Discovery of functional motifs in h-regions of trypanosome signal sequences

2010 ◽  
Vol 426 (2) ◽  
pp. 135-145 ◽  
Author(s):  
Josh Duffy ◽  
Bhargavi Patham ◽  
Kojo Mensa-Wilmot
Keyword(s):  
Amino Acids ◽  
Signal Sequence ◽  
Bioinformatic Analysis ◽  
Surface Glycoprotein ◽  
Secretory Proteins ◽  
Signal Peptides ◽  
Hydrophobic Core ◽  
Signal Sequences ◽  
Peptide Motifs ◽  
Physiological Relevance

N-terminal signal peptides direct secretory proteins into the ER (endoplasmic reticulum) of eukaryotes or the periplasmic space of prokaryotes. A hydrophobic core (h-region) is important for signal sequence function; however, the mechanism of h-region action is not resolved. To gain new insight into signal sequences, bioinformatic analysis of h-regions from humans, Saccharomyces cerevisiae, Trypanosoma brucei and Escherichia coli was performed. Each species contains a unique set of peptide motifs (h-motifs) characterized by identity components (i.e. sequence of conserved amino acids) joined by spacers. Human h-motifs have four identity components, whereas those from the other species utilize three identity components. Example of h-motifs are human Hs3 {L-x(2)-[AGILPV]-L-x(0,2)-L}, S. cerevisiae Sc1 [L-x(0,2)-S-x(0,3)-A], T. brucei Tb2 {L-x(1,2)-L-[AILV]} and E. coli Ec1 [A-x(0,2)-L-x(0,3)-A]. The physiological relevance of h-motifs was tested with a T. brucei microsomal system for translocation of a VSG (variant surface glycoprotein)-117 signal peptide. Disruption of h-motifs by scrambling of sequences in h-regions produced defective signal peptides, although the hydrophobicity of the peptide was not altered. We conclude that: (i) h-regions harbour h-motifs, and are not random hydrophobic amino acids; (ii) h-regions from different species contain unique sets of h-motifs; and (iii) h-motifs contribute to the biological activity of ER signal peptides. h-Regions are ‘scaffolds’ in which functional h-motifs are embedded. A hypothetical model for h-motif interactions with a Sec61p protein translocon is presented.

scholarly journals Species-specificity in endoplasmic reticulum signal peptide utilization revealed by proteins from Trypanosoma brucei and Leishmania

1998 ◽  
Vol 331 (2) ◽  
pp. 521-529 ◽  
Author(s):  
Ahmed AL-QAHTANI ◽  
Meredith TEILHET ◽  
Kojo MENSA-WILMOT
Keyword(s):  
Endoplasmic Reticulum ◽  
Trypanosoma Brucei ◽  
Signal Peptide ◽  
Protein Import ◽  
Signal Sequence ◽  
Surface Glycoprotein ◽  
Signal Peptides ◽  
Species Barrier ◽  
Signal Sequences ◽  
Nuclear Polyhedrosis

N-Terminal signal peptides direct secretory and most membrane proteins into the exocytic pathway at the endoplasmic reticulum. Signal sequences can function across kingdoms. However, our attempts at translocating variant surface glycoprotein (VSG) 117, VSG MVAT7, VSG 221 and BiP from Trypanosoma brucei and gp63 from Leishmania chagasi into canine pancreas microsomes failed. On replacing the signal peptide of VSG 117 with that from yeast prepro-α-mating factor (ppαMF) the chimaeric protein was imported, indicating that the signal sequence of VSG 117 was incompatible with the protein-import machinery of mammalian microsomes. Replacement of the gp63-h-region with a hybrid composed of the N-terminal nine residues from the h-region of gp67 from Autographa californica nuclear polyhedrosis virus and the C-terminal 10 residues from the h-region of gp63 from L. major produced a functional signal peptide. Thus, the h-region of kinetoplastid signal peptides appears to be the subdomain that is non-functional at the mammalian translocon. The calculated biophysical properties and computed discriminant scores (predictive of importability of signal peptides into mammalian microsomes) of the kinetoplastid signal sequences nevertheless are similar to those of ppαMF and Escherichia coliβ-lactamase both of which were imported. These signal peptides are the first collection from one biological family that have been found to fail to function across a species barrier. They indicate that signal peptides are not as universally interchangeable as previously believed. Intriguingly, endoplasmic reticulum signal peptides from Leishmania and Crithidia fasciculata are reminiscent of signal peptides from Gram-positive bacteria.

