The N-domain of the signal recognition particle 54-kDa subunit promotes efficient signal sequence binding

The signal recognition particle 54-kDa subunit (SRP54) binds to the signal sequences of nascent presecretory and transmembrane proteins. Previous studies have shown that signal sequences bind to the C-terminal methionine-rich domain of the protein (M-domain), but have raised the possibility that either the N-terminal domain (N-domain) or the central guanosine triphosphatase module (GTPase-domain) also contribute to signal-sequence-binding activity. We have generated a series of N-domain and GTPase-domain mutants to investigate this issue further. Mutations in a conserved N-domain motif (ALLEADV) produced significant defects in signal sequence binding that correlate with the severity of the mutation. The magnitude of the defect was independent of the preprotein substrate, which suggested that the mutations do not alter the specificity of signal sequence recognition. The N-domain mutants also showed defects in promoting the translocation of presecretory proteins across the membrane of microsomal vesicles, but these defects appeared to be a direct consequence of the reduction in signal-sequence-binding activity and not separate effects of the mutations. By contrast, mutations in the guanosine triphosphatase consensus sequence had no effect on signal sequence binding, but instead severely impaired protein translocation activity. These results indicate that a principal function of the SRP54 N-domain is to promote efficient signal sequence binding. These data also suggest that the SRP54 GTPase regulates the cycle of signal sequence binding and release, perhaps by modulating the relative orientation of the N- and M-domains.     

Assembly of the 68- and 72-kD proteins of signal recognition particle with 7S RNA

Signal recognition particle (SRP), the cytoplasmic ribonucleoprotein particle that mediates the targeting of proteins to the ER, consists of a 7S RNA and six different proteins. The 68- (SRP68) and 72- (SRP72) kD proteins of SRP are bound to the 7S RNA of SRP as a heterodimeric complex (SRP68/72). Here we describe the primary structure of SRP72 and the assembly of SRP68, SRP72 and 7S RNA into a ribonucleoprotein particle. The amino acid sequence deduced from the cDNA of SRP72 reveals a basic protein of 671 amino acids which shares no sequence similarity with any protein in the sequence data libraries. Assembly of SRP72 into a ribonucleoprotein particle required the presence of 7S RNA and SRP68. In contrast, SRP68 alone specifically bound to 7S RNA. SRP68 contacts the 7S RNA via its NH2-terminal half while COOH-terminal portions of SRP68 and SRP72 are in contact with each other in SRP. SRP68 thus serves as a link between 7S RNA and SRP72. As a large NH2-terminal domain of SRP72 is exposed on SRP it may be a site of contact to other molecules involved in the SRP cycle between the ribosome and the ER membrane.     

A chromodomain protein encoded by the arabidopsis CAO gene is a plant-specific component of the chloroplast signal recognition particle pathway that is involved in LHCP targeting

Adjustment outcomes of 224 transracial and inracial adoptees were investigated using data collected over 17 years. Findings reveal an association between adoptees' outcomes and their race, gender, and adoptive family structure. Placement history was not significant. Implications for policy and practice are discussed, as are future directions for research.     

Another factor besides hydrophobicity can affect signal peptide interaction with signal recognition particle

Translocation of alkaline extracellular protease (AEP) into the endoplasmic reticulum of Yarrowia lipolytica is cotranslational and signal recognition particle (SRP)-dependent, whereas translocation of P17M AEP (proline to methionine at position 17, second amino acid in the pro-region) is posttranslational and SRP-independent. P17M signal peptide mutations that resulted in more rapid SRP-dependent translocation of AEP precursor were isolated. Most of these mutations significantly increased hydrophobicity, but the A12P/P17M mutation did not. The switch from SRP-dependent to SRP-independent translocation without a decrease in hydrophobicity (wild type to P17M) and restoration of SRP-dependent translocation without an increase in hydrophobicity (P17M to A12P/P17M) indicate that some factor(s) in addition to hydrophobicity determines selection of targeting pathway. Models of extended forms of wild type and A12P/P17M signal peptides are kinked, whereas the P17M signal peptide is relatively straight. Possibly the conformation/orientation of signal peptides at the ribosomal surface affects SRP binding and consequently the targeting route to the endoplasmic reticulum. Kinked signal peptides might approach SRP more closely more often. Most likely, these effects were only detectable because of the short length and low average hydrophobicity of the AEP signal peptide.     

