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Mycobacterium tuberculosis (M. tuberculosis H37Rv) utilizes the signal recognition particle pathway (SRP pathway) system for secretion of various proteins from ribosomes to the extracellular surface which plays an important role in the machinery running inside the bacterium. This system comprises of three major components FtsY, FfH and 4.5S rRNA. This manuscript highlights essential factors responsible for the optimized enzymatic activity of FtsY. Kinetic parameters include Vmax and Km for the hydrolysis of GTP by ftsY which were 20.25±5.16 μM/min/mg and 39.95±7.7 μM respectively. kcat and catalytic efficiency of the reaction were 0.012±0.003 s−1 and 0.00047±0.0001 μM/s−1 respectively. These values were affected upon changing the standard conditions. Cations (Mg2+ and Mn2+) play important role in FtsY enzymatic activity as increasing Mg2+ decrease the activity. Mn2+on the other hand is required at higher concentration around 60 mM for carrying optimum GTPase activity. FtsY is hydrolyzing ATP and GDP as well and GDP acts as an inhibitor of the reaction. MD simulation shows effective binding and stabilization of the FtsY complexed structure with GTP, GDP and ATP. Mutational analysis was done at two important residues of GTP binding motif of FtsY, namely, GXXXXGK (K236) and DXXG (D367) and showed that these mutations significantly decrease FtsY GTPase activity. FtsY is comprised of α helices, but this structural pattern was shown to change with increasing concentrations of GTP and ATP which symbolize that these ligands cause significant conformational change by variating the secondary structure to transduce signals required by downstream effectors. This binding favors the functional stabilization of FtsY by destabilization of α-helix integrity. Revealing the hidden aspects of the functioning of FtsY might be an essential part for the understanding of the SRP pathway which is one of the important contributors of M. tuberculosis virulence.

Chloroplast development and chlorophyll biosynthesis are affected by temperature. However, the underlying molecular mechanism of this phenomenon remains elusive. Here, we isolated and characterized a thermosensitive yellow-green leaf mutant named tsyl1 (thermosensitive yellow leaf 1) from an ethylmethylsulfone (EMS)-mutagenized pool of rice. The mutant exhibits a yellow-green leaf phenotype and decreased leaf chlorophyll contents throughout development. At the mature stage of the tsyl1 mutant, the plant height, tiller number, number of spikelets per panicle and 1000 seed weight were decreased significantly compared to those of wild-type plants, but the seed setting rate and panicle length were not. The mutant phenotype was controlled by a single recessive nuclear gene on the short arm of rice chromosome 11. Map-based cloning of TSYL1, followed by a complementation experiment, showed a G base deletion at the coding region of LOC_Os11g05552, leading to the yellow-green phenotype. The TSYL1 gene encodes a signal recognition particle 54 kDa (SRP54) protein that is conserved in all organisms. The expression of tsyl1 was induced by high temperature. Furthermore, the expression of chlorophyll biosynthesis- and chloroplast development-related genes was influenced in tsyl1 at different temperatures. These results indicated that the TSYL1 gene plays a key role in chlorophyll biosynthesis and is affected by temperature at the transcriptional level.

International audience; The signal recognition particle (SRP) is a universally conserved non-coding ribonucleoprotein complex that is essential for targeting transmembrane and secretory proteins to the endoplasmic reticulum. Its composition and size varied during evolution. In mammals, SRP contains one RNA molecule, 7SL RNA, and six proteins: SRP9, 14, 19, 54, 68 and 72. Despite a very good understanding of the SRP structure and of the SRP assembly in vitro, how SRP is assembled in vivo remains largely enigmatic. Here we review current knowledge on how the 7SL RNA is assembled with core proteins to form functional RNP particles in cells. SRP biogenesis is believed to take place both in the nucleolus and in the cytoplasm and to rely on the survival of motor neuron complex, whose defect leads to spinal muscular atrophy.

Signal recognition particle (SRP) recognizes signal sequences of secretory proteins and targets them to the endoplasmic reticulum membrane for translocation. Many human diseases are connected with defects in signal sequences. The current dogma states that the molecular basis of the disease-associated mutations in the secretory proteins is connected with defects in their transport. Here, we demonstrate for several secretory proteins with disease-associated mutations that the molecular mechanism is different from the dogma. Positively charged or helix-breaking mutations in the signal sequence hydrophobic core prevent synthesis of the aberrant proteins and lead to degradation of their mRNAs. The degree of mRNA depletion depends on the location and severity of the mutation in the signal sequence and correlates with inhibition of SRP interaction. Thus, SRP protects secretory protein mRNAs from degradation. The data demonstrate that if disease-associated mutations obstruct SRP interaction, they lead to silencing of the mutated protein expression.

Background Protein secretion is a driving force in evolution and tissue specialization. Plasma cells (PC), responsible for intense antibody (Ab) production, are paradigmatic professional secretors whose differentiation entails rapid endoplasmic reticulum (ER) expansion and concerted expression of the protein translocation, folding and trafficking machinery. A novel player in PC differentiation, recently implicated in Ab immunity is the non-canonical poly(A) polymerase FAM46C/TENT5C. Methods In developing PCs, FAM46C is induced to polyadenylate and enhance the translation of a number of mRNAs encoding Igs and ER-targeted proteins [1,2]. Being deleted or mutated in up to 20% multiple myeloma (MM) patients, but intact in other cancers, the FAM46C gene is among the most frequent genomic hits in MM and a myeloma-specific onco-suppressor [3,4]. Results We have recently shown that the ER-specific action of FAM46C depends on its interaction with the ER transmembrane FNDC3 proteins. As a result, FAM46C concertedly boosts the expression of ER and Golgi proteins, potently upsizing the secretory apparatus and Ig secretion [5]. Our data suggest that in MM the FAM46C gene is under selective pressure to contain the proteosynthetic, oxidative and metabolic stress associated with Ig secretion, favoring myeloma cell survival and growth. In keeping with this, exogenous re-expression of FAM46C in mutated MM cells raised Ig secretory capacity beyond sustainability, causing ATP shortage, ROS accumulation and apoptosis. Interestingly, although able to increase the secretory capacity across different cell types, FAM46C overexpression induced apoptosis exclusively in MM cells, being well tolerated in non-professional secretors, suggesting a key role of the secretory cargo in FAM46C toxicity. Moreover, we discovered a broader function of FAM46C in ER homeostasis beyond mRNA stabilization. Indeed, we found that FAM46C is part of a novel integrated network coordinating the biogenesis of ribonucleoprotein complexes, ER protein translation and import, and ER expansion, to harmonize massive Ab production with protein homeostasis. Finally, our proteomic analysis unveiled a surprising opposite modulation of signal recognition particle (SRP) proteins mediated by FAM46C. Indeed, while 4 out of 6 SRP proteins were positively regulated by FAM46C, the heterodimeric members SRP9 and SRP14, mediating translational arrest, increased upon FAM46C loss. Conclusions We infer that PCs have the ability to sense FAM46C-induced ER expansion and modulate SRP composition to minimize translational pausing and maximize Ab manufacture. This novel integrated network offers a framework to identify unprecedented highly specific therapeutic targets against PC dyscrasias.