scholarly journals Interaction of wild-type signal sequences and their charged variants with model and natural membranes

1993 ◽  
Vol 293 (1) ◽  
pp. 43-49 ◽  
Author(s):  
N M Rao ◽  
R Nagaraj
Keyword(s):  
Amino Acids ◽  
Synthetic Peptides ◽  
Model Membranes ◽  
Signal Peptides ◽  
Wild Type ◽  
Signal Sequences ◽  
Hydrophobic Region ◽  
Phospholipases A2 ◽  
Charged Amino Acids

The interaction of synthetic peptides corresponding to wild-type signal sequences, and their mutants having charged amino acids in the hydrophobic region, with model and natural membranes has been studied. At high peptide concentrations, i.e. low lipid/peptide ratios, the signal peptides cause release of carboxyfluorescein (CF) from model membranes with lipid compositions corresponding to those of translocation-competent as well as translocation-incompetent membranes. Interestingly, mutant sequences, which were non-functional in vivo, caused considerable release of CF compared with the wild-type sequences. Both wild-type and mutant signal sequences perturb model membranes even at lipid/peptide ratios of 1000:1, as indicated by the activities of phospholipases A2, C and D. These studies indicate that such mutant signals are non-functional not because of their inability to interact with membranes, but due to defective targeting to the membrane. The signal peptides inhibit phospholipase C activity in microsomes, uncouple oxidative phosphorylation in mitochondria and increase K+ efflux from erythrocytes, and one of the mutant sequences is a potent degranulator of the mast cells. Both wild-type and mutant signal sequences have the ability to perturb vesicles of various lipid compositions. With respect to natural membranes, the peptides do not show any bias towards translocation-competent membranes.

scholarly journals Measuring Endoplasmic Reticulum Signal Sequences Translocation Efficiency Using the Xbp1 Arrest Peptide

2018 ◽  
Author(s):  
Theresa Kriegler ◽  
Anastasia Magoulopoulou ◽  
Rocio Amate Marchal ◽  
Tara Hessa
Keyword(s):  
Signal Sequence ◽  
Dominant Role ◽  
Complex Signal ◽  
Secretory Proteins ◽  
Signal Sequences ◽  
Functional Efficiency ◽  
Nascent Chain ◽  
Er Targeting ◽  
Translational Arrest ◽  
Two Phases

SummarySecretory proteins translocate across the mammalian ER membrane co-translationally via the ribosome-sec61 translocation channel complex. Signal sequences within the polypeptide, which guide this event, are diverse in their hydrophobicity, charge, length, and amino acid composition. Despite the known sequence diversity in the ER-targeting signals, it is generally assumed that they have a dominant role in determining co-translational targeting and translocation initiation process. We have analyzed co-translational events experienced by secretory proteins carrying efficient, versus inefficient (poorly hydrophobic) signal sequences, using an assay based on Xbp1 peptide-mediated translational arrest. With this method we were able to measure the functional efficiency of ER signal sequences. We show that an efficient signal sequence experiences a two-phases event in which the nascent chain is pulled from the ribosome during its translocation, thus resuming translation and yielding full-length products. Conversely, the inefficient signal sequence experiences a single weaker pulling event, suggesting inadequate engagement by the translocation machinery of these marginally hydrophobic signal sequences.

scholarly journals Glycine Residues in the Hydrophobic Core of the GspB Signal Sequence Route Export toward the Accessory Sec Pathway

