The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Peter Walter - One of the best experts on this subject based on the ideXlab platform.
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real time observation of Signal Recognition Particle binding to actively translating ribosomes
eLife, 2014Co-Authors: Thomas R Noriega, Peter Walter, Jin Chen, Joseph D PuglisiAbstract:The Signal Recognition Particle (SRP) directs translating ribosome-nascent chain complexes (RNCs) that display a Signal sequence to protein translocation channels in target membranes. All previous work on the initial step of the targeting reaction, when SRP binds to RNCs, used stalled and non-translating RNCs. This meant that an important dimension of the co-translational process remained unstudied. We apply single-molecule fluorescence measurements to observe directly and in real-time E. coli SRP binding to actively translating RNCs. We show at physiologically relevant SRP concentrations that SRP-RNC association and dissociation rates depend on nascent chain length and the exposure of a functional Signal sequence outside the ribosome. Our results resolve a long-standing question: how can a limited, sub-stoichiometric pool of cellular SRP effectively distinguish RNCs displaying a Signal sequence from those that are not? The answer is strikingly simple: as originally proposed, SRP only stably engages translating RNCs exposing a functional Signal sequence.
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structures of srp54 and srp19 the two proteins that organize the ribonucleic core of the Signal Recognition Particle from pyrococcus furiosus
PLOS ONE, 2008Co-Authors: Pascal F Egea, Peter Walter, Johanna Napetschnig, Robert M StroudAbstract:In all organisms the Signal Recognition Particle (SRP), binds to Signal sequences of proteins destined for secretion or membrane insertion as they emerge from translating ribosomes. In Archaea and Eucarya, the conserved ribonucleoproteic core is composed of two proteins, the accessory protein SRP19, the essential GTPase SRP54, and an evolutionarily conserved and essential SRP RNA. Through the GTP-dependent interaction between the SRP and its cognate receptor SR, ribosomes harboring nascent polypeptidic chains destined for secretion are dynamically transferred to the protein translocation apparatus at the membrane. We present here high-resolution X-ray structures of SRP54 and SRP19, the two RNA binding components forming the core of the Signal Recognition Particle from the hyper-thermophilic archaeon Pyrococcus furiosus (Pfu). The 2.5 A resolution structure of free Pfu-SRP54 is the first showing the complete domain organization of a GDP bound full-length SRP54 subunit. In its ras-like GTPase domain, GDP is found tightly associated with the protein. The flexible linker that separates the GTPase core from the hydrophobic Signal sequence binding M domain, adopts a purely α-helical structure and acts as an articulated arm allowing the M domain to explore multiple regions as it scans for Signal peptides as they emerge from the ribosomal tunnel. This linker is structurally coupled to the GTPase catalytic site and likely to propagate conformational changes occurring in the M domain through the SRP RNA upon Signal sequence binding. Two different 1.8 A resolution crystal structures of free Pfu-SRP19 reveal a compact, rigid and well-folded protein even in absence of its obligate SRP RNA partner. Comparison with other SRP19•SRP RNA structures suggests the rearrangement of a disordered loop upon binding with the RNA through a reciprocal induced-fit mechanism and supports the idea that SRP19 acts as a molecular scaffold and a chaperone, assisting the SRP RNA in adopting the conformation required for its optimal interaction with the essential subunit SRP54, and proper assembly of a functional SRP.
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The Signal Recognition Particle (SRP) RNA Links Conformational Changes in the SRP to Protein Targeting
Molecular Biology of the Cell, 2007Co-Authors: Niels Bradshaw, Peter WalterAbstract:The RNA component of the Signal Recognition Particle (SRP) is universally required for cotranslational protein targeting. Biochemical studies have shown that SRP RNA participates in the central step of protein targeting by catalyzing the interaction of the SRP with the SRP receptor (SR). SRP RNA also accelerates GTP hydrolysis in the SRP·SR complex once formed. Using a reverse-genetic and biochemical analysis, we identified mutations in the E. coli SRP protein, Ffh, that abrogate the activity of the SRP RNA and cause corresponding targeting defects in vivo. The mutations in Ffh that disrupt SRP RNA activity map to regions that undergo dramatic conformational changes during the targeting reaction, suggesting that the activity of the SRP RNA is linked to the major conformational changes in the Signal sequence-binding subunit of the SRP. In this way, the SRP RNA may coordinate the interaction of the SRP and the SR with ribosome recruitment and transfer to the translocon, explaining why the SRP RNA is an indispensable component of the protein targeting machinery.
