scholarly journals Proteomics identifies signal peptide features determining the substrate specificity in human Sec62/Sec63-dependent ER protein import

2019 ◽  
Author(s):  
Stefan Schorr ◽  
Duy Nguyen ◽  
Sarah Haßdenteufel ◽  
Nagarjuna Nagaraj ◽  
Adolfo Cavalié ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Mammalian Cells ◽  
Protein Import ◽  
Supplementary Information ◽  
Signal Peptides ◽  
Proteomics Data ◽  
Amino Terminal ◽  
Link Type ◽  
Sec61 Channel

AbstractIn mammalian cells one-third of all polypeptides are integrated into the membrane or translocated into the lumen of the endoplasmic reticulum (ER) via the Sec61-channel. While the Sec61-complex facilitates ER-import of most precursor polypeptides, the Sec61-associated Sec62/Sec63-complex supports ER-import in a substrate-specific manner. So far, mainly posttranslationally imported precursors and the two cotranslationally imported precursors of ERj3 and prion protein were found to depend on the Sec62/Sec63-complex in vitro. Therefore, we determined the rules for engagement of Sec62/Sec63 in ER-import in intact human cells using a recently established unbiased proteomics approach. In addition to confirming ERj3, we identified twenty-two novel Sec62/Sec63-substrates under these in vivo-like conditions. As a common feature, those previously unknown substrates share signal peptides with comparatively longer but less hydrophobic H-region and lower C-region polarity. Further analyses with four substrates, and ERj3 in particular, revealed the combination of a slowly-gating signal peptide and a downstream translocation-disruptive positively charged cluster of amino acid residues as decisive for the Sec62-/Sec63-requirement. In the case of ERj3, these features were found to be responsible for an additional BiP-requirement and to correlate with sensitivity towards the Sec61-channel inhibitor CAM741. Thus, the human Sec62/Sec63-complex may support Sec61-channel opening for precursor polypeptides with slowly-gating signal peptides by direct interaction with the cytosolic amino-terminal peptide of Sec61α or via recruitment of BiP and its interaction with the ER-lumenal loop 7 of Sec61α. These novel insights into the mechanism of human ER protein import contribute to our understanding of the etiology of SEC63-linked Polycystic Liver Disease.DatabasesThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (http://www.ebi.ac.uk/pride/archive/projects/Identifiers) with the dataset identifiers: PXD008178, PXD011993, and PXD012078. Supplementary information was deposited at Mendeley Data under the DOI:10.17632/6s5hn73jcv.1 (http://dx.doi.or/10.17632/6s5hn73jcv.1).

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

scholarly journals Quickomics: exploring omics data in an intuitive, interactive and informative manner

2021 ◽  
Author(s):  
Benbo Gao ◽  
Jing Zhu ◽  
Soumya Negi ◽  
Xinmin Zhang ◽  
Stefka Gyoneva ◽  
...  
Keyword(s):  
Modular Design ◽  
Functional Module ◽  
Supplementary Information ◽  
Data Sets ◽  
Omics Data ◽  
Proteomics Data ◽  
Primary Analysis ◽  
Link Type ◽  
R Shiny ◽  
Advanced Analysis

AbstractSummaryWe developed Quickomics, a feature-rich R Shiny-powered tool to enable biologists to fully explore complex omics data and perform advanced analysis in an easy-to-use interactive interface. It covers a broad range of secondary and tertiary analytical tasks after primary analysis of omics data is completed. Each functional module is equipped with customized configurations and generates both interactive and publication-ready high-resolution plots to uncover biological insights from data. The modular design makes the tool extensible with ease.AvailabilityResearchers can experience the functionalities with their own data or demo RNA-Seq and proteomics data sets by using the app hosted at http://quickomics.bxgenomics.com and following the tutorial, https://bit.ly/3rXIyhL. The source code under GPLv3 license is provided at https://github.com/interactivereport/[email protected], [email protected] informationSupplementary materials are available at https://bit.ly/37HP17g.

Fibrinogen Biosynthesis: In Vitro Translation. Glycosylation And Translocation Of Fibrinogen Peptide Chains

1981 ◽  
Author(s):  
G M Fuller ◽  
J M Nickerson
Keyword(s):  
Signal Peptide ◽  
Hepatoma Cell ◽  
Temporal Analysis ◽  
In Vitro Translation ◽  
Translation System ◽  
Hepatoma Cell Line ◽  
Amino Terminal ◽  
Cellular Processes ◽  
Microsomal Membranes

Fibrinogen is a hepatically derived plasma glycoprotein that is composed of three pairs of nonidentical chains linked together by complex sets of disulfide bridges. In an effort to understand the molecular and cellular processes of translating and assembling this important multichained protein we have utilized an in vitro translating system using mRNA’s for rat fibrinogen. Highly specific antibodies to fibrinogen and to each chain have been developed and used to immunoprecipitate the nascent Aα, Bβ, and γ polypeptides. We have also used a rat hepatoma cell line which synthesizes and secretes fibrinogen to prepare nonglycosylated but processed fibrinogen subunits. SDS/PAGE analysis of the translation products clearly show that each polypeptide has a “signal” peptide located at its amino terminal end. The size of the signal peptide is different for each chain. These results demonstrate that separate mRNA’s exist for each of the fibrinogen subunits. Temporal analysis of the glycosylation of the Bβ and γ chain reveal that the γ chain receives its Asn-linked carbohydrate as an early cotranslational event. The Bβ chain’s core carbohydrate moiety is near the end of the polypeptide and our evidence shows that the glycosylation event likely occurs posttranslationally. When microsomal membranes are added to an on-going translation system, all three of fibrinogen's polypeptides translocate into the cisternal space, with an apparent equal stiochiometry. Additional experiments suggest that fibrinogen assembly occurs as a cotranslational process.These studies have been supported in part by NIH HL - 16445 and HL 00162.

