Co-translational protein targeting by the signal recognition particle

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.     

GTP hydrolysis by complexes of the signal recognition particle and the signal recognition particle receptor

Translocation of proteins across the endoplasmic reticulum membrane is a GTP-dependent process. The signal recognition particle (SRP) and the SRP receptor both contain subunits with GTP binding domains. One GTP- dependent reaction during protein translocation is the SRP receptor- mediated dissociation of SRP from the signal sequence of a nascent polypeptide. Here, we have assayed the SRP and the SRP receptor for GTP binding and hydrolysis activities. GTP hydrolysis by SRP was not detected, so the maximal GTP hydrolysis rate for SRP was estimated to be < 0.002 mol GTP hydrolyzed x mol of SRP-1 x min-1. The intrinsic GTP hydrolysis activity of the SRP receptor ranged between 0.02 and 0.04 mol GTP hydrolyzed x mol of SRP receptor-1 x min-1. A 40-fold enhancement of GTP hydrolysis activity relative to that observed for the SRP receptor alone was obtained when complexes were formed between SRP and the SRP receptor. GTP hydrolysis activity was inhibited by GDP, but not by ATP. Extended incubation of the SRP or the SRP receptor with GTP resulted in substoichiometric quantities of protein-bound ribonucleotide. SRP-SRP receptor complexes engaged in GTP hydrolysis were found to contain a minimum of one bound guanine ribonucleotide per SRP-SRP receptor complex. We conclude that the GTP hydrolysis activity described here is indicative of one of the GTPase cycles that occur during protein translocation across the endoplasmic reticulum.     

Lipid activation of the signal recognition particle receptor provides spatial coordination of protein targeting

The signal recognition particle (SRP) and SRP receptor comprise the major cellular machinery that mediates the cotranslational targeting of proteins to cellular membranes. It remains unclear how the delivery of cargos to the target membrane is spatially coordinated. We show here that phospholipid binding drives important conformational rearrangements that activate the bacterial SRP receptor FtsY and the SRP–FtsY complex. This leads to accelerated SRP–FtsY complex assembly, and allows the SRP–FtsY complex to more efficiently unload cargo proteins. Likewise, formation of an active SRP–FtsY GTPase complex exposes FtsY’s lipid-binding helix and enables stable membrane association of the targeting complex. Thus, membrane binding, complex assembly with SRP, and cargo unloading are inextricably linked to each other via conformational changes in FtsY. These allosteric communications allow the membrane delivery of cargo proteins to be efficiently coupled to their subsequent unloading and translocation, thus providing spatial coordination during protein targeting.     

Conformation of the signal recognition particle in ribosomal targeting complexes

The bacterial signal recognition particle (SRP) binds to ribosomes synthesizing inner membrane proteins and, by interaction with the SRP receptor, FtsY, targets them to the translocon at the membrane. Here we probe the conformation of SRP and SRP protein, Ffh, at different stages of targeting by measuring fluorescence resonance energy transfer (FRET) between fluorophores placed at various positions within SRP. Distances derived from FRET indicate that SRP binding to nontranslating ribosomes triggers a global conformational change of SRP that facilitates binding of the SRP receptor, FtsY. Binding of SRP to a signal-anchor sequence exposed on a ribosome-nascent chain complex (RNC) causes a further change of the SRP conformation, involving the flexible part of the Ffh(M) domain, which increases the affinity for FtsY of ribosome-bound SRP up to the affinity exhibited by the isolated NG domain of Ffh. This indicates that in the RNC–SRP complex the Ffh(NG) domain is fully exposed for binding FtsY to form the targeting complex. Binding of FtsY to the RNC–SRP complex results in a limited conformational change of SRP, which may initiate subsequent targeting steps.     

Co-translational protein targeting catalyzed by the Escherichia coli signal recognition particle and its receptor.

The Ffh-4.5S ribonucleoprotein particle (RNP) and FtsY from Escherichia coli are homologous to essential components of the mammalian signal recognition particle (SRP) and SRP receptor, respectively. The ability of these E. coli components to function in a bona fide co-translational targeting pathway remains unclear. Here we demonstrate that the Ffh-4.5S RNP and FtsY can efficiently replace their mammalian counterparts in targeting nascent secretory proteins to microsomal membranes in vitro. Targeting in the heterologous system requires a hydrophobic signal sequence, utilizes GTP and, moreover, occurs co-translationally. Unlike mammalian SRP, however, the Ffh-4.5S RNP is unable to arrest translational elongation, which results in a narrow time window for the ribosome nascent chain to interact productively with the membrane-bound translocation machinery. The highly negatively charged N-terminal domain of FtsY, which is a conserved feature among prokaryotic SRP receptor homologs, is important for translocation and acts to localize the protein to the membrane. Our data illustrate the extreme functional conservation between prokaryotic and eukaryotic SRP and SRP receptors and suggest that the basic mechanism of co-translational protein targeting is conserved between bacteria and mammals.     

