Signal Recognition Particle Receptor Research Articles

Enhancing surfactin production in Bacillus subtilis: Insights from proteomic analysis of nitrate-induced overproduction and strategies for combinatorial metabolic engineering

Surfactin biosynthesis in Bacillus subtilis is intricately regulated by environmental conditions. In the present study, addition of nitrate, a nitrogen source, increased the production of surfactin in B. subtilis ATCC 21332, whereas its absence resulted in minimal or no surfactin production. Proteomics revealed the mechanism underlying nitrate-induced surfactin overproduction, identifying three key differential proteins (preprotein translocase subunit SecA, signal recognition particle receptor FtsY, and cell division adenosine triphosphate-binding protein FtsE) relevant to surfactin transport and regulation. Combinatorial metabolic engineering strategies (enhanced nitrate reduction, fatty acid hydroxylation, rational transporter engineering, and feeding) led to a 41.4-fold increase in surfactin production compared with the initial production in the wild-type strain. This study provides insights into the molecular mechanism of nitrate-induced surfactin overproduction and strategies to enhance the performance of surfactin-producing strains.

Cryo-EMstructure of SRP68/72 reveals an extended dimerization domain with RNA-binding activity.

The signal recognition particle (SRP) is a critical component in protein sorting pathways in all domains of life. Human SRP contains six proteins bound to the 7S RNA and their structures and functions have been mostly elucidated. The SRP68/72 dimer is the largest SRP component and is essential for SRP function. Although the structures of the SRP68/72 RNA binding and dimerization domains have been previously reported, the structure and function of large portions of the SRP68/72 dimer remain unknown. Here, we analyse full-length SRP68/72 using cryo-EM and report that SRP68/72 depend on each other for stability and form an extended dimerization domain. This newly observed dimerization domain is both a protein- and RNA-binding domain. Comparative analysis with current structural models suggests that this dimerization domain undergoes dramatic translocation upon SRP docking onto SRP receptor and eventually comes close to the Alu domain. We propose that the SRP68/72 dimerization domain functions by binding and detaching the Alu domain and SRP9/14 from the ribosomal surface, thus releasing elongation arrest upon docking onto the ER membrane.

A signal recognition particle receptor gene from the sea cucumber, Apostichopus japonicas

The signal recognition particle (SRP) system delivers approximately 30% of the proteome to the endoplasmic reticulum (ER) membrane. SRP receptor alpha (SRα) binds to SRP for targeting nascent secreted proteins to the ER membrane in eukaryotic cells. In this study, the SRα homologous gene was identified in the sea cucumber, Apostichopus japonicus (AjSRα). AjSRα codes for 641 amino acids and has 54.94% identity with its mammalian homologs. Like mammalian SRα, it is expected to contain the SRP-alpha N domain, SRP54_N domain, and SRP54 domain. In addition, AjSRα is ubiquitously expressed in adult tissues and exhibits a sexually dimorphic expression pattern, with significantly higher expression in ovaries compared to testes. As a maternal factor, AjSRα can be continuously detected during embryonic development. Importantly, we first attempted to investigate its function by using lentiviral vectors for delivering SRα gene-specific shRNA, and we revealed that lentiviral vectors do not induce an upregulation of immune-related enzymes in sea cucumbers. However, compared to the dsRNA-based RNA interference (RNAi) method, lentivirus-mediated RNAi caused dynamic changes in gene expression at a later time. This study supplied the technical support for studying the functional mechanism of SRα in sea cucumbers.

Examining SRP pathway function in mRNA localization to the endoplasmic reticulum.

