Bacillus subtilis histone-like protein, HBsu, is an integral component of a SRP-like particle that can bind the Alu domain of small cytoplasmic RNA

Small cytoplasmic RNA (scRNA) is metabolically stable and abundant in Bacillus subtilis cells. Consisting of 271 nucleotides, it is structurally homologous to mammalian signal recognition particle RNA. In contrast to 4.5 S RNA of Escherichia coli, B. subtilis scRNA contains an Alu domain in addition to the evolutionarily conserved S domain. In this study, we show that a 10-kDa protein in B. subtilis cell extracts has scRNA binding activity at the Alu domain. The in vitro binding selectivity of the 10-kDa protein shows that it recognizes the higher structure of the Alu domain of scRNA caused by five consecutive complementary sequences in the two loops. Purification and subsequent analyses demonstrated that the 10-kDa protein is HBsu, which was originally identified as a member of the histone-like protein family. By constructing a HBsu-deficient B. subtilis mutant, we showed that HBsu is essential for normal growth. Immunoprecipitating cell lysates using anti-HBsu antibody yielded scRNA. Moreover, the co-precipitation of HBsu with (His)6-tagged Ffh depended on the presence of scRNA, suggesting that HBsu, Ffh, and scRNA make a ternary complex and that scRNA serves as a functional unit for binding. These results demonstrated that HBsu is the third component of a signal recognition particle-like particle in B. subtilis that can bind the Alu domain of scRNA.     

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.     

Structures of SRP54 and SRP19, the two proteins that organize the ribonucleic core of the signal recognition particle from Pyrococcus furiosus

In all organisms the Signal Recognition Particle (SRP), binds to signal sequences of proteins destined for secretion or membrane insertion as they emerge from translating ribosomes. In Archaea and Eucarya, the conserved ribonucleoproteic core is composed of two proteins, the accessory protein SRP19, the essential GTPase SRP54, and an evolutionarily conserved and essential SRP RNA. Through the GTP-dependent interaction between the SRP and its cognate receptor SR, ribosomes harboring nascent polypeptidic chains destined for secretion are dynamically transferred to the protein translocation apparatus at the membrane. We present here high-resolution X-ray structures of SRP54 and SRP19, the two RNA binding components forming the core of the signal recognition particle from the hyper-thermophilic archaeon Pyrococcus furiosus (Pfu). The 2.5 A resolution structure of free Pfu-SRP54 is the first showing the complete domain organization of a GDP bound full-length SRP54 subunit. In its ras-like GTPase domain, GDP is found tightly associated with the protein. The flexible linker that separates the GTPase core from the hydrophobic signal sequence binding M domain, adopts a purely alpha-helical structure and acts as an articulated arm allowing the M domain to explore multiple regions as it scans for signal peptides as they emerge from the ribosomal tunnel. This linker is structurally coupled to the GTPase catalytic site and likely to propagate conformational changes occurring in the M domain through the SRP RNA upon signal sequence binding. Two different 1.8 A resolution crystal structures of free Pfu-SRP19 reveal a compact, rigid and well-folded protein even in absence of its obligate SRP RNA partner. Comparison with other SRP19*SRP RNA structures suggests the rearrangement of a disordered loop upon binding with the RNA through a reciprocal induced-fit mechanism and supports the idea that SRP19 acts as a molecular scaffold and a chaperone, assisting the SRP RNA in adopting the conformation required for its optimal interaction with the essential subunit SRP54, and proper assembly of a functional SRP.     

Structural requirements of Bacillus subtilis small cytoplasmic RNA for cell growth, sporulation, and extracellular enzyme production.

