Signal Recognition Particle Protein Research Articles

Small ribonucleoproteins in Schizosaccharomyces pombe and Yarrowia lipolytica homologous to signal recognition particle.

We have partially purified ribonucleoproteins (RNPs) from Schizosaccharomyces pombe and Yarrowia lipolytica with properties resembling those of mammalian signal recognition particle (SRP). In both species of yeast we have identified a single major RNA species in the size range of SRP RNA (256 nucleotides in S. pombe and 270 nucleotides in Y. lipolytica) present in postribosomal salt extracts of the cytoplasm. The RNPs containing these RNAs sediment in sucrose gradients at 11 S and 10 S for S. pombe and Y. lipolytica, respectively. Analysis of genomic clones of these RNAs has revealed that (i) they are encoded by single copy genes; (ii) they share two short conserved sequences that match the A and B boxes defined for polymerase III promoters; (iii) they can be folded into secondary structures that closely match that defined by phylogenetic analysis of higher eukaryotic SRP RNAs; and (iv) they show primary sequence conservation in short regions predicted to be single stranded. Both of the yeast RNAs bind under stringent conditions to canine SRP proteins. Most importantly, RNase protection of the S. pombe RNA by the individual canine SRP proteins, p19 and p68/72, shows that the proteins recognize homologous elements of the mammalian and yeast RNA. Taken together these data suggest strongly that we have identified yeast SRP homologues.

Binding sites of the 19-kDa and 68/72-kDa signal recognition particle (SRP) proteins on SRP RNA as determined in protein-RNA "footprinting".

We have used the nuclease alpha-sarcin to map the binding sites of the 19-kDa and the 68/72-kDa proteins of signal recognition particle (SRP) on SRP RNA. We found that the regions of protection to nuclease afforded by the two proteins were distinct. p19 protected primarily the two tips in the RNA secondary structure. p68/72 protected a large region extending across the center of the particle and altered the nuclease pattern in the regions that p19 would bind, suggesting that these two proteins may be in close proximity in the particle. The protection afforded by the two proteins in combination was equal to the sum of the individual protections. We have not observed cooperativity in the binding of these two proteins as assessed by the protection assay; nor do we have any evidence that the structure becomes more compact as it assembles. The map derived from this "footprint" analysis places the signal recognition domain (p54 bound to the RNA via the 19-kDa protein) and the elongation arrest domain (associated with the Alu end of the particle) on opposite ends of the particle. Thus, it is possible that SRP recognizes signals by the direct interaction of p54 with the signal sequence at the nascent chain exit site and simultaneously blocks elongation by the entrance of p9/14 into the aminoacyl tRNA site 16 nm away.

Isolation and characterization of a cDNA clone encoding the 19 kDa protein of signal recognition particle (SRP): expression and binding to 7SL RNA

Signal recognition particle (SRP) consists of a 7SL RNA molecule and 6 protein subunits. We have isolated and characterized cDNA clones from human liver which encode the 19kDa protein subunit (SRP19). This subunit binds to the RNA directly and mediates binding of a second polypeptide, the 54kDa subunit which is involved in signal sequence recognition. Amino acid sequences deduced from the human cDNA sequence were identical to amino acid sequences of tryptic peptides from canine pancreatic SRP19. In vitro transcription and translation of the human cDNA resulted in a protein product the same size as canine SRP19 which could be immunoprecipitated by an antiserum raised against canine SRP19. SRP19 synthesized in a cell-free system specifically bound to 7SL RNA. The sequence of SRP19 is discussed with respect to its binding to 7SL RNA.

Each of the activities of signal recognition particle (SRP) is contained within a distinct domain: Analysis of biochemical mutants of SRP

Signal recognition particle (SRP), a small ribonucleoprotein required for targeting secretory proteins to the ER, has three known functions: signal recognition, elongation arrest, and translocation promotion. Because SRP is inactivated by the sulfhydryl alkylating reagent N-ethylmaleimide (NEM), we have attempted to establish structure-function relationships within SRP by assembling particles in which a single protein is modified. Alkylation of the 68 72 kd protein of SRP yields a particle that arrests elongation but fails to promote translocation and no longer interacts with SRP receptor. Alkylation of the 54 kd protein yields a particle that fails to recognize signal sequences. This approach has allowed us to map activities to specific protein domains on SRP, and should be generally useful for analyzing other ribonucleoproteins.

