Signal Recognition Particle Cycle Research Articles

Spatiotemporal kinetics of the SRP pathway in live E. coli cells

Mechanistic details of the signal recognition particle (SRP)-mediated insertion of membrane proteins have been described from decades of invitro biochemical studies. However, the dynamics of the pathway inside the living cell remain obscure. By combining invivo single-molecule tracking with numerical modeling and simulated microscopy, we have constructed a quantitative reaction-diffusion model of the SRP cycle. Our results suggest that the SRP-ribosome complex finds its target, the membrane-bound translocon, through a combination of three-dimensional (3D) and 2D diffusional search, together taking on average 750 ms. During this time, the nascent peptide is expected to be elongated only 12 or 13 amino acids, which explains why, in Escherichia coli, no translation arrest is needed to prevent incorrect folding of the polypeptide in the cytosol. We also found that a remarkably high proportion (75%) of SRP bindings to ribosomes occur in the cytosol, suggesting that the majority of target ribosomes bind SRP before reaching the membrane. In combination with the average SRP cycling time, 2.2 s, this result further shows that the SRP pathway is capable of targeting all substrate ribosomes to translocons.

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

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

Structural Basis for Conserved Regulation and Adaptation of the Signal Recognition Particle Targeting Complex

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.

Dynamics of the SRP cycle: From scanning to targeting

Inner‐membrane proteins in Escherichia coli are synthesized on ribosomes that are bound to the translocation pore (translocon) and enter the membrane co‐translationally. Ribosomes are targeted to the translocon by the signal recognition particle (SRP) and the SRP receptor, FtsY. Targeting is initiated when SRP is recruited to ribosomes that expose a signal‐anchor sequence (SAS). Here we report the kinetics of the interaction of SRP with ribosomes in different functional states and with FtsY, as followed by stopped‐flow monitoring FRET between fluorophores in SRP and the ribosome or FtsY. Rapid SRP binding to the ribosome is followed by two rearrangements the forward rate constants of which are independent of the functional state of the ribosomes. Conversely, ribosome‐SRP complex dissociation is very slow with ribosomes exposing an SAS, whereas the complex with non‐translating ribosomes dissociates rapidly. SAS‐induced stabilization of SRP on the ribosome is correlated with an acceleration of receptor binding. Thus, SRP can rapidly scan all ribosomes until it encounters a ribosome exposing an SAS, where SRP binding is strongly stabilized and membrane targeting is promoted by accelerating targeting complex formation with FtsY and, presumably, the translocon. The formation of the targeting complex is correlated with the lengthening of the nascent peptide within the exit tunnel of the ribosome.

Signal sequence–independent SRP-SR complex formation at the membrane suggests an alternative targeting pathway within the SRP cycle

Protein targeting by the signal recognition particle (SRP) and the bacterial SRP receptor FtsY requires a series of closely coordinated steps that monitor the presence of a substrate, the membrane, and a vacant translocon. Although the influence of substrate binding on FtsY-SRP complex formation is well documented, the contribution of the membrane is largely unknown. In the current study, we found that negatively charged phospholipids stimulate FtsY-SRP complex formation. Phospholipids act on a conserved positively charged amphipathic helix in FtsY and induce a conformational change that strongly enhances the FtsY-lipid interaction. This membrane-bound, signal sequence-independent FtsY-SRP complex is able to recruit RNCs to the membrane and to transfer them to the Sec translocon. Significantly, the same results were also observed with an artificial FtsY-SRP fusion protein, which was tethered to the membrane via a transmembrane domain. This indicates that substrate recognition by a soluble SRP is not essential for cotranslational targeting in Escherichia coli. Our findings reveal a remarkable flexibility of SRP-dependent protein targeting, as they indicate that substrate recognition can occur either in the cytosol via ribosome-bound SRP or at the membrane via a preassembled FtsY-SRP complex.