Bacillus subtilis small cytoplasmic RNA (scRNA; 271 nucleotides) is a member of the signal recognition particle (SRP) RNA family, which has evolutionarily conserved primary and secondary structures. The scRNA consists of three domains corresponding to domains I, II, and IV of human SRP 7S RNA. To identify the structural determinants required for its function, we constructed mutant scRNAs in which individual domains or conserved nucleotides were deleted, and their importance was assayed in vivo. The results demonstrated that domain IV of scRNA is necessary to maintain cell viability. On the other hand, domains I and II were not essential for vegetative growth but were preferentially required for the RNA to achieve its active structure, and assembled ribonucleoprotein between Ffh and scRNA is required for sporulation to proceed. This view is highly consistent with the fact that the presence of domains I and II is restricted to sporeforming B. subtilis scRNA among eubacterial SRP RNA-like RNAs.     

RNA interference of signal peptide-binding protein SRP54 elicits deleterious effects and protein sorting defects in trypanosomes

Trypanosomes are protozoan parasites that have a major impact on health. This family diverged very early from the eukaryotic lineage and possesses unique RNA processing mechanisms such as trans-splicing and RNA editing. The trypanosome signal recognition particle (SRP) has a unique composition compared with all known SRP complexes, because it contains two RNA molecules, the 7SL RNA and a tRNA-like molecule. RNA interference was utilized to elucidate the essentiality of the SRP pathway and its role in protein translocation in Trypanosoma brucei. The production of double stranded RNA specific for the signal peptide-binding protein SRP54 induced the degradation of the mRNA and a loss of the SRP54 protein. SRP54 depletion elicited inhibition in growth and cytokinesis, suggesting that the SRP pathway is essential. The translocation of four signal peptide-containing proteins was examined. Surprisingly, the proteins were translocated to the endoplasmic reticulum and properly processed. However, the surface EP procyclin, the lysosomal protein p67, and the flagellar pocket protein CRAM were mislocalized and accumulated in megavesicles, most likely because of a secondary effect on protein sorting. The translocation of these proteins to the endoplasmic reticulum under SRP54 depletion suggests that an alternative pathway for protein translocation exists in trypanosomes.     

A complex of the signal sequence binding protein and the SRP RNA promotes translocation of nascent proteins.

Translocation of proteins across the endoplasmic reticulum membrane is initiated by the signal recognition particle (SRP), a cytoplasmic ribonucleoprotein complex consisting of a 7S RNA and six polypeptides. To investigate the functions of the SRP components, we have tested the activities of several SRP subparticles. We show that the SRP GTPase (SRP54) alone binds a signal sequence and discriminates it from a non-signal sequence. Although SRP54 alone is unable to promote translocation, SRP54 in a complex with SRP RNA is both necessary and sufficient to promote translocation of an elongation-arrested nascent protein in a GTP-regulated manner. For co-translational translocation, additional SRP components are required. We discuss the implications of our results for the function of the Escherichia coli SRP which is homologous to the SRP54/SRP-RNA complex.     

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.     

Signal recognition particle-dependent protein targeting, universal to all kingdoms of life

The signal recognition particle (SRP) and its membrane-bound receptor represent a ubiquitous protein-targeting device utilized by organisms as different as bacteria and humans, archaea and plants. The unifying concept of SRP-dependent protein targeting is that SRP binds to signal sequences of newly synthesized proteins as they emerge from the ribosome. In eukaryotes this interaction arrests or retards translation elongation until SRP targets the ribosome-nascent chain complexes via the SRP receptor to the translocation channel. Such channels are present in the endoplasmic reticulum of eukaryotic cells, the thylakoids of chloroplasts, or the plasma membrane of prokaryotes. The minimal functional unit of SRP consists of a signal sequence-recognizing protein and a small RNA. The as yet most complex version is the mammalian SRP whose RNA, together with six proteinaceous subunits, undergo an intricate assembly process. The preferential substrates of SRP possess especially hydrophobic signal sequences. Interactions between SRP and its receptor, the ribosome, the signal sequence, and the target membrane are regulated by GTP hydrolysis. SRP-dependent protein targeting in bacteria and chloroplasts slightly deviate from the canonical mechanism found in eukaryotes. Pro- and eukaryotic cells harbour regulatory mechanisms to prevent a malfunction of the SRP pathway. Electronic Publication     

