Signal Recognition Particle Receptor Research Articles

Electrostatics and Intrinsic Disorder Drive Translocon Binding of the SRP Receptor FtsY.

Integral membrane proteins in bacteria are co‐translationally targeted to the SecYEG translocon for membrane insertion via the signal recognition particle (SRP) pathway. The SRP receptor FtsY and its N‐terminal A domain, which is lacking in any structural model of FtsY, were studied using NMR and fluorescence spectroscopy. The A domain is mainly disordered and highly flexible; it binds to lipids via its N terminus and the C‐terminal membrane targeting sequence. The central A domain binds to the translocon non‐specifically and maintains disorder. Translocon targeting and binding of the A domain is driven by electrostatic interactions. The intrinsically disordered A domain tethers FtsY to the translocon, and because of its flexibility, allows the FtsY NG domain to scan a large area for binding to the NG domain of ribosome‐bound SRP, thereby promoting the formation of the quaternary transfer complex at the membrane.

Electrostatics and Intrinsic Disorder Drive Translocon Binding of the SRP Receptor FtsY

AbstractIntegral membrane proteins in bacteria are co‐translationally targeted to the SecYEG translocon for membrane insertion via the signal recognition particle (SRP) pathway. The SRP receptor FtsY and its N‐terminal A domain, which is lacking in any structural model of FtsY, were studied using NMR and fluorescence spectroscopy. The A domain is mainly disordered and highly flexible; it binds to lipids via its N terminus and the C‐terminal membrane targeting sequence. The central A domain binds to the translocon non‐specifically and maintains disorder. Translocon targeting and binding of the A domain is driven by electrostatic interactions. The intrinsically disordered A domain tethers FtsY to the translocon, and because of its flexibility, allows the FtsY NG domain to scan a large area for binding to the NG domain of ribosome‐bound SRP, thereby promoting the formation of the quaternary transfer complex at the membrane.

Variable protein profiles in extracellular products of the protistan parasite Perkinsus olseni among regions of the Spanish coast

The variability of the protein expression profiling in the extracellular products (ECPs) of in vitro cultured Perkinsus olseni deriving from 4 regions of the Spanish coast was evaluated. The regions involved were the rías of Arousa and Pontevedra (Galicia, NW Spain), Carreras River (Andalusia, SW Spain) and Delta de l’Ebre (Catalonia, NE Spain). P. olseni in vitro clonal cultures were produced from parasite isolates from four clams from each region. Proteins released by the in vitro cultured parasites were isolated and separated by two dimensional electrophoresis (2DE). Qualitative comparison of protein expression profiles in the P. olseni ECPs among clones from all the regions was performed with PD Quest software. Around 130 spots were counted in the gels from ECPs of P. olseni clones from each region, of which 23 spots were shared by clones from all the regions and various spots were representative from clones of one region (appear in every clonal culture from that region but did not in every one of the other regions). A total of 34 spots were excised from the gels and analysed for sequencing. The protein cathepsin B, involved in proteolysis, the signal recognition particle receptor subunit β, involved in protein transport through membranes, and a protein belonging to N-acetyl transferase superfamily, involved in biosynthesis, were identified in spots shared by P. olseni ECPs from all regions. Pepsin A precursor, involved in proteolysis; heat shock protein (HSP) 60; and phosphoserine aminotransferase, involved in biosynthesis, were representative of P. olseni ECPs from Ría de Arousa, while peroxiredoxin V, involved in oxidation–reduction, was representative of P. olseni ECPs from Ría de Pontevedra. Differences in released proteins suggest different virulence or resistance to host attack between parasites from different locations.