A signal-anchor sequence stimulates signal recognition particle binding to ribosomes from inside the exit tunnel

Sorting of eukaryotic membrane and secretory proteins depends on recognition of ribosome-bound nascent chain signal sequences by the signal recognition particle (SRP). The current model suggests that the SRP cycle is initiated when a signal sequence emerges from the ribosomal tunnel and binds to SRP. Then elongation is slowed until the SRP-bound ribosome-nascent chain complex (RNC) is targeted to the SRP receptor in the endoplasmic reticulum (ER) membrane. The RNC is then transferred to the translocon, SRP is released, and translation resumes. Because RNCs do not target to the translocon efficiently if nascent chains become too long, the window for SRP to identify its substrates is short. We now show that a transmembrane signal-anchor sequence (SA) significantly enhances binding of SRP to RNCs even before the SA emerges from the ribosomal tunnel. In this mode, SRP does not contact the SA directly but is in close proximity to the portion of the nascent polypeptide that has already left the ribosomal tunnel. Early recruitment of SRP provides a mechanism to expand the window for substrate identification. We suggest that the dynamics of the SRP-ribosome interaction is affected not only by the direct binding of SRP to an exposed signal sequence but also by properties of the translating ribosome that are triggered from within the tunnel.

Use of endogenous signal sequences for transient production and efficient secretion by moss (Physcomitrella patens) cells

BackgroundEfficient targeting to appropriate cell organelles is one of the bottlenecks for the production of recombinant proteins in plant systems. A common practice is to use the native secretory signal peptide of the heterologous protein to be produced. Though general features of secretion signals are conserved between plants and animals, the broad sequence variability among signal peptides suggests differing efficiency of signal peptide recognition.ResultsAiming to improve secretion in moss bioreactors, we quantitatively compared the efficiency of two human signal peptides and six signals from recently isolated moss (Physcomitrella patens) proteins. We therefore used fusions of the different signals to heterologous reporter sequences for transient transfection of moss cells and measured the extra- and intracellular accumulation of the recombinant proteins rhVEGF and GST, respectively. Our data demonstrates an up to fivefold higher secretion efficiency with endogenous moss signals compared to the two utilised human signal peptides.ConclusionFrom the distribution of extra- and intracellular recombinant proteins, we suggest translational inhibition during the signal recognition particle-cycle (SRP-cycle) as the most probable of several possible explanations for the decreased extracellular accumulation with the human signals. In this work, we report on the supremacy of moss secretion signals over the utilised heterologous ones within the moss-bioreactor system. Though the molecular details of this effect remain to be elucidated, our results will contribute to the improvement of molecular farming systems.

FtsY, the bacterial signal‐recognition particle receptor, interacts functionally and physically with the SecYEG translocon

Co-translational membrane targeting of proteins by the bacterial signal-recognition particle (SRP) requires the specific interaction of the SRP-ribosome nascent chain complex with FtsY, the bacterial SRP receptor (SR). FtsY is homologous to the SRalpha-subunit of the eukaryotic SR, which is tethered to the endoplasmic-reticulum membrane by its interaction with the integral SRbeta-subunit. In contrast to SRalpha, FtsY is partly membrane associated and partly located in the cytosol. However, the mechanisms by which FtsY associates with the membrane are unclear. No gene encoding an SRbeta homologue has been found in bacterial genomes, and the presence of an FtsY-specific membrane receptor has not been shown so far. We now provide evidence for the direct interaction between FtsY and the SecY translocon. This interaction offers an explanation of how the bacterial SRP cycle is regulated in response to available translocation channels.

A structural step into the SRP cycle.

Co-translational targeting of secretory and membrane proteins to the translocation machinery is mediated by the signal recognition particle (SRP) and its membrane-bound receptor (SR) in all three domains of life. Although the overall composition of the SRP system differs, the central ribonucleoprotein core and the general mechanism of GTP-dependent targeting are highly conserved. Recently, structural studies have contributed significantly to our understanding of the molecular organization of SRP. SRP appears as a structurally flexible particle modulated and regulated by its interactions with the ribosome-nascent chain complex, the translocon and the SR. The SRP core (SRP54 with its cognate RNA binding site) plays a central role in these interactions and communicates the different binding states by long-range interdomain communication. Based on recent structures of SRP54, a model for signal peptide binding stimulating the GTP affinity during the first step of the SRP cycle is presented. The model is placed in the context of the recent structures of mammalian SRP bound to a ribosome-nascent chain complex and of a subcomplex of SRP-SR.

Determinants of a protein-induced RNA switch in the large domain of signal recognition particle identified by systematic-site directed mutagenesis.