The 54-kD protein of signal recognition particle contains a methionine- rich RNA binding domain

Signal recognition particle (SRP) plays the key role in targeting secretory proteins to the membrane of the endoplasmic reticulum (Walter, P., and V. R. Lingappa. 1986. Annu. Rev. Cell Biol. 2:499- 516). It consists of SRP7S RNA and six proteins. The 54-kD protein of SRP (SRP54) recognizes the signal sequence of nascent polypeptides. The 19-kD protein of SRP (SRP19) binds to SRP7S RNA directly and is required for the binding of SRP54 to the particle. We used deletion mutants of SRP19 and SRP54 and an in vitro assembly assay in the presence of SRP7S RNA to define the regions in both proteins which are required to form a ribonucleoprotein particle. Deletion of the 21 COOH- terminal amino acids of SRP19 does not interfere with its binding to SRP7S RNA. Further deletions abolish SRP19 binding to SRP7S RNA. The COOH-terminal 207 amino acids of SRP54 (M domain) were found to be necessary and sufficient for binding to the SRP19/7S RNA complex in vitro. Limited protease digestion of purified SRP confirmed our results for SRP54 from the in vitro binding assay. The SRP54M domain could also bind to Escherichia coli 4.5S RNA that is homologous to part of SRP7S RNA. We suggest that the methionine-rich COOH terminus of SRP54 is a RNA binding domain and that SRP19 serves to establish a binding site for SRP54 on the SRP7S RNA.     

SRP-dependent co-translational targeting and SecA-dependent translocation analyzed as individual steps in the export of a bacterial protein

Recently it has been recognized that the signal recognition particle (SRP) of Escherichia coli represents a specific targeting device for hydrophobic inner membrane proteins. It has remained unclear, however, whether the bacterial SRP functions in concert with SecA, which is required for the translocation of secretory proteins across the inner membrane. Here, we have analyzed a hybrid protein constructed by fusing the signal anchor sequence of an SRP-dependent inner membrane protein (MtlA) to the mature part of an exclusively SecA-requiring secretory protein (OmpA). We show that the signal anchor sequence of MtlA confers the novel properties onto nascent chains of OmpA of being co-translationally recognized and targeted to SecY by SRP. Once targeted to SecY, ribosome-associated nascent chains of the hybrid protein, however, remain untranslocated unless SecA is present. These results indicate that SRP and SecA cooperate in a sequential, non-overlapping manner in the topogenesis of those membrane proteins which, in addition to a signal anchor sequence, harbor a substantial hydrophilic domain to be translocated into the periplasm.     

Insertion of a multispanning membrane protein occurs sequentially and requires only one signal sequence

To study the insertion of multispanning membrane proteins into the endoplasmic reticulum, we constructed novel proteins on the cDNA level by repeating, up to four times, the internal signal-anchor domain of the asialoglycoprotein receptor H1. Upon in vitro translation in the presence of microsomes, these polypeptides are indeed inserted as polytopic membrane proteins. The first hydrophobic domain functions as a signal and the second as a stop-transfer sequence, while the third initiates a second translocation process, halted again by the fourth. We were able to demonstrate that insertion occurs sequentially, starting with the first apolar segment from the amino terminus. By replacing the original signal-anchor domains by a mutant sequence not recognized by signal recognition particle (SRP), it was shown that only the first hydrophobic domain needs to be a signal sequence and that the second translocation event does not require SRP.     

The signal sequence interacts with the methionine-rich domain of the 54- kD protein of signal recognition particle

The signal sequence of nascent preprolactin interacts with the 54-kD protein of the signal recognition particle (SRP54). To identify the domain or site on SRP54 that interacts with the signal sequence we used a photocross-linking approach followed by limited proteolysis and immunoprecipitation using anti-peptide antibodies specific for defined regions of SRP54. We found that the previously identified methionine-rich RNA-binding domain of SRP54 (SRP54M domain) also interacts with the signal sequence. The smallest fragment that was found to be crosslinked to the signal sequence comprised the COOH-terminal 6-kD segment of the SRP54M domain. No cross-link to the putative GTP-binding domain of SRP54 (SRP54G domain) was found. Proteolytic cleavage between the SRP54M domain and SRP54G domain did not impair the subsequent interaction between the signal sequence and the SRP54M domain. Our results show that both the RNA binding and signal sequence binding functions of SRP54 are performed by the SRP54M domain.     

