Signal Recognition Particle Components Research Articles

The RNA Helicase Ded1 from Yeast Is Associated with the Signal Recognition Particle and Is Regulated by SRP21.

The DEAD-box RNA helicase Ded1 is an essential yeast protein involved in translation initiation that belongs to the DDX3 subfamily. The purified Ded1 protein is an ATP-dependent RNA-binding protein and an RNA-dependent ATPase, but it was previously found to lack substrate specificity and enzymatic regulation. Here we demonstrate through yeast genetics, yeast extract pull-down experiments, in situ localization, and in vitro biochemical approaches that Ded1 is associated with, and regulated by, the signal recognition particle (SRP), which is a universally conserved ribonucleoprotein complex required for the co-translational translocation of polypeptides into the endoplasmic reticulum lumen and membrane. Ded1 is physically associated with SRP components in vivo and in vitro. Ded1 is genetically linked with SRP proteins. Finally, the enzymatic activity of Ded1 is inhibited by SRP21 in the presence of SCR1 RNA. We propose a model where Ded1 actively participates in the translocation of proteins during translation. Our results provide a new understanding of the role of Ded1 during translation.

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.

Engineering protein translocation and unfolded protein response enhanced human PH-20 secretion in Pichia pastoris.

Hyaluronidases catalyze the degradation of hyaluronan (HA), which is finding rising applications in medicine, cosmetic, and food industries. Recombinant expression of hyaluronidases in microbial hosts has been given special attention as a sustainable way to substitute animal tissue-derived hyaluronidases. In this study, we focused on optimizing the secretion of hyaluronidase from Homo sapiens in Pichia pastoris by secretion pathway engineering. The recombinant hyaluronidase was first expressed under the control of a constitutive promoter PGCW14. Then, two endoplasmic reticulum-related secretory pathways were engineered to improve the secretion capability of the recombinant strain. Signal peptide optimization suggested redirecting the protein into co-translational translocation using the ost1-proα signal sequence improved the secretion level by 20%. Enhancing the co-translational translocation by overexpressing signal recognition particle components further enhanced the secretory capability by 48%. Then, activating the unfolded protein response by overexpressing a transcriptional factor ScHac1p led to a secreted hyaluronidase activity of 4.06 U/mL, which was 2.1-fold higher than the original strain. Finally, fed-batch fermentation elevated the production to 19.82 U/mL. The combined engineering strategy described here could be applied to enhance the secretion capability of other proteins in yeast hosts. KEY POINTS: • Improving protein secretion by enhancing co-translational translocation in P. pastoris was reported for the first time. • Overexpressing Hac1p homologous from different origins improved the rhPH-20 secretion. • A 4.9-fold increase in rhPH-20 secretion was achieved after fermentation optimization and fed-batch fermentation.

Nuclear SRP9/SRP14 heterodimer transcriptionally regulates 7SL and BC200 RNA expression.

The SRP9/SRP14 heterodimer is a central component of signal recognition particle (SRP) RNA (7SL) processing and Alu retrotransposition. In this study, we sought to establish the role of nuclear SRP9/SRP14 in the transcriptional regulation of 7SL and BC200 RNA. 7SL and BC200 RNA steady-state levels, rate of decay, and transcriptional activity were evaluated under SRP9/SRP14 knockdown conditions. Immunofluorescent imaging, and subcellular fractionation of MCF-7 cells, revealed a distinct nuclear localization for SRP9/SRP14. The relationship between this localization and transcriptional activity at 7SL and BC200 genes was also examined. These findings demonstrate a novel nuclear function of SRP9/SRP14 establishing that this heterodimer transcriptionally regulates 7SL and BC200 RNA expression. We describe a model in which SRP9/SRP14 cotranscriptionally regulate 7SL and BC200 RNA expression. Our model is also a plausible pathway for regulating Alu RNA transcription and is consistent with the hypothesized roles of SRP9/SRP14 transporting 7SL RNA into the nucleolus for posttranscriptional processing, and trafficking of Alu RNA for retrotransposition.