tRNA gene transcription by RNA polymerase III (Pol III) is a tightly regulated process, but dysregulated Pol III transcription is widely observed in cancers. Approximately 75% of all breast cancers are positive for expression of Estrogen Receptor alpha (ERα), which acts as a key driver of disease. MCF-7 cells rapidly upregulate tRNA gene transcription in response to estrogen and ChIP-PCR demonstrated ERα enrichment at tRNALeu and 5S rRNA genes in this breast cancer cell line. While these data implicate the ERα as a Pol III transcriptional regulator, how widespread this regulation is across the 631 tRNA genes has yet to be revealed. Through analyses of ERα ChIP-seq datasets, we show that ERα interacts with hundreds of tRNA genes, not only in MCF-7 cells, but also in primary human breast tumours and distant metastases. The extent of ERα association with tRNA genes varies between breast cancer cell lines and does not correlate with levels of ERα binding to its canonical target gene GREB1. Amongst other Pol III-transcribed genes, ERα is consistently enriched at the long non-coding RNA gene RMRP, a positive regulator of cell cycle progression that is subject to focal amplification in tumours. Another Pol III template targeted by ERα is the RN7SL1 gene, which is strongly implicated in breast cancer pathology by inducing inflammatory responses in tumours. Our data indicate that Pol III-transcribed non-coding genes should be added to the list of ERα targets in breast cancer.

Idiopathic inflammatory myopathies are a heterogeneous group of potentially treatable myopathies. They are classified, on the basis of clinical and histopathological features, into four subtypes: dermatomyositis, polymyositis, necrotizing autoimmune myositis and inclusion-body myositis. Myositis-associated antibodies and myositis-specific autoantibodies are frequently found in patients with idiopathic inflammatory myopathies, and are useful in the diagnosis and classification. Anti-histidyl transfer RNA synthetase antibody is the most widely prevalent and is highly specific for polymyositis. Signal recognition particle antibody is also a specific autoantibody for polymyositis, but it is infrequent and rarely found in patients having other myositis-specific autoantibodies. We present a man with polymyositis who had both antibodies in serum, which is considered an extremely rare clinical situation. Here we analyze the clinical course and findings, and examine the effect of the coexistence and possible interaction on prognosis.

RNA has an important role not only as the messenger of genetic information but also as a regulator of gene expression. Given its central role in cell biology, there is significant interest in studying the structural and dynamic behavior of RNA in relation to other biomolecules. Coarse-grain molecular dynamics simulations are a key tool to that end. Here, we have extended the coarse-grain Martini force field to include RNA after our recent extension to DNA. In the same way DNA was modeled, the tertiary structure of RNA is constrained using an elastic network. This model, therefore, is not designed for applications involving RNA folding but rather offers a stable RNA structure for studying RNA interactions with other (bio) molecules. The RNA model is compatible with all other Martini models and opens the way to large-scale explicit-solvent molecular dynamics simulations of complex systems involving RNA.

The universally conserved signal recognition particle (SRP) co-translationally delivers newly synthesized membrane and secretory proteins to the target cellular membrane. The only exception is found in the chloroplast of green plants, where the chloroplast SRP (cpSRP) post-translationally targets light-harvesting chlorophyll a/b-binding proteins (LHCP) to the thylakoid membrane. The mechanism and regulation of this post-translational mode of targeting by cpSRP remain unclear. Using biochemical and biophysical methods, here we show that anionic phospholipids activate the cpSRP receptor cpFtsY to promote rapid and stable cpSRP54·cpFtsY complex assembly. Furthermore, the stromal domain of the Alb3 translocase binds with high affinity to and regulates GTP hydrolysis in the cpSRP54·cpFtsY complex, suggesting that cpFtsY is primarily responsible for initial recruitment of the targeting complex to Alb3. These results suggest a new model for the sequential recruitment, remodeling, and unloading of the targeting complex at membrane translocase sites in the post-translational cpSRP pathway.

Necrotizing autoimmune myopathy (NAM) is a rare and emerging entity of idiopathic inflammatory myopathy (IIM). They have been associated with connective tissue disorders, viral infections, malignancy, anti-signal recognition particle (SRP) and anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase protein (with or without the use of statins). Anti-SRP associated NAM has different clinical and histological characteristics that differentiate them from other IIM, resulting in a poor prognosis. Very few cases treated with rituximab have been published, with varying clinical response. Here we describe a case of anti-SRP associated NAM refractory to conventional immunosuppressants and its successful long-term management with the combination of rituximab, corticosteroids and methotrexate.

