RESUMO
During cell division, newly replicated DNA is actively segregated to the daughter cells. In most bacteria, this process involves the DNA-binding protein ParB, which condenses the centromeric regions of sister DNA molecules into kinetochore-like structures that recruit the DNA partition ATPase ParA and the prokaroytic SMC/condensin complex. Here, we report the crystal structure of a ParB-like protein (PadC) that emerges to tightly bind the ribonucleotide CTP. The CTP-binding pocket of PadC is conserved in ParB and composed of signature motifs known to be essential for ParB function. We find that ParB indeed interacts with CTP and requires nucleotide binding for DNA condensation in vivo. We further show that CTP-binding modulates the affinity of ParB for centromeric parS sites, whereas parS recognition stimulates its CTPase activity. ParB proteins thus emerge as a new class of CTP-dependent molecular switches that act in concert with ATPases and GTPases to control fundamental cellular functions.
Assuntos
Proteínas de Bactérias/química , Citidina Trifosfato/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Motivos de Nucleotídeos , Ligação ProteicaRESUMO
Eukaryotic 60S ribosomal subunits are comprised of three rRNAs and â¼50 ribosomal proteins. The initial steps of their formation take place in the nucleolus, but, owing to a lack of structural information, this process is poorly understood. Using cryo-EM, we solved structures of early 60S biogenesis intermediates at 3.3 Å to 4.5 Å resolution, thereby providing insights into their sequential folding and assembly pathway. Besides revealing distinct immature rRNA conformations, we map 25 assembly factors in six different assembly states. Notably, the Nsa1-Rrp1-Rpf1-Mak16 module stabilizes the solvent side of the 60S subunit, and the Erb1-Ytm1-Nop7 complex organizes and connects through Erb1's meandering N-terminal extension, eight assembly factors, three ribosomal proteins, and three 25S rRNA domains. Our structural snapshots reveal the order of integration and compaction of the six major 60S domains within early nucleolar 60S particles developing stepwise from the solvent side around the exit tunnel to the central protuberance.
Assuntos
Chaetomium/química , Biogênese de Organelas , Subunidades Ribossômicas Maiores de Eucariotos/química , Chaetomium/citologia , Microscopia Crioeletrônica , Redes e Vias Metabólicas , Modelos Moleculares , Dobramento de RNA , Ribonucleoproteínas/químicaRESUMO
Fractals are patterns that are self-similar across multiple length-scales1. Macroscopic fractals are common in nature2-4; however, so far, molecular assembly into fractals is restricted to synthetic systems5-12. Here we report the discovery of a natural protein, citrate synthase from the cyanobacterium Synechococcus elongatus, which self-assembles into Sierpinski triangles. Using cryo-electron microscopy, we reveal how the fractal assembles from a hexameric building block. Although different stimuli modulate the formation of fractal complexes and these complexes can regulate the enzymatic activity of citrate synthase in vitro, the fractal may not serve a physiological function in vivo. We use ancestral sequence reconstruction to retrace how the citrate synthase fractal evolved from non-fractal precursors, and the results suggest it may have emerged as a harmless evolutionary accident. Our findings expand the space of possible protein complexes and demonstrate that intricate and regulatable assemblies can evolve in a single substitution.
Assuntos
Citrato (si)-Sintase , Evolução Molecular , Fractais , Multimerização Proteica , Synechococcus , Microscopia Crioeletrônica , Modelos Moleculares , Synechococcus/enzimologia , Citrato (si)-Sintase/química , Citrato (si)-Sintase/metabolismo , Citrato (si)-Sintase/ultraestruturaRESUMO
ParB-like CTPases mediate the segregation of bacterial chromosomes and low-copy number plasmids. They act as DNA-sliding clamps that are loaded at parS motifs in the centromere of target DNA molecules and spread laterally to form large nucleoprotein complexes serving as docking points for the DNA segregation machinery. Here, we solve crystal structures of ParB in the pre- and post-hydrolysis state and illuminate the catalytic mechanism of nucleotide hydrolysis. Moreover, we identify conformational changes that underlie the CTP- and parS-dependent closure of ParB clamps. The study of CTPase-deficient ParB variants reveals that CTP hydrolysis serves to limit the sliding time of ParB clamps and thus drives the establishment of a well-defined ParB diffusion gradient across the centromere whose dynamics are critical for DNA segregation. These findings clarify the role of the ParB CTPase cycle in partition complex assembly and function and thus advance our understanding of this prototypic CTP-dependent molecular switch.
