Inhibition of the biosynthesis of SRP polypeptides and secretory proteins by aflatoxin B1 can disrupt protein targeting

Cell culture and western blotting studies revealed that aflatoxin B(1) (AFB(1)) inhibits the biosynthesis of two of the constituent polypeptides of signal recognition particle (SRP) (SRP54 and 72). SRP escorts polyribosomes carrying signal peptides from free form in the cytosol to the bound form on endoplasmic reticulum (ER) membrane during protein targeting. These effects of AFB(1) on SRP biosynthesis may inhibit the formation of functional SRP. Our experiments have further shown that AFB(1) also inhibits the biosynthesis/translocation of a secretory protein, preprolactin, which fails to appear in the lumen of ER consequent to the treatment with this hepatocarcinogen. The results of the experiments presented in this article therefore enable us to infer for the first time that aflatoxin B(1) may inhibit the functioning of SRP as an escort and deplete the ER of polyribosomes for secretory protein synthesis. As these secretory proteins are important components of the plasma membrane, gap junctions and intercellular matrix, their absence from these locations could disturb cell to cell communication leading to tumorigenesis.     

Protein SRP68 of human signal recognition particle: identification of the RNA and SRP72 binding domains

The signal recognition particle (SRP) plays an important role in the delivery of secretory proteins to cellular membranes. Mammalian SRP is composed of six polypeptides among which SRP68 and SRP72 form a heterodimer that has been notoriously difficult to investigate. Human SRP68 was purified from overexpressing Escherichia coli cells and was found to bind to recombinant SRP72 as well as in vitro-transcribed human SRP RNA. Polypeptide fragments covering essentially the entire SRP68 molecule were generated recombinantly or by proteolytic digestion. The RNA binding domain of SRP68 included residues from positions 52 to 252. Ninety-four amino acids near the C terminus of SRP68 mediated the binding to SRP72. The SRP68-SRP72 interaction remained stable at elevated salt concentrations and engaged approximately 150 amino acids from the N-terminal region of SRP72. This portion of SRP72 was located within a predicted tandem array of four tetratricopeptide (TPR)-like motifs suggested to form a superhelical structure with a groove to accommodate the C-terminal region of SRP68.     

Signal recognition particle triggers the translocation of storage globulin polypeptides from field beans (Vicia faba L.) across mammalian endoplasmic reticulum membrane

Hybridization-selected mRNAs coding for individual storage globulin polypeptides of field beans (Vicia faba L.) were translated in a cell-free system. Added mammalian signal recognition particle (SRP) recognizes cleavable signal peptides of the major vicilin and both legumin polypeptide precursors and induces translational arrest. The latter can be released by potassium-washed membranes (K-RM) leading to shortened polypeptides protected against proteases. Thus, SRP and K-RM function in a similar way with plant polypeptides as described for mammalian secretory proteins [(1981) J. Cell Biol. 91, 557-561]. Obviously, the initial steps in the biosynthesis and processing of plant storage globulin polypeptides are principally identical to those of animal secretory proteins.     

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.     

Characterization of the SRP68/72 interface of human signal recognition particle by systematic site‐directed mutagenesis

The signal recognition particle (SRP) is a ribonucleoprotein complex which is crucial for the delivery of proteins to cellular membranes. Among the six proteins of the eukaryotic SRP, the two largest, SRP68 and SRP72, form a stable SRP68/72 heterodimer of unknown structure which is required for SRP function. Fragments 68e′ (residues 530 to 620) and 72b′ (residues 1 to 166) participate in the SRP68/72 interface. Both polypeptides were expressed in Escherichia coli and assembled into a complex which was stable at high ionic strength. Disruption of 68e′/72b′ and SRP68/72 was achieved by denaturation using moderate concentrations of urea. The four predicted tetratricopeptide repeats (TPR1 to TPR4) of 72b′ were required for stable binding of 68e′. Site‐directed mutagenesis suggested that they provide the structural framework for the binding of SRP68. Deleting the region between TPR3 and TPR4 (h120) also prevented the formation of a heterodimer, but this predicted alpha‐helical region appeared to engage several of its amino acid residues directly at the interface with 68e′. A 39‐residue polypeptide (68h, residues 570–605), rich in prolines and containing an invariant aspartic residue at position 585, was found to be active. Mutagenesis scanning of the central region of 68h demonstrated that D585 was solely responsible for the formation of the heterodimer. Coexpression experiments suggested that 72b′ protects 68h from proteolytic digestion consistent with the assertion that 68h is accommodated inside a groove formed by the superhelically arranged four TPRs of the N‐terminal region of SRP72.     

