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.     

An Ipomoea batatas Iron-Sulfur Cluster Scaffold Protein Gene,IbNFU1, Is Involved in Salt Tolerance

Iron-sulfur cluster biosynthesis involving the nitrogen fixation (Nif) proteins has been proposed as a general mechanism acting in various organisms. NifU-like protein may play an important role in protecting plants against abiotic and biotic stresses. An iron-sulfur cluster scaffold protein gene, IbNFU1, was isolated from a salt-tolerant sweetpotato (Ipomoea batatas (L.) Lam.) line LM79 in our previous study, but its role in sweetpotato stress tolerance was not investigated. In the present study, the IbNFU1 gene was introduced into a salt-sensitive sweetpotato cv. Lizixiang to characterize its function in salt tolerance. The IbNFU1-overexpressing sweetpotato plants exhibited significantly higher salt tolerance compared with the wild-type. Proline and reduced ascorbate content were significantly increased, whereas malonaldehyde (MDA) content was significantly decreased in the transgenic plants. The activities of superoxide dismutase (SOD) and photosynthesis were significantly enhanced in the transgenic plants. H₂O₂ was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbNFU1 up-regulated pyrroline-5-carboxylate synthase (P5CS) and pyrroline-5-carboxylate reductase (P5CR) genes under salt stress. The systemic up-regulation of reactive oxygen species (ROS) scavenging genes was found in the transgenic plants under salt stress. These findings suggest that IbNFU1gene is involved in sweetpotato salt tolerance and enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and activating ROS scavenging system.     

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.     

Bacillus subtilis Vegetative Catalase Is an Extracellular Enzyme

Strong catalase activity was secreted by Bacillus subtilis cells during stationary growth phase in rich medium but not in sporulation-inducing medium. N-terminal sequencing indicated that the secreted activity was due to the vegetative catalase KatA, previously considered an endocellular enzyme. Extracellular catalase protected B. subtilis cells from oxidative assault.     

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.     

Cell Wall Protein in Bacillus subtilis

The cell wall of Bacillus subtilis 168 contains protein that is refractory to removal by salts, detergents, and denaturants.     

Defining the Region of Bacillus subtilis SpoIIIJ That Is Essential for Its Sporulation-Specific Function

Proteins of the YidC/OxaI/Alb3 family play a crucial role in the insertion, folding, and/or assembly of membrane proteins in prokaryotes and eukaryotes. Bacillus subtilis has two YidC-like proteins, denoted SpoIIIJ and YqjG. SpoIIIJ and YqjG are largely exchangeable in function, but SpoIIIJ has a unique role in sporulation, while YqjG stimulates competence development. To obtain more insight into the regions important for the sporulation specificity of SpoIIIJ, a series of SpoIIIJ/YqjG chimeras was constructed. These chimeras were tested for functionality during vegetative growth and for their ability to complement the sporulation defect of a spoIIIJ deletion strain. The data suggest an important role for the domain comprising transmembrane segment 2 (TMS2) and its flanking loops in sporulation specificity, with lesser contributions to specificity by TMS1 and TMS3.     

Mechanistic characterization of sulfur transfer from cysteine desulfurase SufS to the iron-sulfur scaffold SufU in Bacillus subtilis

Iron–sulfur cluster biosynthesis in Gram-positive bacteria is mediated by the SUF system. The transfer of sulfide from the cysteine desulfurase SufS to the scaffold protein SufU is one of the first steps within the assembly process. In this study, we analyzed the interaction between Bacillus subtilis SufS and its scaffold SufU. The activity of SufS represents a Ping-Pong mechanism leading to successive sulfur loading of the conserved cysteine residues in SufU. Cysteine 41 of SufU is shown to be essential for receiving sulfide from SufS, while cysteines 66 and 128 are needed for SufS/SufU interaction. In conclusion, we present the first step-by-step model for loading of the essential scaffold component SufU by its sulfur donor SufS.

Structured summary

SufS and SufUbind by molecular sieving(View interaction)SufSbinds to SufS by molecular sieving(View interaction)SufS and SufUredox react by enzymatic study (View Interaction 1, 2, 3, 4, 5)SufUphysically interacts with SufS by pull down (View Interaction 1, 2)     

The Suf Iron-Sulfur Cluster Synthesis Pathway Is Required for Apicoplast Maintenance in Malaria Parasites

The apicoplast organelle of the malaria parasite Plasmodium falciparum contains metabolic pathways critical for liver-stage and blood-stage development. During the blood stages, parasites lacking an apicoplast can grow in the presence of isopentenyl pyrophosphate (IPP), demonstrating that isoprenoids are the only metabolites produced in the apicoplast which are needed outside of the organelle. Two of the isoprenoid biosynthesis enzymes are predicted to rely on iron-sulfur (FeS) cluster cofactors, however, little is known about FeS cluster synthesis in the parasite or the roles that FeS cluster proteins play in parasite biology. We investigated two putative FeS cluster synthesis pathways (Isc and Suf) focusing on the initial step of sulfur acquisition. In other eukaryotes, these proteins can be located in multiple subcellular compartments, raising the possibility of cross-talk between the pathways or redundant functions. In P. falciparum, SufS and its partner SufE were found exclusively the apicoplast and SufS was shown to have cysteine desulfurase activity in a complementation assay. IscS and its effector Isd11 were solely mitochondrial, suggesting that the Isc pathway cannot contribute to apicoplast FeS cluster synthesis. The Suf pathway was disrupted with a dominant negative mutant resulting in parasites that were only viable when supplemented with IPP. These parasites lacked the apicoplast organelle and its organellar genome – a phenotype not observed when isoprenoid biosynthesis was specifically inhibited with fosmidomycin. Taken together, these results demonstrate that the Suf pathway is essential for parasite survival and has a fundamental role in maintaining the apicoplast organelle in addition to any role in isoprenoid biosynthesis.     