Ribosome binding induces repositioning of the signal recognition particle receptor on the translocon

Cotranslational protein targeting delivers proteins to the bacterial cytoplasmic membrane or to the eukaryotic endoplasmic reticulum membrane. The signal recognition particle (SRP) binds to signal sequences emerging from the ribosomal tunnel and targets the ribosome-nascent-chain complex (RNC) to the SRP receptor, termed FtsY in bacteria. FtsY interacts with the fifth cytosolic loop of SecY in the SecYEG translocon, but the functional role of the interaction is unclear. By using photo-cross-linking and fluorescence resonance energy transfer measurements, we show that FtsY-SecY complex formation is guanosine triphosphate independent but requires a phospholipid environment. Binding of an SRP-RNC complex exposing a hydrophobic transmembrane segment induces a rearrangement of the SecY-FtsY complex, which allows the subsequent contact between SecY and ribosomal protein uL23. These results suggest that direct RNC transfer to the translocon is guided by the interaction between SRP and translocon-bound FtsY in a quaternary targeting complex.

Structure and Switch Cycle of SRβ as Ancestral Eukaryotic GTPase Associated with Secretory Membranes

G proteins of the Ras-family of small GTPases trace the evolution of eukaryotes. The earliest branching involves the closely related Arf, Sar1, and SRβ GTPases associated with secretory membranes. SRβ is an integral membrane component of the signal recognition particle (SRP) receptor that targets ribosome-nascent chain complexes to the ER. How SRβ integrates into the regulation of SRP-dependent membrane protein biogenesis is not known. Here we show that SRβ-GTP interacts with ribosomes only in presence of SRα and present crystal structures of SRβ in complex with the SRX domain of SRα in the GTP-bound state at 3.2 Å, and of GDP- and GDP · Mg(2+)-bound SRβ at 1.9 Å and 2.4 Å, respectively. We define the GTPase switch cycle of SRβ and identify specific differences to the Arf and Sar1 families with implications for GTPase regulation. Our data allow a better integration of SRβ into the scheme of protein targeting.

Signal-sequence induced conformational changes in the signal recognition particle.

Co-translational protein targeting is an essential, evolutionarily conserved pathway for delivering nascent proteins to the proper cellular membrane. In this pathway, the signal recognition particle (SRP) first recognizes the N-terminal signal sequence of nascent proteins and subsequently interacts with the SRP receptor. For this, signal sequence binding in the SRP54 M domain must be effectively communicated to the SRP54 NG domain that interacts with the receptor. Here we present the 2.9 Å crystal structure of unbound- and signal sequence bound SRP forms, both present in the asymmetric unit. The structures provide evidence for a coupled binding and folding mechanism in which signal sequence binding induces the concerted folding of the GM linker helix, the finger loop, and the C-terminal alpha helix αM6. This mechanism allows for a high degree of structural adaptability of the binding site and suggests how signal sequence binding in the M domain is coupled to repositioning of the NG domain.

Regulation of Structural Dynamics within a Signal Recognition Particle Promotes Binding of Protein Targeting Substrates

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 among these proteins are 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 multidomain 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 interdomain dynamics that are reduced 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.

A functional link between the co-translational protein translocation pathway and the UPR

Upon endoplasmic reticulum (ER) stress, the transmembrane endoribonuclease Ire1α performs mRNA cleavage reactions to increase the ER folding capacity. It is unclear how the low abundant Ire1α efficiently finds and cleaves the majority of mRNAs at the ER membrane. Here, we reveal that Ire1α forms a complex with the Sec61 translocon to cleave its mRNA substrates. We show that Ire1α's key substrate, XBP1u mRNA, is recruited to the Ire1α-Sec61 translocon complex through its nascent chain, which contains a pseudo-transmembrane domain to utilize the signal recognition particle (SRP)-mediated pathway. Depletion of SRP, the SRP receptor or the Sec61 translocon in cells leads to reduced Ire1α-mediated splicing of XBP1u mRNA. Furthermore, mutations in Ire1α that disrupt the Ire1α-Sec61 complex causes reduced Ire1α-mediated cleavage of ER-targeted mRNAs. Thus, our data suggest that the Unfolded Protein Response is coupled with the co-translational protein translocation pathway to maintain protein homeostasis in the ER during stress conditions.