FtsY, the prokaryotic signal recognition particle receptor homologue, is essential for biogenesis of membrane proteins

In mammalian cells, many secretory proteins are targeted to the endoplasmic reticulum co-translationally, by the signal recognition particle (SRP) and its receptor. In Escherichia coli, the targeting of secretory proteins to the inner membrane can be accomplished post-translationally. Unexpectedly, despite this variance, E. coli contains essential genes encoding Ffh and FtsY with a significant similarity to proteins of the eukaryotic SRP machinery. In this study, we investigated the possibility that the prokaryotic SRP-like machinery is involved in biogenesis of membrane proteins in E. coli. The data presented here demonstrate that the SRP-receptor homologue, FtsY, is indeed essential for expression of integral membrane proteins in E. coli, indicating that, in the case of this group of proteins, FtsY and the mammalian SRP receptor have similar functions.     

The nascent polypeptide-associated complex modulates interactions between the signal recognition particle and the ribosome

BACKGROUND: The first step in the co-translational targeting of secretory proteins to the endoplasmic reticulum membrane involves the recognition of signal sequences by the 54 kDa subunit of the signal recognition particle (SRP) as they emerge from the ribosome. It has recently been proposed that the nascent polypeptide-associated complex (NAC) contributes to the fidelity of targeting by modulating interactions that occur between the ribosome-nascent chain complex, the SRP and the endoplasmic reticulum membrane. Precisely how NAC influences SRP function is presently unclear. RESULTS: We have used immunoblotting experiments to monitor interactions between the SRP and the ribosome-nascent chain complex, in the absence and presence of NAC. In the absence of NAC, SRP binds in a high-salt-resistant manner only to ribosomes that contain a signal sequence, confirming the specificity of SRP for signal sequences. Binding of SRP to signalless ribosome nascent chains is observed at lower salt concentrations; however, the amount of SRP bound to this complex is indistinguishable from that bound to ribosomes lacking nascent chains. Thus, this salt-sensitive binding is likely to be the result of interactions between SRP and the ribosome that occur independently of the nascent chain. A minimal particle consisting of SRP54 and SRP RNA is sufficient to confer salt-resistant binding to ribosomes that contain signal sequences, whereas all of the SRP subunits are required for salt-sensitive binding to ribosomes that lack nascent chains. This salt-sensitive binding by SRP is inhibited by the addition of purified NAC. CONCLUSIONS: Based on our results, we define two distinct modes of interaction between SRP and the ribosome-nascent chain complex: salt-resistant interactions between SRP54 and signal sequences, and salt-sensitive interactions between additional components of SRP and the ribosome. We conclude that NAC does not directly influence signal sequence recognition by SRP but, rather, that it negatively modulates interactions that occur between SRP and the ribosome itself. These results are discussed in terms of a model wherein SRP and NAC regulate each others' activity during protein targeting.     

Interaction of guanine nucleotides with the signal recognition particle from Escherichia coli

