S-domain assembly of the signal recognition particle

The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein that associates with ribosomes to mediate the targeting of membrane and secretory proteins to biological membranes. In higher eukaryotes, SRP biogenesis involves the sequential binding of SRP19 and SRP54 proteins to the S domain of 7S RNA. The recently determined crystal structures of SRP19 in complex with the S domain, and that of the ternary complex of SRP19, the S domain and the M domain of SRP54, provide insight into the molecular basis of S-domain assembly and SRP function.     

Compartmentalization directs assembly of the signal recognition particle

Many ribonucleoprotein complexes assemble stepwise in distinct cellular compartments, a process that usually involves bidirectional transport of both RNA and proteins between the nucleus and cytoplasm. The biological rationale for such complex transport steps in RNP assembly is obscure. One important example is the eukaryotic signal recognition particle (SRP), a cytoplasmic RNP consisting of one RNA and six proteins. Prior in vivo studies support an "SRP54-late" assembly model in which all SRP proteins, except SRP54, are imported from the cytoplasm to the nucleus to bind SRP RNA. This partially assembled complex is then exported to the cytoplasm where SRP54 binds and forms the SRP holocomplex. Here we show that native SRP assembly requires segregated and ordered binding by its protein components. A native ternary complex forms in vitro when SRP19 binds the SRP RNA prior to binding by SRP54, which approximates the eukaryotic cellular pathway. In contrast, the presence of SRP54 disrupts native assembly of SRP19, such that two RNA-binding loops in SRP19 misfold. These results imply that SRP54 must be sequestered during early SRP assembly steps, as apparently occurs in vivo, for proper assembly of the SRP to occur. Our findings emphasize that spatial compartmentalization provides an additional level of regulation that prevents competition among components and can function to promote native assembly of the eukaryotic SRP.     

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.     

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.     

Evidence for a two-step mechanism involved in the formation of covalent HC x TSG-6 complexes

IalphaI and TSG-6 interact to form a covalent bond between the C-terminal Asp alpha-carbon of an IalphaI heavy chain (HC) and an unknown component of TSG-6. This event disrupts the protein-glycosaminoglycan-protein (PGP) cross-link and dissociates IalphaI. In simple terms the interaction involves 5 components: (i) the IalphaI HCs, (ii) bikunin, (iii) chondroitin sulfate chain, (iv) TSG-6, and (v) divalent cations. To understand the molecular mechanism of complex formation, the effect of these were separately examined. The data show that although the mature covalent cross-link between the HCs and TSG-6 only involves the C-terminal Asp residue, the native fold of both IalphaI and TSG-6 was essential for the reaction to occur. Similarly, complex formation was prevented if the chondroitin sulfate chain was cleaved, releasing bikunin but maintaining the HC1 and HC2 PGP cross-links. In contrast, releasing the majority of the bikunin protein moiety by limited proteolysis did not prevent complex formation. An analysis of the divalent-cation requirements revealed two distinct interactions between IalphaI and TSG-6: (i) a noncovalent manganese, magnesium, or calcium-independent interaction between TSG-6 and the chondroitin sulfate chain (Kd 180 nM) and (ii) a covalent manganese, magnesium, or calcium-dependent interaction generating HC1 x TSG-6, HC2 x TSG-6, and high molecular weight (HMW) IalphaI. Significantly, both free TSG-6 and HC x TSG-6 complexes were able to bind the chondroitin sulfate chain suggesting that the sites on TSG-6 were distinct. On the basis of these findings, we propose a two-step reaction mechanism involving two putative binding sites. Initially, a cation-independent interaction between TSG-6 and the chondroitin sulfate chain is formed at site 1. Subsequently, a cation-dependent transesterification occurs, generating the covalent HC x TSG-6 cross-link at another site, site 2.     

Genetic evidence for functional interaction of the Escherichia coli signal recognition particle receptor with acidic lipids in vivo

The mechanism underlying the interaction of the Escherichia coli signal recognition particle receptor FtsY with the cytoplasmic membrane has been studied in detail. Recently, we proposed that FtsY requires functional interaction with inner membrane lipids at a late stage of the signal recognition particle pathway. In addition, an essential lipid-binding α-helix was identified in FtsY of various origins. Theoretical considerations and in vitro studies have suggested that it interacts with acidic lipids, but this notion is not yet fully supported by in vivo experimental evidence. Here, we present an unbiased genetic clue, obtained by serendipity, supporting the involvement of acidic lipids. Utilizing a dominant negative mutant of FtsY (termed NG), which is defective in its functional interaction with lipids, we screened for E. coli genes that suppress the negative dominant phenotype. In addition to several unrelated phenotype-suppressor genes, we identified pgsA, which encodes the enzyme phosphatidylglycerophosphate synthase (PgsA). PgsA is an integral membrane protein that catalyzes the committed step to acidic phospholipid synthesis, and we show that its overexpression increases the contents of cardiolipin and phosphatidylglycerol. Remarkably, expression of PgsA also stabilizes NG and restores its biological function. Collectively, our results strongly support the notion that FtsY functionally interacts with acidic lipids.     

