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.     

Nucleotide-dependent binding of the GTPase domain of the signal recognition particle receptor beta-subunit to the alpha-subunit

The signal recognition particle (SRP) receptor (SR) is a heterodimer of two polypeptides (SRalpha and SRbeta) that each contain a GTP-binding domain. The GTP-binding domain in the peripheral membrane SRalpha subunit has a well defined role in regulating targeting of SRP-ribosome-nascent chain complexes to the translocon. The only well established function for the transmembrane SRbeta subunit is anchoring SRalpha on the endoplasmic reticulum membrane. Deletion of the amino-terminal transmembrane domain of SRbeta did not affect receptor dimerization, but revealed a cryptic translocation signal that overlaps the GTPase domain. We demonstrate that the domain of SRalpha that binds SRbeta does so by binding directly to the nucleotide-bound form of the GTPase domain of SRbeta. An SRbeta mutant containing an amino acid substitution that allows the GTPase domain to bind XTP dimerized with SRalpha most efficiently in the presence of XTP or XDP, but not ATP. Our results suggest an additional level of regulation of SRP receptor function based on regulated dissociation of the receptor subunits.     

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.     

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 Streptomyces lividans cytoplasmic signal recognition particle receptor FtsY is involved in protein secretion

The bacterial version of the mammalian signal recognition particle (SRP) and its receptor alpha-subunit (FtsY) is well conserved and essential to all known bacteria. In gram-negative bacteria, the SRP pathway mediates a co-translational targeting of most inner membrane proteins. Additionally, in Streptomyces lividans, a gram-positive bacterium, SRP also targets secretory proteins to the translocon. The role of S. lividans FtsY has been assessed in this work. Co-immunoprecipitation studies confirmed that FtsY is associated with the S. lividans SRP in the cytoplasm and that this complex also co-immunoprecipitated with pre-agarase, suggesting that the SRP receptor is involved in SRP-mediated targeting of secretory proteins in S. lividans. Furthermore, the SRP remains attached for the most part to the cellular membrane when the cleavage of pre-secretory proteins is severely reduced in a strain lacking the gene coding for the major type-I signal peptidase.     

The beta subunit of the signal recognition particle receptor is a transmembrane GTPase that anchors the alpha subunit, a peripheral membrane GTPase, to the endoplasmic reticulum membrane

The signal recognition particle receptor (SR) is required for the cotranslational targeting of both secretory and membrane proteins to the endoplasmic reticulum (ER) membrane. During targeting, the SR interacts with the signal recognition particle (SRP) which is bound to the signal sequence of the nascent protein chain. This interaction catalyzes the GTP-dependent transfer of the nascent chain from SRP to the protein translocation apparatus in the ER membrane. The SR is a heterodimeric protein comprised of a 69-kD subunit (SR alpha) and a 30- kD subunit (SR beta) which are associated with the ER membrane in an unknown manner. SR alpha and the 54-kD subunits of SRP (SRP54) each contain related GTPase domains which are required for SR and SRP function. Molecular cloning and sequencing of a cDNA encoding SR beta revealed that SR beta is a transmembrane protein and, like SR alpha and SRP54, is a member of the GTPase superfamily. Although SR beta defines its own GTPase subfamily, it is distantly related to ARF and Sar1. Using UV cross-linking, we confirm that SR beta binds GTP specifically. Proteolytic digestion experiments show that SR alpha is required for the interaction of SRP with SR. SR alpha appears to be peripherally associated with the ER membrane, and we suggest that SR beta, as an integral membrane protein, mediates the membrane association of SR alpha. The discovery of its guanine nucleotide-binding domain, however, makes it likely that its role is more complex than that of a passive anchor for SR alpha. These findings suggest that a cascade of three directly interacting GTPases functions during protein targeting to the ER membrane.     

The signal recognition particle receptor is a complex that contains two distinct polypeptide chains

Signal recognition particle (SRP) and SRP receptor are known to be essential components of the cellular machinery that targets nascent secretory proteins to the endoplasmic reticulum (ER) membrane. Here we report that the SRP receptor contains, in addition to the previously identified and sequenced 69-kD polypeptide (alpha-subunit, SR alpha), a 30-kD beta-subunit (SR beta). When SRP receptor was purified by SRP-Sepharose affinity chromatography, we observed the co-purification of two other ER membrane proteins. Both proteins are approximately 30 kD in size and are immunologically distinct from each other, as well as from SR alpha and SRP proteins. One of the 30-kD proteins (SR beta) forms a tight complex with SR alpha in detergent solution that is stable to high salt and can be immunoprecipitated with antibodies to either SR alpha or SR beta. Both subunits are present in the ER membrane in equimolar amounts and co-fractionate in constant stoichiometry when rough and smooth liver microsomes are separated on sucrose gradients. We therefore conclude that SR beta is an integral component of SRP receptor. The presence of SR beta was previously masked by proteolytic breakdown products of SR alpha observed by others and by the presence of another 30-kD ER membrane protein (mp30) which co-purifies with SR alpha. Mp30 binds to SRP-Sepharose directly and is present in the ER membrane in several-fold molar excess of SR alpha and SR beta. The affinity of mp30 for SRP suggests that it may serve a yet unknown function in protein translocation.     