Summary The series of RNA folding events that occur during transcription can critically influence cellular RNA function. Here, we present reconstructing RNA dynamics from data (R2D2), a method to uncover details of cotranscriptional RNA folding. We model the folding of the Escherichia coli signal recognition particle (SRP) RNA and show that it requires specific local structural fluctuations within a key hairpin to engender efficient cotranscriptional conformational rearrangement into the functional structure. All-atom molecular dynamics simulations suggest that this rearrangement proceeds through an internal toehold-mediated strand-displacement mechanism, which can be disrupted with a point mutation that limits local structural fluctuations and rescued with compensating mutations that restore these fluctuations. Moreover, a cotranscriptional folding intermediate could be cleaved in vitro by recombinant E. coli RNase P, suggesting potential cotranscriptional processing. These results from experiment-guided multi-scale modeling demonstrate that even an RNA with a simple functional structure can undergo complex folding and processing during synthesis.

Protein synthesis must be finely tuned in the developing nervous system as the final essential step of gene expression. This study investigates the architecture of ribosomes from the neocortex during neurogenesis, revealing Ebp1 as a high-occupancy 60S peptide tunnel exit (TE) factor during protein synthesis at near-atomic resolution by cryoelectron microscopy (cryo-EM). Ribosome profiling demonstrated Ebp1-60S binding is highest during start codon initiation and N-terminal peptide elongation, regulating ribosome occupancy of these codons. Membrane-targeting domains emerging from the 60S tunnel, which recruit SRP/Sec61 to the shared binding site, displace Ebp1. Ebp1 is particularly abundant in the early-born neural stem cell (NSC) lineage and regulates neuronal morphology. Ebp1 especially impacts the synthesis of membrane-targeted cell adhesion molecules (CAMs), measured by pulsed stable isotope labeling by amino acids in cell culture (pSILAC)/bioorthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry (MS). Therefore, Ebp1 is a central component of protein synthesis, and the ribosome TE is a focal point of gene expression control in the molecular specification of neuronal morphology during development.

Background: In eukaryotic cells, many proteins have to be transported across or inserted into the endoplasmic reticulum membrane during their biogenesis on the ribosome. This process is facilitated by the protein translocon, a highly dynamic multi-subunit membrane protein complex. Scope of review: The aim of this review is to summarize the current structural knowledge about protein translocon components in mammals. Major conclusions: Various structural biology approaches have been used in synergy to characterize the translocon in recent years. X-ray crystallography and cryoelectron microscopy single particle analysis have yielded highly detailed insights into the structure and functional mechanism of the protein-conducting channel Sec61, which constitutes the functional core of the translocon. Cryoelectron tomography and subtomogram analysis have advanced our understanding of the overall structure, molecular organization and compositional heterogeneity of the translocon in a native membrane environment. Tomography densities at subnanometer resolution revealed an intricate network of interactions between the ribosome, Sec61 and accessory translocon components that assist in protein transport, membrane insertion and maturation. General significance: The protein translocon is a gateway for approximately one third of all synthesized proteins and numerous human diseases are associated with malfunctioning of its components. Thus, detailed insights into the structure and molecular organization of the translocon will not only advance our understanding of membrane protein biogenesis in general, but they can potentially pave the way for novel therapeutic approaches against human diseases. (C) 2016 Elsevier B.V. All rights reserved.

Translocation into the endoplasmic reticulum (ER) is the first step in the biogenesis of thousands of eukaryotic endomembrane proteins. Although functional ER translocation has been avidly studied, little is known about the quality control mechanisms that resolve faulty translocational states. One such faulty state is translocon clogging, in which the substrate fails to properly translocate and obstructs the translocon pore. To shed light on the machinery required to resolve clogging, we carried out a systematic screen in Saccharomyces cerevisiae that highlighted a role for the ER metalloprotease Ste24. We could demonstrate that Ste24 approaches the translocon upon clogging, and it interacts with and generates cleavage fragments of the clogged protein. Importantly, these functions are conserved in the human homolog, ZMPSTE24, although disease-associated mutant forms of ZMPSTE24 fail to clear the translocon. These results shed light on a new and critical task of Ste24, which safeguards the essential process of translocation.

The Alu element is the most successful human genomic parasite affecting development and causing disease. It originated as a retrotransposon during early primate evolution of the gene encoding the signal recognition particle (SRP) RNA. We defined a minimal Alu RNA sufficient for effective retrotransposition and determined a high-resolution structure of its complex with the SRP9/14 proteins. The RNA adopts a compact, closed conformation that matches the envelope of the SRP Alu domain in the ribosomal translation elongation factor-binding site. Conserved structural elements in SRP RNAs support an ancient function of the closed conformation that predates SRP9/14. Structure-based mutagenesis shows that retrotransposition requires the closed conformation of the Alu ribonucleoprotein particle and is consistent with the recognition of stalled ribosomes. We propose that ribosome stalling is a common cause for the cis-preference of the mammalian L1 retrotransposon and for the efficiency of the Alu RNA in hijacking nascent L1 reverse transcriptase.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recognition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2 infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. Our results uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to suppress host defenses., Graphical Abstract, Highlights • NSP16 binds mRNA recognition domains of U1/U2 snRNAs and disrupts mRNA splicing • NSP1 binds in the mRNA entry channel of the ribosome to disrupt protein translation • NSP8 and NSP9 bind the signal recognition particle and disrupt protein trafficking • These disruptions of protein production suppress the interferon response to infection, SARS-CoV-2 proteins directly engage host RNAs to dysregulate essential steps of protein production and suppress the interferon response.

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 A), 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.

Protein targeting is critical in all living organisms and involves a signal recognition particle (SRP), an SRP receptor and a translocase. In co-translational targeting, interactions between these proteins is mediated by the ribosome. In chloroplasts, the light-harvesting chlorophyll-binding protein (LHCP) in the thylakoid membrane is targeted post-translationally without a ribosome. A multi-domain chloroplast-specific subunit of the SRP, cpSRP43, is proposed to take on the role of coordinating the sequence of targeting events. Here, we demonstrate that cpSRP43 exhibits significant inter-domain dynamics, which reduce upon binding its SRP binding partner, cpSRP54. We showed that the affinity of cpSRP43 for the binding motif of LHCP (L18) increases when cpSRP43 is complexed to the binding motif of cpSRP54 (cpSRP54pep). These results support the conclusion that substrate binding to the chloroplast SRP is modulated by protein structural dynamics, in which a major role of cpSRP54 is to improve substrate-binding efficiency to the cpSRP.

Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders characterized by cytopenias, ineffective hematopoiesis, myelodysplasia, and an increased risk of acute myeloid leukemia (AML). While sporadic MDS is primarily a disease of the elderly, MDS in children and young and middle-aged adults is frequently associated with underlying genetic predisposition syndromes. In addition to the classic hereditary bone marrow failure syndromes (BMFS) such as Fanconi Anemia and Dyskeratosis Congenita, in recent years there has been an increased awareness of non-syndromic familial MDS/AML predisposition syndromes such as those caused by mutations in GATA2, RUNX1, CEBPA, and SRP72 genes. Here, we will discuss the importance of recognizing an underlying genetic predisposition syndrome a patient with MDS, will review clinical scenarios when genetic predisposition should be considered, and will provide a practical overview of the common BMFS and familial MDS/AML syndromes which may be encountered in adult patients with MDS.

In this issue, Ahl et al. (2015) and Doucet et al. (2015) illuminate structural and functional features of substrates that promote integration of RNA molecules into the human genome by LINE retrotransposons, contributing to the ∼ 50% of the human genome that has been colonized by mobile genetic elements.

Virus-encoded molecular signatures, such as cytosolic double-stranded or otherwise biochemically distinct RNA species, trigger cellular antiviral signaling. Cytoplasmic proteins recognize these non-self RNAs and activate signal transduction pathways that drive the expression of virus-induced genes, including the primary antiviral cytokine, IFNβ, and diverse direct and indirect antiviral effectors [1–4]. One important group of cytosolic RNA sensors known as the RIG-I like receptors (RLRs) is comprised of three proteins that are similar in structure and function. The RLR proteins, RIG-I, MDA5, and LGP2, share the ability to recognize nucleic acid signatures produced by virus infections and activate antiviral signaling. Emerging evidence indicates that RNA detection by RLRs culminates in the assembly of dynamic multimeric ribonucleoprotein (RNP) complexes. These RNPs can act as signaling platforms that are capable of propagating and amplifying antiviral signaling responses. Despite their common domain structures and similar abilities to induce antiviral responses, the RLRs differ in their enzymatic properties, their intrinsic abilities to recognize RNA, and their ability to assemble into filamentous complexes. This molecular specialization has enabled the RLRs to recognize and respond to diverse virus infections, and to mediate both unique and overlapping functions in immune regulation [5, 6].

Of 207 adult patients with idiopathic inflammatory myopathies, detection of autoantibodies by RNA immunoprecipitation showed that 99 patients (48%) were antibody-positive. We divided these 99 into five subgroups: anti-signal recognition particle (SRP), anti-aminoacyl transfer RNA synthetase, anti-Ku, anti-U1RNP, and anti-SSA/B. Younger age at onset, severe weakness, muscle atrophy, elevated creatine kinase, and necrosis in muscle fibers without inflammatory cell infiltration were found significantly more frequently among the patients with anti-SRP antibodies (n=41) compared to the antibody-negative patients (n=108). Autoantibody detection by RNA immunoprecipitation can provide useful information associated with clinical and histological findings.

More than 30years of research have revealed that the dynamic nanomotor SecA is a central player in bacterial protein secretion. SecA associates with the SecYEG channel and transports polypeptides post-translationally to the trans side of the cytoplasmic membrane. It comprises a helicase-like ATPase core coupled to two domains that provide specificity for preprotein translocation. Apart from SecYEG, SecA associates with multiple ligands like ribosomes, nucleotides, lipids, chaperones and preproteins. It exerts its essential contribution in two phases. First, SecA, alone or in concert with chaperones, helps mediate the targeting of the secretory proteins from the ribosome to the membrane. Next, at the membrane it converts chemical energy to mechanical work and translocates preproteins through the SecYEG channel. SecA is a highly dynamic enzyme, it exploits disorder–order kinetics, swiveling and dissociation of domains and dimer to monomer transformations that are tightly coupled with its catalytic function. Preprotein signal sequences and mature domains exploit these dynamics to manipulate the nanomotor and thus achieve their export at the expense of metabolic energy. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

Myopathy with anti-signal recognition particle antibodies (SRP) is generally thought to be immune-mediated necrotic myopathy in previous studies. We report the clinical and histopathological features of myopathy with anti-SRP antibodies in Chinese patients. Muscle biopsy and immunoblots for myositis antibodies were carried out in 123 patients with idiopathic inflammatory myopathy. Among them, 16 (13.0%) patients had anti-SRP antibodies. Age of onset ranged from 24 to 77 years, and the disease began insidiously. Fourteen of 16 patients presented with chronic progression of proximal limb weakness, with 6 having myalgia. Serum creatine kinase levels ranged from 400 to 9082 IU/L. Muscle biopsies showed necrotic and/or regenerative muscle fibers in all 16, infiltrates of lymphocytes in 11 and morphological features of muscular dystrophy in 7. Eleven patients showed focal or diffuse major histocompatibility complex class 1 expression in sarcolemma or cytoplasm of muscle fibers, with 9 showing deposition of membrane attack complex in necrotic muscle fibers and 2 around capillaries. These findings indicate that anti-SRP antibodies are most likely to be related to IMNM. Myopathy with anti-SRP antibodies is not infrequent in Chinese patients with idiopathic inflammatory myopathy.

The ability to efficiently produce recombinant proteins in a secreted form is highly desirable and cultured mammalian cells such as CHO cells have become the preferred host as they secrete proteins with human-like post-translational modifications. However, attempts to express high levels of particular proteins in CHO cells may consistently result in low yields, even for non-engineered proteins such as immunoglobulins. In this study, we identified the responsible faulty step at the stage of translational arrest, translocation and early processing for such a "difficult-to-express" immunoglobulin, resulting in improper cleavage of the light chain and its precipitation in an insoluble cellular fraction unable to contribute to immunoglobulin assembly. We further show that proper processing and secretion were restored by over-expressing human signal receptor protein SRP14 and other components of the secretion pathway. This allowed the expression of the difficult-to-express protein to high yields, and it also increased the production of an easy-to-express protein. Our results demonstrate that components of the secretory and processing pathways can be limiting, and that engineering of the secretory pathway may be used to improve the secretion efficiency of therapeutic proteins from CHO cells.