Import of honeybee prepromelittin into the endoplasmic reticulum: structural basis for independence of SRP and docking protein.

Honeybee prepromelittin is correctly processed and imported by dog pancreas microsomes. Insertion of prepromelittin into microsomal membranes, as assayed by signal sequence removal, does not depend on signal recognition particle (SRP) and docking protein. We addressed the question as to how prepromelittin bypasses the SRP/docking protein system. Hybrid proteins between prepromelittin, or carboxy-terminally truncated derivatives, and the cytoplasmic protein dihydrofolate reductase from mouse were constructed. These hybrid proteins were analysed for membrane insertion and sequestration into microsomes. The results suggest the following: (i) The signal sequence of prepromelittin is capable of interacting with the SRP/docking protein system, but this interaction is not mandatory for membrane insertion; this is related to the small size of prepromelittin. (ii) In prepromelittin a cluster of negatively charged amino acids must be balanced by a cluster of positively charged amino acids in order to allow membrane insertion. (iii) In general, a signal sequence can be sufficient to mediate membrane insertion independently of SRP and docking protein in the case of short precursor proteins; however, the presence and distribution of charged amino acids within the mature part of these precursors can play distinct roles.

Characterization of human autoantibodies that selectively precipitate the 7SL RNA component of the signal recognition particle.

The signal recognition particle (SRP), which consists of the 7SL RNA molecule associated with six polypeptides ranging between 9,000 and 72,000 m.w., mediates the translocation of newly synthesized proteins across the endoplasmic reticulum. We have characterized autoantibodies that are directed against this particle from two patients with rheumatic diseases. These sera immunoprecipitated the 7SL RNA from whole extracts of HeLa cells radiolabeled with 32P, but no RNA from deproteinized cell extracts. From 35S-methionine-labeled cell extracts, they immunoprecipitated a single polypeptide of 54,000 m.w. that is consistent with a known SRP component. Sucrose density gradient studies confirmed that this protein co-migrated with the 7SL RNA, indicating the likelihood that it is physically associated with this RNA. Thus, the 54,000 m.w. SRP protein, which is essential for the SRP functions of elongation arrest and translocation, appears to be a preferential target for human autoimmune responses. Human autoantibodies that recognize the SRP should be useful adjuncts to animal antisera for studies of the structure and function of this particle.

Disassembly and domain structure of the proteins in the signal-recognition particle.

The signal-recognition particle (SRP) is a ribonucleoprotein (RNP) complex consisting of six different polypeptide chains and a 7SL RNA. It participates in initiating the translocation of proteins across the membrane of the endoplasmic reticulum. SRP was disassembled in 2 M KCl into three components, one RNP composed of 7SL RNA and the 54-kDa and 19-kDa proteins, and two heterodimers consisting of the 72/68-kDa and the 14/9-kDa proteins respectively. The 54-kDa protein could be released from the RNP subparticle by chromatography on DEAE-Sepharose in Mg2+-depleted buffer, while the 19-kDa protein remained bound to the 7SL RNA. The domain structure of SRP proteins was probed by using mild elastase treatment and protein-specific antibodies. It was found that the 72, 68, 54 and 19-kDa SRP proteins were proteolytically processed in distinct steps. Most remarkably a protein fragment of 55-kDa, generated from the 72-kDa SRP protein, and a 35-kDa fragment from the 54-kDa SRP protein were both released from the RNP particle. Fragments generated from the 68-kDa protein and detectable with the anti-(68-kDa protein) antibody remained associated with the RNP particle. Cleavage of the SRP proteins by elastase at 2.5 micrograms/ml resulted in partial loss of activity, while 10 micrograms/ml caused complete inactivation of the particle. Neither the elongation arrest of IgG light chain nor its translocation across SRP-depleted microsomal membranes was promoted. The implications of these results on the possible interaction between the SRP subunits are discussed.

Signal recognition particle arrests elongation of nascent secretory and membrane proteins at multiple sites in a transient manner.