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.     

The beta-subunit of the protein-conducting channel of the endoplasmic reticulum functions as the guanine nucleotide exchange factor for the beta-subunit of the signal recognition particle receptor

Cotranslational protein transport to the endoplasmic reticulum is controlled by the concerted interaction of three GTPases: the SRP54 subunit of the signal recognition particle (SRP) and the alpha- and beta-subunits of the SRP receptor (SR). SRbeta is related to ADP-ribosylation factor (ARF)-type GTPases, and the recently published crystal structure of SRbeta-GTP in complex with the binding domain of SRalpha suggested that SRbeta, like all ARF-type GT-Pases, requires a guanine nucleotide exchange factor (GEF) for function. Searching the sequence data base, we identified significant sequence similarity between the Sec7 domain of ARF-GEFs and the cytosolic domains of the beta-subunits of the two homologous heterotrimeric protein-conducting channels in yeast. Using a fluorescence nucleotide exchange assay, we show that the beta-subunits of the heterotrimeric protein-conducting channels function as the GEFs for SRbeta. Both the cytosolic domain of Sec61beta as well as the holo-Sec61beta, when part of the isolated trimeric Sec61p complex, function as the GEF for SRbeta, whereas the same Sec61beta, when part of the heptameric complex that facilitates posttranslational protein transport, is inactive as the GEF for SRbeta     

GTPase domain of the 54-kD subunit of the mammalian signal recognition particle is required for protein translocation but not for signal sequence binding

The 54-kD subunit of the signal recognition particle (SRP54) binds to signal sequences of nascent secretory and transmembrane proteins. SRP54 consists of two separable domains, a 33-kD amino-terminal domain that contains a GTP-binding site (SRP54G) and a 22-kD carboxy-terminal domain (SRP54M) containing binding sites for both the signal sequence and SRP RNA. To examine the function of the two domains in more detail, we have purified SRP54M and used it to assemble a partial SRP that lacks the amino-terminal domain of SRP54 [SRP(-54G)]. This particle recognized signal sequences in two independent assays, albeit less efficiently than intact SRP. Analysis of the signal sequence binding activity of free SRP54 and SRP54M supports the conclusion that SRP54M binds signal sequences with lower affinity than the intact protein. In contrast, when SRP(-54G) was assayed for its ability to promote the translocation of preprolactin across microsomal membranes, it was completely inactive, apparently because it was unable to interact normally with the SRP receptor. These results imply that SRP54G plays an essential role in SRP-mediated targeting of nascent chain-ribosome complexes to the ER membrane and also influences signal sequence recognition, possibly by promoting a tighter association between signal sequences and SRP54M.     

Rhodopsin-enhanced GTPase activity of the inhibitory GTP-binding protein of adenylate cyclase

Work in several laboratories has shown that Gi, the inhibitory guanyl nucleotide-binding protein of the adenylate cyclase system, is similar in many ways to transducin, the guanyl nucleotide-binding protein of the retinal light-activated cGMP phosphodiesterase system. Separated subunits of purified transducin, T alpha (approximately 39 kDa) and T beta gamma (approximately 35 and approximately 10 kDa), do not exhibit GTPase activity; GTPase activity is observed when the subunits are combined in the presence of rhodopsin ( Fung , B. K.-K. (1983) J. Biol. Chem. 258, 10495-10502). Subunits of Gi, Gi alpha (approximately 41 kDa), and Gi beta gamma (approximately 35 and approximately 10 kDa) were prepared from rabbit liver membranes. It was found that Gi beta gamma could replace T beta gamma in reconstituting the rhodopsin-stimulated GTPase activity of T alpha. Gi alpha exhibited rhodopsin-stimulated GTPase activity when reconstituted with Gi beta gamma or T beta gamma. GTPase activity was a function of Gi alpha concentration when Gi beta gamma or T beta gamma was constant, and the GTPase activity of a given amount of Gi alpha was dependent on Gi beta gamma concentration. These studies demonstrate that the GTPase activity of Gi resides in Gi alpha and further establish that Gi alpha and Gi beta gamma are functionally analogous to T alpha and T beta gamma, respectively.     