The concept of the Truncated Light-harvesting chlorophyll Antenna (TLA) size, as a tool by which to maximize sunlight utilization and photosynthetic productivity in microalgal mass cultures or high-density plant canopies, is discussed. TLA technology is known to improve sunlight-to-product energy conversion efficiencies and is hereby exemplified by photosynthetic productivity estimates of wild type and a TLA strain under simulated mass culture conditions. Recent advances in the generation of TLA-type mutants by targeting genes of the chloroplast signal-recognition particle (CpSRP) pathway, affecting the thylakoid membrane assembly of light-harvesting proteins, are also summarized. Two distinct CpSRP assembly pathways are recognized, one entailing post-translational, the other a co-translational mechanism. Differences between the post-translational and co-translational integration mechanisms are outlined, as these pertain to the CpSRP-mediated assembly of thylakoid membrane protein complexes in higher plants and green microalgae. The applicability of the CpSRP pathway genes in efforts to generate TLA-type strains with enhanced solar energy conversion efficiency in photosynthesis is evaluated.

Most newly synthesized proteins destined for the secretory pathway contain a signal peptide (SP) that triggers cotranslational translocation into the endoplasmic reticulum (ER). However, how small polypeptides undergo ER translocation is not fully understood. In this issue of JBC, Guo et al. describe a mechanism for posttranslational translocation of small secretory proteins featuring a positive charge within the SP N-terminal region. Defects in this element disrupt proper secretion and explain the effects of genetic mutations associated with one type of diabetes.

Signal Recognition Particle secretory system is one of the major secretory system organized in Mycobacterium tuberculosis H37Rv (M. tuberculosis). SRP system consists of three major components that work in a coordinated manner to produce a significant result in form of SRP secretion system. ftsY gene is essential part of the SRP secretary system of M. tuberculosis therefore might be an important part for its pathogenesis also. Mutation in ftsY gene might be responsible for decrease in pathogenesis of this bacterium. Here, the manuscript highlights the impact of various point mutations on different sites of ftsY protein. For comparing the level of mutations impact on its structure and functions, various bioinformatics approaches had been applied. Mutation on this gene had been searched by collecting their sequential and structural information. EASE-MM server predicted the sequence based single point mutation and select top ten hits which are W273G, F386G, W155G, F362G, L347G, L256G, I384G, F416G, L345G and L179G with maximum decrease in stability. These mutations then further checked by different servers PROVEAN, iStable in sequential manner and also done by considering structural information by using SDM, DUET, and DynaMut. I-Mutant Suit 3.0 and STRUM were used for subsequent analysis of mutations based on its sequence and structure. All mutations show deleterious effect or decreases stability to some extent. DynaMut server was also used for understanding the effect of mutations on interatomic interaction which shows that these mutations disrupts these interactions. As M. tuberculosis has a rising effect on TB day by day, therefore it is necessary to take a serious accomplishment against this disease. Considering these mutations and understanding their molecular effects might provide essential facts regarding M. tuberculosis regulation and thus can help in generating effective treatment.

The universally conserved signal recognition particle (SRP) system mediates the targeting of membrane proteins to the translocon in a multistep process controlled by GTP hydrolysis. Here we present the 2.6 Å crystal structure of the GTPase domains of the E. coli SRP protein (Ffh) and its receptor (FtsY) in complex with the tetraloop and the distal region of SRP-RNA, trapped in the activated state in presence of GDP:AlF_4. The structure reveals the atomic details of FtsY recruitment and, together with biochemical experiments, pinpoints G83 as the key RNA residue that stimulates GTP hydrolysis. Insertion of G83 into the FtsY active site orients a single glutamate residue provided by Ffh (E277), triggering GTP hydrolysis and complex disassembly at the end of the targeting cycle. The complete conservation of the key residues of the SRP-RNA and the SRP protein implies that the suggested chemical mechanism of GTPase activation is applicable across all kingdoms.

The mechanism of necrotizing myopathy associated with antibodies to signal recognition particle (SRP) remains unclear. We investigated the effect of anti-SRP+serum and complement on cell viability in myoblast cultures. Cell viability was only slightly reduced by incubation with anti-SRP+serum compared with control serum. However, the addition of fresh complement resulted in a marked reduction in cell survival. Surface immunostaining for SRP, C3c and C5b-9 was demonstrated in cultures pre-incubated with anti-SRP+serum and complement, and in muscle biopsies from patients with myopathy. These findings provide further support for a complement-dependent antibody-mediated mechanism in anti-SRP associated myopathy.

Nascent chains are known to be targeted to the endoplasmic reticulum membrane either by a signal recognition particle (SRP)-dependent co-translational or by an SRP-independent post-translational translocation route depending on signal sequences. Using a set of model and cellular proteins carrying an N-terminal signal anchor sequence of controlled hydrophobicity and yeast mutant strains defective in SRP or Sec62 function, the hydrophobicity-dependent targeting efficiency and targeting pathway preference were systematically evaluated. Our results suggest that an SRP-dependent co-translational and an SRP-independent post-translational translocation are not mutually exclusive for signal anchor proteins and that moderately hydrophobic ones require both SRP and Sec62 for proper targeting and translocation to the endoplasmic reticulum. Further, defect in Sec62 selectively reduced signal sequences inserted in an N(in)-C(out) (type II) membrane topology, implying an undiscovered role of Sec62 in regulating the orientation of the signal sequence in an early stage of translocation.