Signal recognition particle (SRP) pathway function in protein translocation across the endoplasmic reticulum (ER) is well established; its role in RNA localization to the ER remains, however, unclear. In current models, mRNAs undergo translation- and SRP-dependent trafficking to the ER, with ER localization mediated via interactions between SRP-bound translating ribosomes and the ER-resident SRP receptor (SR), a heterodimeric complex comprising SRA, the SRP-binding subunit, and SRB, an integral membrane ER protein. To study SRP pathway function in RNA localization, SR knockout (KO) mammalian cell lines were generated and the consequences of SR KO on steady-state and dynamic mRNA localization examined. CRISPR/Cas9-mediated SRPRB KO resulted in profound destabilization of SRA. Pairing siRNA silencing of SRPRA in SRPRB KO cells yielded viable SR KO cells. Steady-state mRNA compositions and ER-localization patterns in parental and SR KO cells were determined by cell fractionation and deep sequencing. Notably, steady-state cytosol and ER mRNA compositions and partitioning patterns were largely unaltered by loss of SR expression. To examine SRP pathway function in RNA localization dynamics, the subcellular trafficking itineraries of newly exported mRNAs were determined by 4-thiouridine (4SU) pulse-labeling/4SU-seq/cell fractionation. Newly exported mRNAs were distinguished by high ER enrichment, with ER localization being SR-independent. Intriguingly, under conditions of translation initiation inhibition, the ER was the default localization site for all newly exported mRNAs. These data demonstrate that mRNA localization to the ER can be uncoupled from the SRP pathway function and reopen questions regarding the mechanism of RNA localization to the ER.

Signal recognition particle receptor-β (SR-β) coordinates cotranslational N-glycosylation

Proteins destined for the secretory compartment of the cell are cotranslationally translocated into the endoplasmic reticulum. The majority of these proteins are N-glycosylated, a co- and posttranslational modification that ensures proper protein folding, stability, solubility, and cellular localization. Here, we show that the [Formula: see text] subunit of the signal recognition particle receptor (SR) is required for assembly of the N-glycosylation-competent translocon. We report that guanine analog chemical probes identified by high-throughput screening or mutation of the SR-[Formula: see text] guanosine triphosphate binding site cause an N-glycosylation-deficient phenotype. Neither method alters the association of SR-[Formula: see text] with SR-[Formula: see text], but both approaches reduce the association of SR-[Formula: see text] with the oligosaccharyltransferase complex. These experiments demonstrate that SR-[Formula: see text] has a previously unrecognized function coordinating endoplasmic reticulum translation with N-glycosylation.

Loss of CpFTSY Reduces Photosynthetic Performance and Affects Insertion of PsaC of PSI in Diatoms.

The chloroplast signal recognition particle (CpSRP) receptor (CpFTSY) is a component of the CpSRP pathway that post-translationally targets light-harvesting complex proteins (LHCPs) to the thylakoid membranes in plants and green algae containing chloroplasts derived from primary endosymbiosis. In plants, CpFTSY also plays a major role in the co-translational incorporation of chloroplast-encoded subunits of photosynthetic complexes into the thylakoids. This role has not been demonstrated in green algae. So far, its function in organisms with chloroplasts derived from secondary endosymbiotic events has not been elucidated. Here, we report the generation and characterization of mutants lacking CpFTSY in the diatom Phaeodactylum tricornutum. We found that this protein is not involved in inserting LHCPs into thylakoid membranes, indicating that the post-translational part of the CpSRP pathway is not active in this group of microalgae. The lack of CpFTSY caused an increased level of photoprotection, low electron transport rates, inefficient repair of photosystem II (PSII), reduced growth, a strong decline in the PSI subunit PsaC and upregulation of proteins that might compensate for a non-functional co-translational CpSRP pathway during light stress conditions. The phenotype was highly similar to the one described for diatoms lacking another component of the co-translational CpSRP pathway, the CpSRP54 protein. However, in contrast to cpsrp54 mutants, only one thylakoid membrane protein, PetD of the Cytb6f complex, was downregulated in cpftsy. Our results point to a minor role for CpFTSY in the co-translational CpSRP pathway, suggesting that other mechanisms may partially compensate for the effect of a disrupted CpSRP pathway.