Down-regulation of the trypanosomatid signal recognition particle affects the biogenesis of polytopic membrane proteins but not of signal peptide-containing proteins

Protein translocation across the endoplasmic reticulum is mediated by the signal recognition particle (SRP). In this study, the SRP pathway in trypanosomatids was down-regulated by two approaches: RNA interference (RNAi) silencing of genes encoding SRP proteins in Trypanosoma brucei and overexpression of dominant-negative mutants of 7SL RNA in Leptomonas collosoma. The biogenesis of both signal peptide-containing proteins and polytopic membrane proteins was examined using endogenous and green fluorescent protein-fused proteins. RNAi silencing of SRP54 or SRP68 in T. brucei resulted in reduced levels of polytopic membrane proteins, but no effect on the level of signal peptide-containing proteins was observed. When SRP deficiency was achieved in L. collosoma by overexpression of dominant-negative mutated 7SL RNA, a major effect was observed on polytopic membrane proteins but not on signal peptide-containing proteins. This study included two trypanosomatid species, tested various protein substrates, and induced depletion of the SRP pathway by affecting either the levels of SRP binding proteins or that of SRP RNA. Our results demonstrate that, as in bacteria but in contrast to mammalian cells, the trypanosome SRP is mostly essential for the biogenesis of membrane proteins.     

Signal recognition particle receptor is important for cell growth and protein secretion in Saccharomyces cerevisiae.

In mammalian cells, the signal recognition particle (SRP) receptor is required for the targeting of nascent secretory proteins to the endoplasmic reticulum (ER) membrane. We have identified the Saccharomyces cerevisiae homologue of the alpha-subunit of the SRP receptor (SR alpha) and characterized its function in vivo. S. cerevisiae SR alpha is a 69-kDa peripheral membrane protein that is 32% identical (54% chemically similar) to its mammalian homologue and, like mammalian SR alpha, is predicted to contain a GTP binding domain. Yeast cells that contain the SR alpha gene (SRP101) under control of the GAL1 promoter show impaired translocation of soluble and membrane proteins across the ER membrane after depletion of SR alpha. The degree of the translocation defect varies for different proteins. The defects are similar to those observed in SRP deficient cells. Disruption of the SRP101 gene results in an approximately sixfold reduction in the growth rate of the cells. Disruption of the gene encoding SRP RNA (SCR1) or both SCR1 and SRP101 resulted in an indistinguishable growth phenotype, indicating that SRP receptor and SRP function in the same pathway. Taken together, these results suggest that the components and the mechanism of the SRP-dependent protein targeting pathway are evolutionarily conserved yet not essential for cell growth. Surprisingly, cells that are grown for a prolonged time in the absence of SRP or SRP receptor no longer show pronounced protein translocation defects. This adaptation is a physiological process and is not due to the accumulation of a suppressor mutation. The degree of this adaptation is strain dependent.     

The methionine-rich domain of the 54 kd protein subunit of the signal recognition particle contains an RNA binding site and can be crosslinked to a signal sequence.