Visualizing induced fit in early assembly of the human signal recognition particle

Assembly of almost all ribonucleoprotein complexes involves induced fit in the RNA and, thus, formation of one or more intermediate states. In assembly of the human signal recognition particle (SRP), we show that SRP19 binding to SRP RNA involves obligatory intermediates. An apparent discrepancy exists between the ratio of dissociation and association rate constants, determined in a partitioning experiment, and the equilibrium binding constant; this kinetic signature reflects formation of a stable intermediate in assembly of the ribonucleoprotein complex. Assembly intermediates were observed directly by time-resolved footprinting. SRP19 binds rapidly to SRP RNA to form an initial labile, but structurally specific, encounter complex involving both helices III and IV. Two subsequent steps of structural consolidation yield the native RNA-protein interface. SRP19 binding stabilizes helix IV in the region recognized by SRP54, consistent with protein-protein cooperativity mediated in part by mutual recognition of similar RNA structures. This mechanism illustrates principles general to ribonucleoprotein assembly reactions that rely on recruitment of architectural RNA binding proteins.     

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.     

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.     

Thermal activation of the purified rat hepatic glucocorticoid receptor. Evidence for a two-step mechanism

Thermal "activation" or "transformation" of rat hepatic [6,7-3H]triamcinolone acetonide (TA)-receptor complexes purified in the unactivated state to near homogeneity (Grandics, P., Miller, A., Schmidt, T. J., Mittman, D., and Litwack, G. (1984) J. Biol. Chem. 259, 3173-3180) has been further investigated. The data generated in reconstitution experiments demonstrate that warming (25 degrees C for 30 min) of the purified unactivated complexes promotes their activation as judged by an increase in DNA-cellulose binding, but to a lower extent than that observed after warming of glucocorticoid-receptor complexes in crude cytosols. However, maximal DNA-cellulose binding capacity can be detected in reconstituted systems (also heated at 25 degrees C for 30 min) consisting of purified unactivated [3H]TA-receptor complexes and a cytoplasmic "stimulator(s)." This cytoplasmic factor(s), which does not copurify with the receptor, is heat-stable (90 degrees C for 30 min), excluded from Sephadex G-25, and trypsin-sensitive and stimulates DNA-cellulose binding in a dose-dependent manner. The ability of Na2MoO4 to block thermal activation of the highly purified receptor complexes suggests that this transition metal anion interacts directly with the receptor protein itself. The fact that the cytoplasmic stimulator(s) enhances DNA-cellulose binding of the [3H]TA-receptor complexes without increasing the proportion of those complexes eluted in the activated (low salt) position from DEAE-cellulose is consistent with a proposed two-step model of in vitro activation. During the Na2MoO4-sensitive Step 1, elevated temperature (25 degrees C for 30 min) may directly alter the conformation of the purified receptor complexes (i.e. subunit dissociation or disaggregation), resulting in the appropriate shift in the elution profile of the [3H]TA-receptor complexes on DEAE-cellulose but only in a minimal (approximately 2-3-fold) increase in the binding of these complexes to DNA-cellulose. During the Na2MoO4-insensitive and temperature-independent Step 2, a heat-stable cytoplasmic protein(s) may interact with these thermally activated [3H]TA-receptor complexes and enhance their ability to bind to DNA-cellulose without further increasing the percentage of those complexes which elute from DEAE-cellulose in the activated position. In crude cytosols these two steps would presumably occur simultaneously, and addition of Na2MoO4 prior to warming would block Step 1 and hence Step 2 would not occur.     

Role of signal recognition particle in the membrane assembly of Sindbis viral glycoproteins

We have investigated the role of signal recognition particle (SRP) in the biosynthesis of Sindbis glycoproteins by translating the viral 26S mRNA in a wheat-germ cell-free system. SRP was shown to have no effect on the synthesis or proteolytic processing of the cytoplasmic C protein. In contrast, the membrane integration and the proteolytic processing of the viral glycoproteins PE2 and E1 were demonstrated to be SRP-dependent. In the absence of microsomal membranes, SRP caused an arrest of the synthesis of the viral glycoproteins. This arrest could be released by the addition of salt-extracted microsomal membranes. Synchronization experiments indicated that the uncleaved signal sequence of PE2 was recognized by SRP after at most 130 amino acids of PE2 had been polymerized. No apparent interaction of SRP with a putative signal sequence of E1 and/or a 6-kDa peptide could be detected.     