1208. Polymorphic Locus SerMT1 for Sequence-based Typing of Serratia marcescens

Abstract Background Outbreaks of pathogenic agents within healthcare facilities may have sporadic or nosocomial sources. Effective intervention requires distinguishing between these sources, which in turn requires epidemiological investigation in conjunction with typing of pathogen isolates. For the latter, established methods range from ribotyping and pulse-field gel electrophoresis to multilocus sequence typing and whole genome sequencing. Due to technical complexities and costs associated with these methods, however, typing is rarely pursued. Polymorphic locus sequence typing (PLST) addresses these issue by employing conventional PCR and Sanger sequencing; the key to PLST is its focus on a tandem repeat-containing locus exhibiting maximal variation due to combinations of single nucleotide polymorphisms and insertions/deletions. Serratia marcescens has been implicated in numerous nosocomial outbreaks, and relatedly has the ability to colonize the hospital environment (e.g., sinks), although few studies have definitively established an outbreak-environment link. Methods To extend PLST to S. marcescens, tandem repeats were bioinformatically identified in the genome sequence of strain WW4 (accession CP003959) and used with flanking sequences as queries in BLASTN searches of the GenBank nr/nt Serratia genome database including 97 S. marcescens strains. One locus designated SerMT1 (bp 243212-244195) was present in all strains and exhibited high variability associated with 6-16 copies of a PVVEPE-encoding repeat within signal recognition particle receptor gene ftsY. Results SerMT1 was downloaded from all 97 strains, aligned using Clustal Omega, and phylogenetically analyzed using DNA parsimony. This analysis resolved 91 total alleles and yielded an impressively high Simpson’s Diversity Index of 0.998. Six clusters of 2 to 4 strains shared SerMT1 sequence; their epidemiologic relatedness was supported by their GenBank annotations (e.g., UMH-10 and UMH-11 isolated the same month at the same facility). Conclusion SerMT1 PLST provides an affordable, user-friendly new tool for epidemiologic investigation of S. marcescens outbreaks. Disclosures Tom Edlind, PhD, MicrobiType LLC: Ownership Interest.

Fine mapping of CscpFtsY, a gene conferring the yellow leaf phenotype in cucumber (Cucumis sativus L.)

BackgroundLeaf color mutants are ideal materials to study pigment metabolism and photosynthesis. Leaf color variations are mainly affected by chlorophylls (Chls) and carotenoid contents and chloroplast development in higher plants. However, the regulation of chlorophyll metabolism remains poorly understood in many plant species. The chloroplast signal-recognition particle system is responsible for the insertion of the light-harvesting chlorophyll a/b proteins (LHCPs) to thylakoid membranes, which controls the chloroplast development as well as the regulation of Chls biosynthesis post-translationally in higher plants.ResultsIn this study, the yellow leaf cucumber mutant, named yl, was found in an EMS-induced mutant library, which exhibited a significantly reduced chlorophyll content, abnormal chloroplast ultrastructure and decreased photosynthetic capacity. Genetic analysis demonstrated that the phenotype of yl was controlled by a recessive nuclear gene. Using BSA-seq technology combined with the map-based cloning method, we narrowed the locus to a 100 kb interval in chromosome 3. Linkage analysis and allelism test validated the candidate SNP residing in CsaV3_3G009150 encoding one homolog of chloroplast signal-recognition particle (cpSRP) receptor in Arabidopsis, cpFtsY, could be responsible for the yellow leaf phenotype of yl. The relative expression of CscpFtsY was significantly down-regulated in different organs except for the stem, of yl compared with that in the wild type (WT). Subcellular localization result showed that CscpFtsY located in the chloroplasts of mesophyll cells.ConclusionsThe yl mutant displayed Chls-deficient, impaired chloroplast ultrastructure with intermittent grana stacks and significantly decreased photosynthetic capacity. The isolation of CscpFtsY in cucumber could accelerate the progress on chloroplast development by cpSRP-dependant LHCP delivery system and regulation of Chls biosynthesis in a post-translational way.

Human genetic defects in SRP19 and SRPRA cause severe congenital neutropenia with distinctive proteome changes

AbstractThe mechanisms of coordinated changes in proteome composition and their relevance for the differentiation of neutrophil granulocytes are not well studied. Here, we discover 2 novel human genetic defects in signal recognition particle receptor alpha (SRPRA) and SRP19, constituents of the mammalian cotranslational targeting machinery, and characterize their roles in neutrophil granulocyte differentiation. We systematically study the proteome of neutrophil granulocytes from patients with variants in the SRP genes, HAX1, and ELANE, and identify global as well as specific proteome aberrations. Using in vitro differentiation of human induced pluripotent stem cells and in vivo zebrafish models, we study the effects of SRP deficiency on neutrophil granulocyte development. In a heterologous cell–based inducible protein expression system, we validate the effects conferred by SRP dysfunction for selected proteins that we identified in our proteome screen. Thus, SRP-dependent protein processing, intracellular trafficking, and homeostasis are critically important for the differentiation of neutrophil granulocytes.