A reciprocal feedback loop between HIF-1α and HPIP controls phenotypic plasticity in breast cancer cells.

While phenotypic plasticity is a critical factor contributing to tumor heterogeneity, molecular mechanisms underlying this process are largely unknown. Here we report that breast cancer cells display phenotypic diversity in response to hypoxia or normoxia microenvironments by operating a reciprocal positive feedback regulation of HPIP and HIF-1α. We show that under hypoxia, HIF-1α induces HPIP expression that establishes cell survival, and also promotes cell migration/invasion, EMT and metastatic phenotypes in breast cancer cells. Mechanistic studies revealed that HPIP interacts with SRP14, a component of signal recognition particle, and stimulates MMP9 synthesis under hypoxic stress. Whereas, in normoxia, HPIP stabilizes HIF-1α, causing the Warburg effect to support cell growth. Concurrently, mathematical modelling corroborates this reciprocal feedback loop in enabling cell-state transitions in cancer cells. Clinical data indicate that elevated levels of HPIP and HIF-1α correlate with unfavorable prognosis and shorter survival rates in breast cancer subjects. Together, this data shows a reciprocal positive feedback loop between HPIP and HIF-1α that was unknown hitherto. It unveils how the tumor microenvironment influences phenotypic plasticity that has an impact on tumor growth and metastasis and, further signifies considering this pathway as a potential therapeutic target in breast cancer.

Noncanonical Functions and Cellular Dynamics of the Mammalian Signal Recognition Particle Components.

The signal recognition particle (SRP) is a ribonucleoprotein complex fundamental for co-translational delivery of proteins to their proper membrane localization and secretory pathways. Literature of the past two decades has suggested new roles for individual SRP components, 7SL RNA and proteins SRP9, SRP14, SRP19, SRP54, SRP68 and SRP72, outside the SRP cycle. These noncanonical functions interconnect SRP with a multitude of cellular and molecular pathways, including virus-host interactions, stress response, transcriptional regulation and modulation of apoptosis in autoimmune diseases. Uncovered novel properties of the SRP components present a new perspective for the mammalian SRP as a biological modulator of multiple cellular processes. As a consequence of these findings, SRP components have been correlated with a growing list of diseases, such as cancer progression, myopathies and bone marrow genetic diseases, suggesting a potential for development of SRP-target therapies of each individual component. For the first time, here we present the current knowledge on the SRP noncanonical functions and raise the need of a deeper understanding of the molecular interactions between SRP and accessory cellular components. We examine diseases associated with SRP components and discuss the development and feasibility of therapeutics targeting individual SRP noncanonical functions.

Interactions of protein translocation machinery components of Streptococcus mutans