Assuntos
Proteínas de Bactérias/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos , Citidina Trifosfato/metabolismo , DNA Bacteriano/metabolismo , Myxococcus xanthus/enzimologia , Proteínas de Bactérias/genética , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Hidrólise , Mutação , Myxococcus xanthus/genética , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Fatores de TempoRESUMO
Peroxisomes are organelles with crucial functions in oxidative metabolism. To correctly target to peroxisomes, proteins require specialized targeting signals. A mystery in the field is the sorting of proteins that carry a targeting signal for peroxisomes and as well as for other organelles, such as mitochondria or the endoplasmic reticulum (ER). Exploring several of these proteins in fungal model systems, we observed that they can act as tethers bridging organelles together to create contact sites. We show that in Saccharomyces cerevisiae this mode of tethering involves the peroxisome import machinery, the ER-mitochondria encounter structure (ERMES) at mitochondria and the guided entry of tail-anchored proteins (GET) pathway at the ER. Our findings introduce a previously unexplored concept of how dual affinity proteins can regulate organelle attachment and communication.
Assuntos
Mitocôndrias , Peroxissomos , Retículo Endoplasmático , Movimento Celular , Respiração Celular , Saccharomyces cerevisiaeRESUMO
Under stressful growth conditions and nutrient starvation, bacteria adapt by synthesizing signaling molecules that profoundly reprogram cellular physiology. At the onset of this process, called the stringent response, members of the RelA/SpoT homolog (RSH) protein superfamily are activated by specific stress stimuli to produce several hyperphosphorylated forms of guanine nucleotides, commonly referred to as (p)ppGpp. Some bifunctional RSH enzymes also harbor domains that allow for degradation of (p)ppGpp by hydrolysis. (p)ppGpp synthesis or hydrolysis may further be executed by single-domain alarmone synthetases or hydrolases, respectively. The downstream effects of (p)ppGpp rely mainly on direct interaction with specific intracellular effectors, which are widely used throughout most cellular processes. The growing number of identified (p)ppGpp targets allows us to deduce both common features of and differences between gram-negative and gram-positive bacteria. In this review, we give an overview of (p)ppGpp metabolism with a focus on the functional and structural aspects of the enzymes involved and discuss recent findings on alarmone-regulated cellular effectors.
Assuntos
Regulação Bacteriana da Expressão Gênica , Guanosina Pentafosfato , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Guanosina Pentafosfato/metabolismo , Sistemas do Segundo MensageiroRESUMO
Biogenesis of ribosomes is one of the most cost- and resource-intensive processes in all living cells. In bacteria, ribosome biogenesis is rate-limiting for growth and must be tightly coordinated to yield maximum fitness of the cells. Since bacteria are continuously facing environmental changes and stress conditions, they have developed sophisticated systems to sense and regulate their nutritional status. Amino acid starvation leads to the synthesis and accumulation of the nucleotide-based second messengers ppGpp and pppGpp [(p)ppGpp], which in turn function as central players of a pleiotropic metabolic adaptation mechanism named the stringent response. Here, we review our current knowledge on the multiple roles of (p)ppGpp in the stress-related modulation of the prokaryotic protein biosynthesis machinery with the ribosome as its core constituent. The alarmones ppGpp/pppGpp act as competitors of their GDP/GTP counterparts, to affect a multitude of ribosome-associated P-loop GTPases involved in the translation cycle, ribosome biogenesis and hibernation. A similar mode of inhibition has been found for the GTPases of the proteins involved in the SRP-dependent membrane-targeting machinery present in the periphery of the ribosome. In this sense, during stringent conditions, binding of (p)ppGpp restricts the membrane insertion and secretion of proteins. Altogether, we highlight the enormously resource-intensive stages of ribosome biogenesis as a critical regulatory hub of the stringent response that ultimately tunes the protein synthesis capacity and consequently the survival of the cell.