The 54 kd protein subunit of the signal recognition particle (SRP54) has been shown to bind signal sequences by UV crosslinking. Primary structure analysis and phylogenetic comparisons have suggested that SRP54 is composed of two domains: an amino-terminal domain that contains a putative GTP-binding site (G-domain) and a carboxy-terminal domain that contains a high abundance of methionine residues (M-domain). Partial proteolysis of SRP revealed that the two proposed domains of SRP54 indeed represent structurally discrete entities. Upon proteolysis the intact G-domain was released from SRP, whereas the M-domain remained attached to the core of the particle. Reconstitution experiments demonstrated that the isolated M-domain associates with 7SL RNA in the presence of SRP19. In addition, we observed a specific binding of the M-domain directly to 4.5S RNA of Escherichia coli, which contains a structural motif also present in 7SL RNA. This shows that the M-domain contains an RNA binding site, and suggests that SRP54 may be linked to the rest of SRP through this domain by a direct interaction with 7SL RNA. Using UV crosslinking, we found that in an in vitro translation system the preprolactin signal sequence contacts SRP through the M-domain of SRP54. These results imply that the M-domain contains the signal sequence binding site of SRP54, although we cannot exclude that the G-domain may also be in proximity to bound signal sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unraveling the components of protein translocation pathway in human malaria parasite Plasmodium falciparum

The targeting and translocation of proteins is an essentially required and conserved process in all the living organisms. This complex process involves multiple steps and requires a variety of factors before the protein reaches its final destination. The major components of translocation machinery are signal recognition particle (SRP) and secretory (Sec) complex. These are composed of highly conserved components. SRP contains SRP RNA and other polypeptides such as SRP9, SRP14, SRP19 and SRP54. Sec complex is composed of Sec61αβγ, Sec62 and Sec63. In this review using bioinformatics approach we have shown that the P. falciparum genome contains the homologues for all of these and other factors such as SRP receptor, and TRAM (translocation associated membrane protein), which are required for post- and co-translational protein translocation. We have also shown the various steps of translocation in a hypothetical model.     

Role of protein translocation pathways across the endoplasmic reticulum in Trypanosoma brucei

The translocation of secretory and membrane proteins across the endoplasmic reticulum (ER) membrane is mediated by co-translational (via the signal recognition particle (SRP)) and post-translational mechanisms. In this study, we investigated the relative contributions of these two pathways in trypanosomes. A homologue of SEC71, which functions in the post-translocation chaperone pathway in yeast, was identified and silenced by RNA interference. This factor is essential for parasite viability. In SEC71-silenced cells, signal peptide (SP)-containing proteins traversed the ER, but several were mislocalized, whereas polytopic membrane protein biogenesis was unaffected. Surprisingly trypanosomes can interchangeably utilize two of the pathways to translocate SP-containing proteins except for glycosylphosphatidylinositol-anchored proteins, whose level was reduced in SEC71-silenced cells but not in cells depleted for SRP68, an SRP-binding protein. Entry of SP-containing proteins to the ER was significantly blocked only in cells co-silenced for the two translocation pathways (SEC71 and SRP68). SEC63, a factor essential for both translocation pathways in yeast, was identified and silenced by RNA interference. SEC63 silencing affected entry to the ER of both SP-containing proteins and polytopic membrane proteins, suggesting that, as in yeast, this factor is essential for both translocation pathways in vivo. This study suggests that, unlike bacteria or other eukaryotes, trypanosomes are generally promiscuous in their choice of mechanism for translocating SP-containing proteins to the ER, although the SRP-independent pathway is favored for glycosylphosphatidylinositol-anchored proteins, which are the most abundant surface proteins in these parasites.     

Structural and energetic analysis of RNA recognition by a universally conserved protein from the signal recognition particle

The signal recognition particle (SRP) is a ribonucleoprotein complex responsible for targeting proteins to the endoplasmic reticulum in eukarya or to the inner membrane in prokarya. The crystal structure of the universally conserved RNA-protein core of the Escherichia coli SRP, refined here to 1.5 A resolution, revealed minor groove recognition of the 4.5 S RNA component by the M domain of the Ffh protein. Within the RNA, nucleotides comprising two phylogenetically conserved internal loops create a unique surface for protein recognition. To determine the energetic importance of conserved nucleotides for SRP assembly, we measured the affinity of the M domain for a series of RNA mutants. This analysis reveals how conserved nucleotides within the two internal loop motifs establish the architecture of the macromolecular interface and position essential functional groups for direct recognition by the 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.