BackgroundPivotal virulence properties of the cariogenic bacterium Streptococcus mutans depend on secreted and integral membrane proteins. Bacterial protein trafficking involves the signal recognition particle (SRP) pathway components Ffh, scRNA and FtsY, the SecYEG translocon, the SecA motor protein, and membrane‐localized YidC chaperone/insertases. Unlike Escherichia coli, S. mutans survives loss of the SRP pathway. In addition, S. mutans has two yidC paralogs. The phenotype of a DyidC2 mutant largely parallels that of Dffh, while that of DyidC1 is far less severe. Also in S. mutans, YlxM acts as an accessory factor of the SRP that modulates GTP hydrolysis activities of Ffh and FtsY. The goal of this study is to identify interactions among S. mutans transport machinery components to better understand the respective functions of YidC1 and YidC2 alone and in concert with the SRP, ribosome, and/or Sec translocon that can affect the destiny of translocated proteins.MethodsA chemical cross‐linking approach was employed, whereby whole cell lysates (wcl) were treated with formaldehyde followed by Western blotting using anti‐Ffh, FtsY, YidC1 and YidC2 antibodies and mass spectrometry (MS) analysis of corresponding gel‐shifted bands. Wcl from WT and DyidC2 strains were also reacted with a‐YidC2 antibodies coupled to magnetic Dynabeads™ and co‐captured proteins identified by MS. In addition, biolayer interferometry (BLI) was used to evaluate specific interactions of the C‐terminal tails of YidC1 and YidC2, engineered as glutathione‐S‐transferase fusion proteins (GST‐C1 and GST‐C2), with the SRP components Ffh and YlxM. The same GST‐constructs were also subjected to 2D Difference Gel Electrophoresis (DIGE), in which each was reacted with wcl, captured with glutathione affinity resin, and labelled individually with fluorescent dyes. Spots unique to the GST‐C1 and/or GST‐C2 samples were analysed by MS.Results and ConclusionBand‐shift patterns detected with anti‐Ffh, FtsY and YidC2 antisera were similar to one another and distinct from that of anti‐YidC1. Cross‐linked proteins included elongation factor Tu, DNA‐directed RNA polymerase subunit b, and HSP 70. Immunocaptured proteins from anti‐YidC2 Dynabeads™ included two small, transmembrane proteins, LemA and SMU_1719c, which may represent translocation substrates. Both GST‐C1 and GST‐C2 interacted with Ffh by BLI with binding influenced by YlxM. Over one hundred spots were identified by DIGE. Among differentially identified proteins in the GST‐C1 sample were DnaK, RNA polymerase subunit d, and cell division protein FtsH. Ribosomal proteins S7 and L13 were prevalent in spots of the GST‐C2 sample, whereas ribosomal protein L2, DNA polymerase III g/t subunit, and the Cas5 homolog were all more abundant in spots from GST‐C1 and GST‐C2 samples compared to GST. This information suggests a coupled cellular machinery that integrates transcription and translation with protein transport and folding.Support or Funding InformationNIH R01DE008007This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reconstitution of the human SRP system and quantitative and systematic analysis of its ribosome interactions.

Co-translational protein targeting to membranes depends on the regulated interaction of two ribonucleoprotein particles (RNPs): the ribosome and the signal recognition particle (SRP). Human SRP is composed of an SRP RNA and six proteins with the SRP GTPase SRP54 forming the targeting complex with the heterodimeric SRP receptor (SRαβ) at the endoplasmic reticulum membrane. While detailed structural and functional data are available especially for the bacterial homologs, the analysis of human SRP was impeded by the unavailability of recombinant SRP. Here, we describe the large-scale production of all human SRP components and the reconstitution of homogeneous SRP and SR complexes. Binding to human ribosomes is determined by microscale thermophoresis for individual components, assembly intermediates and entire SRP, and binding affinities are correlated with structural information available for all ribosomal contacts. We show that SRP RNA does not bind to the ribosome, while SRP binds with nanomolar affinity involving a two-step mechanism of the key-player SRP54. Ultrasensitive binding of SRP68/72 indicates avidity by multiple binding sites that are dominated by the C-terminus of SRP72. Our data extend the experimental basis to understand the mechanistic principles of co-translational targeting in mammals and may guide analyses of complex RNP–RNP interactions in general.

Characterization of FtsY, its interaction with Ffh, and proteomic identification of their potential substrates in Mycobacterium tuberculosis.

The universally conserved signal recognition particle (SRP) pathway that mediates co-translational targeting of membrane and secretory proteins is essential for eukaryotic and prokaryotic cells. The Mycobacterium tuberculosis SRP pathway consists of 2 proteins, Ffh and FtsY, and a 4.5S RNA molecule. Although the Escherichia coli SRP pathway is well studied, understanding of the M. tuberculosis SRP pathway components is very limited. In this study, we have overexpressed and characterized the M. tuberculosis SRP receptor (SR) FtsY as a GTP binding protein. Further, we established the direct protein-protein interaction between Ffh and FtsY. The Ffh-FtsY complex formation resulted in mutual stimulation of their GTP hydrolysis activity. We also attempted to biochemically characterize the SRP components by constructing the antisense gene knockdown strains of ffh and ftsY in M. tuberculosis. Loss of ffh and ftsY resulted in a decreased in vitro growth rate of the antisense ffh strain as compared with the antisense ftsY strain. Finally, 2-D gel electrophoresis of antisense depleted ffh and ftsY strains identified differential expression of 14 proteins.