Assuntos
Guanosina Pentafosfato , Guanosina Tetrafosfato , Guanosina Tetrafosfato/metabolismo , Guanosina Pentafosfato/metabolismo , Proteínas de Bactérias/metabolismo , Ribossomos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Bactérias/metabolismoRESUMO
Bacterial lifestyles depend on conditions encountered during colonization. The transition between planktonic and biofilm growth is dependent on the intracellular second messenger c-di-GMP. High c-di-GMP levels driven by diguanylate cyclases (DGCs) activity favor biofilm formation, while low levels were maintained by phosphodiesterases (PDE) encourage planktonic lifestyle. The activity of these enzymes can be modulated by stimuli-sensing domains such as Per-ARNT-Sim (PAS). In Pseudomonas aeruginosa, more than 40 PDE/DGC are involved in c-di-GMP homeostasis, including 16 dual proteins possessing both canonical DGC and PDE motifs, that is, GGDEF and EAL, respectively. It was reported that deletion of the EAL/GGDEF dual enzyme PA0285, one of five c-di-GMP-related enzymes conserved across all Pseudomonas species, impacts biofilms. PA0285 is anchored in the membrane and carries two PAS domains. Here, we confirm that its role is conserved in various P. aeruginosa strains and in Pseudomonas putida. Deletion of PA0285 impacts the early stage of colonization, and RNA-seq analysis suggests that expression of cupA fimbrial genes is involved. We demonstrate that the C-terminal portion of PA0285 encompassing the GGDEF and EAL domains binds GTP and c-di-GMP, respectively, but only exhibits PDE activity in vitro. However, both GGDEF and EAL domains are important for PA0285 PDE activity in vivo. Complementation of the PA0285 mutant strain with a copy of the gene encoding the C-terminal GGDEF/EAL portion in trans was not as effective as complementation with the full-length gene. This suggests the N-terminal transmembrane and PAS domains influence the PDE activity in vivo, through modulating the protein conformation.
Assuntos
Proteínas de Bactérias , Pseudomonas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , GMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Fósforo-Oxigênio Liases/genética , Fósforo-Oxigênio Liases/metabolismo , Pseudomonas/enzimologiaRESUMO
In eukaryotes, most secretory and membrane proteins are targeted by an N-terminal signal sequence to the endoplasmic reticulum, where the trimeric Sec61 complex serves as protein-conducting channel (PCC). In the post-translational mode, fully synthesized proteins are recognized by a specialized channel additionally containing the Sec62, Sec63, Sec71, and Sec72 subunits. Recent structures of this Sec complex in the idle state revealed the overall architecture in a pre-opened state. Here, we present a cryo-EM structure of the yeast Sec complex bound to a substrate, and a crystal structure of the Sec62 cytosolic domain. The signal sequence is inserted into the lateral gate of Sec61α similar to previous structures, yet, with the gate adopting an even more open conformation. The signal sequence is flanked by two Sec62 transmembrane helices, the cytoplasmic N-terminal domain of Sec62 is more rigidly positioned, and the plug domain is relocated. We crystallized the Sec62 domain and mapped its interaction with the C-terminus of Sec63. Together, we obtained a near-complete and integrated model of the active Sec complex.