Signal recognition particle (SRP) is a ribonucleoprotein complex that associates with ribosomes to promote the co-translational translocation of proteins across biological membranes. Human SRP RNA molecules exist in two distinct conformations, SR-A and SR-B, which may exchange during the assembly of the particle or could play a functional role in the SRP cycle. We have used systematic site-directed mutagenesis of the SRP RNA to determine the electrophoretic mobilities of altered RNA molecules, and we have identified the nucleotides that avert the formation of the two conformers. The conformer behavior of the various RNAs was addressed quantitatively by calculating a value zeta as an indicator of conformational variability. Single loose A-like forms were induced by changes in helix 5 [nucleotides (nt) at positions 111-128 or 222-231], helix 6 (nt at positions 141-150) and helix 7 (nt at position 169 and 170), whereas other mutations in helix 6 and helix 8 preserved the conformational variability of the mutant RNA molecules. The more compact B-like form was induced only when a small region (129-CAAUAU-134), located in the 5'-proximal portion of helix 6, was altered. Since this region is part of the binding site for SRP19, we suggest that protein SRP19 uses nucleotides at 129-134 to trigger the formation of the favored SR-B-form, and thus directs an early step in the hierarchical assembly of the large SRP domain.

The Srp54 GTPase Is Essential for Protein Export in the Fission Yeast Schizosaccharomyces pombe

Signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein required for targeting a subset of presecretory proteins to the endoplasmic reticulum (ER) membrane. Here we report the results of a series of experiments to define the function of the Schizosaccharomyces pombe homolog of the 54-kDa subunit of mammalian SRP. One-step gene disruption reveals that the Srp54 protein, like SRP RNA, is essential for viability in S. pombe. Precursor to the secretory protein acid phosphatase accumulates in cells in which Srp54 synthesis has been repressed under the control of a regulated promoter, indicating that S. pombe SRP functions in protein targeting. In common with other Srp54 homologs, the S. pombe protein has a modular structure consisting of an amino-terminal G (GTPase) domain and a carboxyl-terminal M (methionine-rich) domain. We have analyzed the effects of 17 site-specific mutations designed to alter the function of each of the four GTPase consensus motifs individually. Several alleles, including some with relatively conservative amino acid substitutions, confer lethal or conditional phenotypes, indicating that GTP binding and hydrolysis are critical to the in vivo role of the protein. Two mutations (R to L at position 194 [R194L] and R194H) which were designed, by analogy to oncogenic mutations in rats, to dramatically decrease the catalytic rate and one (T248N) predicted to alter nucleotide binding specificity produce proteins that are unable to support growth at 18 degrees C. Consistent with its design, the R194L mutant hydrolyzes GTP at a reduced rate relative to wild-type Srp54 in enzymatic assays on immunoprecipitated proteins. In strains that also contain wild-type srp54, this mutant protein, as well as others designed to be locked in a GTP-bound conformation, exhibits temperature-dependent dominant inhibitory effects on growth, while a mutant predicted to be GDP locked does not interfere with the function of the wild-type protein. These results form the basis of a simple model for the role of GTP hydrolysis by Srp54 during the SRP cycle.

The Srp54 GTPase is essential for protein export in the fission yeast Schizosaccharomyces pombe.

Signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein required for targeting a subset of presecretory proteins to the endoplasmic reticulum (ER) membrane. Here we report the results of a series of experiments to define the function of the Schizosaccharomyces pombe homolog of the 54-kDa subunit of mammalian SRP. One-step gene disruption reveals that the Srp54 protein, like SRP RNA, is essential for viability in S. pombe. Precursor to the secretory protein acid phosphatase accumulates in cells in which Srp54 synthesis has been repressed under the control of a regulated promoter, indicating that S. pombe SRP functions in protein targeting. In common with other Srp54 homologs, the S. pombe protein has a modular structure consisting of an amino-terminal G (GTPase) domain and a carboxyl-terminal M (methionine-rich) domain. We have analyzed the effects of 17 site-specific mutations designed to alter the function of each of the four GTPase consensus motifs individually. Several alleles, including some with relatively conservative amino acid substitutions, confer lethal or conditional phenotypes, indicating that GTP binding and hydrolysis are critical to the in vivo role of the protein. Two mutations (R to L at position 194 [R194L] and R194H) which were designed, by analogy to oncogenic mutations in rats, to dramatically decrease the catalytic rate and one (T248N) predicted to alter nucleotide binding specificity produce proteins that are unable to support growth at 18 degrees C. Consistent with its design, the R194L mutant hydrolyzes GTP at a reduced rate relative to wild-type Srp54 in enzymatic assays on immunoprecipitated proteins. In strains that also contain wild-type srp54, this mutant protein, as well as others designed to be locked in a GTP-bound conformation, exhibits temperature-dependent dominant inhibitory effects on growth, while a mutant predicted to be GDP locked does not interfere with the function of the wild-type protein. These results form the basis of a simple model for the role of GTP hydrolysis by Srp54 during the SRP cycle.

