scholarly journals Signal Peptides Bind and Aggregate RNA

2001 ◽  
Vol 276 (15) ◽  
pp. 12222-12227 ◽  
Author(s):  
Joanna Feltham Swain ◽  
Lila M. Gierasch
Keyword(s):  
Signal Recognition Particle ◽  
Signal Peptides ◽  
Signal Recognition ◽  
Gtpase Activity ◽  
Hydrophobic Core ◽  
Signal Sequences ◽  
Functional Peptide ◽  
Negative Controls

N-terminal signal sequences can direct nascent protein chains to the inner membrane of prokaryotes and the endoplasmic reticulum of eukaryotes by interacting with the signal recognition particle. In this study, we show that isolated peptides corresponding to several bacterial signal sequences inhibit the GTPase activity of theEscherichia colisignal recognition particle, as previously reported (Miller, J. D., Bernstein, H. D., and Walter, P. (1994)Nature367, 657–659), but not by the direct mechanism proposed. Instead, isolated signal peptides bind nonspecifically to the RNA component and aggregate the entire signal recognition particle, leading to a loss of its intrinsic GTPase activity. Surprisingly, only “functional” peptide sequences aggregate RNA; the peptides in general use as “nonfunctional” negative controls (e.g.those with deletions or charged substitutions within the hydrophobic core), are sufficiently different in physical character that they do not aggregate RNA and thus have no effect on the GTPase activity of the signal recognition particle. We propose that the reported effect of functional signal peptides on the GTPase activity of the signal recognition particle is an artifact of the high peptide concentrations and low salt conditions used in thesein vitrostudies and that signal sequences at the N terminus of nascent chainsin vivodo not exhibit this activity.

scholarly journals Signal sequences specify the targeting route to the endoplasmic reticulum membrane.

1996 ◽  
Vol 134 (2) ◽  
pp. 269-278 ◽  
Author(s):  
D T Ng ◽  
J D Brown ◽  
P Walter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Structural Information ◽  
Signal Recognition Particle ◽  
Endoplasmic Reticulum Membrane ◽  
Signal Recognition ◽  
Hydrophobic Core ◽  
Signal Sequences ◽  
Yeast Saccharomyces Cerevisiae

In the yeast Saccharomyces cerevisiae, only a subset of preproteins that are translocated across the ER membrane require the function of the signal recognition particle (SRP), suggesting that an alternative, SRP-independent pathway must exist (Hann, B.C., and P. Walter. 1991. Cell. 67:131-144). We have established that the two targeting pathways function in parallel. Mutant alleles of SEC62 and SEC63 were isolated that specifically impaired the translocation of SRP-independent preproteins in vivo and in vitro, whereas SRP-dependent preproteins were unaffected. Based on this analysis, preproteins fall into three distinct classes: SRP dependent, SRP independent, and those that can use both pathways. Pathway specificity is conferred by the hydrophobic core of signal sequences. Our studies show a previously unrecognized diversity in ER-directed signal sequences, that carry structural information that serves to identify the route taken.

scholarly journals In Vivo Analysis of an Essential Archaeal Signal Recognition Particle in Its Native Host

2002 ◽  
Vol 184 (12) ◽  
pp. 3260-3267 ◽  
Author(s):  
R. Wesley Rose ◽  
Mechthild Pohlschröder
Keyword(s):  
Protein Targeting ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Sequence Comparisons ◽  
Protein Insertion ◽  
Native Host

ABSTRACT The evolutionarily conserved signal recognition particle (SRP) plays an integral role in Sec-mediated cotranslational protein translocation and membrane protein insertion, as it has been shown to target nascent secretory and membrane proteins to the bacterial and eukaryotic translocation pores. However, little is known about its function in archaea, since characterization of the SRP in this domain of life has thus far been limited to in vitro reconstitution studies of heterologously expressed archaeal SRP components identified by sequence comparisons. In the present study, the genes encoding the SRP54, SRP19, and 7S RNA homologs (hv54h, hv19h, and hv7Sh, respectively) of the genetically and biochemically tractable archaeon Haloferax volcanii were cloned, providing the tools to analyze the SRP in its native host. As part of this analysis, an hv54h knockout strain was created. In vivo characterization of this strain revealed that the archaeal SRP is required for viability, suggesting that cotranslational protein translocation is an essential process in archaea. Furthermore, a method for the purification of this SRP employing nickel chromatography was developed in H. volcanii, allowing the successful copurification of (i) Hv7Sh with a histidine-tagged Hv54h, as well as (ii) Hv54h and Hv7Sh with a histidine-tagged Hv19h. These results provide the first in vivo evidence that these components interact in archaea. Such copurification studies will provide insight into the significance of the similarities and differences of the protein-targeting systems of the three domains of life, thereby increasing knowledge about the recognition of translocated proteins in general.