Network models reveal stability and structural rearrangement of signal recognition particle

The signal recognition particle (SRP) and its receptors (SR) mediate the cotranslational targeting of the membrane and secretory proteins in all cells. In Escherichia coli, SRP is composed of the Ffh protein and the 4.5S SRP RNA. Ffh is a multidomain protein comprising a methionine-rich (M) domain, a helical N domain, and a Ras-like guanine triphosphatase (GTPase) (G) domain. The N and G domains are commonly referred to as one structural unit, the NG domain. In this article, the complex structure of SRP and SR is investigated with the Gaussian network model (GNM) and anisotropic network model (ANM). GNM provides the information of structure stability. It is found that the intermolecular interactions between SRP and SR can obviously decrease the fluctuation of NG domains. Nevertheless, the large structural rearrangement will take place during the cotranslational protein targeting cycle. Hence, the moving directions of fluctuation regions are further ascertained by using cross-correlation analysis and the ANM. The NG domain of Ffh undergoes a clockwise rotation around the GM linker and the M domain of Ffh shows an opposite direction to the NG domain. These functional movements will facilitate the SRP structure to transform into the free form and the sequence-bound form. These simple coarse-grained analyses can be used as a general and quick method for the mechanism studies of protein assembly and supramolecular systems.     

Dual recognition of the ribosome and the signal recognition particle by the SRP receptor during protein targeting to the endoplasmic reticulum

We have analyzed the interactions between the signal recognition particle (SRP), the SRP receptor (SR), and the ribosome using GTPase assays, biosensor experiments, and ribosome binding assays. Possible mechanisms that could contribute to an enhanced affinity between the SR and the SRP-ribosome nascent chain complex to promote protein translocation under physiological ionic strength conditions have been explored. Ribosomes or 60S large ribosomal subunits activate the GTPase cycle of SRP54 and SRalpha by providing a platform for assembly of the SRP-SR complex. Biosensor experiments revealed high-affinity, saturable binding of ribosomes or large ribosomal subunits to the SR. Remarkably, the SR has a 100-fold higher affinity for the ribosome than for SRP. Proteoliposomes that contain the SR bind nontranslating ribosomes with an affinity comparable to that shown by the Sec61 complex. An NH2-terminal 319-residue segment of SRalpha is necessary and sufficient for binding of SR to the ribosome. We propose that the ribosome-SR interaction accelerates targeting of the ribosome nascent chain complex to the RER, while the SRP-SR interaction is crucial for maintaining the fidelity of the targeting reaction.     

Evidence for coupling of membrane targeting and function of the signal recognition particle (SRP) receptor FtsY

Recent studies have indicated that FtsY, the signal recognition particle receptor of Escherichia coli, plays a central role in membrane protein biogenesis. For proper function, FtsY must be targeted to the membrane, but its membrane-targeting pathway is unknown. We investigated the relationship between targeting and function of FtsY in vivo, by separating its catalytic domain (NG) from its putative targeting domain (A) by three means: expression of split ftsY, insertion of various spacers between A and NG, and separation of A and NG by in vivo proteolysis. Proteolytic separation of A and NG does not abolish function, whereas separation by long linkers or expression of split ftsY is detrimental. We propose that proteolytic cleavage of FtsY occurs after completion of co-translational targeting and assembly of NG. In contrast, separation by other means may interrupt proper synchronization of co-translational targeting and membrane assembly of NG. The co-translational interaction of FtsY with the membrane was confirmed by in vitro experiments.     

Advances in the structure and functions of signal recognition particle in protein targeting

The signal recognition particle (SRP) is a unique moiety in living cells, which has been conserved during evolution for protein targeting and translocation across membranes in collaboration with its receptor (SR). The structural and functional features of its components, (six polypeptides and RNA) are being rapidly elucidated. We have endeavored in this review to epitomize most recent advances in this field. Its two domains (S and Alu) play important roles in signal recognition, elongation arrest and protein targeting of the polypeptide being synthesized in the cytoplasm. SRP14 and SRP9 help in the elongation arrest by interacting with signal peptide. GTPase activity of SRP54 releases SRP from SR. In addition, alpha and beta subunits of SR also possess GTPase activities and the three GTPases help in docking of nascent peptide chain-ribosome complex to the translocation site. Further strides in proteomics employing mass spectrometry and X-ray crystallography are expected to throw more light on the molecular events occurring during protein targeting and translocation.     