The bacterial signal recognition particle (SRP) is an RNA-protein complex. In Escherichia coli, the particle consists of a 114 nt RNA, a 4.5S RNA, and a 48 kDa GTP-binding protein, Ffh. GDP-GTP exchange on, and GTP hydrolysis by, Ffh are thought to regulate SRP function in membrane targeting of translating ribosomes. In the present paper, we report the equilibrium and kinetic constants of guanine nucleotide binding to Ffh in different functional complexes. The association and dissociation rate constants of GTP/GDP binding to Ffh were measured using a fluorescent analogue of GTP/GDP, mant-GTP/GDP. For both nucleotides, association and dissociation rate constants were about 10(6) M-1 s-1 and 10 s-1, respectively. The equilibrium constants of nonmodified GTP and GDP binding to Ffh alone and in SRP, and in the complex with the ribosomes were measured by competition with mant-GDP. In all cases, the same 1-2 microM affinity for GTP and GDP was observed. Binding of both GTP and GDP to Ffh was independent of Mg2+ ions. The data suggest that, at conditions in vivo, (i) there will be rapid spontaneous GDP-GTP exchange, and (ii) the GTP-bound form of Ffh, or of SRP, will be predominant.     

The 40-kDa subunit of DNA fragmentation factor induces DNA fragmentation and chromatin condensation during apoptosis

We report here the reconstitution of a pathway that leads to the apoptotic changes in nuclei by using recombinant DNA fragmentation factor (DFF), a heterodimeric protein of 40 and 45 kDa. Coexpression of DFF40 and DFF45 is required to generate recombinant DFF, which becomes activated when DFF45 is cleaved by caspase-3. The cleaved fragments of DFF45 dissociate from the DFF40, the active component of DFF. Purified DFF40 exhibited an intrinsic DNase activity that was markedly stimulated by chromatin-associated proteins histone H1 and high mobility group proteins. DFF40 also triggered chromatin condensation when incubated with nuclei. These data suggest that DFF40 is sufficient to trigger both DNA fragmentation and chromatin condensation during apoptosis.     

Dual signal peptides mediate the signal recognition particle/Sec-independent insertion of a thylakoid membrane polyprotein, PsbY

The nuclear psbY gene (formerly ycf32) encodes two distinct single-spanning chloroplast thylakoid membrane proteins in Arabidopsis thaliana. After import into the chloroplast, the precursor protein is processed to a polyprotein in which each "mature" protein is preceded by an additional hydrophobic region; we show that these regions function as signal peptides that are cleaved after insertion into the thylakoid membrane. Inhibition of the first or second signal cleavage reaction by enlargement of the -1 residues leads in each case to the accumulation of a thylakoid-integrated intermediate containing three hydrophobic regions after import into chloroplasts; a double mutant is converted to a protein containing all four hydrophobic regions. We propose that the overall insertion process involves (i) insertion as a double-loop structure, (ii) two cleavages by the thylakoidal processing peptidase on the lumenal face of the membrane, and (iii) cleavage by an unknown peptidase on the stromal face on the membrane between the first mature protein and the second signal peptide. We also show that this polyprotein can insert into the thylakoid membrane in the absence of stromal factors, nucleoside triphosphates, or a functional Sec apparatus; this effectively shows for the first time that a multispanning protein can insert posttranslationally without the aid of signal recognition particle, SecA, or the membrane-bound Sec machinery.     

A homologue of the 19 kDa signal recognition particle protein locus in Drosophila melanogaster

AIMS: To determine the localisation of human cytomegalovirus (CMV) DNA in abdominal aorta, spleen, and transplantable organs, such as kidney, pancreas, and liver, obtained from healthy individuals; to characterise the cell type(s) in these tissues that serve as a reservoir for latent CMV. METHODS: CMV DNA was detected by dot blot DNA hybridisation and in situ DNA hybridisation with a probe for CMV major immediate early sequences (UL123) and nested PCR with primers derived from the CMV major immediate early (IE) gene exon 4 (UL123ex4). Samples of liver, abdominal aorta, spleen, kidney, and pancreas were obtained at necropsy or from donor kidneys from healthy subjects. RESULTS: CMV DNA was detected in most tissue samples using dot blot hybridisation and nested PCR. In situ hybridisation demonstrated that, in addition to smooth muscle cells in the arterial wall, hepatocytes, tubular and glomerular kidney cells, splenic red pulp cells, and pancreatic acinar cells also harboured CMV DNA. CMV DNA was detected in seropositive and in some seronegative subjects. CONCLUSION: CMV DNA is widely distributed in organs of healthy subjects. CMV DNA was found in various cell types in several organs, suggesting that during latency, CMV DNA is present thoughout the body.     