Mutations in SRP54 Gene Cause Severe Primary Neutropenia As Well As Shwachman-Diamond-like Syndrome

Context. Congenital neutropenia (CN) is a heterogeneous group of diseases characterized by low neutrophil count, severe bacterial infections, increased risk of leukemic transformation and various extra-hematopoietic organ dysfunctions. Even if 24 different genes have been linked to the etiology of CN, in many patients the genetic causes of CN remain unknown.Methods. Whole-exome sequencing (WES) was performed on a trio-based approach in 8 sporadic cases and 6 multiplex families. Sanger sequencing was performed in a second phase on 66 additional patients from the French CN registry. Structural and functional studies were performed using primary cells from patients including fibroblasts and hematopoietic cells. In vitro granulocytic differentiation was conducted by culturing CD34+ in serum-free medium with SCF, IL-3 and G-CSF for 21 days.Results. WES analysis identified a heterozygous mutation in the SRP54 gene, encoding the signal recognition particle (SRP) 54 GTPase protein, in 3 sporadic cases and an autosomal dominant family. Considering these results we directly sequenced SRP54 in the French CN cohort and identified 13 additional sporadic cases and 2 multiplex families, thus reaching a total of 23 cases carrying a SRP54 mutation (19 families, Figure 1A). The Thr117del in-frame deletion was found in 12 probands. In all patients, neutropenia was profound (mean neutrophil absolute count 0.23x109/L), diagnosed in the neonate period or during childhood (mean age 4.2 months), and required long-term G-CSF therapy (mean dose 9 µg/kg/day). Bone marrow examination showed a maturation arrest at the promyelocytic stage. In contrast to CN associated with ELANE mutations, SRP54-mutated patients presented an important degree of dysgranulopoiesis (abnormal localization and number of mature granules) and enlarged endoplasmic reticulum (ER) in promyelocytes as well as dystrophic neutrophils (Figure 1B-1C). No evolution into acute myeloid leukemia was observed in this cohort after a median follow-up period of 14.8 years despite high doses of G-CSF. Six out of the 23 patients had extra-hematopoietic manifestations comprising severe neurodevelopmental delay (n=5) and/or exocrine pancreatic insufficiency (n=3), and/or bone abnormalities (n=2) (Figure 1A).The SRP54 protein is a key component of the ribonucleoprotein complex SRP that mediates the co-translational targeting and the insertion of secretory and membrane proteins to the ER. We identified 7 distinct mutations that affect highly conserved residues within or interacting with G motifs involved in the GTPase activity of SRP54. Of note, 17 out of 18 patients with mutations located within the G1 element and predicted to affect the structure and/or stability of the NG domain presented only a severe neutropenia. In contrast, the other 5 patients with mutations affecting either the magnesium-binding or the guanine-binding site and/or implied in the heterodimeric association with the receptor of SRP54, were associated with Shwachman-Diamond-like syndrome features. During the in vitro granulocytic differentiation of both normal and mutated hematopoietic progenitors, we found a strong increase in SRP54 mRNA expression levels associated with a slight decrease in the protein level in patients compared to controls. Moreover, SRP54 mutations induced a major deleterious effect on proliferation (Figure 1D) and a delay in the differentiation associated with increased apoptosis. Using both in vitro-derived granulocytic cells and primary fibroblasts, we also observed that SRP54 mutations led to ER stress (increased eIF2a phosphorylation, XBP1 splicing, ATF4 and CHOP expression) and enhanced autophagy (higher levels of LC3-II and ULK1 expression).Conclusions. This study thus identifies a novel pathological pathway implicating the co-translational process of protein targeting. This new genetic subtype which represents the second cause of CN in the French registry (prevalence 6.9%), is characterized by a promyelocytic maturation arrest with dysgranulopoiesis leading to a profound neutropenia, with a poor response to G-CSF, and in few patients, is associated with severe neurodevelopmental delay and exocrine pancreatic insufficiency. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Co-evolution of Two GTPases Enables Efficient Protein Targeting in an RNA-less Chloroplast Signal Recognition Particle Pathway