Molecular evolution of SRP cycle components: functional implications.

Signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein that targets a subset of nascent presecretory proteins to the endoplasmic reticulum membrane. We have considered the SRP cycle from the perspective of molecular evolution, using recently determined sequences of genes or cDNAs encoding homologs of SRP (7SL) RNA, the Srp54 protein (Srp54p), and the alpha subunit of the SRP receptor (SR alpha) from a broad spectrum of organisms, together with the remaining five polypeptides of mammalian SRP. Our analysis provides insight into the significance of structural variation in SRP RNA and identifies novel conserved motifs in protein components of this pathway. The lack of congruence between an established phylogenetic tree and size variation in 7SL homologs implies the occurrence of several independent events that eliminated more than half the sequence content of this RNA during bacterial evolution. The apparently non-essential structures are domain I, a tRNA-like element that is constant in archaea, varies in size among eucaryotes, and is generally missing in bacteria, and domain III, a tightly base-paired hairpin that is present in all eucaryotic and archeal SRP RNAs but is invariably absent in bacteria. Based on both structural and functional considerations, we propose that the conserved core of SRP consists minimally of the 54 kDa signal sequence-binding protein complexed with the loosely base-paired domain IV helix of SRP RNA, and is also likely to contain a homolog of the Srp68 protein. Comparative sequence analysis of the methionine-rich M domains from a diverse array of Srp54p homologs reveals an extended region of amino acid identity that resembles a recently identified RNA recognition motif. Multiple sequence alignment of the G domains of Srp54p and SR alpha homologs indicates that these two polypeptides exhibit significant similarity even outside the four GTPase consensus motifs, including a block of nine contiguous amino acids in a location analogous to the binding site of the guanine nucleotide dissociation stimulator (GDS) for E. coli EF-Tu. The conservation of this sequence, in combination with the results of earlier genetic and biochemical studies of the SRP cycle, leads us to hypothesize that a component of the Srp68/72p heterodimer serves as the GDS for both Srp54p and SR alpha. Using an iterative alignment procedure, we demonstrate similarity between Srp68p and sequence motifs conserved among GDS proteins for small Ras-related GTPases. The conservation of SRP cycle components in organisms from all three major branches of the phylogenetic tree suggests that this pathway for protein export is of ancient evolutionary origin.

Assembly of the 68- and 72-kD proteins of signal recognition particle with 7S RNA.

Signal recognition particle (SRP), the cytoplasmic ribonucleoprotein particle that mediates the targeting of proteins to the ER, consists of a 7S RNA and six different proteins. The 68- (SRP68) and 72- (SRP72) kD proteins of SRP are bound to the 7S RNA of SRP as a heterodimeric complex (SRP68/72). Here we describe the primary structure of SRP72 and the assembly of SRP68, SRP72 and 7S RNA into a ribonucleoprotein particle. The amino acid sequence deduced from the cDNA of SRP72 reveals a basic protein of 671 amino acids which shares no sequence similarity with any protein in the sequence data libraries. Assembly of SRP72 into a ribonucleoprotein particle required the presence of 7S RNA and SRP68. In contrast, SRP68 alone specifically bound to 7S RNA. SRP68 contacts the 7S RNA via its NH2-terminal half while COOH-terminal portions of SRP68 and SRP72 are in contact with each other in SRP. SRP68 thus serves as a link between 7S RNA and SRP72. As a large NH2-terminal domain of SRP72 is exposed on SRP it may be a site of contact to other molecules involved in the SRP cycle between the ribosome and the ER membrane.

Changes in 7SL RNA conformation during the signal recognition particle cycle.

The structure of 7SL RNA has been probed by chemical modification followed by primer extension, using four substrates: (i) naked 7SL RNA; (ii) free signal recognition particle (SRP); (iii) polysome bound SRP; and (iv) membrane bound SRP. Decreasing sensitivity to chemical modification between these different substrates suggests regions on 7SL RNA that: bind proteins associated with SRP might interact with ribosomes; and are protected by binding to membranes. Other areas increase in chemical sensitivity, exemplified by a tertiary interaction present in naked 7SL RNA but not in free SRP. Such changes suggest that 7SL RNA changes its conformation during the SRP cycle. These conformational changes could be a necessary component to move through the SRP cycle from one stage to the next.