The signal recognition particle receptor alpha subunit of the hyperthermophilic archaeon Acidianus ambivalens exhibits an intrinsic GTP-hydrolyzing activity

Two adjacent genes of the acidophilic and hyperthermophilic crenarchaeon Acidianus ambivalens were cloned and sequenced. The 1.6 kb genomic nucleotide sequence under investigation consists of the 1.12 kb SRa gene encoding the putative signal recognition particle receptor alpha subunit (SR alpha, 42.2 kDa) and the 186 basepair secE gene coding for the putative secretory component secE subunit (6800 Da). The SR alpha protein is structured by three distinct regions: the N-terminal hydrophilic H-region, the following X-region and the C-terminal GTP-binding domain. A polyclonal anti-E. coli lacZ/A. ambivalens SR alpha antiserum detects a 51 kDa cell protein (p51) on immunoblots. Proteolysis of the recombinant SR alpha protein by Proteinase K produces a 31.6 kDa protease-resistant protein fragment comprising X-region and G-domain. The protein binds tightly to the GTP-agarose affinity matrix in a temperature-dependent manner. It hydrolyzes GTP readily at higher temperatures only in the presence of Mg2+. Point mutations (T326N) and (D329A) in the G-4 element of A. ambivalens SR alpha G-domain diminish the GTPase activity significantly. In contrast, the deletion mutant protein SR alpha (delta1-92) lacking the hydrophilic H-region displays a higher GTP-hydrolyzing activity when compared to the unmodified recombinant protein. Addition of GDP greatly inhibits GTP hydrolysis in mutant and unmodified A. ambivalens SR alpha.     

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

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

Activity of 72-kDa and 92-kDa matrix metalloproteinases in placental tissues of cows with and without retained fetal membranes

We produced hypertrophic and metaplastic changes of goblet cells in rat nasal respiratory epithelium by the intranasal instillation of endotoxin (ETN). In the present study, we examined in vivo effects of indomethacin (IND), dexamethasone (DEX), and erythromycin (EM) on intraepithelial mucus production using this animal model. Intraperitoneal injection of IND (2 to 4 mg/kg body weight x 4 days) or DEX (4 to 8 mg/kg body weight x 4 days) significantly inhibited intraepithelial mucus production induced after 3 days of ETN instillations. Intraperitoneal injection of EM (100 mg/kg body weight x 8 days), aminobenzylpenicillin (ABPC, 200 mg/kg body weight x 8 days), and cephalothin (CET, 200 mg/kg body weight x 8 days) also inhibited intraepithelial mucus production induced after 7 days of ETN instillations. When compared with ABPC and CET, EM had a greater inhibitory effect. These results indicate that ETN-induced intraepithelial mucus production can be inhibited by treatment with the anti-inflammatory drugs IND and DEX. Antibiotics such as EM, ABPC, and CET will also be effective, probably by preventing secondary bacterial infection, and EM has an additional inhibitory effect on intraepithelial mucus production.     

The signal recognition particle receptor of Escherichia coli (FtsY) has a nucleotide exchange factor built into the GTPase domain