The signal recognition particle (SRP) is an essential ribonucleoprotein particle that mediates the co-translational targeting of newly synthesized proteins to cellular membranes. The SRP RNA is a universally conserved component of SRP that mediates key interactions between two GTPases in SRP and its receptor, thus enabling rapid delivery of cargo to the target membrane. Notably, this essential RNA is bypassed in the chloroplast (cp) SRP of green plants. Previously, we showed that the cpSRP and cpSRP receptor GTPases (cpSRP54 and cpFtsY, respectively) interact efficiently by themselves without the SRP RNA. Here, we explore the molecular mechanism by which this is accomplished. Fluorescence analyses showed that, in the absence of SRP RNA, the M-domain of cpSRP54 both accelerates and stabilizes complex assembly between cpSRP54 and cpFtsY. Cross-linking coupled with mass spectrometry and mutational analyses identified a new interaction between complementarily charged residues on the cpFtsY G-domain and the vicinity of the cpSRP54 M-domain. These residues are specifically conserved in plastids, and their evolution coincides with the loss of SRP RNA in green plants. These results provide an example of how proteins replace the functions of RNA during evolution.

The Archaeal Signal Recognition Particle: Present Understanding and Future Perspective.

The signal recognition particle (SRP) and its receptor constitute universally conserved and essential cellular machinery that controls the proper membrane localization of nascent polypeptides with the transmembrane domain. In the past decade, there has been an immense advancement in our understanding of this targeting machine in all three domains of life. A significant portion of such progress came from the structural analysis of archaeal SRP components. Despite the availability of structural insights from different archaeal SRP components, little is known about protein translocation in this domain of life compared to either bacteria or eukaryotes. One of the primary reasons being limited availability of the genetic and cell biological tools in archaea. In the present review, an attempt has been made to explore the structural information available for archaeal SRP components to gain insights into the protein translocation mechanism of this group of organisms. Besides, many exciting avenues of archaeal research possible using the recently developed genetic and cell biological tools for some species have been identified.

Noncanoncial signal recognition particle RNAs in a major eukaryotic phylum revealed by purification of SRP from the human pathogen Cryptococcus neoformans.

Despite conservation of the signal recognition particle (SRP) from bacteria to man, computational approaches have failed to identify SRP components from genomes of many lower eukaryotes, raising the possibility that they have been lost or altered in those lineages. We report purification and analysis of SRP in the human pathogen Cryptococcus neoformans, providing the first description of SRP in basidiomycetous yeast. The C. neoformans SRP RNA displays a predicted structure in which the universally conserved helix 8 contains an unprecedented stem-loop insertion. Guided by this sequence, we computationally identified 152 SRP RNAs throughout the phylum Basidiomycota. This analysis revealed additional helix 8 alterations including single and double stem-loop insertions as well as loop diminutions affecting RNA structural elements that are otherwise conserved from bacteria to man. Strikingly, these SRP RNA features in Basidiomycota are accompanied by phylum-specific alterations in the RNA-binding domain of Srp54, the SRP protein subunit that directly interacts with helix 8. Our findings reveal unexpected fungal SRP diversity and suggest coevolution of the two most conserved SRP features—SRP RNA helix 8 and Srp54—in basidiomycetes. Because members of this phylum include important human and plant pathogens, these noncanonical features provide new targets for antifungal compound development.