Ribosome–SRP–FtsY cotranslational targeting complex in the closed state

The signal recognition particle (SRP)-dependent pathway is essential for correct targeting of proteins to the membrane and subsequent insertion in the membrane or secretion. In Escherichia coli, the SRP and its receptor FtsY bind to ribosome-nascent chain complexes with signal sequences and undergo a series of distinct conformational changes, which ensures accurate timing and fidelity of protein targeting. Initial recruitment of the SRP receptor FtsY to the SRP-RNC complex results in GTP-independent binding of the SRP-FtsY GTPases at the SRP RNA tetraloop. In the presence of GTP, a closed state is adopted by the SRP-FtsY complex. The cryo-EM structure of the closed state reveals an ordered SRP RNA and SRP M domain with a signal sequence-bound. Van der Waals interactions between the finger loop and ribosomal protein L24 lead to a constricted signal sequence-binding pocket possibly preventing premature release of the signal sequence. Conserved M-domain residues contact ribosomal RNA helices 24 and 59. The SRP-FtsY GTPases are detached from the RNA tetraloop and flexible, thus liberating the ribosomal exit site for binding of the translocation machinery.

Co-translational membrane association of the Escherichia coli SRP receptor.

The signal recognition particle (SRP) receptor is a major player in the pathway of membrane protein biogenesis in all organisms. The receptor functions as a membrane-bound entity but very little is known about its targeting to the membrane. Here, we demonstrate in vivo that the Escherichia coli SRP receptor targets the membrane co-translationally. This requires emergence from the ribosome of the four-helix-long N-domain of the receptor, of which only helices 2-4 are required for co-translational membrane attachment. The results also suggest that the targeting might be regulated co-translationally. Taken together, our in vivo studies shed light on the biogenesis of the SRP receptor and its hypothetical role in targeting ribosomes to the E. coli membrane.

Dynamic Interaction of the Sec Translocon with the Chaperone PpiD

The Sec translocon constitutes a ubiquitous protein transport channel that consists in bacteria of the three core components: SecY, SecE, and SecG. Additional proteins interact with SecYEG during different stages of protein transport. During targeting, SecYEG interacts with SecA, the SRP receptor, or the ribosome. Protein transport into or across the membrane is then facilitated by the interaction of SecYEG with YidC and the SecDFYajC complex. During protein transport, SecYEG is likely to interact also with the protein quality control machinery, but details about this interaction are missing. By in vivo and in vitro site-directed cross-linking, we show here that the periplasmic chaperone PpiD is located in front of the lateral gate of SecY, through which transmembrane domains exit the SecY channel. The strongest contacts were found to helix 2b of SecY. Blue native PAGE analyses verify the presence of a SecYEG-PpiD complex in native Escherichia coli membranes. The PpiD-SecY interaction was not influenced by the addition of SecA and only weakly influenced by binding of nontranslating ribosomes to SecYEG. In contrast, PpiD lost contact to the lateral gate of SecY during membrane protein insertion. These data identify PpiD as an additional and transient subunit of the bacterial SecYEG translocon. The data furthermore demonstrate the highly modular and versatile composition of the Sec translocon, which is probably essential for its ability to transport a wide range of substrates across membranes in bacteria and eukaryotes.

The Code for Directing Proteins for Translocation across ER Membrane: SRP Cotranslationally Recognizes Specific Features of a Signal Sequence

The signal recognition particle (SRP) cotranslationally recognizes signal sequences of secretory proteins and targets ribosome-nascent chain complexes to the SRP receptor in the endoplasmic reticulum membrane, initiating translocation of the nascent chain through the Sec61 translocon. Although signal sequences do not have homology, they have similar structural regions: a positively charged N-terminus, a hydrophobic core and a more polar C-terminal region that contains the cleavage site for the signal peptidase. Here, we have used site-specific photocrosslinking to study SRP–signal sequence interactions. A photoreactive probe was incorporated into the middle of wild-type or mutated signal sequences of the secretory protein preprolactin by in vitro translation of mRNAs containing an amber-stop codon in the signal peptide in the presence of the Nε-(5-azido-2 nitrobenzoyl)-Lys-tRNAamb amber suppressor. A homogeneous population of SRP–ribosome-nascent chain complexes was obtained by the use of truncated mRNAs in translations performed in the presence of purified canine SRP. Quantitative analysis of the photoadducts revealed that charged residues at the N-terminus of the signal sequence or in the early part of the mature protein have only a mild effect on the SRP–signal sequence association. However, deletions of amino acid residues in the hydrophobic portion of the signal sequence severely affect SRP binding. The photocrosslinking data correlate with targeting efficiency and translocation across the membrane. Thus, the hydrophobic core of the signal sequence is primarily responsible for its recognition and binding by SRP, while positive charges fine-tune the SRP–signal sequence affinity and targeting to the translocon.