Because of the lack of post-translational glycosylation, Escherichia coli is not a preferable host for immunoglobulin G (IgG) production. However, recent successes in the developments of aglycosylated IgG variants that do not require glycosylation for effector functions have increased the likelihood of using E. coli for IgG production. Here, we have developed a new E. coli host-vector system for enhanced production of recombinant IgG using: (i) a combination of SRP/Sec-dependent pathways for the efficient secretion of heavy and light chains in the periplasm; (ii) co-expression of periplasmic foldase (DsbC) for efficient assembly of IgG in the periplasm; and (iii) co-expression of Ffh for enhancing the SRP machinery. Finally, with engineered host-vector system, fed-batch cultivations were conducted at four different conditions, and under an optimized condition, up to 62 mg/L of active full-length IgG was produced during a 28-h cultivation.

Protein aggregation is detrimental to the maintenance of proper protein homeostasis in all cells. To overcome this problem, cells have evolved a network of molecular chaperones to prevent protein aggregation and even reverse existing protein aggregates. The most extensively studied disaggregase systems are ATP-driven macromolecular machines. Recently, we reported an alternative disaggregase system in which the 38-kDa subunit of chloroplast signal recognition particle (cpSRP43) efficiently reverses the aggregation of its substrates, the light-harvesting chlorophyll a/b-binding (LHC) proteins, in the absence of external energy input. To understand the molecular mechanism of this novel activity, here we used biophysical and biochemical methods to characterize the structure and nature of LHC protein aggregates. We show that LHC proteins form micellar, disc-shaped aggregates that are kinetically stable and detergent-resistant. Despite the nonamyloidal nature, the LHC aggregates have a defined global organization, displaying the chaperone recognition motif on its solvent-accessible surface. These findings suggest an attractive mechanism for recognition of the LHC aggregate by cpSRP43 and provide important constraints to define the capability of this chaperone.

TatC, a subunit of the twin arginine translocase, is a 6-membrane-spanning protein exposing three periplasmic loops. We have used TatC as a model system to examine how multispanning proteins insert into the membrane. To assay translocation of each of the three loops of TatC across the membrane, we used trypsin mapping, proteinase K mapping, and chemical modification methods. Here, we show that the signal recognition particle is required for targeting TatC to the inner membrane of Escherichiacoli. While translocation of loops 1 and 2 is strictly dependent on the Sec translocase and the YidC insertase, translocation of loop 3 does not depend on the translocase or insertase. None of the periplasmic loops require SecA or the proton motive force for membrane translocation. This work demonstrates a strategy where all the loops of a multispanning membrane protein can be monitored individually. The membrane translocation mechanism of each periplasmic loop can be complex with different energy and translocase requirements for a multispanning membrane protein.

We report the case of a 15-year-old Japanese girl with myopathy associated with antibodies to a signal recognition particle (anti-SRP myopathy). The patient presented with progressive symmetrical proximal muscle weakness that caused difficulty in walking within 3 months, and marked elevation of the serum creatine kinase levels. A skeletal muscle biopsy revealed active necrotic and regenerating processes, with mild inflammatory changes. Based on the above findings, the patient was diagnosed as having anti-SRP myopathy. Only a limited number of pediatric patients with anti-SRP myopathy has been reported previously, with usually a poor prognosis. Early diagnosis is important for obtaining a better prognosis in patients with anti-SRP myopathy.

Aplastic anemia (AA) and myelodysplasia (MDS) are forms of bone marrow failure that are often part of the same progressive underlying disorder. While most cases are simplex and idiopathic, some show a clear pattern of inheritance; therefore, elucidating the underlying genetic cause could lead to a greater understanding of this spectrum of disorders. We used a combination of exome sequencing and SNP haplotype analysis to identify causative mutations in a family with a history of autosomal-dominant AA/MDS. We identified a heterozygous mutation in SRP72, a component of the signal recognition particle (SRP) that is responsible for the translocation of nascent membrane-bound and excreted proteins to the endoplasmic reticulum. A subsequent screen revealed another autosomal-dominant family with an inherited heterozygous SRP72 mutation. Transfection of these sequences into mammalian cells suggested that these proteins localize incorrectly within the cell. Furthermore, coimmunoprecipitation of epitope-tagged SRP72 indicated that the essential RNA component of the SRP did not fully associate with one of the SRP72 variants. These results suggest that inherited mutations in a component of the SRP have a role in the pathophysiology of AA/MDS, identifying a third pathway for developing these disorders alongside transcription factor and telomerase mutations.

The mechanosensitive channel MscL in the inner membrane of Escherichia coli is a homopentameric complex involved in homeostasis when cells are exposed to hypoosmotic conditions. The E. coli MscL protein is synthesized as a polypeptide of 136 amino acid residues and uses the bacterial signal recognition particle for membrane targeting. The protein is inserted into the membrane independently of the Sec translocon but requires YidC. Depletion of YidC inhibits translocation of the protein across the membrane. Insertion of MscL occurs primarily in a proton motive force-independent manner. The hydrophilic loop region of MscL has 29 residues that include 5 charged residues. Altering the charges in the periplasmic loop of MscL affects the requirements for membrane insertion. The introduction of one, two or three negatively charged amino acids makes the insertion dependent on the electrochemical membrane potential and gradually dependent on the Sec translocon, whereas the addition of five negatively charged residues as well as the addition of three positively charged residues inhibits membrane insertion of MscL. However, we find that the mutant with three uncharged residues requires both the SecYEG complex and YidC but not SecA for membrane insertion. In vivo cross-linking data showed that the newly synthesized MscL interacts with YidC and with SecY. Therefore, the MscL mutants use a membrane insertion mechanism that involves SecYEG and YidC simultaneously.

The signal recognition particle (SRP) is a universally conserved cellular machinery responsible for delivering membrane and secretory proteins to the proper cellular destination. The precise mechanism by which fidelity is achieved by the SRP pathway within the in vivo environment is yet to be understood. Previous studies have focused on the SRP pathway in isolation. Here we describe another important factor that modulates substrate selection by the SRP pathway: the ongoing synthesis of the nascent polypeptide chain by the ribosome. A slower translation elongation rate rescues the targeting defect of substrate proteins bearing mutant, suboptimal signal sequences both in vitro and in vivo. Consistent with a kinetic origin of this effect, similar rescue of protein targeting was also observed with mutant SRP receptors or SRP RNAs that specifically compromise the kinetics of SRP-receptor interaction during protein targeting. These data are consistent with a model in which ongoing protein translation is in constant kinetic competition with the targeting of the nascent proteins by the SRP and provides an important factor to regulate the fidelity of substrate selection by the SRP.