The Archaeal Signal Recognition Particle: Steps Toward Membrane Binding

Signal recognition particles and their receptors target ribosome nascent chain complexes of preproteins toward the protein translocation apparatus of the cell. The discovery of essential SRP components in the third urkingdom of the phylogenetic tree, the archaea (Woese, C. R., and Fox, G. E. (1977). Proc. Natl. Acad. Sci. U.S.A. 74, 5088-5090) raises questions concerning the structure and composition of the archaeal signal recognition particle as well as the functions that route nascent prepoly peptide chains to the membrane. Investigations of the archaeal SRP pathway could therefore identify novel aspects of this process not previously reported or unique to archaea when compared with the respective eukaryal and bacterial systems.     

TLR序列在SRP54蛋白与SRPRNA和信号肽结合中的作用

SRP54蛋白是信号识别颗粒(signal recognition particle)的一个关键组分.对人SRP54蛋白328～330位的TLR3个氨基酸进行人工诱变,在大肠杆菌BL21(DE3)pLysS中表达了A3突变体,并对A3突变体进行纯化和Superdex75凝胶过滤分析.观察到A3突变体丧失了与SRPRNA结合的能力,其自身也不能形成二聚体.结果证明,TLR这3个氨基酸残基与二聚体结构的形成有关,TLR是SRP54蛋白结合SRPRNA和新生蛋白质信号肽所必需的关键性氨基酸序列.     

Does a deficiency of the signal recognition particle (SRP)-pathway affect the biosynthesis of its components in Saccharomyces cerevisiae and Escherichia coli?

We studied the behavior of the signal recognition particle (SRP) components in Saccharomyces cerevisiae upon deficiencies of the protein transport caused by the absence of the SRP membrane receptor alpha-subunit. A decrease in the concentration of the SRP membrane receptor alpha-subunit in the cell significantly decreased the level of an SRP component, protein SRP72, as well as the levels of mRNAs of SRP protein components and the SRP receptor beta-subunit. But the amount of 7SL RNA remained unchanged. In contrast, in Escherichia coli cells the gradual decrease in the level of the protein FtsY (a homolog of the SRP membrane receptor alpha-subunit) was not associated with changes in the Ffh protein level.     

Signal recognition particle mediates post-translational targeting in eukaryotes

Signal recognition particle (SRP) plays a central role in the delivery of classical secretory and membrane proteins to the endoplasmic reticulum (ER). All nascent chains studied to date dissociate from SRP once released from the ribosome, thereby supporting a strictly cotranslational mode of action for eukaryotic SRP. We now report a novel post-translational function for SRP in the targeting of tail-anchored (TA) proteins to the ER. TA proteins possess a hydrophobic membrane insertion sequence at their C-terminus such that it can only emerge from the ribosome after translation is terminated. We show that SRP can associate post-translationally with this type of ER-targeting signal, and deliver newly synthesised TA proteins to the ER membrane by a pathway dependent upon GTP and the SRP receptor. We find that dependency upon this SRP-dependent route is precursor specific, and propose a unifying model to describe the biogenesis of TA proteins in vivo.     

Signal Recognition Particle-ribosome Binding Is Sensitive to Nascent Chain Length

The signal recognition particle (SRP) directs ribosome-nascent chain complexes (RNCs) displaying signal sequences to protein translocation channels in the plasma membrane of prokaryotes and endoplasmic reticulum of eukaryotes. It was initially proposed that SRP binds the signal sequence when it emerges from an RNC and that successful binding becomes impaired as translation extends the nascent chain, moving the signal sequence away from SRP on the ribosomal surface. Later studies drew this simple model into question, proposing that SRP binding is unaffected by nascent chain length. Here, we reinvestigate this issue using two novel and independent fluorescence resonance energy transfer assays. We show that the arrival and dissociation rates of SRP binding to RNCs vary according to nascent chain length, resulting in the highest affinity shortly after a functional signal sequence emerges from the ribosome. Moreover, we show that SRP binds RNCs in multiple and interconverting conformations, and that conversely, RNCs exist in two conformations distinguished by SRP interaction kinetics.     

Identification of an RNA-binding domain in human SRP72

The signal recognition particle (SRP) is a ribonucleoprotein complex that plays a crucial role during the delivery of secretory proteins from the ribosome to the cell membrane. Among the six proteins of the eukaryotic SRP, the 72 kDa protein (SRP72) is the largest and least characterized. Polypeptides corresponding to various regions of the entire human SRP72 sequence were expressed in Escherichia coli, purified, and partially proteolyzed. Human SRP RNA bound with high affinity to a 63 amino acid residue region near the C terminus of SRP72. Mild treatment of the fragment with chymotrypsin abolished its RNA-binding activity. A conserved sequence with the consensus PDPXRWLPXXER was identified within a 56 amino acid residue RNA-binding domain. Sucrose gradient centrifugation and filter-binding analysis using mutant SRP RNAs showed that SRP72 bound to the moderately conserved portion of SRP RNA helix 5. Nine tetratricopeptide-like repeats (TPRs) poised to interact with other SRP or ribosomal proteins were predicted in the NH2-terminal region. These identifications assign two important functions to a large portion of SRP72 and demonstrate the RNA-binding capacity of the protein.     

