scholarly journals Subcellular distribution of signal recognition particle and 7SL-RNA determined with polypeptide-specific antibodies and complementary DNA probe.

1983 ◽  
Vol 97 (6) ◽  
pp. 1693-1699 ◽  
Author(s):  
P Walter ◽  
G Blobel
Keyword(s):  
Protein Translocation ◽  
Solid Phase ◽  
Signal Recognition Particle ◽  
Endoplasmic Reticulum Membrane ◽  
Signal Recognition ◽  
Specific Antibodies ◽  
7Sl Rna ◽  
Radioimmune Assay ◽  
Cell Fractions

Signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of small cytoplasmic 7SL-RNA. The particle was previously shown to function in protein translocation across and protein integration into the endoplasmic reticulum membrane. Polypeptide specific antibodies were raised in rabbits against the 72,000-, 68,000-, and 54,000-mol-wt polypeptide of SRP. All three antibodies are shown to neutralize SRP activity in vitro. A solid phase radioimmune assay is described and used to follow SRP in various cell fractions. The partitioning of SRP is shown to be dependent on the ionic conditions of the fractionation. Under conditions approximating physiological ionic strength, SRP is found to be about equally distributed between a membrane associated (38%) and a free (15%) or ribosome associated (47%) state. Furthermore, it is shown that greater than 75% of the total cellular 7SL-RNA is associated with SRP polypeptide in these fractions. Thus it is likely that the major--if not the only--cellular function of 7SL-RNA is as a part of SRP.

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 Protein translocation across the endoplasmic reticulum membrane: identification by photocross-linking of a 39-kD integral membrane glycoprotein as part of a putative translocation tunnel.

1989 ◽  
Vol 109 (5) ◽  
pp. 2033-2043 ◽  
Author(s):  
U C Krieg ◽  
A E Johnson ◽  
P Walter
Keyword(s):  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
In Vitro Translation ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Membrane Glycoprotein ◽  
Messenger Rnas ◽  
Er Membrane

The molecular environment of secretory proteins during translocation across the ER membrane was examined by photocross-linking. Nascent preprolactin chains of various lengths, synthesized by in vitro translation of truncated messenger RNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA, signal recognition particle, and microsomal membranes, were used to position photoreactive probes at various locations within the membrane. Upon photolysis, each nascent chain species was cross-linked to an integral membrane glycoprotein with a deduced mass of 39 kD (mp39) via photoreactive lysines located in either the signal sequence or the mature prolactin sequence. Thus, different portions of the nascent preprolactin chain are in close proximity to the same membrane protein during the course of translocation, and mp39 therefore appears to be part of the translocon, the specific site of protein translocation across the ER membrane. The similarity of the molecular and cross-linking properties of mp39 and the glyco-protein previously identified as a signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature [Lond.]. 328: 830-833) suggests that these two proteins may be identical. Our data indicate, however, that mp39 does not (or not only) function as a signal sequence receptor, but rather may be part of a putative translocation tunnel.

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 Down-Regulation of the Trypanosomatid Signal Recognition Particle Affects the Biogenesis of Polytopic Membrane Proteins but Not of Signal Peptide-Containing Proteins

2007 ◽  
Vol 6 (10) ◽  
pp. 1865-1875 ◽  
Author(s):  
Yaniv Lustig ◽  
Yaron Vagima ◽  
Hanoch Goldshmidt ◽  
Avigail Erlanger ◽  
Vered Ozeri ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Signal Peptide ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Protein Translocation ◽  
Signal Recognition Particle ◽  
Dominant Negative ◽  
Signal Recognition ◽  
Rnai Silencing ◽  
7Sl Rna

ABSTRACT Protein translocation across the endoplasmic reticulum is mediated by the signal recognition particle (SRP). In this study, the SRP pathway in trypanosomatids was down-regulated by two approaches: RNA interference (RNAi) silencing of genes encoding SRP proteins in Trypanosoma brucei and overexpression of dominant-negative mutants of 7SL RNA in Leptomonas collosoma. The biogenesis of both signal peptide-containing proteins and polytopic membrane proteins was examined using endogenous and green fluorescent protein-fused proteins. RNAi silencing of SRP54 or SRP68 in T. brucei resulted in reduced levels of polytopic membrane proteins, but no effect on the level of signal peptide-containing proteins was observed. When SRP deficiency was achieved in L. collosoma by overexpression of dominant-negative mutated 7SL RNA, a major effect was observed on polytopic membrane proteins but not on signal peptide-containing proteins. This study included two trypanosomatid species, tested various protein substrates, and induced depletion of the SRP pathway by affecting either the levels of SRP binding proteins or that of SRP RNA. Our results demonstrate that, as in bacteria but in contrast to mammalian cells, the trypanosome SRP is mostly essential for the biogenesis of membrane proteins.

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 Direct probing of the interaction between the signal sequence of nascent preprolactin and the signal recognition particle by specific cross-linking.

