufA or IscA can bind [2Fe-2S] clusters in their monomeric state and were found to be involved in the maturation of [4Fe-4S] proteins such as aconitase (17, 32, 47). Overproduced IscA was also shown to bind mononuclear iron which could be used for Fe/S cluster assembly on IscU in vitro.The ISC system characteristically contains the molecular chaperone pair HscA and HscB that are involved in Fe/S cluster transfer from the IscU scaffold protein to the target proteins. The Hsp70-type HscA specifically binds to a highly conserved LPPVK motif located near the third strictly conserved cluster-binding cysteine in IscU. Specific IscU-HscA complex formation was found to be necessary and sufficient to stimulate the ATPase activity of HscA (12, 21, 48). The SUF system, in contrast, does not contain HscA and HscB chaperones in the suf gene cluster. Instead it comprises the SufBCD proteins. The SufC protein has intrinsic ATPase activity and forms a complex with SufB, a putative scaffold protein, and SufD (29). The precise molecular function of the ATP-hydrolyzing SufBCD complex is not yet clear.The best-characterized SUF system from E. coli contains the gene cluster sufABCDSE (5). However, many bacteria, in particular members of the phylum Firmicutes, contain a different suf gene cluster encoding sufCDSUB, which has been studied so far only by bioinformatic approaches (39, 46). While SufS and SufBCD in the two gene clusters appear to be similar proteins, SufE is lacking in most Firmicutes and also in the mycobacterial and Thermotoga maritima SUF machineries (22, 24). The additionally present SufU shares similarities with IscU of the ISC system (39). The protein contains all three conserved cysteine residues involved in Fe/S cluster association and yet characteristically lacks the LPPVK motif, consistent with the absence of HscA and HscB proteins in sufCDSUB species.In this study, we made use of the Gram-positive bacterium Bacillus subtilis to initiate functional analysis of the sufCDSUB genes in Fe/S cluster biosynthesis by genetic and biochemical approaches. In particular, since no functional information is available for SufU, we tested its putative role as an Fe/S scaffold protein. SufU was found to be crucial for cell viability and for Fe/S-dependent enzyme activities in crude cell lysates. In vitro cluster reconstitution with recombinant SufU indicated that SufU binds a labile Fe/S cluster which can be transferred to apo-Leu1 in a fast and efficient way, fully activating its catalytic function as a [4Fe-4S] cluster-dependent isopropylmalate isomerase. The B. subtilis SufU was found to activate the desulfurase activity of purified B. subtilis SufS. Our results suggest that SufS and the SufU scaffold protein closely act together in the Fe/S cluster biogenesis in B. subtilis.     

The Yeast Nbp35-Cfd1 Cytosolic Iron-Sulfur Cluster Scaffold Is an ATPase

Nbp35 and Cfd1 are prototypical members of the MRP/Nbp35 class of iron-sulfur (FeS) cluster scaffolds that function to assemble nascent FeS clusters for transfer to FeS-requiring enzymes. Both proteins contain a conserved NTPase domain that genetic studies have demonstrated is essential for their cluster assembly activity inside the cell. It was recently reported that these proteins possess no or very low nucleotide hydrolysis activity in vitro, and thus the role of the NTPase domain in cluster biogenesis has remained uncertain. We have reexamined the NTPase activity of Nbp35, Cfd1, and their complex. Using in vitro assays and site-directed mutagenesis, we demonstrate that the Nbp35 homodimer and the Nbp35-Cfd1 heterodimer are ATPases, whereas the Cfd1 homodimer exhibited no or very low ATPase activity. We ruled out the possibility that the observed ATP hydrolysis activity might result from a contaminating ATPase by showing that mutation of key active site residues reduced activity to background levels. Finally, we demonstrate that the fluorescent ATP analog 2′/3′-O-(N′-methylanthraniloyl)-ATP (mantATP) binds stoichiometrically to Nbp35 with a K_D = 15.6 μm and that an Nbp35 mutant deficient in ATP hydrolysis activity also displays an increased K_D for mantATP. Together, our results demonstrate that the cytosolic iron-sulfur cluster assembly scaffold is an ATPase and pave the way for interrogating the role of nucleotide hydrolysis in cluster biogenesis by this large family of cluster scaffolding proteins found across all domains of life.     