scholarly journals The Signal Recognition Particle Pathway Is Required for Virulence in Streptococcus pyogenes

2008 ◽  
Vol 76 (6) ◽  
pp. 2612-2619 ◽  
Author(s):  
Jason W. Rosch ◽  
Luis Alberto Vega ◽  
John M. Beyer ◽  
Ada Lin ◽  
Michael G. Caparon
Keyword(s):  
Streptococcus Pyogenes ◽  
Signal Recognition Particle ◽  
Growth Defect ◽  
Signal Recognition ◽  
Human Pathogens ◽  
Gram Positive ◽  
Zebrafish Model ◽  
Gram Positive Bacteria

ABSTRACT The signal recognition particle (SRP) pathway is a universally conserved pathway for targeting polypeptides for secretion via the cotranslational pathway. In particular, the SRP pathway is thought to be the main mechanism for targeting polypeptides in gram-positive bacteria, including a number of important human pathogens. Though widely considered to be an essential cellular component, recent advances have indicated this pathway may be dispensable in gram-positive bacteria of the genus Streptococcus under in vitro conditions. However, its importance for the pathogenesis of streptococcal disease is unknown. In this study, we investigated the importance of the SRP pathway for virulence factor secretion in the human pathogen Streptococcus pyogenes. While the SRP pathway was not found to be essential for viability in vitro, SRP mutants demonstrated a medium-specific growth defect that could be rescued by the addition of glucose. We also observed that a distinct subset of virulence factors were dependent upon the SRP pathway for secretion, whereas others were completely independent of this pathway. Significantly, deletion of the SRP pathway resulted in mutants that were highly attenuated in both a zebrafish model of necrotic myositis and a murine subcutaneous ulcer model, highlighting the importance of this pathway in vivo. These studies emphasize the importance of the SRP pathway for the in vivo survival and pathogenesis of S. pyogenes.

scholarly journals Interactions of signal peptides with signal-recognition particle

1990 ◽  
Vol 266 (1) ◽  
pp. 149-156 ◽  
Author(s):  
A Robinson ◽  
O M R Westwood ◽  
B M Austen
Keyword(s):  
Signal Peptide ◽  
Wheat Germ ◽  
Signal Recognition Particle ◽  
Translation System ◽  
Secretory Proteins ◽  
Signal Peptides ◽  
Signal Recognition ◽  
Synthetic Signal ◽  
Cross Links

The mechanisms whereby isolated or synthetic signal peptides inhibit processing of newly synthesized prolactin in microsome-supplemented lysates from reticulocytes and wheat-germ were investigated. At a concentration of 5 microM, a consensus signal peptide reverses the elongation arrest imposed by the signal-recognition particle (SRP), and at higher concentrations in addition inhibits elongation of both secretory and non-secretory proteins. A photoreactive form of a synthetic signal peptide cross-links under u.v. illumination to the 54 kDa and 68 kDa subunits of SRP, whereas the major cross-linked protein produced after photoreaction of rough microsomes is of 45 kDa. As SRP-mediated elongation arrest is unlikely to be essential for translocation, it is suggested that signal peptides may interact with components other than SRP in the translation system in vitro.

scholarly journals Neisseria gonorrhoeae PilA Is an FtsY Homolog

1999 ◽  
Vol 181 (3) ◽  
pp. 731-739 ◽  
Author(s):  
Cindy Grove Arvidson ◽  
Ted Powers ◽  
Peter Walter ◽  
Magdalene So
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Amino Acid Level ◽  
Signal Recognition Particle ◽  
Normal Growth ◽  
Gtpase Activity ◽  
Conditional Mutant ◽  
Major Structural Component ◽  
Translocation Assay