Assuntos
Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , Retículo Endoplasmático/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/químicaRESUMO
Fungi-induced plant diseases affect global food security and plant ecology. The biotrophic fungus Ustilago maydis causes smut disease in maize (Zea mays) plants by secreting numerous virulence effectors that reprogram plant metabolism and immune responses1,2. The secreted fungal chorismate mutase Cmu1 presumably affects biosynthesis of the plant immune signal salicylic acid by channelling chorismate into the phenylpropanoid pathway3. Here we show that one of the 20 maize-encoded kiwellins (ZmKWL1) specifically blocks the catalytic activity of Cmu1. ZmKWL1 hinders substrate access to the active site of Cmu1 through intimate interactions involving structural features that are specific to fungal Cmu1 orthologues. Phylogenetic analysis suggests that plant kiwellins have a versatile scaffold that can specifically counteract pathogen effectors such as Cmu1. We reveal the biological activity of a member of the kiwellin family, a widely conserved group of proteins that have previously been recognized only as important human allergens.
Assuntos
Antígenos de Plantas/metabolismo , Doenças das Plantas/microbiologia , Ustilago/metabolismo , Ustilago/patogenicidade , Fatores de Virulência/metabolismo , Zea mays/metabolismo , Zea mays/microbiologia , Corismato Mutase/antagonistas & inibidores , Corismato Mutase/química , Corismato Mutase/metabolismo , Ácido Corísmico/metabolismo , Modelos Moleculares , Filogenia , Doenças das Plantas/imunologia , Ácido Salicílico/imunologia , Ustilago/enzimologia , Zea mays/imunologiaRESUMO
CRISPR-Cas systems are prokaryotic immune systems against invading nucleic acids. Type I CRISPR-Cas systems employ highly diverse, multi-subunit surveillance Cascade complexes that facilitate duplex formation between crRNA and complementary target DNA for R-loop formation, retention, and DNA degradation by the subsequently recruited nuclease Cas3. Typically, the large subunit recognizes bona fide targets through the PAM (protospacer adjacent motif), and the small subunit guides the non-target DNA strand. Here, we present the Apo- and target-DNA-bound structures of the I-Fv (type I-F variant) Cascade lacking the small and large subunits. Large and small subunits are functionally replaced by the 5' terminal crRNA cap Cas5fv and the backbone protein Cas7fv, respectively. Cas5fv facilitates PAM recognition from the DNA major groove site, in contrast to all other described type I systems. Comparison of the type I-Fv Cascade with an anti-CRISPR protein-bound I-F Cascade reveals that the type I-Fv structure differs substantially at known anti-CRISPR protein target sites and might therefore be resistant to viral Cascade interception.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA/metabolismo , Endonucleases/metabolismo , Ácidos Nucleicos Heteroduplexes/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Cristalografia por Raios X , DNA/química , DNA/genética , Endonucleases/química , Endonucleases/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/genética , Ligação Proteica , Conformação Proteica , Capuzes de RNA/metabolismo , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/genética , Shewanella putrefaciens/enzimologia , Shewanella putrefaciens/genética , Relação Estrutura-AtividadeRESUMO
Prophages control their lifestyle to either be maintained within the host genome or enter the lytic cycle. Bacillus subtilis contains the SPß prophage whose lysogenic state depends on the MrpR (YopR) protein, a key component of the lysis-lysogeny decision system. Using a historic B. subtilis strain harboring the heat-sensitive SPß c2 mutant, we demonstrate that the lytic cycle of SPß c2 can be induced by heat due to a single nucleotide exchange in the mrpR gene, rendering the encoded MrpRG136E protein temperature-sensitive. Structural characterization revealed that MrpR is a DNA-binding protein resembling the overall fold of tyrosine recombinases. MrpR has lost its recombinase function and the G136E exchange impairs its higher-order structure and DNA binding activity. Genome-wide profiling of MrpR binding revealed its association with the previously identified SPbeta repeated element (SPBRE) in the SPß genome. MrpR functions as a master repressor of SPß that binds to this conserved element to maintain lysogeny. The heat-inducible excision of the SPß c2 mutant remains reliant on the serine recombinase SprA. A suppressor mutant analysis identified a previously unknown component of the lysis-lysogeny management system that is crucial for the induction of the lytic cycle of SPß.