Binding of the Chaperone Jac1 Protein and Cysteine Desulfurase Nfs1 to the Iron-Sulfur Cluster Scaffold Isu Protein Is Mutually Exclusive

Biogenesis of mitochondrial iron-sulfur (Fe/S) cluster proteins requires the interaction of multiple proteins with the highly conserved 14-kDa scaffold protein Isu, on which clusters are built prior to their transfer to recipient proteins. For example, the assembly process requires the cysteine desulfurase Nfs1, which serves as the sulfur donor for cluster assembly. The transfer process requires Jac1, a J-protein Hsp70 cochaperone. We recently identified three residues on the surface of Jac1 that form a hydrophobic patch critical for interaction with Isu. The results of molecular modeling of the Isu1-Jac1 interaction, which was guided by these experimental data and structural/biophysical information available for bacterial homologs, predicted the importance of three hydrophobic residues forming a patch on the surface of Isu1 for interaction with Jac1. Using Isu variants having alterations in residues that form the hydrophobic patch on the surface of Isu, this prediction was experimentally validated by in vitro binding assays. In addition, Nfs1 was found to require the same hydrophobic residues of Isu for binding, as does Jac1, suggesting that Jac1 and Nfs1 binding is mutually exclusive. In support of this conclusion, Jac1 and Nfs1 compete for binding to Isu. Evolutionary analysis revealed that residues involved in these interactions are conserved and that they are critical residues for the biogenesis of Fe/S cluster protein in vivo. We propose that competition between Jac1 and Nfs1 for Isu binding plays an important role in transitioning the Fe/S cluster biogenesis machinery from the cluster assembly step to the Hsp70-mediated transfer of the Fe/S cluster to recipient proteins.     

A Medicago truncatula EF-Hand Family Gene,MtCaMP1, Is Involved in Drought and Salt Stress Tolerance

Background

Calcium-binding proteins that contain EF-hand motifs have been reported to play important roles in transduction of signals associated with biotic and abiotic stresses. To functionally characterize gens of EF-hand family in response to abiotic stress, an MtCaMP1 gene belonging to EF-hand family from legume model plant Medicago truncatula was isolated and its function in response to drought and salt stress was investigated by expressing MtCaMP1 in Arabidopsis.

Methodology/Principal Findings

Transgenic Arabidopsis seedlings expressing MtCaMP1exhibited higher survival rate than wild-type seedlings under drought and salt stress, suggesting that expression of MtCaMP1 confers tolerance of Arabidopsis to drought and salt stress. The transgenic plants accumulated greater amounts of Pro due to up-regulation of P5CS1 and down-regulation of ProDH than wild-type plants under drought stress. There was a less accumulation of Na⁺ in the transgenic plants than in WT plants due to reduced up-regulation of AtHKT1 and enhanced regulation of AtNHX1 in the transgenic plants compared to WT plants under salt stress. There was a reduced accumulation of H₂O₂ and malondialdehyde in the transgenic plants than in WT plants under both drought and salt stress.

Conclusions/Significance

The expression of MtCaMP1 in Arabidopsis enhanced tolerance of the transgenic plants to drought and salt stress by effective osmo-regulation due to greater accumulation of Pro and by minimizing toxic Na⁺ accumulation, respectively. The enhanced accumulation of Pro and reduced accumulation of Na⁺ under drought and salt stress would protect plants from water default and Na⁺ toxicity, and alleviate the associated oxidative stress. These findings demonstrate that MtCaMP1 encodes a stress-responsive EF-hand protein that plays a regulatory role in response of plants to drought and salt stress.     

The OsDHODH1 Gene is Involved in Salt and Drought Tolerance in Rice

In the present paper, we identified and cloned OsDHODH1 encoding a putative cytosolic dihydroorotate dehydrogenase （DHODH） in rice. Expression analysis indicated that OsDHODH1 is upregulated by salt, drought and exogenous abscisic acid （ABA）, but not by cold. By prokaryotic expression, we determined the enzymatic activity of OsDHODH1 and found that overproduction of OsDHODH1 significantly improved the tolerance of Escherichia coil cells to salt and osmotic stresses. Overexpression of the OsDHODH1 gene in rice increased the DHODH activity and enhanced plant tolerance to salt and drought stresses as compared with wild type and OsDHODHl-antisense transgenic plants. Our findings reveal, for the first time, that cytosolic dihydroorotate dehydrogenase is involved in plant stress response and that OsDHODH1 could be used in engineering crop plants with enhanced tolerance to salt and drought.     

The OsDHODH1 Gene is Involved in Salt and Drought Tolerance in Rice

In the present paper, we identified and cloned OsDHODH1 encoding a putative cytosolic dihydroorotate dehydrogenase (DHODH) in rice. Expression analysis indicated that OsDHODH1 is upregulated by salt, drought and exogenous abscisic acid (ABA), but not by cold. By prokaryotic expression, we determined the enzymatic activity of OsDHODH1 and found that overproduction of OsDHODH1 significantly improved the tolerance of Escherichia coli cells to salt and osmotic stresses. Overexpression of the OsDHODH1 gene in rice increased the DHODH activity and enhanced plant tolerance to salt and drought stresses as compared with wild type and OsDHODH1 -antisense transgenic plants. Our findings reveal, for the first time, that cytosolic dihydroorotate dehydrogenase is involved in plant stress response and that OsDHODH1 could be used in engineering crop plants with enhanced tolerance to salt and drought.     