Role of SRP19 in assembly of the Archaeoglobus fulgidus signal recognition particle

Previous studies have shown that SRP19 promotes association of the highly conserved signal peptide-binding protein, SRP54, with the signal recognition particle (SRP) RNA in both archaeal and eukaryotic model systems. In vitro characterization of this process is now reported using recombinantly expressed components of SRP from the hyperthermophilic, sulfate-reducing archaeon Archaeoglobus fulgidis. A combination of native gel mobility shift, filter binding, and Ni-NTA agarose bead binding assays were used to determine the binding constants for binary and ternary complexes of SRP proteins and SRP RNA. Archaeal SRP54, unlike eukaryotic homologues, has significant intrinsic affinity for 7S RNA (K(D) approximately 15 nM), making it possible to directly compare particles formed in the presence and absence of SRP19 and thereby assess the precise role of SRP19 in the assembly process. Chemical modification studies using hydroxyl radicals and DEPC identify nonoverlapping primary binding sites for SRP19 and SRP54 corresponding to the tips of helix 6 and helix 8 (SRP19) and the distal loop and asymmetric bulge of helix 8 (SRP54). SRP19 additionally induces conformational changes concentrated in the proximal asymmetric bulge of helix 8. Selected nucleotides in this bulge become modified as a result of SRP19 binding but are subsequently protected from modification by formation of the complete complex with SRP54. Together these results suggest a model for assembly in which bridging the ends of helix 6 and helix 8 by SRP19 induces a long-range structural change to present the proximal bulge in a conformation compatible with high-affinity SRP54 binding.     

Hierarchical assembly of the Alu domain of the mammalian signal recognition particle

The mammalian signal recognition particle (SRP) catalytically promotes cotranslational translocation of signal sequence containing proteins across the endoplasmic reticulum membrane. While the S-domain of SRP binds the N-terminal signal sequence on the nascent polypeptide, the Alu domain of SRP temporarily interferes with the ribosomal elongation cycle until the translocation pore in the membrane is correctly engaged. Here we present biochemical and biophysical evidence for a hierarchical assembly pathway of the SRP Alu domain. The proteins SRP9 and SRP14 first heterodimerize and then initially bind to the Alu RNA 5' domain. This creates the binding site for the Alu RNA 3' domain. Alu RNA then undergoes a large conformational change with the flexibly linked 3' domain folding back by 180 degrees onto the 5' domain complex to form the final compact Alu ribonucleoprotein particle (Alu RNP). We discuss the possible mechanistic consequences of the likely reversibility of this final step with reference to translational regulation by the SRP Alu domain and with reference to the structurally similar Alu RNP retroposition intermediates derived from Alu elements in genomic DNA.     

A threefold RNA-protein interface in the signal recognition particle gates native complex assembly

Intermediate states play well-established roles in the folding and misfolding reactions of individual RNA and protein molecules. In contrast, the roles of transient structural intermediates in multi-component ribonucleoprotein (RNP) assembly processes and their potential for misassembly are largely unexplored. The SRP19 protein is unstructured but forms a compact core domain and two extended RNA-binding loops upon binding the signal recognition particle (SRP) RNA. The SRP54 protein subsequently binds to the fully assembled SRP19-RNA complex to form an intimate threefold interface with both SRP19 and the RNA and without significantly altering the structure of SRP19. We show, however, that the presence of SRP54 during SRP19-RNA assembly dramatically alters the folding energy landscape to create a non-native folding pathway that leads to an aberrant SRP19-RNA conformation. The misassembled complex arises from the surprising ability of SRP54 to bind rapidly to an SRP19-RNA assembly intermediate and to interfere with subsequent folding of one of the RNA binding loops at the three-way protein-RNA interface. An incorrect temporal order of assembly thus readily yields a non-native three-component ribonucleoprotein particle. We propose there may exist a general requirement to regulate the order of interaction in multi-component RNP assembly reactions by spatial or temporal compartmentalization of individual constituents in the cell.     

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.     

Evidence for an extended 7SL RNA structure in the signal recognition particle.

The signal recognition particle (SRP) functions in conjunction with the SRP receptor to target nascent ectoplasmic proteins to the protein translocation machinery of the endoplasmic reticulum membrane. SRP is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of 7SL RNA 300 nucleotides long. SRP has previously been visualized by a variety of electron microscopic techniques as a rod-shaped particle 24 nm long and 6 nm wide. We report here microanalysis by electron spectroscopic imaging which localizes the RNA molecule in SRP to primarily the two ends of the particle. These results suggest that the single 7SL RNA molecule spans the length of the particle. Micrographs from a scanning transmission electron microscope permit visualization of unstained SRP with low electron exposure, as well as the direct measurement of the mol. wt of the particle. These micrographs confirm our earlier suggestion that SRP is divided into three structural domains and allow discrimination of the two ends of the structure. The results of both techniques have been combined in a model for the structure of SRP in which we propose the basic orientation of the 7SL RNA. The structure proposed is consistent with the secondary structure predicted for the RNA and with biochemical data.     

