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 Signal recognition particle mediates a transient elongation arrest of preprolactin in reticulocyte lysate.

1989 ◽  
Vol 109 (6) ◽  
pp. 2617-2622 ◽  
Author(s):  
S L Wolin ◽  
P Walter
Keyword(s):  
Wheat Germ ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
Translation Arrest ◽  
Microsomal Membranes ◽  
Reticulocyte Lysate ◽  
Reticulocyte Lysates ◽  
Ribosome Pausing

Signal recognition particle (SRP) is a ribonucleoprotein that functions in the targeting of ribosomes synthesizing presecretory proteins to the ER. SRP binds to the signal sequence as it emerges from the ribosome, and in wheat germ extracts, arrests further elongation. The translation arrest is released when SRP interacts with its receptor on the ER membrane. We show that the delay of elongation mediated by SRP is not unique to wheat germ translation extracts. Addition of mammalian SRP to reticulocyte lysates resulted in a delay of preprolactin synthesis due to increased ribosome pausing at specific sites on preprolactin mRNA. Addition of canine pancreatic microsomal membranes to reticulocyte lysates resulted in an acceleration of preprolactin synthesis, suggesting that the endogenous SRP present in the reticulocyte lysate also delays synthesis of secretory proteins.

Assembly of the Alu domain of the signal recognition particle (SRP): dimerization of the two protein components is required for efficient binding to SRP RNA

1990 ◽  
Vol 10 (2) ◽  
pp. 777-784
Author(s):  
K Strub ◽  
P Walter
Keyword(s):  
Cdna Clone ◽  
Repetitive Sequences ◽  
Signal Recognition Particle ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Cdna Clones ◽  
Functional Domain ◽  
Signal Recognition ◽  
Srp Rna

The signal recognition particle (SRP), a cytoplasmic ribonucleoprotein, plays an essential role in targeting secretory proteins to the rough endoplasmic reticulum membrane. In addition to the targeting function, SRP contains an elongation arrest or pausing function. This function is carried out by the Alu domain, which consists of two proteins, SRP9 and SRP14, and the portion of SRP (7SL) RNA which is homologous to the Alu family of repetitive sequences. To study the assembly pathway of the components in the Alu domain, we have isolated a cDNA clone of SRP9, in addition to a previously obtained cDNA clone of SRP14. We show that neither SRP9 nor SRP14 alone interacts specifically with SRP RNA. Rather, the presence of both proteins is required for the formation of a stable RNA-protein complex. Furthermore, heterodimerization of SRP9 and SRP14 occurs in the absence of SRP RNA. Since a partially reconstituted SRP lacking SRP9 and SRP14 [SRP(-9/14)] is deficient in the elongation arrest function, it follows from our results that both proteins are required to assemble a functional domain. In addition, SRP9 and SRP14 synthesized in vitro from synthetic mRNAs derived from their cDNA clones restore elongation arrest activity to SRP(-9/14).

scholarly journals In Vitro Studies with Purified Components Reveal Signal Recognition Particle (SRP) and SecA/SecB as Constituents of Two Independent Protein-targeting Pathways of Escherichia coli

1999 ◽  
Vol 10 (7) ◽  
pp. 2163-2173 ◽  
Author(s):  
Hans-Georg Koch ◽  
Thomas Hengelage ◽  
Christoph Neumann-Haefelin ◽  
Juan MacFarlane ◽  
Hedda K. Hoffschulte ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Signal Recognition Particle ◽  
Membrane Vesicles ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
Free System ◽  
E Coli

The molecular requirements for the translocation of secretory proteins across, and the integration of membrane proteins into, the plasma membrane of Escherichia coli were compared. This was achieved in a novel cell-free system from E. coliwhich, by extensive subfractionation, was simultaneously rendered deficient in SecA/SecB and the signal recognition particle (SRP) components, Ffh (P48), 4.5S RNA, and FtsY. The integration of two membrane proteins into inside-out plasma membrane vesicles of E. coli required all three SRP components and could not be driven by SecA, SecB, and ΔμH+. In contrast, these were the only components required for the translocation of secretory proteins into membrane vesicles, a process in which the SRP components were completely inactive. Our results, while confirming previous in vivo studies, provide the first in vitro evidence for the dependence of the integration of polytopic inner membrane proteins on SRP in E. coli. Furthermore, they suggest that SRP and SecA/SecB have different substrate specificities resulting in two separate targeting mechanisms for membrane and secretory proteins in E. coli. Both targeting pathways intersect at the translocation pore because they are equally affected by a blocked translocation channel.

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

1990 ◽  
Vol 111 (5) ◽  
pp. 1793-1802 ◽  
Author(s):  
K Römisch ◽  
J Webb ◽  
K Lingelbach ◽  
H Gausepohl ◽  
B Dobberstein
Keyword(s):  
Amino Acids ◽  
Rna Binding ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Binding Domain ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
Rna Binding Domain ◽  
Necessary And Sufficient

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.

scholarly journals Signal recognition particle causes a transient arrest in the biosynthesis of prepromelittin and mediates its translocation across mammalian endoplasmic reticulum.