Abstract 3981: Targeting SRP receptor-beta reduces protein glycosylation and cell growth in lung cancer

Abstract PURPOSE: Lung cancer is a major cause of morbidity and mortality world wide. Signaling receptors, adhesion molecules, and surface antigens that drive lung cancer progression are regulated by N-glycosylation, a post-translational protein modification initiated in the ER. Strategies for inhibition of N-glycosylation have recently been refined through the discovery of small molecules that partially disrupt glycan transfer. Here we screened for the target of a novel small molecule that reduces protein glycosylation with the goal of identifying new therapeutic targets for lung cancer. METHODS: Pooled results from chemical library HTS efforts identified hit compounds with activity. Counter-screening was performed in A549 cells using a fluorescence-based reporter (Halo1N) that detects aberrant N-glycosylation. The cellular targets for one class of inhibitors were evaluated by combining a whole-genome CRISPR-Cas9 screen with FACS sorting for loss of pharmacologic function. Significant changes in gRNA between populations was determined by MAGeCK analysis. The interaction between a compound and its target was determined by CETSA, and mechanism of action was verified by gene knockout and rescue experiments. Glycosylation status of glycoproteins were measured by western blot and cell compartment fluorescence. Predicted compound binding sites on the protein were mutated using site-directed mutagenesis. RESULTS: The lead tool compound (CP-9) has an IC50 in uM range and was used as a probe for target identification. CP-9 activates Halo-1N fluorescence, and whole genome CRISPR-Cas9 screening was used to identify gRNAs associated with loss of CP-9 activity. MAGeCK analysis demonstrated significant enrichment of genes associated with the translocon and ribosome (FDR P < 0.01) in the non-fluorescent population. We tested components of the signal recognition particle-receptor complex for direct interactions using the CETSA and demonstrate CP-9 binding to SR-β but not SR-α. Knockout experiments confirm that inhibition of glycosylation by CP-9 is dependent on SR-β expression, and CP-9’s effect on glycosylation and was rescued by SR-β re-expression. Mutated SR-β diminished CP-9’s effects on glycosylation and reduced SR-β thermal stabilization. CP-9 disrupted an interaction between SR-β/SR-α and the ribosome-translocon-OST complex suggesting a mechanism of action. Interestingly, a transmembrane domain mutant (ΔTM) of SR-β reduced the CP-9 effect and glycoprotein expression. ΔTM-SR-β also significantly reduced cell proliferation in A459 lung adenocarcinoma cells. CONCLUSION: CP-9 partially inhibits N-glycosylation by interfering with SR-β/SR-α and the translocon complex, leading to the disruption of protein import and glycosylation in the ER. Transmembrane domain mutants of SR-β reduce lung cancer cell growth and suggest this protein as a potential target for lung cancer drug development in the future. Citation Format: Chatchai Phoomak, Joseph N. Contessa. Targeting SRP receptor-beta reduces protein glycosylation and cell growth in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3981.

Expression and purification of the NG domain from human SRα, a key component of the Signal Recognition Particle (SRP) receptor

The Signal Recognition Particle (SRP) and the SRP receptor (SR) are responsible for protein targeting to the plasma membrane and the protein secretory pathway. Eukaryotic SRα, one of the two proteins that form the SR, is composed of the NG, MoRF and X domains. The SRα-NG domain is responsible for binding to SRP proteins such as SRP54, interacting with RNA, binding and hydrolysing GTP. The ability to produce folded SRα-NG is a prerequisite for structural studies directed towards a better understanding of its molecular mechanism and function, as well as in (counter-)screening assays for potential binders in the drug development pipeline. However, previously reported SRα-NG constructs and purification methods only used a truncated version, lacking the first N-terminal helix. This helix in other NG domains (e.g., SRP54) has been shown to be important for protein:protein interactions but its importance in SRα remains unknown. Here, we present the cloning as well as optimised expression and purification protocols of the whole SRα-NG domain including the first N-terminal helix. We have also expressed and purified isotopically labelled SRα-NG to facilitate Nuclear Magnetic Resonance (NMR) studies.