Concerted complex assembly and GTPase activation in the chloroplast signal recognition particle

The universally conserved signal recognition particle (SRP) and SRP receptor (SR) mediate the cotranslational targeting of proteins to cellular membranes. In contrast, a unique chloroplast SRP in green plants is primarily dedicated to the post-translational targeting of light harvesting chlorophyll a/b binding (LHC) proteins. In both pathways, dimerization and activation between the SRP and SR GTPases mediate the delivery of cargo; whether and how the GTPase cycle in each system adapts to its distinct substrate proteins were unclear. Here, we show that interactions at the active site essential for GTPase activation in the chloroplast SRP and SR play key roles in the assembly of the GTPase complex. In contrast to their cytosolic homologues, GTPase activation in the chloroplast SRP-SR complex contributes marginally to the targeting of LHC proteins. These results demonstrate that complex assembly and GTPase activation are highly coupled in the chloroplast SRP and SR and suggest that the chloroplast GTPases may forego the GTPase activation step as a key regulatory point. These features may reflect adaptations of the chloroplast SRP to the delivery of their unique substrate protein.     

RNA-mediated interaction between the peptide-binding and GTPase domains of the signal recognition particle

The signal recognition particle (SRP) targets nascent proteins to cellular membranes for insertion or secretion by recognizing polypeptides containing an N-terminal signal sequence as they emerge from the ribosome. GTP-dependent binding of SRP to its receptor protein leads to controlled release of the nascent chain into a membrane-spanning translocon pore. Here we show that the association of the SRP with its receptor triggers a marked conformational change in the complex, localizing the SRP RNA and the adjacent signal peptide-binding site at the SRP-receptor heterodimer interface. The orientation of the RNA suggests how peptide binding and GTP hydrolysis can be coupled through direct structural contact during cycles of SRP-directed protein translocation.     

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.     

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.     

Predominant membrane localization is an essential feature of the bacterial signal recognition particle receptor

Background  

The signal recognition particle (SRP) receptor plays a vital role in co-translational protein targeting, because it connects the soluble SRP-ribosome-nascent chain complex (SRP-RNCs) to the membrane bound Sec translocon. The eukaryotic SRP receptor (SR) is a heterodimeric protein complex, consisting of two unrelated GTPases. The SRβ subunit is an integral membrane protein, which tethers the SRP-interacting SRα subunit permanently to the endoplasmic reticulum membrane. The prokaryotic SR lacks the SRβ subunit and consists of only the SRα homologue FtsY. Strikingly, although FtsY requires membrane contact for functionality, cell fractionation studies have localized FtsY predominantly to the cytosolic fraction of Escherichia coli. So far, the exact function of the soluble SR in E. coli is unknown, but it has been suggested that, in contrast to eukaryotes, the prokaryotic SR might bind SRP-RNCs already in the cytosol and only then initiates membrane targeting.     

The signal recognition particle and its interactions during protein targeting

The synthesis of secretory or integral membrane proteins can be directly coupled to their translocation across or insertion into membranes. In co-translational targeting, the translation machine, the ribosome, is transferred to the respective membrane by the signal recognition particle (SRP) and its receptor (SR) as soon as a signal sequence emerges. Protein synthesis can continue at the membrane, with the nascent peptide chain directly inserting into the ribosome-bound protein-conducting channel, the Sec61 complex. During the past two years, several structures have been solved by crystallography and cryo-electron microscopy that represent distinct functional states of the SRP cycle. On this basis, the first structure-based models can be suggested that explain important aspects of protein targeting, such as the SRP-ribosome and SRP-SR interactions.     

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.     

The structure of the chloroplast signal recognition particle (SRP) receptor reveals mechanistic details of SRP GTPase activation and a conserved membrane targeting site

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.     

The signal recognition particle of Archaea

It is becoming increasingly clear that similarities exist in the manner in which extracytoplasmic proteins are targeted to complexes responsible for translocating these proteins across membranes in each of the three domains of life. In Eukarya and Bacteria, the signal recognition particle (SRP) directs nascent polypeptides to membrane-embedded translocation sites. In Archaea, the SRP protein targeting pathway apparently represents an intermediate between the bacterial and eukaryal systems. Understanding the archaeal SRP pathway could therefore reveal universal aspects of targeting not detected in current comparisons of the eukaryal and bacterial systems while possibly identifying aspects of the process either not previously reported or unique to Archaea.