Regulation of cargo recognition, commitment, and unloading drives cotranslational protein targeting.

Efficient and accurate protein localization is essential to cells and requires protein-targeting machineries to both effectively capture the cargo in the cytosol and productively unload the cargo at the membrane. To understand how these challenges are met, we followed the interaction of translating ribosomes during their targeting by the signal recognition particle (SRP) using a site-specific fluorescent probe in the nascent protein. We show that initial recruitment of SRP receptor (SR) selectively enhances the affinity of SRP for correct cargos, thus committing SRP-dependent substrates to the pathway. Real-time measurement of cargo transfer from the targeting to translocation machinery revealed multiple factors that drive this event, including GTPase rearrangement in the SRP-SR complex, stepwise displacement of SRP from the ribosome and signal sequence by SecYEG, and elongation of the nascent polypeptide. Our results elucidate how active and sequential regulation of the SRP-cargo interaction drives efficient and faithful protein targeting.

Fidelity of cotranslational protein targeting by the signal recognition particle.

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.

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

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

Heat Shock Response Regulator Is Pinned to the Membrane

Heat is the enemy of stability, and nowhere is this more true than in living cells, whose delicately folded proteins fall apart as temperatures rise. To combat this crisis, cells mount a “heat shock response,” a central feature of which is the coordinated production of chaperone proteins, to refold proteins that can be salvaged, and proteases, to degrade those that can't. In the Escherichia coli bacterium, the heat shock response is coordinated by sigma-32, a protein that binds to RNA polymerase and directs it to the promoters of chaperone and protease genes. Much is known about the regulation of sigma-32, including a variety of positive and negative feedback loops that govern its expression and activity (increasing free chaperones, for instance, reduces sigma-32 expression). But it has been clear from some disparities between prediction and reality that some critical element of sigma-32 activity has remained elusive. In a new study in PLOS Biology, Bentley Lim, Carol Gross, and colleagues pin down that missing element, showing that, rather than being dispersed in the cytoplasm as previous work had assumed, sigma-32 must be tethered to the bacterial membrane to be properly regulated. The σ32 protein can promote transcription of chaperones and proteases while in the cytosol or associated with the inner membrane, but is properly regulated only when membrane associated. Green arrows denote transcription; red arrows denote negative ... The authors began by performing a genetic screen for new regulators of sigma-32. They found that reduction in expression of the signal recognition particle (SRP) receptor increased the activity of sigma-32, but also prevented its normal inhibition by chaperones. They used immunoprecipitation to show that sigma-32 co-precipitated with SRP, and gel filtration to show a direct interaction. These results immediately implied that sigma-32 localized to the inner bacterial membrane, since the function of SRP and its receptor is to target proteins there. The interaction of sigma-32 with SRP, and its localization to the membrane, could be reduced by mutation of sigma-32's so-called homeostatic control region, a small cluster of amino acids known to be critical for its regulation, further implicating membrane localization via SRP as a key to normal sigma-32 function. Finally, they artificially tethered a mutant in the homeostatic control region to the inner membrane using a viral coat protein. This single modification was able to confer full regulatory control of the sigma-32 heat shock response, including responsiveness to elevation of chaperones, even when SRP and the SRP receptor levels were reduced. The placement of sigma-32 at the inner membrane may allow it to sense the state of protein folding both in the cytoplasm and in the membrane, the authors argue. The SRP system targets a wide variety of proteins to the membrane, and is thought to minimize protein misfolding by making translation coincident with membrane insertion. The SRP-dependent presence of sigma-32 at the membrane may further reduce or mitigate the potential for accumulation of misfolded proteins during translation through its control of chaperones and proteases. Lim B, Miyazaki R, Neher S, Siegele DA, Ito K, et al. (2013) Heat Shock Transcription Factor σ32 Co-opts the Signal Recognition Particle to Regulate Protein Homeostasis in E. coli. doi:10.1371/journal.pbio.1001735

Molecular Mechanism of GTPase Activation at the Signal Recognition Particle (SRP) RNA Distal End

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.