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targeting proteins to membranes structure of the Signal Recognition Particle
Current Opinion in Structural Biology, 2005Co-Authors: Pascal F Egea, Robert M Stroud, Peter WalterAbstract:In all three kingdoms of life, co-translational targeting of secretory and membrane proteins to the prokaryotic plasma membrane or eukaryotic endoplasmic reticulum is mediated by a ribonucleoprotein complex, the Signal Recognition Particle (SRP), and its membrane-associated receptor (SR). SRP binds to Signal sequences of nascent proteins as they emerge from the exit tunnel of the ribosome. The resulting targeting complex, composed of the SRP and the ribosome-nascent chain complex (RNC), then docks with the SR in a GTP-dependent manner. Passing through a complex series of conformational states, SRP and SR deliver the RNC to the translocon, which in turn mediates protein translocation across or integration into the membrane. The core structural and mechanistic principles of SRP-dependent protein targeting are universally conserved. Recent structural investigations combining X-ray crystallography and cryo-electron microscopy have provided new insights into three essentials steps of the SRP-dependent protein targeting cycle: the assembly and interaction of the SRP ribonucleoprotein core, the GTP-dependent SRP-SR association, and the interaction between SRP and the ribosome.
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co translational protein targeting by the Signal Recognition Particle
FEBS Letters, 2005Co-Authors: Shu-ou Shan, Peter WalterAbstract:The Signal Recognition Particle (SRP) mediates the co-translational targeting of nascent proteins to the eukaryotic endoplasmic reticulum membrane, or the bacterial plasma membrane. During this process, two GTPases, one in the SRP and one in the SRP receptor (SR), form a complex in which both proteins reciprocally activate the GTPase reaction of one another. The recent crystal structures of the T. aquaticus SRP · SR complex show that the two GTPases associate via an unusually extensive and highly cooperative interaction surface, and form a composite active site at the interface. GTPase activation proceeds through a unique mechanism, stimulated by both interactions between the twinned GTP molecules across the dimer interface and by conformational rearrangements that position catalytic residues in each active site with respect to the bound substrates. Distinct classes of mutations have been isolated that inhibit specific stages during SRP–SR complex formation and activation, suggesting discrete conformational stages during formation of the active SRP · SR complex. Each stage provides a potential control point in the targeting reaction at which regulation by additional components can be exerted, thus ensuring the binding and release of cargo at the appropriate time.
Irmgard Sinning - One of the best experts on this subject based on the ideXlab platform.
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structural basis for conserved regulation and adaptation of the Signal Recognition Particle targeting complex
Journal of Molecular Biology, 2016Co-Authors: Klemens Wild, Gert Bange, Domantas Motiejunas, Judith F Kribelbauer, Astrid Hendricks, Bernd Segnitz, Rebecca C Wade, Irmgard SinningAbstract:The Signal Recognition Particle (SRP) is a ribonucleoprotein complex with a key role in targeting and insertion of membrane proteins. The two SRP GTPases, SRP54 (Ffh in bacteria) and FtsY (SRα in eukaryotes), form the core of the targeting complex (TC) regulating the SRP cycle. The architecture of the TC and its stimulation by RNA has been described for the bacterial SRP system while this information is lacking for other domains of life. Here, we present the crystal structures of the GTPase heterodimers of archaeal (Sulfolobus solfataricus), eukaryotic (Homo sapiens), and chloroplast (Arabidopsis thaliana) SRP systems. The comprehensive structural comparison combined with Brownian dynamics simulations of TC formation allows for the description of the general blueprint and of specific adaptations of the quasi-symmetric heterodimer. Our work defines conserved external nucleotide-binding sites for SRP GTPase activation by RNA. Structural analyses of the GDP-bound, post-hydrolysis states reveal a conserved, magnesium-sensitive switch within the I-box. Overall, we provide a general model for SRP cycle regulation by RNA.