ABSTRACT The pilA gene of Neisseria gonorrhoeae was initially identified in a screen for transcriptional regulators ofpilE, the expression locus for pilin, the major structural component of gonococcal pili. The predicted protein sequence for PilA has significant homology to two GTPases of the mammalian signal recognition particle (SRP), SRP54 and SRα. Homologs of SRP54 and SRα were subsequently identified in bacteria (Ffh and FtsY, respectively) and appear to form an SRP-like apparatus in prokaryotes. Of the two proteins, PilA is the most similar to FtsY (47% identical and 67% similar at the amino acid level). Like FtsY, PilA is essential for viability and hydrolyzes GTP. The similarities between PilA and the bacterial FtsY led us to ask whether PilA might function as the gonococcal FtsY. In this work, we show that overproduction of PilA inEscherichia coli leads to an accumulation of pre-β-lactamase, similar to previous observations with other bacterial SRP components. Low-level expression of pilA in an ftsY conditional mutant can complement theftsY mutation and restore normal growth to this strain under nonpermissive conditions. In addition, purified PilA can replace FtsY in an in vitro translocation assay using purified E. coli SRP components. A PilA mutant that is severely affected in its GTPase activity cannot replace FtsY in vivo or in vitro. However, overexpression of the GTPase mutant leads to the accumulation of pre-β-lactamase, suggesting that the mutant protein may interact with the SRP apparatus to affect protein maturation. Taken together, these results show that the gonococcal PilA is an FtsY homolog and that the GTPase activity is necessary for its function.

scholarly journals The DEAD-box RNA helicase Ded1 from yeast is associated with the signal recognition particle (SRP), and its enzymatic activity is regulated by SRP21

2020 ◽  
Author(s):  
Hilal Yeter-Alat ◽  
Naïma Belgareh-Touzé ◽  
Emmeline Huvelle ◽  
Molka Mokdadi ◽  
Josette Banroques ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Rna Binding ◽  
Signal Recognition Particle ◽  
Rna Helicase ◽  
Signal Recognition ◽  
Dead Box ◽  
Enzymatic Regulation ◽  
The Dead

ABSTRACTThe DEAD-box RNA helicase Ded1 is an essential yeast protein involved in translation initiation. It belongs to the DDX3 subfamily of proteins implicated in developmental and cell-cycle regulation. In vitro, the purified Ded1 protein is an ATP-dependent RNA binding protein and an RNA-dependent ATPase, but it lacks RNA substrate specificity and enzymatic regulation. Here we demonstrate by yeast genetics, in situ localization and in vitro biochemical approaches that Ded1 is associated with, and regulated by, the signal recognition particle (SRP), which is a universally conserved ribonucleoprotein complex required for the co-translational translocation of polypeptides into the endoplasmic reticulum lumen and membrane. Ded1 is physically associated with SRP components in vivo and in vitro. Ded1 is genetically linked with SRP proteins. Finally, the enzymatic activity of Ded1 is inhibited by SRP21 with SCR1 RNA. We propose a model where Ded1 actively participates in the translocation of proteins during translation. Our results open a new comprehension of the cellular role of Ded1 during translation.

scholarly journals Protein–protein, protein–RNA and protein–lipid interactions of signal-recognition particle components in the hyperthermoacidophilic archaeon Acidianus ambivalens

2003 ◽  
Vol 374 (1) ◽  
pp. 247-254 ◽  
Author(s):  
Ralf G. MOLL
Keyword(s):  
Electrostatic Interactions ◽  
Plasma Membranes ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Independent Manner ◽  
Receptor Component ◽  
Protein Components ◽  
Srp Rna

The signal-recognition particle (SRP) of one of the most acidophilic and hyperthermophilic archaeal cells, Acidianus ambivalens, and its putative receptor component, FtsY (prokaryotic SRP receptor), were investigated in detail. A. ambivalens Ffh (fifty-four-homologous protein) was shown to be a soluble protein with strong affinity to membranes. In its membrane-residing form, Ffh was extracted from plasma membranes with chaotropic agents like urea, but not with agents diminishing electrostatic interactions. Using unilamellar tetraether phospholipid vesicles, both Ffh and FtsY associate independently from each other in the absence of other factors, suggesting an equilibrium of soluble and membrane-bound protein forms under in vivo conditions. The Ffh protein precipitated from cytosolic cell supernatants with anti-Ffh antibodies, together with an 7 S-alike SRP–RNA, suggesting a stable core ribonucleoprotein composed of both components under native conditions. The SRP RNA of A. ambivalens depicted a size of about 309 nucleotides like the SRP RNA of the related organism Sulfolobus acidocaldarius. A stable heterodimeric complex composed of Ffh and FtsY was absent in cytosolic super-natants, indicating a transiently formed complex during archaeal SRP targeting. The FtsY protein precipitated in cytosolic super-natants with anti-FtsY antisera as a homomeric protein lacking accessory protein components. However, under in vitro conditions, recombinantly generated Ffh and FtsY associate in a nucleotide-independent manner, supporting a structural receptor model with two interacting apoproteins.

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