The signal recognition particle (SRP) has been shown to target nascent secretory and membrane proteins to the endoplasmic reticulum. In the wheat germ cell-free system, SRP arrests the elongation of the nascent chains until the translational complex is docked to the endoplasmic reticulum membrane where the interaction between SRP and docking protein causes a release of the nascent chain arrest. For two secretory proteins, arrested peptides of 70 amino acids have been identified (Walter, P., Ibrahimi, I., and Blobel, G. (1981) J. Cell Biol. 91, 545-550; Meyer, D. I., Krause, E., and Dobberstein, B. (1982) Nature 297, 647-650). By using an in vitro coupled transcription-translation system, we have analyzed SRP arrest and the resulting peptides of the two secretory proteins lysozyme and granulocyte-macrophage colony-stimulating factor and the membrane protein invariant chain. SRP arrested the elongation of all three proteins at multiple sites, giving rise to ladders of arrested peptides. The size of the arrested peptides increased with the time of translation, resulting in mostly full-length pre-peptides after about 40 min. This suggests that SRP arrest in transient rather than stable. Upon addition of microsomes, the SRP arrest was released, and all the blocked peptides could be chased into mature proteins or full-length precursors.

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

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.

The signal recognition particle receptor is a complex that contains two distinct polypeptide chains.

Signal recognition particle (SRP) and SRP receptor are known to be essential components of the cellular machinery that targets nascent secretory proteins to the endoplasmic reticulum (ER) membrane. Here we report that the SRP receptor contains, in addition to the previously identified and sequenced 69-kD polypeptide (alpha-subunit, SR alpha), a 30-kD beta-subunit (SR beta). When SRP receptor was purified by SRP-Sepharose affinity chromatography, we observed the co-purification of two other ER membrane proteins. Both proteins are approximately 30 kD in size and are immunologically distinct from each other, as well as from SR alpha and SRP proteins. One of the 30-kD proteins (SR beta) forms a tight complex with SR alpha in detergent solution that is stable to high salt and can be immunoprecipitated with antibodies to either SR alpha or SR beta. Both subunits are present in the ER membrane in equimolar amounts and co-fractionate in constant stoichiometry when rough and smooth liver microsomes are separated on sucrose gradients. We therefore conclude that SR beta is an integral component of SRP receptor. The presence of SR beta was previously masked by proteolytic breakdown products of SR alpha observed by others and by the presence of another 30-kD ER membrane protein (mp30) which co-purifies with SR alpha. Mp30 binds to SRP-Sepharose directly and is present in the ER membrane in several-fold molar excess of SR alpha and SR beta. The affinity of mp30 for SRP suggests that it may serve a yet unknown function in protein translocation.

Elongation arrest is not a prerequisite for secretory protein translocation across the microsomal membrane.

Signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of small cytoplasmic 7SL RNA. It was previously shown to promote the co-translational translocation of secretory proteins across the endoplasmic reticulum by (a) arresting the elongation of the presecretory nascent chain at a specific point, and (b) interacting with the SRP receptor, an integral membrane protein of the endoplasmic reticulum which is active in releasing the elongation arrest. Recently a procedure was designed by which the particle could be disassembled into its protein and RNA components. We have further separated the SRP proteins into four homogeneous fractions. When recombined with each other and with 7SL RNA, they formed fully active SRP. Particles missing specific proteins were assembled in the hope that some of these would retain some functional activity. SRP(-9/14), the particle lacking the 9-kD and 14-kD polypeptides, was fully active in promoting translocation, but was completely inactive in elongation arrest. This implied that elongation arrest is not a prerequisite for protein translocation. SRP receptor was required for SRP(-9/14)-mediated translocation to occur, and thus must play some role in the translocation process in addition to releasing the elongation arrest.

Disassembly and reconstitution of signal recognition particle

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. A rapid procedure was developed to disassemble SRP into native protein and RNA components. The method utilizes unfolding of SRP with EDTA and dissociation on polycationic matrixes. SRP proteins prepared this way sediment below 7S and are inactive in activity assays. When recombined with 7SL-RNA in the presence of magnesium, the proteins are shown to reassociate stoichiometrically with 7SL-RNA to form fully active 11S SRP.