Assuntos
Fagos Bacilares , Bacteriófagos , Proteínas Virais , Fagos Bacilares/genética , Bacillus subtilis/genética , Lisogenia/genética , Prófagos/genética , Recombinases/genética , Proteínas Virais/metabolismoRESUMO
The spatiotemporal regulation of cell division is a fundamental issue in cell biology. Bacteria have evolved a variety of different systems to achieve proper division site placement. In many cases, the underlying molecular mechanisms are still incompletely understood. In this study, we investigate the function of the cell division regulator MipZ from Caulobacter crescentus, a P-loop ATPase that inhibits the polymerization of the treadmilling tubulin homolog FtsZ near the cell poles, thereby limiting the assembly of the cytokinetic Z ring to the midcell region. We show that MipZ interacts with FtsZ in both its monomeric and polymeric forms and induces the disassembly of FtsZ polymers in a manner that is not dependent but enhanced by the FtsZ GTPase activity. Using a combination of biochemical and genetic approaches, we then map the MipZ-FtsZ interaction interface. Our results reveal that MipZ employs a patch of surface-exposed hydrophobic residues to interact with the C-terminal region of the FtsZ core domain. In doing so, it sequesters FtsZ monomers and caps the (+)-end of FtsZ polymers, thereby promoting their rapid disassembly. We further show that MipZ influences the conformational dynamics of interacting FtsZ molecules, which could potentially contribute to modulating their assembly kinetics. Together, our findings show that MipZ uses a combination of mechanisms to control FtsZ polymerization, which may be required to robustly regulate the spatiotemporal dynamics of Z ring assembly within the cell.
Assuntos
Caulobacter crescentus , Proteínas do Citoesqueleto , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/química , Polímeros , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Caulobacter crescentus/genética , Divisão CelularRESUMO
The expression of virulence factors essential for the invasion of host cells by Salmonella enterica is tightly controlled by a network of transcription regulators. The AraC/XylS transcription factor HilD is the main integration point of environmental signals into this regulatory network, with many factors affecting HilD activity. Long-chain fatty acids, which are highly abundant throughout the host intestine, directly bind to and repress HilD, acting as environmental cues to coordinate virulence gene expression. The regulatory protein HilE also negatively regulates HilD activity, through a protein-protein interaction. Both of these regulators inhibit HilD dimerization, preventing HilD from binding to target DNA. We investigated the structural basis of these mechanisms of HilD repression. Long-chain fatty acids bind to a conserved pocket in HilD, in a comparable manner to that reported for other AraC/XylS regulators, whereas HilE forms a stable heterodimer with HilD by binding to the HilD dimerization interface. Our results highlight two distinct, mutually exclusive mechanisms by which HilD activity is repressed, which could be exploited for the development of new antivirulence leads.
Assuntos
Proteínas de Bactérias , Intestinos , Salmonella typhimurium , Proteínas de Bactérias/metabolismo , Ácidos Graxos/metabolismo , Regulação Bacteriana da Expressão Gênica , Intestinos/metabolismo , Intestinos/microbiologia , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Virulência , Animais , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologiaRESUMO
The major pathogen Staphylococcus aureus has to cope with host-derived oxidative stress to cause infections in humans. Here, we report that S. aureus tolerates high concentrations of hypothiocyanous acid (HOSCN), a key antimicrobial oxidant produced in the respiratory tract. We discovered that the flavoprotein disulfide reductase (FDR) MerA protects S. aureus from this oxidant by functioning as a HOSCN reductase, with its deletion sensitizing bacteria to HOSCN. Crystal structures of homodimeric MerA (2.4 Å) with a Cys43 -Cys48 intramolecular disulfide, and reduced MerACys43 S (1.6 Å) showed the FAD cofactor close to the active site, supporting that MerA functions as a group I FDR. MerA is controlled by the redox-sensitive repressor HypR, which we show to be oxidized to intermolecular disulfides under HOSCN stress, resulting in its inactivation and derepression of merA transcription to promote HOSCN tolerance. Our study highlights the HOSCN tolerance of S. aureus and characterizes the structure and function of MerA as a major HOSCN defense mechanism. Crippling the capacity to respond to HOSCN may be a novel strategy for treating S. aureus infections.