Physiological basis for conservation of the signal recognition particle targeting pathway in Escherichia coli

The Escherichia coli signal recognition particle (SRP) is a ribonucleoprotein complex that targets nascent inner membrane proteins (IMPs) to transport sites in the inner membrane (IM). Since SRP depletion only partially inhibits IMP insertion under some growth conditions, however, it is not clear why the particle is absolutely essential for viability. Insights into this question emerged from experiments in which we analyzed the physiological consequences of reducing the intracellular concentration of SRP below the wild-type level. We found that even moderate SRP deficiencies that have little effect on cell growth led to the induction of a heat shock response. Genetic manipulations that suppress the heat shock response were lethal in SRP-deficient cells, indicating that the elevated synthesis of heat shock proteins plays an important role in maintaining cell viability. Although it is conceivable that the heat shock response serves to increase the capacity of cells to target IMPs via chaperone-based mechanisms, SRP-deficient cells did not show an increased dependence on either GroEL or DnaK. By contrast, the heat shock-regulated proteases Lon and ClpQ became essential for viability when SRP levels were reduced. These results suggest that the heat shock response protects SRP-deficient cells by increasing their capacity to degrade mislocalized IMPs. Consistent with this notion, a model IMP that was mislocalized in the cytoplasm as the result of SRP depletion appeared to be more stable in a Deltalon DeltaclpQ strain than in control cells. Taken together, the data provide direct evidence that SRP is essential in E. coli and possibly conserved throughout prokaryotic evolution as well partly because efficient IMP targeting prevents a toxic accumulation of aggregated proteins in the cytoplasm.     

Structures of the signal recognition particle receptor from the archaeon Pyrococcus furiosus: implications for the targeting step at the membrane

In all organisms, a ribonucleoprotein called the signal recognition particle (SRP) and its receptor (SR) target nascent proteins from the ribosome to the translocon for secretion or membrane insertion. We present the first X-ray structures of an archeal FtsY, the receptor from the hyper-thermophile Pyrococcus furiosus (Pfu), in its free and GDP*magnesium-bound forms. The highly charged N-terminal domain of Pfu-FtsY is distinguished by a long N-terminal helix. The basic charges on the surface of this helix are likely to regulate interactions at the membrane. A peripheral GDP bound near a regulatory motif could indicate a site of interaction between the receptor and ribosomal or SRP RNAs. Small angle X-ray scattering and analytical ultracentrifugation indicate that the crystal structure of Pfu-FtsY correlates well with the average conformation in solution. Based on previous structures of two sub-complexes, we propose a model of the core of archeal and eukaryotic SRP*SR targeting complexes.     

Involvement of a chloroplast homologue of the signal recognition particle receptor protein, FtsY, in protein targeting to thylakoids

We isolated an Arabidopsis thaliana cDNA whose translated product shows sequence similarity to the FtsY, a bacterial homologue of SRP receptor protein. The Arabidopsis FtsY homologue contains a typical chloroplast transit peptide. The in vitro-synthesized 37 kDa FtsY homologue was imported into chloroplasts, and the processed 32 kDa polypeptide bound peripherally on the outer surface of thylakoids. Antibodies raised against the FtsY homologue also reacted with a thylakoid-bound 32 kDa protein. The antibodies inhibited the cpSRP-dependent insertion of the light-harvesting chlorophyll alb-binding protein into thylakoid membranes suggesting that the chloroplast FtsY homologue is involved in the cpSRP-dependent protein targeting to the thylakoid membranes.     

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.     

Conformational variability of the GTPase domain of the signal recognition particle receptor FtsY

The prokaryotic signal recognition particle Ffh and its receptor FtsY allow targeting of proteins into or across the plasma membrane. The targeting process is GTP dependent and the two proteins constitute a distinct GTPase family. The receptor FtsY is composed of A and NG domains where the NG's GTPase domain plays a critical role in the targeting process. In this study, we describe two X-ray structures determined independently of each other of the NG domain of FtsY from Mycoplasma mycoides (MmFtsY). The two structures are markedly different in three of the nucleotide-binding segments, GI (P-loop), GII, and GIII, making only one of the structures compatible with nucleotide binding. Interestingly, the two distinct conformations of the nucleotide-binding segments of MmFtsY are similar to the apo- and ADP-loaded forms of certain ATPases. The structure of the extended interface between the A and NG domains of MmFtsY provides new insights into the role of the A domain for phospholipid interaction.     