1987 ◽  
Vol 104 (2) ◽  
pp. 201-208 ◽  
Author(s):  
M Wiedmann ◽  
T V Kurzchalia ◽  
H Bielka ◽  
T A Rapoport
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Cross Linking ◽  
Endoplasmic Reticulum Membrane ◽  
Signal Recognition ◽  
Group 4 ◽  
Lysine Residues ◽  
Nascent Chain

We have studied the interaction between the signal sequence of nascent preprolactin and the signal recognition particle (SRP) during the initial events in protein translocation across the endoplasmic reticulum membrane. A new method of affinity labeling was used, whereby lysine residues, carrying the photoreactive group 4-(3-trifluoromethyldiazirino) benzoic acid in their side chains, are incorporated into a protein by means of modified lysyl-tRNA, and cross-linking to the interacting component is induced by irradiation. SRP interacts through its Mr 54,000 polypeptide component with the signal sequences of nascent preprolactin chains containing about 70 residues, and with decreasing affinity with longer chains as well; it causes inhibition of elongation. Binding of SRP is reversible and requires the nascent chain to be bound to a functional ribosome. SRP cross-linked to the signal sequence still inhibits elongation but does not prevent it completely. We conclude that SRP does not block the exit site of the polypeptide chain on the ribosome. The SRP receptor of the endoplasmic reticulum membrane displaces the signal sequence from SRP and, even if SRP is cross-linked, releases elongation arrest.

scholarly journals Protein translocation across the ER requires a functional GTP binding site in the alpha subunit of the signal recognition particle receptor.

1992 ◽  
Vol 117 (3) ◽  
pp. 493-503 ◽  
Author(s):  
P J Rapiejko ◽  
R Gilmore
Keyword(s):  
Binding Site ◽  
Protein Translocation ◽  
Process Analysis ◽  
Signal Recognition Particle ◽  
In Vitro Translation ◽  
Alpha Subunit ◽  
Signal Recognition ◽  
Wild Type ◽  
Gtp Binding

The signal recognition particle (SRP)-mediated translocation of proteins across the RER is a GTP dependent process. Analysis of the primary amino acid sequence of one protein subunit of SRP (SRP54), as well as the alpha subunit of the SRP receptor (SR alpha), has indicated that these proteins contain predicted GTP binding sites. Several point mutations confined to the GTP binding consensus elements of SR alpha were constructed by site specific mutagenesis to define a role for the GTP binding site in SR alpha during protein translocation. The SR alpha mutants were analyzed using an in vitro system wherein SR alpha-deficient microsomal membranes were repopulated with SR alpha by in vitro translation of wild-type or mutant mRNA transcripts. SRP receptors containing SR alpha point mutants were analyzed for their ability to function in protein translocation and to form guanylyl-5'-imidodiphosphate (Gpp[NH]p) stabilized complexes with the SRP. Mutations in SR alpha produced SRP receptors that were either impaired or inactive in protein translocation. These SRP receptors were likewise unable to form Gpp(NH)p stabilized complexes with the SRP. One SR alpha point mutant, Thr 588 to Asn 588, required 50- to 100-fold higher concentrations of GTP relative to the wild-type SR alpha to function in protein translocation. This mutant has provided information on the reaction step in protein translocation that involves the GTP binding site in the alpha subunit of the SRP receptor.

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).

Identification of an essential Schizosaccharomyces pombe RNA homologous to the 7SL component of signal recognition particle

1988 ◽  
Vol 8 (4) ◽  
pp. 1580-1590
Author(s):  
P Brennwald ◽  
X Liao ◽  
K Holm ◽  
G Porter ◽  
J A Wise
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Rna Polymerase Iii ◽  
Signal Recognition Particle ◽  
Single Copy ◽  
Endoplasmic Reticulum Membrane ◽  
Cell Fractionation ◽  
Signal Recognition ◽  
Gene Encoding ◽  
7Sl Rna

We have cloned the gene encoding a novel small cytoplasmic RNA from the fission yeast Schizosaccharomyces pombe. Four lines of evidence support the idea that this RNA is a homolog of the 7SL RNA component of mammalian signal recognition particle (SRP), which targets presecretory proteins to the endoplasmic reticulum membrane. First, it shares limited but significant primary sequence homology with previously identified 7SL RNAs and can be folded into a similar secondary structure. Second, it possesses the 5' triphosphate characteristic of unprocessed RNA polymerase III transcripts, and moreover, it is the only fission yeast RNA in this size range with such a terminus. Third, its behavior in cell fractionation experiments suggests that it is part of a small ribonucleoprotein which forms salt-labile contacts with larger structures. Fourth, the particle containing S. pombe 7SL RNA resembles mammalian SRP in both size (11S) and affinity for DEAE-Sepharose. Disruption of the single-copy gene, designated slr1+, reveals that the RNA is indispensable for growth in fission yeast. This result is not surprising, since secretion is an essential cellular process.

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