Assuntos
Oxirredutases , Staphylococcus aureus , Humanos , Dissulfetos , Oxidantes , Oxirredutases/metabolismo , Staphylococcus aureus/enzimologia , Staphylococcus aureus/metabolismoRESUMO
In the chloroplast, the 54 kDa subunit of the signal recognition particle (cpSRP54) is involved in the posttranslational transport of the light-harvesting chlorophyll a/b-binding proteins (LHCPs) and the cotranslational transport of plastid-encoded subunits of the photosynthetic complexes to the thylakoid membrane. It forms a high-affinity complex with plastid-specific cpSRP43 for posttranslational transport, while a ribosome-associated pool coordinates its cotranslational function. CpSRP54 constitutes a conserved multidomain protein, comprising a GTPase (NG) and a methionine-rich (M) domain linked by a flexible region. It is further characterized by a plastid-specific C-terminal tail region containing the cpSRP43-binding motif. To characterize the physiological role of the various regions of cpSRP54 in thylakoid membrane protein transport, we generated Arabidopsis thaliana cpSRP54 knockout (ffc1-2) lines producing truncated cpSRP54 variants or a GTPase point mutation variant. Phenotypic characterization of the complementation lines demonstrated that the C-terminal tail region of cpSRP54 plays an important role exclusively in posttranslational LHCP transport. Furthermore, we show that the GTPase activity of cpSRP54 plays an essential role in the transport pathways for both nuclear- as well as plastid-encoded proteins. In addition, our data revealed that plants expressing cpSRP54 without the C-terminal region exhibit a strongly increased accumulation of a photosystem I assembly intermediate.
RESUMO
Biofilms can be viewed as tissue-like structures in which microorganisms are organized in a spatial and functional sophisticated manner. Biofilm formation requires the orchestration of a highly integrated network of regulatory proteins to establish cell differentiation and production of a complex extracellular matrix. Here, we discuss the role of the essential Bacillus subtilis biofilm activator RemA. Despite intense research on biofilms, RemA is a largely underappreciated regulatory protein. RemA forms donut-shaped octamers with the potential to assemble into dimeric superstructures. The presumed DNA-binding mode suggests that RemA organizes its target DNA into nucleosome-like structures, which are the basis for its role as transcriptional activator. We discuss how RemA affects gene expression in the context of biofilm formation, and its regulatory interplay with established components of the biofilm regulatory network, such as SinR, SinI, SlrR, and SlrA. We emphasize the additional role of RemA played in nitrogen metabolism and osmotic-stress adjustment.