Cotranslational Biogenesis of Membrane Proteins in Bacteria.

Nascent polypeptides emerging from the ribosome during translation are rapidly scanned and processed by ribosome-associated protein biogenesis factors (RPBs). RPBs cleave the N-terminal formyl and methionine groups, assist cotranslational protein folding, and sort the proteins according to their cellular destination. Ribosomes translating inner-membrane proteins are recognized and targeted to the translocon with the help of the signal recognition particle, SRP, and SRP receptor, FtsY. The growing nascent peptide is then inserted into the phospholipid bilayer at the translocon, an inner-membrane protein complex consisting of SecY, SecE, and SecG. Folding of membrane proteins requires that transmembrane helices (TMs) attain their correct topology, the soluble domains are inserted at the correct (cytoplasmic or periplasmic) side of the membrane, and – for polytopic membrane proteins – the TMs find their interaction partner TMs in the phospholipid bilayer. This review describes the recent progress in understanding how growing nascent peptides are processed and how inner-membrane proteins are targeted to the translocon and find their correct orientation at the membrane, with the focus on biophysical approaches revealing the dynamics of the process. We describe how spontaneous fluctuations of the translocon allow diffusion of TMs into the phospholipid bilayer and argue that the ribosome orchestrates cotranslational targeting not only by providing the binding platform for the RPBs or the translocon, but also by helping the nascent chains to find their correct orientation in the membrane. Finally, we present the auxiliary role of YidC as a chaperone for inner-membrane proteins. We show how biophysical approaches provide new insights into the dynamics of membrane protein biogenesis and raise new questions as to how translation modulates protein folding.

Co-Translational Membrane Targeting and Holo-Translocon Docking of Ribosomes Translating the SRP Receptor

Many integral membrane proteins are produced by translocon-associated ribosomes. The assembly of ribosomes translating membrane proteins on the translocons is mediated by a conserved system, composed of the signal recognition particle and its receptor (FtsY in Escherichia coli). FtsY is a peripheral membrane protein, and its role late during membrane protein targeting involves interactions with the translocon. However, earlier stages in the pathway have remained obscure, namely, how FtsY targets the membrane in vivo and where it initially docks. Our previous studies have demonstrated co-translational membrane-targeting of FtsY translation intermediates and identified a nascent FtsY targeting-peptide. Here, in a set of in vivo experiments, we utilized tightly stalled FtsY translation intermediates, pull-down assays and site-directed cross-linking, which revealed FtsY-nascent chain-associated proteins in the cytosol and on the membrane. Our results demonstrate interactions between the FtsY-translating ribosomes and cytosolic chaperones, which are followed by directly docking on the translocon. In support of this conclusion, we show that translocon over-expression increases dramatically the amount of membrane associated FtsY-translating ribosomes. The co-translational contacts of the FtsY nascent chains with the translocon differ from its post-translational contacts, suggesting a major structural maturation process. The identified interactions led us to propose a model for how FtsY may target the membrane co-translationally. On top of our past observations, the current results may add another tier to the hypothesis that FtsY acts stoichiometrically in targeting ribosomes to the membrane in a constitutive manner.

Rapid inactivation of the yeast Sec complex selectively blocks transport of post-translationally translocated proteins