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lipids trigger a conformational switch that regulates Signal Recognition Particle srp mediated protein targeting
Journal of Biological Chemistry, 2011Co-Authors: Goran Stjepanovic, Gert Bange, Katja Kapp, Christian Graf, Richard Parlitz, Klemens Wild, Matthias P Mayer, Irmgard SinningAbstract: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.
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Protein targeting by the Signal Recognition Particle
Biological Chemistry, 2009Co-Authors: Przemyslaw Grudnik, Gert Bange, Irmgard SinningAbstract:Protein targeting by the Signal Recognition Particle (SRP) is universally conserved and starts with the Recognition of a Signal sequence in the context of a translating ribo- some. SRP54 and FtsY, two multidomain proteins with guanosine triphosphatase (GTPase) activity, are the cen- tral elements of the SRP system. They have to coordinate the presence of a Signal sequence with the presence of a vacant translocation channel in the membrane. For coordination the two GTPases form a unique, nearly symmetric heterodimeric complex in which the activation of GTP hydrolysis plays a key role for membrane inser- tion of substrate proteins. Recent results are integrated in an updated perception of the order of events in SRP- mediated protein targeting.
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structural basis for specific substrate Recognition by the chloroplast Signal Recognition Particle protein cpsrp43
Science, 2008Co-Authors: Katharina F Stengel, Klemens Wild, I Holdermann, Peter Cain, Colin Robinson, Irmgard SinningAbstract:Secretory and membrane proteins carry amino-terminal Signal sequences that, in cotranslational targeting, are recognized by the Signal Recognition Particle protein SRP54 without sequence specificity. The most abundant membrane proteins on Earth are the light-harvesting chlorophyll a/b binding proteins (LHCPs). They are synthesized in the cytoplasm, imported into the chloroplast, and posttranslationally targeted to the thylakoid membrane by cpSRP, a heterodimer formed by cpSRP54 and cpSRP43. We present the 1.5 angstrom crystal structure of cpSRP43 characterized by a unique arrangement of chromodomains and ankyrin repeats. The overall shape and charge distribution of cpSRP43 resembles the SRP RNA, which is absent in chloroplasts. The complex with the internal Signal sequence of LHCPs reveals that cpSRP43 specifically recognizes a DPLG peptide motif. We describe how cpSPR43 adapts the universally conserved SRP system to posttranslational targeting and insertion of the LHCP family of membrane proteins.
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the structure of the chloroplast Signal Recognition Particle srp receptor reveals mechanistic details of srp gtpase activation and a conserved membrane targeting site
FEBS Letters, 2007Co-Authors: Katharina F Stengel, Klemens Wild, Iris Holdermann, Irmgard SinningAbstract:Two GTPases in the Signal Recognition Particle and its receptor (FtsY) regulate protein targeting to the membrane by formation of a heterodimeric complex. The activation of both GTPases in the complex is essential for protein translocation. We present the crystal structure of chloroplast FtsY (cpFtsY) at 1.75 A resolution. The comparison with FtsY structures in different nucleotide bound states shows structural changes relevant for GTPase activation and provides insights in how cpFtsY is pre-organized for complex formation with cpSRP54. The structure contains an amino-terminal amphipathic helix similar to the membrane targeting sequence of Escherichia coli FtsY. In cpFtsY this motif is extended, which might be responsible for the enhanced attachment of the protein to the thylakoid membrane.
Shu-ou Shan - One of the best experts on this subject based on the ideXlab platform.
