scholarly journals The Hydrophobic Core of Twin-Arginine Signal Sequences Orchestrates Specific Binding to Tat-Pathway Related Chaperones

PLoS ONE ◽  
2012 ◽  
Vol 7 (3) ◽  
pp. e34159 ◽  
Author(s):  
Anitha Shanmugham ◽  
Adil Bakayan ◽  
Petra Völler ◽  
Joost Grosveld ◽  
Holger Lill ◽  
...  
Keyword(s):  
Specific Binding ◽  
Hydrophobic Core ◽  
Signal Sequences ◽  
Tat Pathway

scholarly journals Thylakoid ΔpH-dependent precursor proteins bind to a cpTatC–Hcf106 complex before Tha4-dependent transport

2001 ◽  
Vol 154 (4) ◽  
pp. 719-730 ◽  
Author(s):  
Kenneth Cline ◽  
Hiroki Mori
Keyword(s):  
Specific Binding ◽  
Signal Peptides ◽  
Hydrophobic Core ◽  
Precursor Proteins ◽  
Peptide Precursors ◽  
Folded Proteins ◽  
Dependent Transport ◽  
Dependent Pathway ◽  
Blue Native ◽  
Chemical Cross Linking

The thylakoid ΔpH-dependent pathway transports folded proteins with twin arginine–containing signal peptides. Identified components of the machinery include cpTatC, Hcf106, and Tha4. The reaction occurs in two steps: precursor binding to the machinery, and transport across the membrane. Here, we show that a cpTatC–Hcf106 complex serves as receptor for specific binding of twin arginine–containing precursors. Antibodies to either Hcf106 or cpTatC, but not Tha4, inhibited precursor binding. Blue native gel electrophoresis and coimmunoprecipitation of digitonin-solubilized thylakoids showed that Hcf106 and cpTatC are members of an ∼700-kD complex that lacks Tha4. Thylakoid-bound precursor proteins were also associated with an ∼700-kD complex and were coimmunoprecipitated with antibodies to cpTatC or Hcf106. Chemical cross-linking revealed that precursors make direct contact with cpTatC and Hcf106 and confirmed that Tha4 is not associated with precursor, cpTatC, or Hcf106 in the membrane. Precursor binding to the cpTatC–Hcf106 complex required both the twin arginine and the hydrophobic core of the signal peptide. Precursors remained bound to the complex when Tha4 was sequestered by antibody, even in the presence of ΔpH. These results indicate that precursor binding to the cpTatC–Hcf106 complex constitutes the recognition event for this pathway and that subsequent participation by Tha4 leads to translocation.

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 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.

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 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 ten amino acids of the oxygen-evolving enhancer of tobacco is sufficient as the peptide residues for protein transport to the chloroplast thylakoid

2021 ◽  
Author(s):  
Sang Hoon Ma ◽  
Hyun Min Kim ◽  
Se Hee Park ◽  
Seo Young Park ◽  
Thanh Dat Mai ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Transport ◽  
Amino Acid Sequences ◽  
Hydrophobic Core ◽  
Thylakoid Lumen ◽  
Transit Peptides ◽  
Tat Pathway ◽  
Arginine Residues ◽  
Oxygen Evolving

Abstract Key message The thylakoid transit peptide of tobacco oxygen-evolving enhancer protein contains a minimal ten amino acid sequences for thylakoid lumen transports. This ten amino acids do not contain twin-arginine, which is required for typical chloroplast lumen translocation. Abstract Chloroplasts are intracellular organelles responsible for photosynthesis to produce organic carbon for all organisms. Numerous proteins must be transported from the cytosol to chloroplasts to support photosynthesis. This transport is facilitated by chloroplast transit peptides (TPs). Four chloroplast thylakoid lumen TPs were isolated from Nicotiana tabacum and were functionally analyzed as thylakoid lumen TPs. Typical chloroplast stroma-transit peptides and thylakoid lumen transit peptides (tTPs) are found in N. tabacum transit peptides (NtTPs) and the functions of these peptides are confirmed with TP–GFP fusion proteins under fluorescence microscopy and chloroplast fractionation, followed by Western blot analysis. During the functional analysis of tTPs, we uncovered the minimum 10 amino acid sequence is sufficient for thylakoid lumen transport. These ten amino acids can efficiently translocate GFP protein, even if they do not contain the twin-arginine residues required for the twin-arginine translocation (Tat) pathway, which is a typical thylakoid lumen transport. Further, thylakoid lumen transporting processes through the Tat pathway was examined by analyzing tTP sequence functions and we demonstrate that the importance of hydrophobic core for the tTP cleavage and target protein translocation.

scholarly journals Signal Peptides Bind and Aggregate RNA

2001 ◽  
Vol 276 (15) ◽  
pp. 12222-12227 ◽  
Author(s):  
Joanna Feltham Swain ◽  
Lila M. Gierasch
Keyword(s):  
Signal Recognition Particle ◽  
Signal Peptides ◽  
Signal Recognition ◽  
Gtpase Activity ◽  
Hydrophobic Core ◽  
Signal Sequences ◽  
Functional Peptide ◽  
Negative Controls