Posttranslational targeting of the light-harvesting chlorophyll a,b-binding proteins depends on the function of the chloroplast signal recognition particle, its receptor cpFtsY, and the translocase Alb3. The thylakoid membrane protein Alb3 of Arabidopsis chloroplasts belongs to the evolutionarily conserved YidC/Oxa1/Alb3 protein family; the members of this family facilitate the insertion, folding, and assembly of membrane proteins in bacteria, mitochondria, and chloroplasts. Here, we analyzed the interaction sites of full-length Alb3 with the cpSRP pathway component cpSRP43 by using in vitro and in vivo studies. Bimolecular fluorescence complementation and Alb3 proteoliposome studies showed that the interaction of cpSRP43 is dependent on a binding domain in the C terminus of Alb3 as well as an additional membrane-embedded binding site in the fifth transmembrane domain (TMD5) of Alb3. The C-terminal binding domain was mapped to residues 374–388, and the binding domain within TMD5 was mapped to residues 314–318 located close to the luminal end of TMD5. A direct binding between cpSRP43 and these binding motifs was shown by pepspot analysis. Further studies using blue-native gel electrophoresis revealed that full-length Alb3 is able to form dimers. This finding and the identification of a membrane-embedded cpSRP43 binding site in Alb3 support a model in which cpSRP43 inserts into a dimeric Alb3 translocation pore during cpSRP-dependent delivery of light-harvesting chlorophyll a,b-binding proteins.

Co-translational protein targeting to the membrane is mediated by the signal recognition particle and its receptor (FtsY). Their homologous GTPase domains interact at the membrane and form a heterodimer in which both GTPases are activated. The prerequisite for protein targeting is the interaction of FtsY with phospholipids. However, the mechanism of FtsY regulation by phospholipids remained unclear. Here we show that the N terminus of FtsY (A domain) is natively unfolded in solution and define the complete membrane-targeting sequence. We show that the membrane-targeting sequence is highly dynamic in solution, independent of nucleotides and directly responds to the density of anionic phospholipids by a random coil-helix transition. This conformational switch is essential for tethering FtsY to membranes and activates the GTPase for its subsequent interaction with the signal recognition particle. Our results underline the dynamics of lipid-protein interactions and their importance in the regulation of protein targeting and translocation across biological membranes.

Protein translocation across the endoplasmic reticulum membrane occurs via a "translocon" channel formed by the Sec61p complex. In yeast, two channels exist: the canonical Sec61p channel and a homolog called Ssh1p. Here, we used trapped translocation intermediates to demonstrate that a specific signal recognition particle-dependent substrate, Sec71p, is targeted exclusively to Ssh1p. Strikingly, we found that, in the absence of Ssh1p, precursor could be successfully redirected to canonical Sec61p, demonstrating that the normal targeting reaction must involve preferential sorting to Ssh1p. Our data therefore demonstrate that Ssh1p is the primary translocon for Sec71p and reveal a novel sorting mechanism at the level of the endoplasmic reticulum membrane enabling precursors to be directed to distinct translocons. Interestingly, the Ssh1p-dependent translocation of Sec71p was found to be dependent upon Sec63p, demonstrating a previously unappreciated functional interaction between Sec63p and the Ssh1p translocon.

All living cells have co-translational pathways for targeting membrane proteins. Co-translation pathways for secretory proteins also exist but mostly in eukaryotes. Unlike secretory proteins, the biosynthetic pathway of most membrane proteins is conserved through evolution and these proteins are usually synthesized by membrane-bound ribosomes. Translation on the membrane requires that both the ribosomes and the mRNAs be properly localized. Theoretically, this can be achieved by several means. (i) The current view is that the targeting of cytosolic mRNA-ribosome-nascent chain complexes (RNCs) to the membrane is initiated by information in the emerging hydrophobic nascent polypeptides. (ii) The alternative model suggests that ribosomes may be targeted to the membrane also constitutively, whereas the appropriate mRNAs may be carried on small ribosomal subunits or targeted by other cellular factors to the membrane-bound ribosomes. Importantly, the available experimental data do not rule out the possibility that cells may also utilize both pathways in parallel. In any case, it is well documented that a major player in the targeting pathway is the signal recognition particle (SRP) system composed of the SRP and its receptor (SR). Although the functional core of the SRP system is evolutionarily conserved, its composition and biological practice come with different flavors in various organisms. This review is dedicated mainly to the Escherichia (E.) coli SRP, where the biochemical and structural properties of components of the SRP system have been relatively characterized, yielding essential information about various aspects of the pathway. In addition, several cellular interactions of the SRP and its receptor have been described in E. coli, providing insights into their spatial function. Collectively, these in vitro studies have led to the current view of the targeting pathway [see (i) above]. Interestingly, however, in vivo studies of the role of the SRP and its receptor, with emphasis on the temporal progress of the pathway, elicited an alternative hypothesis [see (ii) above]. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

Secretory proteins destined for the lumen of the endoplasmic reticulum are key regulators of cellular functions and are thus subject to several levels of quality control. A recent study finds that the earliest step in secretory protein biogenesis, i.e. binding of the signal recognition particle to the signal sequence of the nascent peptide, is subject to a quality control process termed RAPP for “regulation of aberrant protein production”. This process involves AGO2 and mRNA degradation.

Summary The Escherichia coli signal recognition particle (SRP) receptor, FtsY, plays a fundamental role in co-translational targeting of membrane proteins via the SRP pathway. Efficient targeting relies on membrane interaction of FtsY and heterodimerization with the SRP protein Ffh, which is driven by detachment of α helix (αN1) in FtsY. Here we show that apart from the heterodimer, FtsY forms a nucleotide-dependent homodimer on the membrane, and upon αN1 removal also in solution. Homodimerization triggers reciprocal stimulation of GTP hydrolysis and occurs in vivo. Biochemical characterization together with integrative modeling suggests that the homodimer employs the same interface as the heterodimer. Structure determination of FtsY NG+1 with GMPPNP shows that a dimerization-induced conformational switch of the γ-phosphate is conserved in Escherichia coli, filling an important gap in SRP GTPase activation. Our findings add to the current understanding of SRP GTPases and may challenge previous studies that did not consider homodimerization of FtsY.

