scholarly journals Ffh and FtsY in a Mycoplasma mycoides signal‐recognition particle pathway: SRP RNA and M domain of Ffh are not required for stimulation of GTPase activity in vitro

1997 ◽  
Vol 24 (3) ◽  
pp. 523-534 ◽  
Author(s):  
Bertil Macao ◽  
Joen Luirink ◽  
Tore Samuelsson
Keyword(s):  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Gtpase Activity ◽  
Mycoplasma Mycoides ◽  
Srp Rna ◽  
Stimulation Of

scholarly journals SRP19 Is a Dispensable Component of the Signal Recognition Particle in Archaea

2006 ◽  
Vol 189 (1) ◽  
pp. 276-279 ◽  
Author(s):  
Sophie Yurist ◽  
Idit Dahan ◽  
Jerry Eichler
Keyword(s):  
Cell Growth ◽  
Membrane Protein ◽  
Protein Secretion ◽  
Signal Recognition Particle ◽  
Haloferax Volcanii ◽  
Signal Recognition ◽  
Protein Insertion ◽  
Membrane Protein Insertion ◽  
Srp Rna

ABSTRACT In vitro, archaeal SRP54 binds SRP RNA in the absence of SRP19, suggesting the latter to be expendable in Archaea. Accordingly, the Haloferax volcanii SRP19 gene was deleted. Although normally transcribed at a level comparable to that of the essential SRP54 gene, SRP19 deletion had no effect on cell growth, membrane protein insertion, protein secretion, or ribosome levels. The absence of SRP19 did, however, increase membrane bacterioruberin levels.

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 Signal recognition particle protein 19 is imported into the nucleus by importin 8 (RanBP8) and transportin

2001 ◽  
Vol 114 (19) ◽  
pp. 3479-3485 ◽  
Author(s):  
Kellie A. Dean ◽  
Oliver von Ahsen ◽  
Dirk Görlich ◽  
Howard M. Fried
Keyword(s):  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Protein Subunit ◽  
Animal Cells ◽  
Nuclear Assembly ◽  
Cytoplasmic Rna ◽  
Cytoplasmic Transport ◽  
Transport Receptors ◽  
Srp Rna

The signal recognition particle (SRP) is a cytoplasmic RNA-protein complex that targets proteins to the rough endoplasmic reticulum. Although SRP functions in the cytoplasm, RNA microinjection and cDNA transfection experiments in animal cells, as well as genetic analyses in yeast, have indicated that SRP assembles in the nucleus. Nonetheless, the mechanisms responsible for nuclear-cytoplasmic transport of SRP RNA and SRP proteins are largely unknown. Here we show that the 19 kDa protein subunit of mammalian SRP, SRP19, was efficiently imported into the nucleus in vitro by two members of the importin β superfamily of transport receptors, importin 8 and transportin; SRP19 was also imported less efficiently by several other members of the importin β family. Although transportin is known to import a variety of proteins, SRP19 import is the first function assigned to importin 8. Furthermore, we show that a significant pool of endogenous SRP19 is located in the nucleus, as well as the nucleolus. Our results show that at least one mammalian SRP protein is specifically imported into the nucleus, by members of the importin β family of transport receptors, and the findings add additional evidence for nuclear assembly of SRP.

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 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.

SRP RNA Remodeling by SRP68 Explains Its Role in Protein Translocation

Science ◽  
2014 ◽  
Vol 344 (6179) ◽  
pp. 101-104 ◽  
Author(s):  
Jan Timo Grotwinkel ◽  
Klemens Wild ◽  
Bernd Segnitz ◽  
Irmgard Sinning
Keyword(s):  
Ribosomal Rna ◽  
Protein Targeting ◽  
Rna Binding ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Structural Basis ◽  
Signal Recognition ◽  
Rna Remodeling ◽  
Srp Rna ◽  
Arginine Rich Motif

The signal recognition particle (SRP) is central to membrane protein targeting; SRP RNA is essential for SRP assembly, elongation arrest, and activation of SRP guanosine triphosphatases. In eukaryotes, SRP function relies on the SRP68-SRP72 heterodimer. We present the crystal structures of the RNA-binding domain of SRP68 (SRP68-RBD) alone and in complex with SRP RNA and SRP19. SRP68-RBD is a tetratricopeptide-like module that binds to a RNA three-way junction, bends the RNA, and inserts an α-helical arginine-rich motif (ARM) into the major groove. The ARM opens the conserved 5f RNA loop, which in ribosome-bound SRP establishes a contact to ribosomal RNA. Our data provide the structural basis for eukaryote-specific, SRP68-driven RNA remodeling required for protein translocation.

scholarly journals Circularization restores signal recognition particle RNA functionality in Thermoproteus

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
André Plagens ◽  
Michael Daume ◽  
Julia Wiegel ◽  
Lennart Randau
Keyword(s):  
Genome Rearrangement ◽  
Signal Recognition Particle ◽  
Circular Rna ◽  
Signal Recognition ◽  
Rna Molecules ◽  
Trna Splicing ◽  
Ribonucleoprotein Complexes ◽  
Domains Of Life ◽  
Thermoproteus Tenax ◽  
Srp Rna

Signal recognition particles (SRPs) are universal ribonucleoprotein complexes found in all three domains of life that direct the cellular traffic and secretion of proteins. These complexes consist of SRP proteins and a single, highly structured SRP RNA. Canonical SRP RNA genes have not been identified for some Thermoproteus species even though they contain SRP19 and SRP54 proteins. Here, we show that genome rearrangement events in Thermoproteus tenax created a permuted SRP RNA gene. The 5'- and 3'-termini of this SRP RNA are located close to a functionally important loop present in all known SRP RNAs. RNA-Seq analyses revealed that these termini are ligated together to generate circular SRP RNA molecules that can bind to SRP19 and SRP54. The circularization site is processed by the tRNA splicing endonuclease. This moonlighting activity of the tRNA splicing machinery permits the permutation of the SRP RNA and creates highly stable and functional circular RNA molecules.

scholarly journals Mechanisms of membrane protein biogenesis

2014 ◽  
Vol 70 (a1) ◽  
pp. C1161-C1161
Author(s):  
Irmgard Sinning
Keyword(s):  
Membrane Proteins ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Protein Biogenesis ◽  
Cargo Proteins ◽  
Target Membrane ◽  
Hydrophobic Nature ◽  
Correct Target ◽  
Srp Rna

More than 25% of the cellular proteome comprise membrane proteins that have to be inserted into the correct target membrane. Most membrane proteins are delivered to the membrane by the signal recognition particle (SRP) pathway which relies on the recognition of an N-terminal signal sequence. In contrast to this co-translational mechanism, which avoids problems due to the hydrophobic nature of the cargo proteins, tail-anchored (TA) membrane proteins utilize a post-translational mechanism for membrane insertion – the GET pathway (guided entry of tail-anchored membrane proteins). The SRP and GET pathways are both regulated by GTP and ATP binding proteins of the SIMIBI family. However, in the SRP pathway the SRP RNA plays a unique regulatory role. Recent insights into eukaryotic SRP will be discussed.

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.

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