N-terminal signal sequences can direct nascent protein chains to the inner membrane of prokaryotes and the endoplasmic reticulum of eukaryotes by interacting with the signal recognition particle. In this study, we show that isolated peptides corresponding to several bacterial signal sequences inhibit the GTPase activity of theEscherichia colisignal recognition particle, as previously reported (Miller, J. D., Bernstein, H. D., and Walter, P. (1994)Nature367, 657–659), but not by the direct mechanism proposed. Instead, isolated signal peptides bind nonspecifically to the RNA component and aggregate the entire signal recognition particle, leading to a loss of its intrinsic GTPase activity. Surprisingly, only “functional” peptide sequences aggregate RNA; the peptides in general use as “nonfunctional” negative controls (e.g.those with deletions or charged substitutions within the hydrophobic core), are sufficiently different in physical character that they do not aggregate RNA and thus have no effect on the GTPase activity of the signal recognition particle. We propose that the reported effect of functional signal peptides on the GTPase activity of the signal recognition particle is an artifact of the high peptide concentrations and low salt conditions used in thesein vitrostudies and that signal sequences at the N terminus of nascent chainsin vivodo not exhibit this activity.

scholarly journals An M protein coiled coil unfurls and exposes its hydrophobic core to capture LL-37

2022 ◽  
Author(s):  
Piotr Kolesinski ◽  
Kuei-Chen Wang ◽  
Yujiro Hirose ◽  
Victor Nizet ◽  
Partho Ghosh
Keyword(s):  
Specific Binding ◽  
Coiled Coil ◽  
M Protein ◽  
Human Populations ◽  
Hydrophobic Core ◽  
Antigenic Variant ◽  
Specific Detail ◽  
Interaction Sites ◽  
Interaction Mode ◽  
M Proteins

Surface-associated, coiled-coil M proteins of Streptococcus pyogenes (Strep A) disable human immunity through interaction with select proteins. However, coiled coils lack features typical of protein-protein interaction sites, and it is therefore challenging to understand how M proteins achieve specific binding, for example, with the human antimicrobial peptide LL-37, which results in its neutralization. The crystal structure of a complex of LL-37 with M87 protein, an antigenic variant from a strain that is an emerging threat, revealed a novel interaction mode. The M87 coiled coil unfurled and asymmetrically exposed its hydrophobic core to capture LL-37. A single LL-37 molecule bound M87 in the crystal, but in solution recruited additional LL-37 molecules, consistent with a protein trap neutralization mechanism. The interaction mode visualized crystallographically was verified to contribute significantly to LL-37 resistance in an M87 Strep A strain, and was identified to be conserved in a number of other M protein types that are prevalent in human populations. Our results provide specific detail for therapeutic inhibition of LL-37 neutralization by M proteins.

scholarly journals Identification of Functional Tat Signal Sequences in Mycobacterium tuberculosis Proteins

2008 ◽  
Vol 190 (19) ◽  
pp. 6428-6438 ◽  
Author(s):  
Justin A. McDonough ◽  
Jessica R. McCann ◽  
Erin McElvania Tekippe ◽  
Jason S. Silverman ◽  
Nathan W. Rigel ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Signal Sequence ◽  
In Silico Analysis ◽  
Full Length ◽  
Specific Factor ◽  
Dependent Manner ◽  
Signal Sequences ◽  
Native Proteins ◽  
Tat Pathway ◽  
In The Wild

ABSTRACT The twin-arginine translocation (Tat) pathway is a system used by some bacteria to export proteins out from the cytosol to the cell surface or extracellular environment. A functional Tat pathway exists in the important human pathogen Mycobacterium tuberculosis. Identification of the substrates exported by the Tat pathway can help define the role that this pathway plays in the physiology and pathogenesis of M. tuberculosis. Here we used a reporter of Tat export, a truncated β-lactamase, ′BlaC, to experimentally identify M. tuberculosis proteins with functional Tat signal sequences. Of the 13 proteins identified, one lacks the hallmark of a Tat-exported substrate, the twin-arginine dipeptide, and another is not predicted by in silico analysis of the annotated M. tuberculosis genome. Full-length versions of a subset of these proteins were tested to determine if the native proteins are Tat exported. For three proteins, expression in a Δtat mutant of Mycobacterium smegmatis revealed a defect in precursor processing compared to expression in the wild type, indicating Tat export of the full-length proteins. Conversely, two proteins showed no obvious Tat export in M. smegmatis. One of this latter group of proteins was the M. tuberculosis virulence factor phospholipase C (PlcB). Importantly, when tested in M. tuberculosis a different result was obtained and PlcB was exported in a twin-arginine-dependent manner. This suggests the existence of an M. tuberculosis-specific factor(s) for Tat export of a proven virulence protein. It also emphasizes the importance of domains beyond the Tat signal sequence and bacterium-specific factors in determining if a given protein is Tat exported.

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