Assuntos
Regulação Bacteriana da Expressão Gênica , Lepidópteros , Animais , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , Fatores de Transcrição/metabolismoRESUMO
Mitochondria harbor the bacteria-inherited iron-sulfur cluster assembly (ISC) machinery to generate [2Fe-2S; iron-sulfur (Fe-S)] and [4Fe-4S] proteins. In yeast, assembly of [4Fe-4S] proteins specifically involves the ISC proteins Isa1, Isa2, Iba57, Bol3, and Nfu1. Functional defects in their human equivalents cause the multiple mitochondrial dysfunction syndromes, severe disorders with a broad clinical spectrum. The bacterial Iba57 ancestor YgfZ was described to require tetrahydrofolate (THF) for its function in the maturation of selected [4Fe-4S] proteins. Both YgfZ and Iba57 are structurally related to an enzyme family catalyzing THF-dependent one-carbon transfer reactions including GcvT of the glycine cleavage system. On this basis, a universally conserved folate requirement in ISC-dependent [4Fe-4S] protein biogenesis was proposed. To test this idea for mitochondrial Iba57, we performed genetic and biochemical studies in Saccharomyces cerevisiae, and we solved the crystal structure of Iba57 from the thermophilic fungus Chaetomium thermophilum. We provide three lines of evidence for the THF independence of the Iba57-catalyzed [4Fe-4S] protein assembly pathway. First, yeast mutants lacking folate show no defect in mitochondrial [4Fe-4S] protein maturation. Second, the 3D structure of Iba57 lacks many of the side-chain contacts to THF as defined in GcvT, and the THF-binding pocket is constricted. Third, mutations in conserved Iba57 residues that are essential for THF-dependent catalysis in GcvT do not impair Iba57 function in vivo, in contrast to an exchange of the invariant, surface-exposed cysteine residue. We conclude that mitochondrial Iba57, despite structural similarities to both YgfZ and THF-binding proteins, does not utilize folate for its function.
Assuntos
Proteínas Ferro-Enxofre , Proteínas de Saccharomyces cerevisiae , Humanos , Proteínas de Transporte/metabolismo , Ácido Fólico/metabolismo , Ferro/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Tetra-Hidrofolatos/metabolismoRESUMO
Bacillus subtilis cells are well suited to study how bacteria sense and adapt to proteotoxic stress such as heat, since temperature fluctuations are a major challenge to soil-dwelling bacteria. Here, we show that the alarmones (p)ppGpp, well known second messengers of nutrient starvation, are also involved in the heat stress response as well as the development of thermo-resistance. Upon heat-shock, intracellular levels of (p)ppGpp rise in a rapid but transient manner. The heat-induced (p)ppGpp is primarily produced by the ribosome-associated alarmone synthetase Rel, while the small alarmone synthetases RelP and RelQ seem not to be involved. Furthermore, our study shows that the generated (p)ppGpp pulse primarily acts at the level of translation, and only specific genes are regulated at the transcriptional level. These include the down-regulation of some translation-related genes and the up-regulation of hpf, encoding the ribosome-protecting hibernation-promoting factor. In addition, the alarmones appear to interact with the activity of the stress transcription factor Spx during heat stress. Taken together, our study suggests that (p)ppGpp modulates the translational capacity at elevated temperatures and thereby allows B. subtilis cells to respond to proteotoxic stress, not only by raising the cellular repair capacity, but also by decreasing translation to concurrently reduce the protein load on the cellular protein quality control system.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Resposta ao Choque Térmico/genética , Ligases/genética , Regulação Bacteriana da Expressão Gênica/genéticaRESUMO
Bacterial flagella differ in their number and spatial arrangement. In many species, the MinD-type ATPase FlhG (also YlxH/FleN) is central to the numerical control of bacterial flagella, and its deletion in polarly flagellated bacteria typically leads to hyperflagellation. The molecular mechanism underlying this numerical control, however, remains enigmatic. Using the model species Shewanella putrefaciens, we show that FlhG links assembly of the flagellar C ring with the action of the master transcriptional regulator FlrA (named FleQ in other species). While FlrA and the flagellar C-ring protein FliM have an overlapping binding site on FlhG, their binding depends on the ATP-dependent dimerization state of FlhG. FliM interacts with FlhG independent of nucleotide binding, while FlrA exclusively interacts with the ATP-dependent FlhG dimer and stimulates FlhG ATPase activity. Our in vivo analysis of FlhG partner switching between FliM and FlrA reveals its mechanism in the numerical restriction of flagella, in which the transcriptional activity of FlrA is down-regulated through a negative feedback loop. Our study demonstrates another level of regulatory complexity underlying the spationumerical regulation of flagellar biogenesis and implies that flagellar assembly transcriptionally regulates the production of more initial building blocks.