Lactobacillus plantarum WCFS1 O-linked protein glycosylation: An extended spectrum of target proteins and modification sites detected by mass spectrometry

It has recently been shown that the major autolysin Acm2 from Lactobacillus plantarum WCFS1 undergoes intracellular O-GlcNAcylation [Fredriksen L, Mathiesen G, Moen A, Bron PA, Kleerebezem M, Eijsink VG, Egge-Jacobsen W. 2012. The major autolysin Acm2 from Lactobacillus plantarum undergoes cytoplasmic O-glycosylation. J Bacteriol. 194(2):325-333]. To gain more insight into the occurrence of this protein modification, methods based on the higher energy collisional fragmentation of the Orbitrap XL mass spectrometer to generate both diagnostic oxonium (glycan) ions and significant peptide sequencing information were used to detect and identify novel glycoproteins. This led to the identification of 10 novel glycoproteins, including four proteins with well-known functions in the cytoplasm, a compartment not previously recognized to contain glycosylated proteins in bacteria: the molecular chaperone DnaK, the E2 subunit of the pyruvate dehydrogenase complex PdhC, the signal recognition particle receptor FtsY and the DNA translocase FtsK1. Among the other, glycosylated proteins were two extracellular peptidoglycan hydrolases and a mucus-binding protein. In total, 49 glycosylation sites for N-acetylhexosamine (HexNAc) were detected in the 11 Lactobacillus glycoproteins found so far. Most of the attached glycans consisted of a single HexNAc per site, whereas hexose moieties were also found in a few cases (in both of the peptidoglycan hydrolases and in DnaK).

Structural basis of signal sequence surveillance and selection by the SRP–FtsY complex

Signal-recognition particle (SRP)-dependent targeting of translating ribosomes to membranes is a multistep quality-control process. Ribosomes that are translating weakly hydrophobic signal sequences can be rejected from the targeting reaction even after they are bound to the SRP. Here we show that the early complex, formed by Escherichia coli SRP and its receptor FtsY with ribosomes translating the incorrect cargo EspP, is unstable and rearranges inefficiently into subsequent conformational states, such that FtsY dissociation is favored over successful targeting. The N-terminal extension of EspP is responsible for these defects in the early targeting complex. The cryo-electron microscopy structure of this 'false' early complex with EspP revealed an ordered M domain of SRP protein Ffh making two ribosomal contacts, and the NG domains of Ffh and FtsY forming a distorted, flexible heterodimer. Our results provide a structural basis for SRP-mediated signal-sequence selection during recruitment of the SRP receptor.

Protein translocation across the inner membrane of Gram-negative bacteria: the Sec and Tat dependent protein transport pathways

Gram negative bacteria possess a large variety of protein transport systems, by which proteins that are synthesised in the cytosol are exported to destinations in the cell envelope or entirely secreted into the extracellular environment. The inner membrane (IM) contains three major transport systems for the translocation and insertion of signal sequence containing proteins: the Sec translocon, the YidC insertase, and the Tat system. The heterotrimeric SecYEG translocon forms a narrow channel in the membrane that serves a dual function; it allows the translocation of unfolded proteins across the pore and the integration of α-helical proteins into the IM. The YidC insertase is a multi-spanning membrane protein that cooperates with the SecYEG translocon during the integration of membrane proteins but also functions as an independent insertase. Depending upon the type of protein cargo that needs to be transported, the Signal Recognition Particle (SRP), the SRP receptor, SecA and chaperones are required to coordinate translation with transport and to target and energise the different transport systems. The Tat system consists of three membrane proteins (TatA, TatB and TatC) which in a still unknown manner accomplish the transmembrane passage of completely folded proteins and protein complexes.