The yeast endoplasmic reticulum has three distinct protein translocation channels. The heterotrimeric Sec61 and Ssh1 complexes, which bind translating ribosomes, mediate cotranslational translocation of proteins targeted to the endoplasmic reticulum by the signal recognition particle (SRP) and SRP receptor targeting pathway, whereas the heptameric Sec complex has been proposed to mediate ribosome-independent post-translational translocation of proteins with less hydrophobic signal sequences that escape recognition by the SRP. However, multiple reports have proposed that the Sec complex may function cotranslationally and be involved in translocation or integration of SRP-dependent protein translocation substrates. To provide insight into these conflicting views, we induced expression of the tobacco etch virus protease to achieve rapid inactivation of the Sec complex by protease-mediated cleavage within the cytoplasmic domain of the Sec63 protein. Protein translocation assays conducted after tobacco etch virus protease induction revealed a complete block in translocation of two well-characterized substrates of the Sec complex, carboxypeptidase Y (CPY) and Gas1p, when the protease cleavage sites were located at structural domain boundaries in Sec63. However, integration of SRP-dependent membrane protein substrates was not detectably impacted. Moreover, redirecting CPY to the cotranslational pathway by increasing the hydrophobicity of the signal sequence rendered translocation of CPY insensitive to inactivation of the Sec complex. We conclude that the Sec complex is primarily responsible for the translocation of yeast secretome proteins with marginally hydrophobic signal sequences.

Transcriptome analyses to reveal the dynamic change mechanism of pigeon magnum during one egg-laying cycle.

We analyzed the transcriptome of pigeon magnum in three stages (C1: pre-ovulation, C2: post-ovulation, C3: 5-6 days after ovulation) to elucidate the molecular and cellular events associated with morphological changes during the laying cycle. We observed that C1 was highly developed, apoptosis rate was highest in C2, and C3 attained the smallest size. Through RNA-sequencing, we obtained 54,764,938 (97.2%) high-quality clean reads that aligned to 20,767 genes. Gene expression profile analysis showed the greatest difference between C1 and C3; 3966 differentially expressed genes (DEGs) were identified, of which 2250 genes were upregulated and 1716 genes were downregulated in C1. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that protein processing and transport activities were prominent in C1, and upregulated genes included those related to signal recognition particle (SRP), signal recognition particle receptor (SRPR), translocon, GRP78, RRBP1, TRAP, TRAM1, and OST. Egg white protein-related gene expression was highest, with OVALY being the most highly expressed. In C2, apoptosis-related gene expression was higher than in C1, and fatty acid metabolism was active, which may be correlated with magnum tissue regression. Collagen- and laminin-related gene expression was prominent in C1 and C3, indicating roles in egg white protein generation and magnum reconstruction. PR gene expression was highest and exhibited drastic change in the three groups, indicating that PR and its regulation may be involved in changes in magnum morphology and function. Through the identification and functional analysis of DEGs and other crucial genes, this may contribute to understand the egg white protein production, magnum tissue regression, and magnum regeneration mechanisms.

Structural and Functional Impact of SRP54 Mutations Causing Severe Congenital Neutropenia

The SRP54 GTPase is a key component of co-translational protein targeting by the signal recognition particle (SRP). Point mutations in SRP54 have been recently shown to lead to a form of severe congenital neutropenia displaying symptoms overlapping with those of Shwachman-Diamond syndrome. The phenotype includes severe neutropenia, exocrine pancreatic deficiency, and neurodevelopmental as well as skeletal disorders. Using a combination of X-ray crystallography, hydrogen-deuterium exchange coupled to mass spectrometry and complementary biochemical and biophysical methods, we reveal extensive structural defects in three disease-causing SRP54 variants resulting in critical protein destabilization. GTP binding is mostly abolished as a consequence of an altered GTPase core. The mutations located in conserved sequence fingerprints of SRP54 eliminate targeting complex formation with the SRP receptor as demonstrated in yeast and human cells. These specific defects critically influence the entire SRP pathway, thereby causing this life-threatening disease.

A molecular recognition feature mediates ribosome-induced SRP-receptor assembly during protein targeting.

Molecular recognition features (MoRFs) provide interaction motifs in intrinsically disordered protein regions to mediate diverse cellular functions. Here we report that a MoRF element, located in the disordered linker domain of the mammalian signal recognition particle (SRP) receptor and conserved among eukaryotes, plays an essential role in sensing the ribosome during cotranslational protein targeting to the endoplasmic reticulum. Loss of the MoRF in the SRP receptor (SR) largely abolishes the ability of the ribosome to activate SRP-SR assembly and impairs cotranslational protein targeting. These results demonstrate a novel role for MoRF elements and provide a mechanism for the ribosome-induced activation of the mammalian SRP pathway. Kinetic analyses and comparison with the bacterial SRP further suggest that the SR MoRF functionally replaces the essential GNRA tetraloop in the bacterial SRP RNA, providing an example for the replacement of RNA function by proteins during the evolution of ancient ribonucleoprotein particles.