Chloroplast SRP54 Was Recruited for Posttranslational Protein Transport via Complex Formation with Chloroplast SRP43 during Land Plant Evolution

In bacteria, membrane proteins are targeted cotranslationally via a signal recognition particle (SRP). During the evolution of higher plant chloroplasts from cyanobacteria, the SRP pathway underwent striking adaptations that enable the posttranslational transport of the abundant light-harvesting chlorophyll-a/b-binding proteins (LHCPs). The conserved 54-kDa SRP subunit in higher plant chloroplasts (cpSRP54) is not bound to an SRP RNA, an essential SRP component in bacteria, but forms a stable heterodimer with the chloroplast-specific cpSRP43. This heterodimeric cpSRP recognizes LHCP and delivers it to the thylakoid membrane whereby cpSRP43 plays a central role. This study shows that the cpSRP system in the green alga Chlamydomonas reinhardtii differs significantly from that of higher plants as cpSRP43 is not complexed to cpSRP54 in Chlamydomonas and cpSRP54 is not involved in LHCP recognition. This divergence is attributed to altered residues within the cpSRP54 tail and the second chromodomain of cpSRP43 that are crucial for the formation of the binding interface in Arabidopsis. These changes are highly conserved among chlorophytes, whereas all land plants contain cpSRP proteins with typical interaction motifs. These data demonstrate that the coevolution of LHCPs and cpSRP43 occurred independently of complex formation with cpSRP54 and that the interaction between cpSRP54 and cpSRP43 evolved later during the transition from chlorophytes to land plants. Furthermore, our data show that in higher plants a heterodimeric form of cpSRP is required for the formation of a low molecular weight transit complex with LHCP.

Regulating maf1 expression and its expanding biological functions.

Regulating maf1 expression and its expanding biological functions.

Integrity of SRP RNA is ensured by La and the nuclear RNA quality control machinery.

The RNA component of signal recognition particle (SRP) is transcribed by RNA polymerase III, and most steps in SRP biogenesis occur in the nucleolus. Here, we examine processing and quality control of the yeast SRP RNA (scR1). In common with other pol III transcripts, scR1 terminates in a U-tract, and mature scR1 retains a U4–5 sequence at its 3′ end. In cells lacking the exonuclease Rex1, scR1 terminates in a longer U5–6 tail that presumably represents the primary transcript. The 3′ U-tract of scR1 is protected from aberrant processing by the La homologue, Lhp1 and overexpressed Lhp1 apparently competes with both the RNA surveillance system and SRP assembly factors. Unexpectedly, the TRAMP and exosome nuclear RNA surveillance complexes are also implicated in protecting the 3′ end of scR1, which accumulates in the nucleolus of cells lacking the activities of these complexes. Misassembled scR1 has a primary degradation pathway in which Rrp6 acts early, followed by TRAMP-stimulated exonuclease degradation by the exosome. We conclude that the RNA surveillance machinery has key roles in both SRP biogenesis and quality control of the RNA, potentially facilitating the decision between these alternative fates.

YlxM Is a Newly Identified Accessory Protein That Influences the Function of Signal Recognition Particle Pathway Components in Streptococcus mutans

Streptococcus mutans is a cariogenic oral pathogen whose virulence is determined largely by its membrane composition. The signal recognition particle (SRP) protein-targeting pathway plays a pivotal role in membrane biogenesis. S. mutans SRP pathway mutants demonstrate growth defects, cannot contend with environmental stress, and exhibit multiple changes in membrane composition. This study sought to define a role for ylxM, which in S. mutans and numerous other bacteria resides directly upstream of the ffh gene, encoding a major functional element of the bacterial SRP. YlxM was observed as a produced protein in S. mutans. Its predicted helix-turn-helix motif suggested that it has a role as a transcriptional regulator of components within the SRP pathway; however, no evidence of transcriptional regulation was found. Instead, capture enzyme-linked immunosorbent assay (ELISA), affinity chromatography, and bio-layer interferometry (BLI) demonstrated that S. mutans YlxM interacts with the SRP components Ffh and small cytoplasmic RNA (scRNA) but not with the SRP receptor FtsY. In the absence of FtsY, YlxM increased the GTP hydrolysis activity of Ffh alone and in complex with scRNA. However, in the presence of FtsY, YlxM caused an overall diminution of net GTPase activity. Thus, YlxM appears to modulate GTP hydrolysis, a process necessary for proper recycling of SRP pathway components. The presence of YlxM conferred a significant competitive growth advantage under nonstress and acid stress conditions when wild-type and ylxM mutant strains were cultured together. Our results identify YlxM as a component of the S. mutans SRP and suggest a regulatory function affecting GTPase activity.