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.     

Nascent membrane and presecretory proteins synthesized in Escherichia coli associate with signal recognition particle and trigger factor

The Escherichia coli signal recognition particle (SRP) and trigger factor are cytoplasmic factors that interact with short nascent polypeptides of presecretory and membrane proteins produced in a heterologous in vitro translation system. In this study, we use an E. coli in vitro translation system in combination with bifunctional cross-linking reagents to investigate these interactions in more detail in a homologous environment. Using this approach, the direct interaction of SRP with nascent polypeptides that expose particularly hydrophobic targeting signals is demonstrated, suggesting that inner membrane proteins are the primary physiological substrate of the E. coli SRP. Evidence is presented that the overproduction of proteins that expose hydrophobic polypeptide stretches, titrates SRP. In addition, trigger factor is efficiently cross-linked to nascent polypeptides of different length and nature, some as short as 57 amino acid residues, indicating that it is positioned near the nascent chain exit site on the E. coli ribosome.     

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.     

Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome

As newly synthesized polypeptides emerge from the ribosome, they interact with chaperones and targeting factors that assist in folding and targeting to the proper location in the cell. In Escherichia coli, the chaperone trigger factor (TF) binds to nascent polypeptides early in biosynthesis facilitated by its affinity for the ribosomal proteins L23 and L29 that are situated around the nascent chain exit site on the ribosome. The targeting factor signal recognition particle (SRP) interacts specifically with the signal anchor (SA) sequence in nascent inner membrane proteins (IMPs). Here, we have used photocross-linking to map interactions of the SA sequence in a short, in vitro-synthesized, nascent IMP. Both TF and SRP were found to interact with the SA with partially overlapping binding specificity. In addition, extensive contacts with L23 and L29 were detected. Both purified TF and SRP could be cross-linked to L23 on nontranslating ribosomes with a competitive advantage for SRP. The results suggest a role for L23 in the targeting of IMPs as an attachment site for TF and SRP that is close to the emerging nascent chain.     

NMR solution structure and SRP54M predicted interaction of the N-terminal sequence (1-30) of the ovine Doppel protein

Prion protein (PrP^C) biosynthesis involves a multi-step process that includes translation and post-translational modifications. While PrP has been widely investigated, for the homolog Doppel (Dpl), limited knowledge is available. In this study, we focused on a vital step of eukaryotic protein biosynthesis: targeting by the signal recognition particle (SRP). Taking the ovine Dpl (OvDpl(1-30)) peptide as a template, we studied its behavior in two different hydrophobic environments using CD and NMR spectroscopy. In both trifluoroethanol (TFE) and dihexanoyl-sn-glycero-3-phosphatidylcholine (DHPC), the OvDpl(1-30) peptide revealed to fold in an alpha-helical conformation with a well-defined central region extending from residue Cys8 until Ser22. The NMR structure was subsequently included in a computational docking complex with the conserved M-domain of SRP54 protein (SRP54M), and further compared with the N-terminal structures of mouse Dpl and bovine PrP^C proteins. This allowed the determination of (i) common predicted N-terminal/SRP54M polar contacts (Asp331, Gln335, Glu365 and Lys432) and (ii) different N–C orientations between prion and Dpl peptides at the SRP54M hydrophobic groove, that are in agreement with each peptide electrostatic potential. Together, these findings provide new insights into the biosynthesis of prion-like proteins. Besides they also show the role of protein conformational switches in signalization toward the endoplasmic membrane, a key event of major significance in the cell cycle. They are thus of general applicability to the study of the biological function of prion-like as well as other proteins.     

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.     

Sequence-specific interactions of nascent Escherichia coli polypeptides with trigger factor and signal recognition particle

As nascent polypeptides exit the ribosomal tunnel they immediately associate with chaperones, folding catalysts, and targeting factors. These interactions are decisive for the future conformation and destination of the protein that is being synthesized. Using Escherichia coli as a model organism, we have systematically analyzed how the earliest contacts of nascent polypeptides with cytosolic factors depend on the nature and future destination of the emerging sequence using a photo cross-linking approach. Together, the data suggest that the chaperone trigger factor is adjacent to emerging sequences by default, consistent with both its placement near the nascent chain exit site and its cellular abundance. The signal recognition particle (SRP) effectively competes the contact with TF when a signal anchor (SA) sequence of a nascent inner membrane protein appears outside the ribosome. The SRP remains in contact with the SA and downstream sequences during further synthesis of approximately 30 amino acids. The contact with trigger factor is then restored unless another transmembrane segment reinitiates SRP binding. Importantly and in contrast to published data, the SRP appears perfectly capable of distinguishing SA sequences from signal sequences in secretory proteins at this early stage in biogenesis.