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molecular mechanism of cargo Recognition and handover by the mammalian Signal Recognition Particle
Cell Reports, 2021Co-Authors: Ahmad Jomaa, Shu-ou Shan, Sowmya Chandrasekar, Simon Eitzinger, Zikun Zhu, Kan Kobayashi, Nenad BanAbstract:Co-translational protein targeting to membranes by the Signal Recognition Particle (SRP) is a universally conserved pathway from bacteria to humans. In mammals, SRP and its receptor (SR) have many additional RNA features and protein components compared to the bacterial system, which were recently shown to play regulatory roles. Due to its complexity, the mammalian SRP targeting process is mechanistically not well understood. In particular, it is not clear how SRP recognizes translating ribosomes with exposed Signal sequences and how the GTPase activity of SRP and SR is regulated. Here, we present electron cryo-microscopy structures of SRP and SRP·SR in complex with the translating ribosome. The structures reveal the specific molecular interactions between SRP and the emerging Signal sequence and the elements that regulate GTPase activity of SRP·SR. Our results suggest the molecular mechanism of how eukaryote-specific elements regulate the early and late stages of SRP-dependent protein targeting.
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sequential activation of human Signal Recognition Particle by the ribosome and Signal sequence drives efficient protein targeting
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Sowmya Chandrasekar, Sangyoon Chung, Yuhsien Hwang Fu, Shimon Weiss, Shu-ou ShanAbstract:Signal Recognition Particle (SRP) is a universally conserved targeting machine that mediates the targeted delivery of ∼30% of the proteome. The molecular mechanism by which eukaryotic SRP achieves efficient and selective protein targeting remains elusive. Here, we describe quantitative analyses of completely reconstituted human SRP (hSRP) and SRP receptor (SR). Enzymatic and fluorescence analyses showed that the ribosome, together with a functional Signal sequence on the nascent polypeptide, are required to activate SRP for rapid recruitment of the SR, thereby delivering translating ribosomes to the endoplasmic reticulum. Single-molecule fluorescence spectroscopy combined with cross-complementation analyses reveal a sequential mechanism of activation whereby the ribosome unlocks the hSRP from an autoinhibited state and primes SRP to sample a variety of conformations. The Signal sequence further preorganizes the mammalian SRP into the optimal conformation for efficient recruitment of the SR. Finally, the use of a Signal sequence to activate SRP for receptor recruitment is a universally conserved feature to enable efficient and selective protein targeting, and the eukaryote-specific components confer upon the mammalian SRP the ability to sense and respond to ribosomes.
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fidelity of cotranslational protein targeting by the Signal Recognition Particle
Annual Review of Biophysics, 2014Co-Authors: Xin Zhang, Shu-ou ShanAbstract:Accurate folding, assembly, localization, and maturation of newly synthesized proteins are essential to all cells and require high fidelity in the protein biogenesis machineries that mediate these processes. Here, we review our current understanding of how high fidelity is achieved in one of these processes, the cotranslational targeting of nascent membrane and secretory proteins by the Signal Recognition Particle (SRP). Recent biochemical, biophysical, and structural studies have elucidated how the correct substrates drive a series of elaborate conformational rearrangements in the SRP and SRP receptor GTPases; these rearrangements provide effective fidelity checkpoints to reject incorrect substrates and enhance the fidelity of this essential cellular pathway. The mechanisms used by SRP to ensure fidelity share important conceptual analogies with those used by cellular machineries involved in DNA replication, transcription, and translation, and these mechanisms likely represent general principles for other complex cellular pathways.
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molecular mechanism of gtpase activation at the Signal Recognition Particle srp rna distal end
Journal of Biological Chemistry, 2013Co-Authors: Kuang Shen, Yaqiang Wang, Qi Zhang, Juli Feigon, Shu-ou ShanAbstract:The Signal Recognition Particle (SRP) RNA is a universally conserved and essential component of the SRP that mediates the co-translational targeting of proteins to the correct cellular membrane. During the targeting reaction, two functional ends in the SRP RNA mediate distinct functions. Whereas the RNA tetraloop facilitates initial assembly of two GTPases between the SRP and SRP receptor, this GTPase complex subsequently relocalizes ∼100 Å to the 5′,3′-distal end of the RNA, a conformation crucial for GTPase activation and cargo handover. Here we combined biochemical, single molecule, and NMR studies to investigate the molecular mechanism of this large scale conformational change. We show that two independent sites contribute to the interaction of the GTPase complex with the SRP RNA distal end. Loop E plays a crucial role in the precise positioning of the GTPase complex on these two sites by inducing a defined bend in the RNA helix and thus generating a preorganized Recognition surface. GTPase docking can be uncoupled from its subsequent activation, which is mediated by conserved bases in the next internal loop. These results, combined with recent structural work, elucidate how the SRP RNA induces GTPase relocalization and activation at the end of the protein targeting reaction.