甘薯盐胁迫响应基因IbMYB3的表达特征及生物信息学分析

MYB转录因子具有多种生物学功能,在植物响应生物和非生物胁迫中发挥重要作用。该文从盐胁迫后的甘薯(Ipomoea batatas)水培苗转录组数据(RNA-seq)中筛选出2个受盐胁迫显著上调表达的MYB基因,分别命名为IbMYB3和IbMYB4。多种非生物胁迫和植物生长物质处理下的基因表达分析显示,IbMYB3受逆境诱导显著上调表达,暗示其可能参与甘薯非生物胁迫响应。生物信息学分析表明,IbMYB3开放阅读框长度为1059 bp,编码353个氨基酸残基,蛋白分子量为39.41 kDa,理论等电点(PI)为5.26,为酸性带负电的亲水性蛋白。亚细胞定位结果表明,IbMYB3蛋白定位于细胞核,具有较强的转录激活活性。上述结果表明,IbMYB3转录因子可能在甘薯非生物胁迫响应过程中发挥重要调控作用,研究结果为进一步探明IbMYB3基因的功能奠定了基础。     

The Mammalian Proteins MMS19, MIP18, and ANT2 Are Involved in Cytoplasmic Iron-Sulfur Cluster Protein Assembly

Iron-sulfur (Fe-S) clusters are essential cofactors of proteins with a wide range of biological functions. A dedicated cytosolic Fe-S cluster assembly (CIA) system is required to assemble Fe-S clusters into cytosolic and nuclear proteins. Here, we show that the mammalian nucleotide excision repair protein homolog MMS19 can simultaneously bind probable cytosolic iron-sulfur protein assembly protein CIAO1 and Fe-S proteins, confirming that MMS19 is a central protein of the CIA machinery that brings Fe-S cluster donor proteins and the receiving apoproteins into proximity. In addition, we show that mitotic spindle-associated MMXD complex subunit MIP18 also interacts with both CIAO1 and Fe-S proteins. Specifically, it binds the Fe-S cluster coordinating regions in Fe-S proteins. Furthermore, we show that ADP/ATP translocase 2 (ANT2) interacts with Fe-S apoproteins and MMS19 in the CIA complex but not with the individual proteins. Together, these results elucidate the composition and interactions within the late CIA complex.     

盐地碱蓬亲环素CYP1基因的克隆、表达及耐盐性分析

为探索盐地碱蓬亲环素与其耐盐性的关系,使用RT-PCR和RACE完成盐地碱蓬亲环素基因的全长克隆与测序,并命名为SsCYP-1.SsCYP-1基因全长为875 bp,编码区为531 bp,编码176个氨基酸.多重序列BLAST结果表明,SsCYP-1基因可归于亲环素家族,并具有特定的肽酰脯氨酸顺反异构酶活性结构域特征.为验证盐地碱蓬亲环素的耐盐特性,构建了SsCYP-1表达载体并在大肠杆菌（E.coli） BL21 （Rosetta）中通过IPTG诱导表达,通过PAGE检测目的蛋白为19 KD,与Editseq软件预测的目的蛋白大小基本一致.对重组菌PET-Ss-CYP1进行耐盐分析表明其在高盐浓度培养下生长速度远高于对照菌pETDuet-1.据此,推测该基因在盐地碱蓬生长中可能具有相关的耐盐性作用.     

An Autophosphorylation Site of the Protein Kinase SOS2 Is Important for Salt Tolerance in Arabidopsis

The protein kinase SOS2 （Salt Overly Sensitive 2） is essential for salt-stress signaling and tolerance in Arabidopsis. SOS2 is known to be activated by calcium-SOS3 and by phosphorylation at its activation loop. SOS2 is autophosphorylated in vitro, but the autophosphorylation site and its role in salt tolerance are not known. In this study, we identified an autophosphorylation site in SOS2 and analyzed its role in the responses of Arabidopsis to salt stress. Mass spectrometry analysis showed that Ser 228 of SOS2 is autophosphorylated. When this site was mutated to Ala, the autophosphorylation rate of SOS2 decreased. The substrate phosphorylation by the mutated SOS2 was also less than that by the wild-type SOS2. In contrast, changing Ser228 to Asp to mimic the autophosphorylation enhanced substrate phosphorylation by SOS2. Complementation tests in a sos2 mutant showed that the S228A but not the $228D mutation partially disrupted the function of SOS2 in salt tolerance. We also show that activation loop phosphorylation at Thr168 and autophosphorylation at Ser228 cannot substitute for each other, suggesting that both are required for salt tolerance. Our results indicate that Ser 228 of SOS2 is autophosphorylated and that this autophosphorylation is important for SOS2 function under salt stress.     