The signal recognition particle (SRP) dependent secretion pathway is as an attractive alternative to Sec-dependent export for the production of disulfide-bonded and/or fast-folding recombinant proteins in the Escherichia coli periplasm. SRP, which shares a ribosomal attachment site with the molecular chaperone trigger factor (TF), recognizes highly hydrophobic signal sequence as they emerge from the ribosome and delivers ribosome nascent chain complexes to FtsY for subsequent cotranslational translocation of target proteins across the SecYEG pore. However, like in the case of Sec-dependent export, secretory yields can be limited by the accumulation of precursor proteins in the cytoplasm. Using leech carboxypeptidase inhibitor (LCI) fused to the SRP-dependent DsbA signal sequence as a model system, we show that a null mutation in the gene encoding TF (Deltatig) or SRP co-expression reduce pre-LCI accumulation by half, and that quantitative export can be achieved by combining the two strategies. Interestingly, enhanced precursor processing did not alter periplasmic LCI levels but increased the amount of protein excreted in the growth medium. All mature LCI was nearly fully active and an 80% increase in productivity was achieved in Deltatig cells alone due to their faster growth. Our results show that competition between SRP and TF can interfere with efficient export of recombinant proteins targeted to the SRP pathway and establish TF-deficient strains and SRP co-expression as a simple solution to improve yields.

Translation-coupled protein translocation requires that mRNAs encoding secreted and membrane proteins (mSMPs) reach the ER membrane. The classical view is that the signal recognition particle (SRP) pathway delivers translating signal sequence-containing proteins to the SRP receptor present on the ER surface and engages the translocation machinery. However, recent studies demonstrate both SRP- and translation-independent mRNA recruitment to the ER, and that mRNAs encoding non-signal sequence-containing cytosolic proteins (mCPs) might be full-time residents of ER membranes. Furthermore, translation-independent cis-acting sequence elements present in both mCPs and mSMPs appear to govern the ability of mRNAs to associate with ER. Thus, a more complex picture of how and why mRNAs target the ER is emerging.

Escherichia colt is one of the preferred bacteria for studies on the energetics and regulation of respiration Respiratory chains consist of primary dehydrogenases and terminal reductases or oxidases linked by quinones. In order to assemble this complex arrangement of protein complexes, synthesis of the subunits occurs in the cytoplasm followed by assembly in the cytoplasm and/or membrane, the incorporation of metal or organic cofactors and the anchoring of the complex to the membrane In the case of exported metalloproteins, synthesis, assembly and incorporation of metal cofactors must be completed before translocation across the cytoplasmic membrane Coordination data on these processes is, however, scarce In this review, we discuss the various processes that respiratory proteins must undergo for correct assembly and functional coupling to the electron transport chain in E colt. Targeting to and translocation across the membrane together with cofactor synthesis and insertion are discussed in a general manner followed by a review of the coordinated biogenesis of individual respiratory enzyme complexes. Lastly, we address the supramolecular organization of respiratory enzymes into supercomplexes and their localization to specialized domains in the membrane (C) 2010 Elsevier B V All rights reserved

Signal recognition particle (SRP) and its receptor (SR) co-translationally target membrane and secretory proteins to membrane specifically and efficiently through multiple layers of regulation. Differential SRP binding to ribosome, SRP-SR association rate, and GTP hydrolysis ensure specificity for substrate over non-substrate. However, there is a debate on whether SRP interacts with its substrate when a target sequence is still buried inside ribosome exit tunnel during the early stage of translation. Previous studies, which only focus on SRP binding to ribosome, produce contradictory results. In this study, we examine the full picture of SRP's multiple layers of regulation at each translation intermediate carrying increasing length of nascent chain. FRET-based SRP-ribosome binding affinity assay and fluorescence detected SRP-SR association rate measurement give the formation of key intermediates in SRP targeting cycle as a function of nascent chain length. Both parameters differ for more than two orders of magnitude between N-terminal and full exposure of targeting sequence from the end of ribosome exit tunnel. Such discrimination is comparable to the difference between substrate and non-substrate. Using e. coli as a model system, our results show that SRP targets its substrate exclusively after the emergence of a transmembrane domain or signal sequence from ribosome exit tunnel.

In living systems, polypeptide chains are synthesised on ribosomes, molecular machines composed of over 50 protein and nucleic acid molecules. As nascent chains emerge from the ribosomal exit tunnel and into the cellular environment, the majority must fold into specific structures in order to function. In this article we discuss recent approaches designed to reveal how such folding occurs and review our current knowledge of this complex self-assembly process.

Recent studies have shown that cytoplasmic proteins are exported efficiently in Escherichia coli only if they are attached to signal peptides that are recognized by the signal recognition particle and are thereby targeted to the SecYEG complex cotranslationally. The evidence suggests that the entry of these proteins into the secretory pathway at an early stage of translation is necessary to prevent them from folding into a translocation-incompetent conformation. We found, however, that several glycolytic enzymes attached to signal peptides that are recognized by the signal recognition particle were exported inefficiently. Based on previous studies of post-translational export, we hypothesized that the export block was due to the presence of basic residues at the extreme N terminus of each enzyme. Consistent with our hypothesis, we found that the introduction of negatively charged residues into this segment increased the efficiency of export. Export efficiency was sensitive to the number, position, and sequence context of charged residues. The importance of charge for efficient export was underscored by an in silico analysis that revealed a conserved negative charge bias at the N terminus of the mature region of bacterial presecretory proteins. Our results demonstrate that cotranslational targeting of a protein to the E. coli SecYEG complex does not ensure its export but that export also depends on a subsequent event (most likely the initiation of translocation) that involves sequences both within and just beyond the signal peptide.