2007 ◽  
Vol 189 (10) ◽  
pp. 3846-3854 ◽  
Author(s):  
Barbara A. Bensing ◽  
Ian R. Siboo ◽  
Paul M. Sullam
Keyword(s):  
Cell Surface ◽  
Signal Sequence ◽  
Human Platelets ◽  
Surface Glycoprotein ◽  
Simultaneous Increase ◽  
Hydrophobic Core ◽  
Cell Surface Glycoprotein ◽  
Sec Pathway ◽  
Amino Terminal ◽  
Sec System

ABSTRACT The Streptococcus gordonii cell surface glycoprotein GspB mediates high-affinity binding to distinct sialylated carbohydrate structures on human platelets and salivary proteins. GspB is glycosylated in the cytoplasm of S. gordonii and is then transported to the cell surface via a dedicated transport system that includes the accessory Sec components SecA2 and SecY2. The means by which the GspB preprotein is selectively recognized by the accessory Sec system have not been characterized fully. GspB has a 90-residue amino-terminal signal sequence that displays a traditional tripartite structure, with an atypically long amino-terminal (N) region followed by hydrophobic (H) and cleavage regions. In this report, we investigate the relative importance of the N and H regions of the GspB signal peptide for trafficking of the preprotein. The results show that the extended N region does not prevent export by the canonical Sec system. Instead, three glycine residues in the H region not only are necessary for export via the accessory Sec pathway but also interfere with export via the canonical Sec route. Replacement of the H-region glycine residues with helix-promoting residues led to a decrease in the efficiency of SecA2-dependent transport of the preprotein and a simultaneous increase in SecA2-independent translocation. Thus, the hydrophobic core of the GspB signal sequence is responsible primarily for routing towards the accessory Sec system.

scholarly journals Signal Sequence Recognition in Cotranslational Translocation by Protein Components of the Endoplasmic Reticulum Membrane

1998 ◽  
Vol 142 (2) ◽  
pp. 355-364 ◽  
Author(s):  
Walther Mothes ◽  
Berit Jungnickel ◽  
Josef Brunner ◽  
Tom A. Rapoport
Keyword(s):  
Endoplasmic Reticulum ◽  
Binding Site ◽  
Signal Sequence ◽  
Specific Binding ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Detergent Solution ◽  
Signal Sequences ◽  
Sequence Recognition ◽  
Protein Components

We have investigated the role of membrane proteins and lipids during early phases of the cotranslational insertion of secretory proteins into the translocation channel of the endoplasmic reticulum (ER) membrane. We demonstrate that all steps, including the one during which signal sequence recognition occurs, can be reproduced with purified translocation components in detergent solution, in the absence of bulk lipids or a bilayer. Photocross-linking experiments with native membranes show that upon complete insertion into the channel signal sequences are both precisely positioned with respect to the protein components of the channel and contact lipids. Together, these results indicate that signal sequences are bound to a specific binding site at the interface between the channel and the surrounding lipids, and are recognized ultimately by protein–protein interactions. Our data also suggest that at least some signal sequences reach the binding site by transfer through the interior of the channel.

scholarly journals The 54-kD protein of signal recognition particle contains a methionine-rich RNA binding domain.

1990 ◽  
Vol 111 (5) ◽  
pp. 1793-1802 ◽  
Author(s):  
K Römisch ◽  
J Webb ◽  
K Lingelbach ◽  
H Gausepohl ◽  
B Dobberstein
Keyword(s):  
Amino Acids ◽  
Rna Binding ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Binding Domain ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
Rna Binding Domain ◽  
Necessary And Sufficient