Targeting of many secretory and membrane proteins to the inner membrane in Escherichia coli is achieved by the signal recognition particle (SRP) and its receptor (FtsY). In E. coli SRP consists of only one polypeptide (Ffh), and a 4.5S RNA. Ffh and FtsY each contain a conserved GTPase domain (G domain) with an alpha-helical domain on its N terminus (N domain). The nucleotide binding kinetics of the NG domain of the SRP receptor FtsY have been investigated, using different fluorescence techniques. Methods to describe the reaction kinetically are presented. The kinetics of interaction of FtsY with guanine nucleotides are quantitatively different from those of other GTPases. The intrinsic guanine nucleotide dissociation rates of FtsY are about 10(5) times higher than in Ras, but similar to those seen in GTPases in the presence of an exchange factor. Therefore, the data presented here show that the NG domain of FtsY resembles a GTPase-nucleotide exchange factor complex not only in its structure but also kinetically. The I-box, an insertion present in all SRP-type GTPases, is likely to act as an intrinsic exchange factor. From this we conclude that the details of the GTPase cycle of FtsY and presumably other SRP-type GTPases are fundamentally different from those of other GTPases.     

The fragile-X-related gene FXR1 is a human autoantigen processed during apoptosis

We describe a new human autoimmune antigen in a patient suffering from scleroderma with high levels of antibodies to nucleolus and cytoplasmic antigens. Using a Chinese hamster ovary cell expression library, we have shown that this antigen corresponds to the autosomal Fragile-X-related gene FXR1. The deduced amino acid sequence from the hamster cDNA is 97, 98, and 58% homologous to the human, mouse, and Xenopus laevis FXR1 genes, respectively. Expression of the hamster cDNA clone in Escherichia coli and antibody production indicates unequivocally the location of the FXR1 protein in the cytoplasm of hamster cells. Affinity chromatography followed by immunofluorescence microscopy analysis and immunoblots demonstrated the presence of autoimmune IgGs to FXR1 in the scleroderma patient. Immunolabeling studies in Jurkat cells, induced to apoptosis by anti-Fas/APO1 serum, indicated that the FXR1 antigens were clearly displaced from their original cytoplasmic location to several punctuated foci, resembling the bleb-like membranous structures characteristic of cells at certain stages of apoptosis. This phenomenon could be part of a putative mechanism in which the FXR1 protein is presented as a target for the autoimmune response in humans.     

The yeast spindle pole body component Spc72p interacts with Stu2p and is required for proper microtubule assembly

We have previously shown that Stu2p is a microtubule-binding protein and a component of the Saccharomyces cerevisiae spindle pole body (SPB). Here we report the identification of Spc72p, a protein that interacts with Stu2p. Stu2p and Spc72p associate in the two-hybrid system and can be coimmunoprecipitated from yeast extracts. Stu2p and Spc72p also interact with themselves, suggesting the possibility of a multimeric Stu2p-Spc72p complex. Spc72p is an essential component of the SPB and is able to associate with a preexisting SPB, indicating that there is a dynamic exchange between soluble and SPB forms of Spc72p. Unlike Stu2p, Spc72p does not bind microtubules in vitro, and was not observed to localize along microtubules in vivo. A temperature-sensitive spc72 mutation causes defects in SPB morphology. In addition, most spc72 mutant cells lack cytoplasmic microtubules; the few cytoplasmic microtubules that are observed are excessively long, and some of these are unattached to the SPB. spc72 cells are able to duplicate and separate their SPBs to form a bipolar spindle, but spindle elongation and chromosome segregation rarely occur. The chromosome segregation block does not arrest the cell cycle; instead, spc72 cells undergo cytokinesis, producing aploid cells and polyploid cells that contain multiple SPBs.     

The role of component function in visual recognition of Chinese characters

M. Taft and X. Zhu (1997) reported that character decision latencies to real Chinese characters containing components that entered into many combinations were faster than decision latencies to characters with components that entered into only a small number of combinations. However, this effect was restricted to components that appeared on the right side of Chinese characters. In written Chinese, phonetic components tend to appear on the right, and semantic components tend to appear on the left. Therefore, in Taft and Zhu's study, there was the possibility of a confound between position (left vs. right) and function (semantic vs. phonetic). Results of the present experiment show combinability effects for components with semantic and with phonetic functions. Counter to the claim by Taft and Zhu that component frequency effects are constrained by position, when component function was considered, character decision latencies varied with component frequency but not reliably with position.