IOP1 Protein Is an External Component of the Human Cytosolic Iron-Sulfur Cluster Assembly (CIA) Machinery and Functions in the MMS19 Protein-dependent CIA Pathway

The emerging link between iron metabolism and genome integrity is increasingly clear. Recent studies have revealed that MMS19 and cytosolic iron-sulfur cluster assembly (CIA) factors form a complex and have central roles in CIA pathway. However, the composition of the CIA complex, particularly the involvement of the Fe-S protein IOP1, is still unclear. The roles of each component are also largely unknown. Here, we show that MMS19, MIP18, and CIAO1 form a tight “core” complex and that IOP1 is an “external” component of this complex. Although IOP1 and the core complex form a complex both in vivo and in vitro, IOP1 behaves differently in vivo. A deficiency in any core component leads to down-regulation of all of the components. In contrast, IOP1 knockdown does not affect the level of any core component. In MMS19-overproducing cells, other core components are also up-regulated, but the protein level of IOP1 remains unchanged. IOP1 behaves like a target protein in the CIA reaction, like other Fe-S helicases, and the core complex may participate in the maturation process of IOP1. Alternatively, the core complex may catch and hold IOP1 when it becomes mature to prevent its degradation. In any case, IOP1 functions in the MMS19-dependent CIA pathway. We also reveal that MMS19 interacts with target proteins. MIP18 has a role to bridge MMS19 and CIAO1. CIAO1 also binds IOP1. Based on our in vivo and in vitro data, new models of the CIA machinery are proposed.     

The Microtubule-Binding Protein Ensconsin Is an Essential Cofactor of Kinesin-1

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Highlights? Ensconsin is required for cargo transport driven by kinesin-1 ? The C terminus of ensconsin activates kinesin-1; its N terminus binds microtubules ? Kinesin-1 mutants without autoinhibitory activity do not require ensconsin     

Essential nature of the mreC determinant of Bacillus subtilis

The mre genes of Escherichia coli and Bacillus subtilis are cell shape determination genes. Mutants affected in mre function are spheres instead of the normal rods. Although the mre determinants are not required for viability in E. coli, the mreB determinant is an essential gene in B. subtilis. Conflicting results have been reported as to whether the two membrane-associated proteins MreC and MreD are essential proteins. Furthermore, although the MreB protein has been studied in some detail, the roles of the MreC and MreD proteins in cell shape determination are unknown. We constructed a strain of B. subtilis in which expression of the mreC determinant is dependent upon the addition of isopropyl-beta-D-thiogalactopyranoside to the culture medium. Utilizing this conditional strain, it was shown that mreC is an essential gene in B. subtilis. Furthermore, it was shown that cells lacking sufficient quantities of MreC undergo morphological changes, namely, swelling and twisting of the cells, which is followed by cell lysis. Electron microscopy was utilized to demonstrate that a polymeric material accumulated at one side of the division septum of the cells and that the presence of this material correlated with the bending of the cell. The best explanation for the results is that the MreC protein is involved in the control of septal versus long-axis peptidoglycan synthesis, that cells lacking MreC perform aberrant septal peptidoglycan synthesis, and that lysis results from a deficiency in long-axis peptidoglycan synthesis.     

Protein secretion by minicells of Bacillus subtilis

Summary The minicell producing strain Bacillus subtilis IA292 was transformed with plasmids encoding the Bacillus enzymes -glucanase, -amylase and neutral protease. Purified minicells were shown to be free of detectable proteolytic activity. Minicells containing plasmids were found to synthesise all three enzymes internally, but evidence of secretion was only observed in the unique case of neutral protease secretion by minicells prepared from cultures grown in BHI medium.     

Protein secretion in phosphate-limited cultures of Bacillus subtilis 168

The secretion of proteins from Bacillus subtilis was studied under physiologically well-defined conditions in continuous cultures at a range of specific growth rates. The kinetics of secretion was analysed by using pulse-chase and immunoprecipitation techniques that allowed both processing and release to be monitored. Growth conditions were selected that were known to lead to significant changes in the anionic polymer composition of the cell wall. Under magnesium limitation only low levels of native proteins were released into the growth medium. In contrast, much higher amounts of released protein were observed under phosphate limitation. Although synthesis of native secretory proteins appeared to be highly regulated, only minor changes in the secretion of heterologous proteins were detected. Comparable kinetics of protein release of cells grown under different conditions indicated similar cell wall permeabilities. The large changes in the amounts of released proteins were not reflected in the production of chaperones and components required for protein secretion. The data suggest that the capacity of the secretion machinery is not a major limiting step in the export of native secretory proteins. Received: 23 September 1997 / Received revision: 10 November 1997 / Accepted: 16 November 1997