Signal recognition particle (SRP) plays the key role in targeting secretory proteins to the membrane of the endoplasmic reticulum (Walter, P., and V. R. Lingappa. 1986. Annu. Rev. Cell Biol. 2:499-516). It consists of SRP7S RNA and six proteins. The 54-kD protein of SRP (SRP54) recognizes the signal sequence of nascent polypeptides. The 19-kD protein of SRP (SRP19) binds to SRP7S RNA directly and is required for the binding of SRP54 to the particle. We used deletion mutants of SRP19 and SRP54 and an in vitro assembly assay in the presence of SRP7S RNA to define the regions in both proteins which are required to form a ribonucleoprotein particle. Deletion of the 21 COOH-terminal amino acids of SRP19 does not interfere with its binding to SRP7S RNA. Further deletions abolish SRP19 binding to SRP7S RNA. The COOH-terminal 207 amino acids of SRP54 (M domain) were found to be necessary and sufficient for binding to the SRP19/7S RNA complex in vitro. Limited protease digestion of purified SRP confirmed our results for SRP54 from the in vitro binding assay. The SRP54M domain could also bind to Escherichia coli 4.5S RNA that is homologous to part of SRP7S RNA. We suggest that the methionine-rich COOH terminus of SRP54 is a RNA binding domain and that SRP19 serves to establish a binding site for SRP54 on the SRP7S RNA.

In Silico Evaluation of Various Signal Peptides to Improve Secretion of Humulin Protein in E. coli Host

2020 ◽  
Vol 17 ◽  
Author(s):  
Soudabe Kavousipour ◽  
Mahadi Barazesh ◽  
Shiva Mohammadi ◽  
Meghdad Abdollahpour- Alitappeh ◽  
Shirzad Fallahi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Peptide ◽  
In Silico ◽  
Signal Sequence ◽  
Expression Vectors ◽  
Secretory Proteins ◽  
Signal Peptides ◽  
Heterologous Proteins ◽  
Genetic Characteristics ◽  
E Coli

Background:: Escherichia coli host has been the workhorse for the production of heterologous proteins due to simplicity of use, low cost, availability of various expression vectors, and widespread knowledge on its genetic characteristics, but without a suitable signal sequence, this host cannot be used for production secretory proteins. Humulin is a form of insulin used to treat hyperglycemia caused by types 1 and 2 diabetes. To improve expression and make a straightforward production of Humulin protein, we chose a series of signal peptides. Objective:: aim our study to predict the most excellent signal peptides to express secretory Humulin in E. coli organisms. Method:: Therefore, to forecast the most excellent signal peptides for expression of Humulin in Escherichia coli, 47 signal sequences from bacteria organisms were elected and the most imperative elements of them were studied. Hence, signal peptide probability along with physicochemical features was evaluated by signal 4.1, and Portparam, PROSO II servers respectively. Later, the in-silico cloning in a known pET28a plasmid system also estimated the possibility of best signal peptide+ Humulin expression in E.Coli. Results:: The outcomes demonstrated among 47 signal peptides only 2 signal peptides can be suggested as suitable signal peptides. Conclusion:: Ultimately protein yebF precursor (YEBF_ECOLI) and protein yebF precursor (YEBF_YERP3) were suggested severally; as the most excellent signal peptides to express Humulin (With D scores 0.812 and 0.623 respectively). Although verification of these results want experimental analysis.

scholarly journals Signal sequence-dependent function of the TRAM protein during early phases of protein transport across the endoplasmic reticulum membrane.

1996 ◽  
Vol 134 (1) ◽  
pp. 25-35 ◽  
Author(s):  
S Voigt ◽  
B Jungnickel ◽  
E Hartmann ◽  
T A Rapoport
Keyword(s):  
Protein Transport ◽  
Signal Sequence ◽  
Chain Complex ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Hydrophobic Core ◽  
Sequence Dependent ◽  
Nascent Chain ◽  
Er Membrane

Cotranslational translocation of proteins across the mammalian ER membrane involves, in addition to the signal recognition particle receptor and the Sec61p complex, the translocating chain-associating membrane (TRAM) protein, the function of which is still poorly understood. Using reconstituted proteoliposomes, we show here that the translocation of most, but not all, secretory proteins requires the function of TRAM. Experiments with hybrid proteins demonstrate that the structure of the signal sequence determines whether or not TRAM is needed. Features that distinguish TRAM-dependent and -independent signal sequences include the length of their charged, NH2-terminal region and the structure of their hydrophobic core. In cases where TRAM is required for translocation, it is not needed for the initial interaction of the ribosome/nascent chain complex with the ER membrane but for a subsequent step inside the membrane in which the nascent chain is inserted into the translocation site in a protease-resistant manner. Thus, TRAM functions in a signal sequence-dependent manner at a critical, early phase of the translocation process.