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Signal Recognition Particle an essential protein targeting machine
Annual Review of Biochemistry, 2013Co-Authors: David Akopian, Xin Zhang, Kuang Shen, Shu-ou ShanAbstract:The Signal Recognition Particle (SRP) and its receptor compose a universally conserved and essential cellular machinery that couples the synthesis of nascent proteins to their proper membrane localization. The past decade has witnessed an explosion in in-depth mechanistic investigations of this targeting machine at increasingly higher resolutions. In this review, we summarize recent work that elucidates how the SRP and SRP receptor interact with the cargo protein and the target membrane, respectively, and how these interactions are coupled to a novel GTPase cycle in the SRP·SRP receptor complex to provide the driving force and enhance the fidelity of this fundamental cellular pathway. We also discuss emerging frontiers in which important questions remain to be addressed.
Joen Luirink - One of the best experts on this subject based on the ideXlab platform.
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posttranslational insertion of small membrane proteins by the bacterial Signal Recognition Particle
PLOS Biology, 2020Co-Authors: Ruth Steinberg, Stephen High, Joen Luirink, Andrea Origi, Ana Natriashvili, Pinku Sarmah, Mariya Licheva, Princess M Walker, Claudine Kraft, Wei ShiAbstract:Small membrane proteins represent a largely unexplored yet abundant class of proteins in pro- and eukaryotes. They essentially consist of a single transmembrane domain and are associated with stress response mechanisms in bacteria. How these proteins are inserted into the bacterial membrane is unknown. Our study revealed that in Escherichia coli, the 27-amino-acid-long model protein YohP is recognized by the Signal Recognition Particle (SRP), as indicated by in vivo and in vitro site-directed cross-linking. Cross-links to SRP were also observed for a second small membrane protein, the 33-amino-acid-long YkgR. However, in contrast to the canonical cotranslational Recognition by SRP, SRP was found to bind to YohP posttranslationally. In vitro protein transport assays in the presence of a SecY inhibitor and proteoliposome studies demonstrated that SRP and its receptor FtsY are essential for the posttranslational membrane insertion of YohP by either the SecYEG translocon or by the YidC insertase. Furthermore, our data showed that the yohP mRNA localized preferentially and translation-independently to the bacterial membrane in vivo. In summary, our data revealed that YohP engages an unique SRP-dependent posttranslational insertion pathway that is likely preceded by an mRNA targeting step. This further highlights the enormous plasticity of bacterial protein transport machineries.
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consequences of depletion of the Signal Recognition Particle in escherichia coli
Journal of Biological Chemistry, 2011Co-Authors: David Wickstrom, Joen Luirink, Samuel Wagner, Louise Baars, Jimmy A Ytterberg, Mirjam Klepsch, Klaas J Van Wijk, Janwillem De GierAbstract:Thus far, the role of the Escherichia coli Signal Recognition Particle (SRP) has only been studied using targeted approaches. It has been shown for a handful of cytoplasmic membrane proteins that their insertion into the cytoplasmic membrane is at least partially SRP-dependent. Furthermore, it has been proposed that the SRP plays a role in preventing toxic accumulation of mistargeted cytoplasmic membrane proteins in the cytoplasm. To complement the targeted studies on SRP, we have studied the consequences of the depletion of the SRP component Fifty-four homologue (Ffh) in E. coli using a global approach. The steady-state proteomes and the proteome dynamics were evaluated using one- and two-dimensional gel analysis, followed by mass spectrometry-based protein identification and immunoblotting. Our analysis showed that depletion of Ffh led to the following: (i) impaired kinetics of the biogenesis of the cytoplasmic membrane proteome; (ii) lowered steady-state levels of the respiratory complexes NADH dehydrogenase, succinate dehydrogenase, and cytochrome bo(3) oxidase and lowered oxygen consumption rates; (iii) increased levels of the chaperones DnaK and GroEL at the cytoplasmic membrane; (iv) a σ(32) stress response and protein aggregation in the cytoplasm; and (v) impaired protein synthesis. Our study shows that in E. coli SRP-mediated protein targeting is directly linked to maintaining protein homeostasis and the general fitness of the cell.
