Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle

Nature ◽  
1989 ◽  
Vol 340 (6233) ◽  
pp. 482-486 ◽  
Author(s):  
Harris D. Bernstein ◽  
Mark A. Poritz ◽  
Katharina Strub ◽  
Patricia J. Hoben ◽  
Sydney Brenner ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Sequence Recognition

scholarly journals GTPase domain of the 54-kD subunit of the mammalian signal recognition particle is required for protein translocation but not for signal sequence binding.

1993 ◽  
Vol 120 (5) ◽  
pp. 1113-1121 ◽  
Author(s):  
D Zopf ◽  
H D Bernstein ◽  
P Walter
Keyword(s):  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Intact Protein ◽  
Signal Recognition ◽  
Signal Sequences ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Sequence Recognition ◽  
Amino Terminal Domain

The 54-kD subunit of the signal recognition particle (SRP54) binds to signal sequences of nascent secretory and transmembrane proteins. SRP54 consists of two separable domains, a 33-kD amino-terminal domain that contains a GTP-binding site (SRP54G) and a 22-kD carboxy-terminal domain (SRP54M) containing binding sites for both the signal sequence and SRP RNA. To examine the function of the two domains in more detail, we have purified SRP54M and used it to assemble a partial SRP that lacks the amino-terminal domain of SRP54 [SRP(-54G)]. This particle recognized signal sequences in two independent assays, albeit less efficiently than intact SRP. Analysis of the signal sequence binding activity of free SRP54 and SRP54M supports the conclusion that SRP54M binds signal sequences with lower affinity than the intact protein. In contrast, when SRP(-54G) was assayed for its ability to promote the translocation of preprolactin across microsomal membranes, it was completely inactive, apparently because it was unable to interact normally with the SRP receptor. These results imply that SRP54G plays an essential role in SRP-mediated targeting of nascent chain-ribosome complexes to the ER membrane and also influences signal sequence recognition, possibly by promoting a tighter association between signal sequences and SRP54M.

Structural basis of signal-sequence recognition by the signal recognition particle

2011 ◽  
Vol 18 (3) ◽  
pp. 389-391 ◽  
Author(s):  
Tobias Hainzl ◽  
Shenghua Huang ◽  
Gitte Meriläinen ◽  
Kristoffer Brännström ◽  
A Elisabeth Sauer-Eriksson
Keyword(s):  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Structural Basis ◽  
Signal Recognition ◽  
Sequence Recognition

scholarly journals Signal sequence recognition and targeting of ribosomes to the endoplasmic reticulum by the signal recognition particle do not require GTP.

1994 ◽  
Vol 5 (8) ◽  
pp. 887-897 ◽  
Author(s):  
P J Rapiejko ◽  
R Gilmore
Keyword(s):  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Beta Subunits ◽  
Signal Recognition ◽  
Binding Assays ◽  
Nascent Polypeptide ◽  
Sequence Recognition ◽  
Gtp Binding ◽  
Microsomal Membranes

The identification of GTP-binding sites in the 54-kDa subunit of the signal recognition particle (SRP) and in both the alpha and beta subunits of the SRP receptor has complicated the task of defining the step in the protein translocation reaction that is controlled by the GTP-binding site in the SRP. Ribonucleotide binding assays show that the purified SRP can bind GDP or GTP. However, crosslinking experiments show that SRP54 can recognize the signal sequence of a nascent polypeptide in the absence of GTP. Targeting of SRP-ribosome-nascent polypeptide complexes, formed in the absence of GTP, to microsomal membranes likewise proceeds normally. To separate the GTPase cycles of SRP54 and the alpha subunit of the SRP receptor (SR alpha), we employed an SR alpha mutant that displays a markedly reduced affinity for GTP. We observed that the dissociation of SRP54 from the signal sequence and the insertion of the nascent polypeptide into the translocation site could only occur when GTP binding to SR alpha was permitted. These data suggest that the GTP binding and hydrolysis cycles of both SRP54 and SR alpha are initiated upon formation of the SRP-SRP receptor complex.

scholarly journals Catalytic and Molecular Properties of the Quinohemoprotein Tetrahydrofurfuryl Alcohol Dehydrogenase from Ralstonia eutropha Strain Bo

2001 ◽  
Vol 183 (6) ◽  
pp. 1954-1960 ◽  
Author(s):  
Grit Zarnt ◽  
Thomas Schräder ◽  
Jan R. Andreesen
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Alcohol Dehydrogenase ◽  
Tertiary Structure ◽  
Ralstonia Eutropha ◽  
Signal Sequence ◽  
Substrate Inhibition ◽  
Catalytic Efficiency ◽  
Gene Encoding

ABSTRACT The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from Ralstonia eutropha strain Bo was investigated for its catalytic properties. The apparentk cat/Km andK i values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with n-pentanol exhibiting a k cat/Km value of 788 × 104 M−1 s−1. The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from R. eutropha strain Bo (tfaA) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases.

scholarly journals Real-time observation of signal recognition particle binding to actively translating ribosomes

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Thomas R Noriega ◽  
Jin Chen ◽  
Peter Walter ◽  
Joseph D Puglisi
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Protein Translocation ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Initial Step ◽  
Signal Recognition ◽  
Fluorescence Measurements ◽  
Nascent Chain ◽  
Time Observation