Evidence for a specific mechanism of laminin assembly

The specificity of laminin chain assembly was investigated using fragments E8 and C8-9, derived from the long arm of the molecule, whose rod-like domain consists of the alpha-helical regions of the A, B1 and B2 chains. Urea-induced chain separation and unfolding were monitored by transverse urea/polyacrylamide gel electrophoresis (PAGE) and circular dichroism. Separation of the A and disulphide-linked B1-B2 chains occurred at 3.5-4.0 M urea and by 7.0 M urea all residual alpha-helicity was lost. Removal of urea by dialysis resulted in high recoveries (87-100%) of renatured protein which in its apparent molecular mass, alpha-helix content, chain composition, degree of association and ultrastructural appearance was indistinguishable from native E8. Reduction or reduction and alkylation of the chains did not lead to a decrease in their ability to reassemble specifically. Reformation of the single interchain disulphide, linking the B1 and B2 chains, clearly demonstrates that these chains are correctly aligned in parallel and in register in E8 renatured from its reduced chains.Renaturation of E8 from its reduced and alkylated chains precludes a role for disulphide formation in determining chain alignment but suggests rather than it is involved in the stabilisation of the correctly assembled molecule. These results, together with recent sequence data, provide evidence for the interaction of the alpha-helical regions of the A, B1 and B2 chains in the formation of a triple coiled-coil within the long arm of the molecule. The highly specific nature of this interaction suggests that it is the mechanism by which laminin is assembled in vivo.     

Laminin polymerization in vitro. Evidence for a two-step assembly with domain specificity

Laminin, a major structural glycoprotein of basement membranes, has been found to self-associate in vitro into large polymers. The formation of these complexes can be followed by the development of turbidity upon incubation in neutral phosphate buffer at 21-35 degrees C and is seen to be time-, concentration-, and temperature-dependent. The process is thermally reversible at 4 degrees C and the protein can be cycled between a dispersed and an aggregated state by alternating between 4 and 35 degrees C. Following incubation at 35 degrees C much of the monomeric laminin, which sediments at 11.4 S, is now seen to sediment at greater than 25 S. Both by turbidometric and sedimentation analysis, an apparent critical concentration for assembly of about 0.1 mg/ml (10(-7) M) is observed and is interpreted as evidence for a nucleation-propagation polymerization mechanism. The relative paucity of intermediates seen in a size-distribution analysis lends further support for this model. On platinum replicas obtained by rotary shadowing analysis, mostly free monomers are seen in the cold while after incubation at 35 degrees C, large multimeric aggregates with smaller amounts of oligomers are observed. The interaction between individual molecules appears to be specific because the dimers, trimers, and smaller oligomers are only associated at the terminal globular domains of the laminin molecules. In addition, removal of the globular domains of laminin with pepsin, which yields fragment P1, abolishes self-association. A divalent cation dependency for polymerization can be demonstrated and incubation in the presence of EDTA stops the polymerization at an oligomeric intermediate step. Hence overall laminin self-assembly can be divided into at least two steps: an initial temperature-dependent, divalent cation independent step followed by a divalent cation-dependent step.     

Protein-induced conformational changes of RNA during the assembly of human signal recognition particle

The human signal recognition particle (SRP) is a large RNA-protein complex that targets secretory and membrane proteins to the endoplasmic reticulum membrane. The S domain of SRP is composed of roughly half of the 7SL RNA and four proteins (SRP19, SRP54, and the SRP68/72 heterodimer). In order to understand how the binding of proteins induces conformational changes of RNA and affects subsequent binding of other protein subunits, we have performed chemical and enzymatic probing of all S domain assembly intermediates. Ethylation interference experiments show that phosphate groups in helices 5, 6 and 7 that are essential for the binding of SRP68/72 are all on the same face of the RNA. Hydroxyl radical footprinting and dimethylsulphate (DMS) modifications show that SRP68/72 brings the lower part of helices 6 and 8 closer. SRP68/72 binding also protects the SRP54 binding site (helix 8 asymmetric loop) from chemical modification and RNase cleavage, whereas, in the presence of both SRP19 and SRP68/72, the long strand of helix 8 asymmetric loop becomes readily accessible to chemical and enzymatic probes. These results indicate that the RNA platform observed in the crystal structure of the SRP19-SRP54M-RNA complex already exists in the presence of SRP68/72 and SRP19. Therefore, SRP68/72, together with SRP19, rearranges the 7SL RNA in an SRP54 binding competent state.