Overlapping Binding Sites of the Frataxin Homologue Assembly Factor and the Heat Shock Protein 70 Transfer Factor on the Isu Iron-Sulfur Cluster Scaffold Protein

In mitochondria FeS clusters, prosthetic groups critical for the activity of many proteins, are first assembled on Isu, a 14-kDa scaffold protein, and then transferred to recipient apoproteins. The assembly process involves interaction of Isu with both Nfs1, the cysteine desulfurase serving as a sulfur donor, and the yeast frataxin homolog (Yfh1) serving as a regulator of desulfurase activity and/or iron donor. Here, based on the results of biochemical experiments with purified wild-type and variant proteins, we report that interaction of Yfh1 with both Nfs1 and Isu are required for formation of a stable tripartite assembly complex. Disruption of either Yfh1-Isu or Nfs1-Isu interactions destabilizes the complex. Cluster transfer to recipient apoprotein is known to require the interaction of Isu with the J-protein/Hsp70 molecular chaperone pair, Jac1 and Ssq1. Here we show that the Yfh1 interaction with Isu involves the PVK sequence motif, which is also the site key for the interaction of Isu with Hsp70 Ssq1. Coupled with our previous observation that Nfs1 and Jac1 binding to Isu is mutually exclusive due to partially overlapping binding sites, we propose that such mutual exclusivity of cluster assembly factor (Nfs1/Yfh1) and cluster transfer factor (Jac1/Ssq1) binding to Isu has functional consequences for the transition from the assembly process to the transfer process, and thus regulation of the biogenesis of FeS cluster proteins.     

转HAL1基因番茄的耐盐性

利用农杆菌介导的叶盘法,把HAL1 基因转入番茄,Southern杂交检测得到转基因植株.耐盐实验表明, T1代转基因番茄在150 mmol/L的NaCl胁迫下仍有43%的发芽率,200 mmol/L的NaCl胁迫下发芽率为6%,而对照种子在100和150 mmol/L的NaCl胁迫下发芽率分别为11.0%和0.转基因番茄的电解质相对外渗率小于对照,而根冠比和叶绿素含量大于对照,转HAL1基因显著提高了番茄的耐盐性.盐胁迫下Na 、K 的累积状况表明,转基因番茄根、茎、叶的K /Na 均有所提高,根系的SK/Na增大,茎、叶的RSK/Na和RLK/Na减小,说明根系对K /Na 离子的选择吸收和运输能力加强.不但选择吸收K /Na ,而且表现出整株水平上的有利于耐盐的K /Na 区域化分配.     

Mdp1, a Saccharomyces Cerevisiae Gene Involved in Mitochondrial/Cytoplasmic Protein Distribution, Is Identical to the Ubiquitin-Protein Ligase Gene Rsp5

Alteration of the subcellular distribution of Mod5p-I, a tRNA modification enzyme, member of the sorting isozyme family, affects tRNA-mediated nonsense suppression. Altered suppression efficiency was used to identify MDP genes, which, when mutant, change the mitochondrial/cytosolic distribution of Mod5p-I,KR6. MDP2 is the previously identified VRP1, which encodes verprolin, required for proper organization of the actin cytoskeleton. MDP3 is identical to PAN1, which encodes a protein involved in initiation of translation and actin cytoskeleton organization. We report here the cloning and characterization of wild-type and mutant MDP1 alleles and the isolation and characterization of a multicopy suppressor of mdp1 mutations. MDP1 is identical to RSP5, which encodes ubiquitin-protein ligase, and mdp1 mutations are suppressed by high copy expression of ubiquitin. All four characterized mdp1 mutations cause missense changes located in the hect domain of Rsp5p that is highly conserved among ubiquitin-protein ligases. In addition to its well-known function in protein turnover, ubiquitination has been proposed to play roles in subcellular sorting of proteins via endocytosis and in delivery of proteins to peroxisomes, the endoplasmic reticulum and mitochondria. mdp1, as well as mdp2/vrp1 and mdp3/pan1 mutations, affect endocytosis. Further, mdp1 mutations show synthetic interactions with mdp2/vrp1 and mdp3/pan1. Identification of MDP1 as RSP5, along with our previous identification of MDP2/VRP1 and MDP3/PAN1, implicate interactions of the ubiquitin system, the actin cytoskeleton and protein synthesis in the subcellular distribution of proteins.     

液泡膜H+ -PPase与植物耐盐性

文章介绍植物液泡膜H^＋-PPase的结构、功能及其分子生物学的研究进展,并着重阐述液泡膜H^＋-PPase在植物耐盐性中的作用。