Assessment of the Requirements for Magnesium Transporters in Bacillus subtilis

Magnesium is the most abundant divalent metal in cells and is required for many structural and enzymatic functions. For bacteria, at least three families of proteins function as magnesium transporters. In recent years, it has been shown that a subset of these transport proteins is regulated by magnesium-responsive genetic control elements. In this study, we investigated the cellular requirements for magnesium homeostasis in the model microorganism Bacillus subtilis. Putative magnesium transporter genes were mutationally disrupted, singly and in combination, in order to assess their general importance. Mutation of only one of these genes resulted in strong dependency on supplemental extracellular magnesium. Notably, this transporter gene, mgtE, is known to be under magnesium-responsive genetic regulatory control. This suggests that the identification of magnesium-responsive genetic mechanisms may generally denote primary transport proteins for bacteria. To investigate whether B. subtilis encodes yet additional classes of transport mechanisms, suppressor strains that permitted the growth of a transporter-defective mutant were identified. Several of these strains were sequenced to determine the genetic basis of the suppressor phenotypes. None of these mutations occurred in transport protein homologues; instead, they affected housekeeping functions, such as signal recognition particle components and ATP synthase machinery. From these aggregate data, we speculate that the mgtE protein provides the primary route of magnesium import in B. subtilis and that the other putative transport proteins are likely to be utilized for more-specialized growth conditions.

Plasmodium falciparum signal recognition particle components and anti-parasitic effect of ivermectin in blocking nucleo-cytoplasmic shuttling of SRP

Signal recognition particle (SRP) is a ubiquitous ribonucleoprotein complex that targets proteins to endoplasmic reticulum (ER) in eukaryotes. Here we report that Plasmodium falciparum SRP is composed of six polypeptides; SRP9, SRP14, SRP19, SRP54, SRP68 and SRP72 and a 303nt long SRP RNA. We generated four transgenic parasite lines expressing SRP-GFP chimeric proteins and co-localization studies showed the nucleo-cytoplasmic localization for these proteins. The evaluation of the effect of known SRP and nuclear import/export inhibitors on P. falciparum revealed that ivermectin, an inhibitor of importin α/β mediated nuclear import inhibited the nuclear import of PfSRP polypeptides at submicromolar concentration, thereby killing the parasites. These findings provide insights into dynamic structure of P. falciparum SRP and also raise the possibility that ivermectin could be used in combination with other antimalarial agents to control the disease.

Evolution from the Prokaryotic to the Higher Plant Chloroplast Signal Recognition Particle: The Signal Recognition Particle RNA Is Conserved in Plastids of a Wide Range of Photosynthetic Organisms

The protein targeting signal recognition particle (SRP) pathway in chloroplasts of higher plants has undergone dramatic evolutionary changes. It disposed of its RNA, which is an essential SRP component in bacteria, and uses a unique chloroplast-specific protein cpSRP43. Nevertheless, homologs of the conserved SRP54 and the SRP receptor, FtsY, are present in higher plant chloroplasts. In this study, we analyzed the phylogenetic distribution of SRP components in photosynthetic organisms to elucidate the evolution of the SRP system. We identified conserved plastid SRP RNAs within all nonspermatophyte land plant lineages and in all chlorophyte branches. Furthermore, we show the simultaneous presence of cpSRP43 in these organisms. The function of this novel SRP system was biochemically and structurally characterized in the moss Physcomitrella patens. We show that P. patens chloroplast SRP (cpSRP) RNA binds cpSRP54 but has lost the ability to significantly stimulate the GTPase cycle of SRP54 and FtsY. Furthermore, the crystal structure at 1.8-Å resolution and the nucleotide specificity of P. patens cpFtsY was determined and compared with bacterial FtsY and higher plant chloroplast FtsY. Our data lead to the view that the P. patens cpSRP system occupies an intermediate position in the evolution from bacterial-type SRP to higher plant-type cpSRP system.