The signal sequence receptor, unlike the signal recognition particle receptor, is not essential for protein translocation

Detergent extracts of canine pancreas rough microsomal membranes were depleted of either the signal recognition particle receptor (SR), which mediates the signal recognition particle (SRP)-dependent targeting of the ribosome/nascent chain complex to the membrane, or the signal sequence receptor (SSR), which has been proposed to function as a membrane bound receptor for the newly targeted nascent chain and/or as a component of a multi-protein translocation complex responsible for transfer of the nascent chain across the membrane. Depletion of the two components was performed by chromatography of detergent extracts on immunoaffinity supports. Detergent extracts lacking either SR or SSR were reconstituted and assayed for activity with respect to SR dependent elongation arrest release, nascent chain targeting, ribosome binding, secretory precursor translocation, and membrane protein integration. Depletion of SR resulted in the loss of elongation arrest release activity, nascent chain targeting, secretory protein translocation, and membrane protein integration, although ribosome binding was unaffected. Full activity was restored by addition of immunoaffinity purified SR before reconstitution of the detergent extract. Surprisingly, depletion of SSR was without effect on any of the assayed activities, indicating that SSR is either not required for translocation or is one of a family of functionally redundant components.     

Regulation of the GTPase cycle in post-translational signal recognition particle-based protein targeting involves cpSRP43

The chloroplast signal recognition particle consists of a conserved 54-kDa GTPase and a novel 43-kDa chromodomain protein (cpSRP43) that together bind light-harvesting chlorophyll a/b-binding protein (LHCP) to form a soluble targeting complex that is subsequently directed to the thylakoid membrane. Homology-based modeling of cpSRP43 indicates the presence of two previously identified chromodomains along with a third N-terminal chromodomain. Chromodomain deletion constructs were used to examine the role of each chromodomain in mediating distinct steps in the LHCP localization mechanism. The C-terminal chromodomain is completely dispensable for LHCP targeting/integration in vitro. The central chromodomain is essential for both targeting complex formation and integration because of its role in binding the M domain of cpSRP54. The N-terminal chromodomain (CD1) is unnecessary for targeting complex formation but is required for integration. This correlates with the ability of CD1 along with the ankyrin repeat region of cpSRP43 to regulate the GTPase cycle of the cpSRP-receptor complex.     

Canonical signal recognition particle components can be bypassed for posttranslational protein targeting in chloroplasts

The chloroplast signal recognition particle (cpSRP) and its receptor (cpFtsY) target proteins both cotranslationally and posttranslationally to the thylakoids. This dual function enables cpSRP to utilize its posttranslational activities for targeting a family of nucleus-encoded light-harvesting chlorophyll binding proteins (LHCPs), the most abundant membrane proteins in plants. Previous in vitro experiments indicated an absolute requirement for all cpSRP pathway soluble components. In agreement, a cpFtsY mutant in Arabidopsis thaliana exhibits a severe chlorotic phenotype resulting from a massive loss of LHCPs. Surprisingly, a double mutant, cpftsy cpsrp54, recovers to a great extent from the chlorotic cpftsy phenotype. This establishes that in plants, a new alternative pathway exists that can bypass cpSRP posttranslational targeting activities. Using a mutant form of cpSRP43 that is unable to assemble with cpSRP54, we complemented the cpSRP43-deficient mutant and found that this subunit is required for the alternative pathway. Along with the ability of cpSRP43 alone to bind the ALBINO3 translocase required for LHCP integration, our results indicate that cpSRP43 has developed features to function independently of cpSRP54/cpFtsY in targeting LHCPs to the thylakoid membranes.     

The ribosome regulates the GTPase of the beta-subunit of the signal recognition particle receptor.

Protein targeting to the membrane of the ER is regulated by three GTPases, the 54-kD subunit of the signal recognition particle (SRP) and the alpha- and beta-subunit of the SRP receptor (SR). Here, we report on the GTPase cycle of the beta-subunits of the SR (SRbeta). We found that SRbeta binds GTP with high affinity and interacts with ribosomes in the GTP-bound state. Subsequently, the ribosome increases the GTPase activity of SRbeta and thus functions as a GTPase activating protein for SRbeta. Furthermore, the interaction between SRbeta and the ribosome leads to a reduction in the affinity of SRbeta for guanine nucleotides. We propose that SRbeta regulates the interaction of SR with the ribosome and thereby allows SRalpha to scan membrane-bound ribosomes for the presence of SRP. Interaction between SRP and SRalpha then leads to release of the signal sequence from SRP and insertion into the translocon. GTP hydrolysis then results in dissociation of SR from the ribosome, and SRP from the SR.