The signal recognition particle (SRP) directs translating ribosome-nascent chain complexes (RNCs) that display a signal sequence to protein translocation channels in target membranes. All previous work on the initial step of the targeting reaction, when SRP binds to RNCs, used stalled and non-translating RNCs. This meant that an important dimension of the co-translational process remained unstudied. We apply single-molecule fluorescence measurements to observe directly and in real-time E. coli SRP binding to actively translating RNCs. We show at physiologically relevant SRP concentrations that SRP-RNC association and dissociation rates depend on nascent chain length and the exposure of a functional signal sequence outside the ribosome. Our results resolve a long-standing question: how can a limited, sub-stoichiometric pool of cellular SRP effectively distinguish RNCs displaying a signal sequence from those that are not? The answer is strikingly simple: as originally proposed, SRP only stably engages translating RNCs exposing a functional signal sequence.

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.

Mouse sperm-egg plasma membrane interactions: analysis of roles of egg integrins and the mouse sperm homologue of PH-30 (fertilin) beta

1995 ◽  
Vol 108 (10) ◽  
pp. 3267-3278 ◽  
Author(s):  
J.P. Evans ◽  
R.M. Schultz ◽  
G.S. Kopf
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Guinea Pig ◽  
Ligand Binding ◽  
Zona Pellucida ◽  
Plasma Membranes ◽  
Signal Sequence ◽  
Rgd Peptides ◽  
Mouse Sperm ◽  
Disintegrin Domain

The guinea pig sperm protein, PH-30 (also known as fertilin), is postulated to participate in the interaction between the sperm and egg plasma membranes. The beta subunit of guinea pig PH-30 (gpPH-30 beta) contains a domain with homology to disintegrins, snake venom proteins that bind to integrins via an integrin-binding domain containing the tripeptide RGD. This raises the question of whether an egg integrin serves as a receptor for PH-30. Although mouse eggs express integrin subunits, their role in mouse fertilization is unresolved. Therefore, we examined fertilization for two different hallmarks of integrin function, namely, dependence of ligand binding on divalent cations and the ability to inhibit ligand binding with RGD peptides. We demonstrate that sperm binding to zona pellucida-free eggs is supported by Ca2+, Mg2+, or Mn2+. Ca2+ was necessary and sufficient for sperm-egg fusion, with 2.5 mM Ca2+ being the most effective concentration. In addition, fertilization could be partially inhibited with various RGD peptides, which caused a decrease in sperm-egg fusion by 30–58%. This partial inhibition of fusion with RGD peptides prompted the cloning of the mouse homologue of gpPH-30 beta (hereafter referred to as mPH-30 beta) to determine if it possessed the tripeptide RGD or a different amino acid sequence in its disintegrin domain. mPH-30 beta, which is expressed during meiotic and post-meiotic phases of spermatogenesis, shares significant similarities to gpPH-30 beta throughout the length of the molecule, from the signal sequence to the cytoplasmic tail. The full-length deduced amino acid sequence of mPH-30 beta. The disintegrin domain of mPH-30 beta has the tripeptide QDE (instead of RGD) in its cell recognition region. Peptides containing this QDE sequence decrease the binding and fusion of sperm with zona pellucida-free eggs by approximately 70%, suggesting that the disintegrin domain of mPH-30 beta participates in the interaction between sperm and egg membranes.

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.

scholarly journals The DsbA Signal Sequence Directs Efficient, Cotranslational Export of Passenger Proteins to the Escherichia coli Periplasm via the Signal Recognition Particle Pathway

2003 ◽  
Vol 185 (19) ◽  
pp. 5706-5713 ◽  
Author(s):  
Clark F. Schierle ◽  
Mehmet Berkmen ◽  
Damon Huber ◽  
Carol Kumamoto ◽  
Dana Boyd ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Fusion ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Signal Recognition ◽  
Sensitive Assay ◽  
Thioredoxin 1 ◽  
Passenger Proteins ◽  
Native Signal Sequence ◽  
Slight Inhibition

ABSTRACT The Escherichia coli cytoplasmic protein thioredoxin 1 can be efficiently exported to the periplasmic space by the signal sequence of the DsbA protein (DsbAss) but not by the signal sequence of alkaline phosphatase (PhoA) or maltose binding protein (MBP). Using mutations of the signal recognition particle (SRP) pathway, we found that DsbAss directs thioredoxin 1 to the SRP export pathway. When DsbAss is fused to MBP, MBP also is directed to the SRP pathway. We show directly that the DsbAss-promoted export of MBP is largely cotranslational, in contrast to the mode of MBP export when the native signal sequence is utilized. However, both the export of thioredoxin 1 by DsbAss and the export of DsbA itself are quite sensitive to even the slight inhibition of SecA. These results suggest that SecA may be essential for both the slow posttranslational pathway and the SRP-dependent cotranslational pathway. Finally, probably because of its rapid folding in the cytoplasm, thioredoxin provides, along with gene fusion approaches, a sensitive assay system for signal sequences that utilize the SRP pathway.

Sign in / Sign up

Export Citation Format

Share Document