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sequence specific interactions of nascent escherichia coli polypeptides with trigger factor and Signal Recognition Particle
Journal of Biological Chemistry, 2006Co-Authors: Ronald S Ullers, Bauke Oudega, Edith N G Houben, Josef Brunner, N Harms, Joen LuirinkAbstract:As nascent polypeptides exit the ribosomal tunnel they immediately associate with chaperones, folding catalysts, and targeting factors. These interactions are decisive for the future conformation and destination of the protein that is being synthesized. Using Escherichia coli as a model organism, we have systematically analyzed how the earliest contacts of nascent polypeptides with cytosolic factors depend on the nature and future destination of the emerging sequence using a photo cross-linking approach. Together, the data suggest that the chaperone trigger factor is adjacent to emerging sequences by default, consistent with both its placement near the nascent chain exit site and its cellular abundance. The Signal Recognition Particle (SRP) effectively competes the contact with TF when a Signal anchor (SA) sequence of a nascent inner membrane protein appears outside the ribosome. The SRP remains in contact with the SA and downstream sequences during further synthesis of approximately 30 amino acids. The contact with trigger factor is then restored unless another transmembrane segment reinitiates SRP binding. Importantly and in contrast to published data, the SRP appears perfectly capable of distinguishing SA sequences from Signal sequences in secretory proteins at this early stage in biogenesis.
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Signal Recognition Particle srp mediated targeting and sec dependent translocation of an extracellular escherichia coli protein
Journal of Biological Chemistry, 2003Co-Authors: Robert Sijbrandi, Malene L Urbanus, Corinne Ten M Hagenjongman, Harris D Bernstein, Bauke Oudega, Ben R Otto, Joen LuirinkAbstract:Hemoglobin protease (Hbp) is a hemoglobin-degrading protein that is secreted by a human pathogenic Escherichia coli strain via the autotransporter mechanism. Little is known about the earliest steps in autotransporter secretion, i.e. the targeting to and translocation across the inner membrane. Here, we present evidence that Hbp interacts with the Signal Recognition Particle (SRP) and the Sec-translocon early during biogenesis. Furthermore, Hbp requires a functional SRP targeting pathway and Sec-translocon for optimal translocation across the inner membrane. SecB is not required for targeting of Hbp but can compensate to some extent for the lack of SRP. Hbp is synthesized with an unusually long Signal peptide that is remarkably conserved among a subset of autotransporters. We propose that these autotransporters preferentially use the cotranslational SRP/Sec route to avoid adverse effects of the exposure of their mature domains in the cytoplasm.
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nascent membrane and presecretory proteins synthesized in escherichia coli associate with Signal Recognition Particle and trigger factor
Molecular Microbiology, 1997Co-Authors: Quido A Valent, Janwillem De Gier, Corinne Ten M Hagenjongman, Bauke Oudega, Gunnar Von Heijne, Debra A Kendall, Joen LuirinkAbstract:The Escherichia coli Signal Recognition Particle (SRP) and trigger factor are cytoplasmic factors that interact with short nascent polypeptides of presecretory and membrane proteins produced in a heterologous in vitro translation system. In this study, we use an E. coli in vitro translation system in combination with bifunctional cross-linking reagents to investigate these interactions in more detail in a homologous environment. Using this approach, the direct interaction of SRP with nascent polypeptides that expose particularly hydrophobic targeting Signals is demonstrated, suggesting that inner membrane proteins are the primary physiological substrate of the E. coli SRP. Evidence is presented that the overproduction of proteins that expose hydrophobic polypeptide stretches, titrates SRP. In addition, trigger factor is efficiently cross-linked to nascent polypeptides of different length and nature, some as short as 57 amino acid residues, indicating that it is positioned near the nascent chain exit site on the E. coli ribosome.