Determining the functionality of putative Tat-dependent signal peptides in Streptomyces coelicolor A3(2) by using two different reporter proteins

Microbiology ◽  
2005 ◽  
Vol 151 (7) ◽  
pp. 2189-2198 ◽  
Author(s):  
Haiming Li ◽  
Pierre-Étienne Jacques ◽  
Mariana Gabriela Ghinet ◽  
Ryszard Brzezinski ◽  
Rolf Morosoli
Keyword(s):  
Signal Sequence ◽  
Streptomyces Coelicolor ◽  
Active Form ◽  
Signal Peptides ◽  
Reporter Protein ◽  
Signal Sequences ◽  
Tat Pathway ◽  
Dependent Signal ◽  
Dependent Protein ◽  
Native Signal Sequence

The availability of the complete genome sequence of Streptomyces coelicolor A3(2) has allowed the prediction of the Tat-exported proteins of this Gram-positive bacterium. To predict secreted proteins that potentially use the Tat pathway for their secretion, the TATscan program was developed. This program identified 129 putative Tat substrates. To test the validity of these predictions, nine signal sequences, including three which were not identified by existing prediction programs, were selected and fused to the structural xlnC gene in place of its native signal sequence. Xylanase C (XlnC) is a cofactorless enzyme which is secreted in an active form exclusively through the Tat-dependent pathway by Streptomyces lividans. Among the nine chosen signal sequences, seven were shown to be Tat-dependent, one was Sec-dependent and one was probably not a signal sequence. The seven Tat-dependent signal sequences comprised two lipoprotein signal sequences and three sequences not predicted by previous programs. Pulse–chase experiments showed that the precursor-processing rate in the seven transformants was generally slower than wild-type XlnC, indicating that these signal peptides were not equivalent in secretion. This suggested that there might be some incompatibility between the signal peptide and the reporter protein fused to it. To test this possibility, the signal peptides were fused to a cofactorless chitosanase (SCO0677), a Tat-dependent protein validated in this work but structurally different from XlnC. With some fluctuations, similar results were obtained with this enzyme, indicating that the type of folding of the reporter protein had little effect on the Tat secretion process.

scholarly journals Molecular components of the signal sequence that function in the initiation of protein export.

1982 ◽  
Vol 95 (3) ◽  
pp. 689-696 ◽  
Author(s):  
S D Emr ◽  
T J Silhavy
Keyword(s):  
Amino Acids ◽  
Signal Sequence ◽  
Protein Localization ◽  
Helical Conformation ◽  
Hydrophobic Core ◽  
Selection Procedures ◽  
Sequence Characterization ◽  
Mutant Strains ◽  
Molecular Components

We are studying the mechanism by which the LamB protein is exported to the outer membrane of Escherichia coli. Using two selection procedures based on gene fusions, we have identified a number of mutations that cause alterations in the LamB signal sequence. Characterization of the mutant strains revealed that although many such mutations block LamB export to greater than 95%, others have essentially no effect. These results allow an analysis of the functions performed by the various molecular components of the signal sequence. Our results suggest that a critical subset of four amino acids is contained within the central hydrophobic core of the LamB signal sequence. If this core can assume an alpha-helical conformation, these four amino acids comprise a recognition site that interacts with a component of the cellular export machinery. Since mechanisms of protein localization appear to have been conserved during evolution, the principles established by these results should be applicable to similar studies in eukaryotic cells.

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