Structural insights into the G-loop dynamics of E. coli FtsY NG domain

The bacterial signal recognition particle (SRP) receptor, FtsY, participates with the SRP in co-translation targeting of proteins. Multiple crystal structures of the NG domain of E. coli FtsYNG have been determined at high-resolution (1.22–1.88 Å), in the nucleotide-free (apo) form as well as bound to GDP and non-hydrolysable GTP analogues. The combination of high-resolution and multiple solved structures of FtsYNG in different states revealed a new sensor-relay system of this unique GTPase receptor. A nucleotide sensing function of the P-loop assists FtsYNG in nucleotide-binding and contributes to modulate nucleotide binding properties in SRP GTPases. A reorganization of the other G-loops and the insertion binding domain (IBD) is observed only upon transition from a diphosphate to a triphosphate nucleotide. The role of a magnesium ion during the GDP and GTP-bound states has also been observed. The binding of magnesium in the nucleotide site causes the reorientation of the β- and γ- phosphate groups toward the jaws of the P-loop and stabilizes the binding of the nucleotide, creating a network of hydrogen and water-bridge interactions.

The SRP signal sequence of KdpD

KdpD is a four-spanning membrane protein that has two large cytoplasmic domains at the amino- and at the carboxyterminus, respectively. During its biogenesis KdpD binds to the signal recognition particle (SRP) of Escherichia coli that consists of a 48-kDa protein Ffh and a 4.5S RNA. The protein is targeted to the inner membrane surface and is released after contacting the SRP receptor protein FtsY. The information within the KdpD protein that confers SRP interaction was found in the amino-terminal cytoplasmic domain of KdpD, particularly at residues 22–48. Within this sequence a Walker A motif is present at residues 30–38. To determine the actual sequence specificity to SRP, a collection of mutants was constructed. When the KdpD peptides (residues 22–48) were fused to sfGFP the targeting to the membrane was observed by fluorescence microscopy. Further, nascent chains of KdpD bound to ribosomes were purified and their binding to SRP was analysed by microscale thermophoresis. We found that the amino acid residues R22, K24 and K26 are important for SRP interaction, whereas the residues G30, G34 and G36, essential for a functional Walker A motif, can be replaced with alanines without affecting the affinity to SRP-FtsY and membrane targeting.

Membrane associated protein flotillin in Litopenaeus vannamei plays a role in WSSV infection

Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), a major disease occurring in these siluriform fish. As the liver is an important organ for defending against bacterial pathogens in fish, this study aimed to determine the liver immune response at the protein level. The differential proteomes of the darkbarbel catfish liver in response to E. ictaluri infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using a 1.2-fold change in expression as a physiologically significant benchmark, a total of 819 differentially expressed proteins were reliably quantified using iTRAQ analysis, including 6 up-regulated proteins and 813 down-regulated proteins. GO enrichment analysis indicated that the “complement activation, alternative pathway” and “complement activation, classical pathway” were significantly enriched. KEGG enrichment analysis indicated the “antigen processing and presentation” and “bacterial secretion system” were significantly enriched. We selected the 6 up-regulated proteins and 10 immune-related down-regulated proteins for validation using real-time PCR. The 10 immune-related proteins included complement component C1r, C3, C5, C7, and C9 and plasma protease C1 inhibitor (C1-INH), signal recognition particle 54 kDa protein (SRP54), SRP receptor, proteasome activator complex subunit 1 (PSME1) and major histocompatibility complex class I (MHC class I) were selected from the GO clusters and KEGG pathways. The variations in mRNA expression for these genes were similar to the results of iTRAQ. This is the first report detailing the proteome response in the darkbarbel catfish liver during E. ictaluri infection and markedly contributes to our understanding of the defense mechanisms in the livers of darkbarbel catfish.