Matthias Müller - One of the best experts on this subject based on the ideXlab platform.
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a derivative of lipid a is involved in Signal Recognition Particle secyeg dependent and independent membrane integrations
Journal of Biological Chemistry, 2006Co-Authors: Michael Moser, Kenichi Nishiyama, Ayao Ikegami, Emile Schiltz, Hajime Tokuda, Matthias MüllerAbstract:A cell-free system was developed that allows the correct integration of single and multispanning membrane proteins of Escherichia coli into proteoliposomes. We found that physiological levels of diacylglycerol were required to prevent spontaneous integration into liposomes even of the polytopic mannitol permease. Using diacylglycerol-containing proteoliposomes, we identified a novel integration-stimulating factor. Integration of mannitol permease was dependent on both the SecYEG translocon and this factor and was mediated by Signal Recognition Particle and Signal Recognition Particle receptor. Integration of M13 procoat, which is independent of both Signal Recognition Particle/Signal Recognition Particle receptor and SecYEG, was also promoted by this factor. Furthermore, the factor stimulated the post-translational translocation of presecretory proteins, suggesting that it also mediates integration of a Signal sequence. This factor was found to be a lipid A-derived membrane component possessing a peptide moiety.
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alternate recruitment of Signal Recognition Particle and trigger factor to the Signal sequence of a growing nascent polypeptide
Journal of Biological Chemistry, 2006Co-Authors: Gottfried Eisner, Michael J Moser, Ute Schafer, Konstanze Beck, Matthias MüllerAbstract:Abstract Different from cytoplasmic membrane proteins, presecretory proteins of bacteria usually do not require the Signal Recognition Particle for targeting to the Sec translocon. Nevertheless Signal sequences of presecretory proteins have been found in close proximity to Signal Recognition Particle immediately after they have emerged from the ribosome. We show here that at the ribosome, the molecular environment of a Signal sequence depends on the nature of downstream sequence elements that can cause an alternate recruitment of Signal Recognition Particle and the ribosome-associated chaperone Trigger factor to a growing nascent chain. While Signal Recognition Particle and Trigger factor might remain bound to the same ribosome, both ligands are clearly able to displace each other from a nascent chain. The data also imply that a Signal sequence owes its molecular environment to the fact that it remains closely apposed to the ribosomal exit site during growth of a nascent secretory protein.
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Signal Recognition Particle-dependent protein targeting, universal to all kingdoms of life.
Reviews of Physiology Biochemistry and Pharmacology, 1Co-Authors: Hans-georg Koch, Michael Moser, Matthias MüllerAbstract:The Signal Recognition Particle (SRP) and its membrane-bound receptor represent a ubiquitous protein-targeting device utilized by organisms as different as bacteria and humans, archaea and plants. The unifying concept of SRP-dependent protein targeting is that SRP binds to Signal sequences of newly synthesized proteins as they emerge from the ribosome. In eukaryotes this interaction arrests or retards translation elongation until SRP targets the ribosome-nascent chain complexes via the SRP receptor to the translocation channel. Such channels are present in the endoplasmic reticulum of eukaryotic cells, the thylakoids of chloroplasts, or the plasma membrane of prokaryotes. The minimal functional unit of SRP consists of a Signal sequence-recognizing protein and a small RNA. The as yet most complex version is the mammalian SRP whose RNA, together with six proteinaceous subunits, undergo an intricate assembly process. The preferential substrates of SRP possess especially hydrophobic Signal sequences. Interactions between SRP and its receptor, the ribosome, the Signal sequence, and the target membrane are regulated by GTP hydrolysis. SRP-dependent protein targeting in bacteria and chloroplasts slightly deviate from the canonical mechanism found in eukaryotes. Pro- and eukaryotic cells harbour regulatory mechanisms to prevent a malfunction of the SRP pathway.
