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.

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.

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 Anti-cooperative assembly of the SRP19 and SRP68/72 components of the signal recognition particle

2008 ◽  
Vol 415 (3) ◽  
pp. 429-437 ◽  
Author(s):  
Tuhin Subhra Maity ◽  
Howard M. Fried ◽  
Kevin M. Weeks
Keyword(s):  
Electrostatic Interactions ◽  
Signal Recognition Particle ◽  
Cooperative Binding ◽  
Signal Recognition ◽  
Parallel Orientation ◽  
Domain Interactions ◽  
Cooperative Assembly ◽  
Srp Rna ◽  
Antagonistic Interactions

The mammalian SRP (signal recognition particle) represents an important model for the assembly and role of inter-domain interactions in complex RNPs (ribonucleoproteins). In the present study we analysed the interdependent interactions between the SRP19, SRP68 and SRP72 proteins and the SRP RNA. SRP72 binds the SRP RNA largely via non-specific electrostatic interactions and enhances the affinity of SRP68 for the RNA. SRP19 and SRP68 both bind directly and specifically to the same two RNA helices, but on opposite faces and at opposite ends. SRP19 binds at the apices of helices 6 and 8, whereas the SRP68/72 heterodimer binds at the three-way junction involving RNA helices 5, 6 and 8. Even though both SRP19 and SRP68/72 stabilize a similar parallel orientation for RNA helices 6 and 8, these two proteins bind to the RNA with moderate anti-cooperativity. Long-range anti-cooperative binding by SRP19 and SRP68/72 appears to arise from stabilization of distinct conformations in the stiff intervening RNA scaffold. Assembly of large RNPs is generally thought to involve either co-operative or energetically neutral interactions among components. By contrast, our findings emphasize that antagonistic interactions can play significant roles in assembly of multi-subunit RNPs.

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).

Identification of RNA sequences and structural elements required for assembly of fission yeast SRP54 protein with signal recognition particle RNA

1993 ◽  
Vol 13 (3) ◽  
pp. 1353-1362
Author(s):  
D Selinger ◽  
P Brennwald ◽  
X Liao ◽  
J A Wise
Keyword(s):  
Fission Yeast ◽  
Base Pair ◽  
Structural Integrity ◽  
Signal Recognition Particle ◽  
Yeast Cells ◽  
Growth Defect ◽  
Signal Recognition ◽  
Nucleotide Substitutions ◽  
Srp Rna

Signal recognition particle (SRP) is a ribonucleoprotein composed of six polypeptides and a single RNA molecule. SRP RNA can be divided into four structural domains, the last of which is the most highly conserved and, in Schizosaccharomyces pombe, is the primary location to which deleterious mutations map. The ability of mammalian SRP54 protein (SRP54p) to bind Escherichia coli 4.5S RNA, a homolog of SRP RNA which contains only domain IV, suggested that SRP54p might interact directly with this region. To determine whether domain IV is critical for SRP54p binding in fission yeast cells, we used a native immunoprecipitation-RNA sequencing assay to test 13 mutant SRP RNAs for the ability to associate with the protein in vivo. The G156A mutation, which alters the 5' residue of the noncanonical first base pair of the domain IV terminal helix and confers a mild conditional growth defect, reduces assembly of the RNA with SRP54p. Mutating either of the two evolutionarily invariant residues in the bulged region 5' to G156 is more deleterious to growth and virtually abolishes SRP54p binding. We conclude that the conservation of nucleotides 154 to 156 is likely to be a consequence of their role as a sequence-specific recognition element for the SRP54 protein. We also tested a series of mutants with nucleotide substitutions in the conserved tetranucleotide loop and adjoining stem of domain IV. Although tetraloop mutations are deleterious to growth, they have little effect on SRP54p binding. Mutations which disrupt the base pair flanking the tetraloop result in conditional growth defects and significantly reduce association with SRP54p. Disruption of the other two base pairs in the short stem adjacent to the tetranucleotide loop has similar but less dramatic effects on SRP54p binding. These data provide the first evidence that both sequence-specific contacts and the structural integrity of domain IV of SRP RNA are important for assembly with SRP54p.

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 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 The SRP9/14 subunit of the signal recognition particle (SRP) is present in more than 20-fold excess over SRP in primate cells and exists primarily free but also in complex with small cytoplasmic Alu RNAs.

1995 ◽  
Vol 6 (4) ◽  
pp. 471-484 ◽  
Author(s):  
F Bovia ◽  
M Fornallaz ◽  
H Leffers ◽  
K Strub
Keyword(s):  
Translational Control ◽  
Small Rnas ◽  
Control Function ◽  
Signal Recognition Particle ◽  
Synthesis Rate ◽  
Signal Recognition ◽  
Control Of Gene Expression ◽  
Fold Excess ◽  
Srp Rna

The heterodimeric protein SRP9/14 bound to the Alu sequences of SRP RNA is essential for the translational control function of the signal recognition particle (SRP). The Alu RNAs of primate cells are believed to be derived from SRP RNA and have been shown to bind to an SRP14-related protein in vitro. We have used antibodies to characterize SRP9/14 and examine its association with small RNAs in vivo. Although SRP9 proteins are the same size in both rodent and primate cells, SRP14 subunits are generally larger in primate cells. An additional alanine-rich domain at the C-terminus accounts for the larger size of one human isoform. Although the other four SRP proteins are largely assembled into SRP in both rodent and primate cells, we found that the heterodimer SRP9/14 is present in 20-fold excess over SRP in primate cells. An increased synthesis rate of both proteins may contribute to their accumulation. The majority of the excess SRP9/14 is cytoplasmic and does not appear to be bound to any small RNAs; however, a significant fraction of a small cytoplasmic Alu RNA is complexed with SRP9/14 in a 8.5 S particle. Our findings that there is a large excess of SRP9/14 in primate cells and that Alu RNAs are bound to SRP9/14 in vivo suggest that this heterodimeric protein may play additional roles in the translational control of gene expression and/or Alu transcript metabolism.

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.

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