1987 ◽  
Vol 104 (1) ◽  
pp. 61-66 ◽  
Author(s):  
I Ibrahimi
Keyword(s):  
Endoplasmic Reticulum ◽  
Wheat Germ ◽  
Signal Recognition Particle ◽  
Chain Elongation ◽  
Translation System ◽  
Signal Recognition ◽  
Nascent Chain ◽  
Reticulocyte Lysate ◽  
The Difference ◽  
Chain Synthesis

The translocation of prepromelittin (pPM) across mammalian endoplasmic reticulum was studied in both wheat germ and reticulocyte lysate. In the wheat germ system, signal recognition particle (SRP) caused a transient arrest in the synthesis of pPM. This was indicated by a slowdown in the rate of synthesis of pPM in the presence of SRP. The arrest was specific, dependent on the concentration of SRP, and more effective at early incubation time. In a tightly synchronized translation system, SRP had no apparent effect on the elongation of pPM, indicating that the effect of SRP on pPM chain synthesis might be at the final stages of chain elongation and release from the ribosome. This was reflected in a transient accumulation of pPM as peptidyl tRNA. Because pPM is composed of only 70 amino acids, arrest by SRP may be very close to chain termination. Arrest at this stage of chain synthesis seems to be unstable and the nascent chain gets terminated and released from the ribosome after a transient delay. The translocation of pPM was shown to be dependent on both SRP and docking protein. The difference in the translocation efficiency of pPM in reticulocyte and wheat germ lysates may reflect a difference in the targeting process in the two systems.

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 Assembly of the Alu domain of the signal recognition particle (SRP): dimerization of the two protein components is required for efficient binding to SRP RNA.

1990 ◽  
Vol 10 (2) ◽  
pp. 777-784 ◽  
Author(s):  
K Strub ◽  
P Walter
Keyword(s):  
Cdna Clone ◽  
Repetitive Sequences ◽  
Signal Recognition Particle ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Cdna Clones ◽  
Functional Domain ◽  
Signal Recognition ◽  
Srp Rna

The signal recognition particle (SRP), a cytoplasmic ribonucleoprotein, plays an essential role in targeting secretory proteins to the rough endoplasmic reticulum membrane. In addition to the targeting function, SRP contains an elongation arrest or pausing function. This function is carried out by the Alu domain, which consists of two proteins, SRP9 and SRP14, and the portion of SRP (7SL) RNA which is homologous to the Alu family of repetitive sequences. To study the assembly pathway of the components in the Alu domain, we have isolated a cDNA clone of SRP9, in addition to a previously obtained cDNA clone of SRP14. We show that neither SRP9 nor SRP14 alone interacts specifically with SRP RNA. Rather, the presence of both proteins is required for the formation of a stable RNA-protein complex. Furthermore, heterodimerization of SRP9 and SRP14 occurs in the absence of SRP RNA. Since a partially reconstituted SRP lacking SRP9 and SRP14 [SRP(-9/14)] is deficient in the elongation arrest function, it follows from our results that both proteins are required to assemble a functional domain. In addition, SRP9 and SRP14 synthesized in vitro from synthetic mRNAs derived from their cDNA clones restore elongation arrest activity to SRP(-9/14).

scholarly journals Evidence for a two-step mechanism involved in assembly of functional signal recognition particle receptor.

1989 ◽  
Vol 108 (3) ◽  
pp. 797-810 ◽  
Author(s):  
D W Andrews ◽  
L Lauffer ◽  
P Walter ◽  
V R Lingappa
Keyword(s):  
Functional Properties ◽  
Cytochrome B5 ◽  
Signal Recognition Particle ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
Cytoplasmic Face ◽  
Signal Recognition Particle Receptor ◽  
A Cell ◽  
Step Mechanism

The signal recognition particle (SRP) and SRP receptor act sequentially to target nascent secretory proteins to the membrane of the ER. The SRP receptor consists of two subunits, SR alpha and SR beta, both tightly associated with the ER membrane. To examine the biogenesis of the SRP receptor we have developed a cell-free assay system that reconstitutes SR alpha membrane assembly and permits both anchoring and functional properties to be assayed independently. Our experiments reveal a mechanism involving at least two distinct steps, targeting to the ER and anchoring of the targeted molecule on the cytoplasmic face of the membrane. Both steps can be reconstituted in vitro to restore translocation activity to ER microsomes inactivated by alkylation with N-ethyl-maleimide. The characteristics elucidated for this pathway distinguish it from SRP-dependent targeting of secretory proteins, SRP-independent ER translocation of proteins such as prepromellitin, and direct insertion mechanisms of the type exemplified by cytochrome b5.

Characterization of FtsY, its interaction with Ffh, and proteomic identification of their potential substrates in Mycobacterium tuberculosis

2018 ◽  
Vol 64 (4) ◽  
pp. 243-251 ◽  
Author(s):  
Arunkumar Venkatesan ◽  
Kannan Palaniyandi ◽  
Divakar Sharma ◽  
Deepa Bisht ◽  
Sujatha Narayanan
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Signal Recognition Particle ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
Antisense Gene ◽  
Gtp Hydrolysis ◽  
Protein Protein Interaction ◽  
Hydrolysis Activity

The universally conserved signal recognition particle (SRP) pathway that mediates co-translational targeting of membrane and secretory proteins is essential for eukaryotic and prokaryotic cells. The Mycobacterium tuberculosis SRP pathway consists of 2 proteins, Ffh and FtsY, and a 4.5S RNA molecule. Although the Escherichia coli SRP pathway is well studied, understanding of the M. tuberculosis SRP pathway components is very limited. In this study, we have overexpressed and characterized the M. tuberculosis SRP receptor (SR) FtsY as a GTP binding protein. Further, we established the direct protein–protein interaction between Ffh and FtsY. The Ffh–FtsY complex formation resulted in mutual stimulation of their GTP hydrolysis activity. We also attempted to biochemically characterize the SRP components by constructing the antisense gene knockdown strains of ffh and ftsY in M. tuberculosis. Loss of ffh and ftsY resulted in a decreased in vitro growth rate of the antisense ffh strain as compared with the antisense ftsY strain. Finally, 2-D gel electrophoresis of antisense depleted ffh and ftsY strains identified differential expression of 14 proteins.

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