scholarly journals Different Minimal Signal Peptide Lengths Recognized by the Archaeal Prepilin-Like Peptidases FlaK and PibD

2008 ◽  
Vol 191 (21) ◽  
pp. 6732-6740 ◽  
Author(s):  
Sandy Y. M. Ng ◽  
David J. VanDyke ◽  
Bonnie Chaban ◽  
John Wu ◽  
Yoshika Nosaka ◽  
...  
Keyword(s):  
Amino Acids ◽  
Signal Peptide ◽  
Signal Peptidase ◽  
Signal Peptides ◽  
Peptidase Activity ◽  
Vitro Assay ◽  
Type Iv ◽  
Peptide Length ◽  
Short Signal

ABSTRACT In Archaea, the preflagellin peptidase (a type IV prepilin-like peptidase designated FlaK in Methanococcus voltae and Methanococcus maripaludis) is the enzyme that cleaves the N-terminal signal peptide from preflagellins. In methanogens and several other archaeal species, the typical flagellin signal peptide length is 11 to 12 amino acids, while in other archaea preflagellins possess extremely short signal peptides. A systematic approach to address the signal peptide length requirement for preflagellin processing is presented in this study. M. voltae preflagellin FlaB2 proteins with signal peptides 3 to 12 amino acids in length were generated and used as a substrate in an in vitro assay utilizing M. voltae membranes as an enzyme source. Processing by FlaK was observed in FlaB2 proteins containing signal peptides shortened to 5 amino acids; signal peptides 4 or 3 amino acids in length were unprocessed. In the case of Sulfolobus solfataricus, where the preflagellin peptidase PibD has broader substrate specificity, some predicted substrates have predicted signal peptides as short as 3 amino acids. Interestingly, the shorter signal peptides of the various mutant FlaB2 proteins not processed by FlaK were processed by PibD, suggesting that some archaeal preflagellin peptidases are likely adapted toward cleaving shorter signal peptides. The functional complementation of signal peptidase activity by FlaK and PibD in an M. maripaludis ΔflaK mutant indicated that processing of preflagellins was detected by complementation with either FlaK or PibD, yet only FlaK-complemented cells were flagellated. This suggested that a block in an assembly step subsequent to signal peptide removal occurred in the PibD complementation.

scholarly journals A Chimeric Protein Containing the N Terminus of the Adeno-Associated Virus Rep Protein Recognizes Its Target Site in an In Vivo Assay

2000 ◽  
Vol 74 (5) ◽  
pp. 2372-2382 ◽  
Author(s):  
Toni Cathomen ◽  
Delphine Collete ◽  
Matthew D. Weitzman
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Target Site ◽  
Yeast Genome ◽  
Chimeric Proteins ◽  
Adeno Associated Virus ◽  
Amino Terminal ◽  
N Terminus

ABSTRACT The Rep78 and Rep68 proteins of adeno-associated virus (AAV) type 2 are involved in DNA replication, regulation of gene expression, and targeting site-specific integration. They bind to a specific Rep recognition sequence (RRS) found in both the viral inverted terminal repeats and the AAVS1 integration locus on human chromosome 19. Previous in vitro studies implied that an N-terminal segment of Rep is involved in DNA recognition, while additional domains might stabilize binding and mediate multimerization. In order to define the minimal requirements for Rep to recognize its target site in the human genome, we developed one-hybrid assays in which DNA-protein interactions are detected in vivo. Chimeric proteins consisting of the N terminus of Rep fused to different oligomerization motifs and a transcriptional activation domain were analyzed for oligomerization, DNA binding, and activation of reporter gene expression. Expression of reporter genes was driven from RRS motifs cloned upstream of minimal promoters and examined in mammalian cells from transfected plasmids and inSaccharomyces cerevisiae from a reporter cassette integrated into the yeast genome. Our results show for the first time that chimeric proteins containing the amino-terminal 244 residues of Rep are able to target the RRS in vitro and in vivo when incorporated into artificial multimers. These studies suggest that chimeric proteins may be used to harness the unique targeting feature of AAV for gene therapy applications.

scholarly journals Unexpected Diversity of Signal Peptides in Prokaryotes

mBio ◽  
2012 ◽  
Vol 3 (6) ◽  
Author(s):  
Samuel H. Payne ◽  
Stefano Bonissone ◽  
Si Wu ◽  
Roslyn N. Brown ◽  
Dmitry N. Ivankov ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Protein Localization ◽  
Cellular Protein ◽  
Peptide Sequence ◽  
Signal Peptides ◽  
Proteomics Data ◽  
Detailed Model ◽  
Signal Peptide Sequence ◽  
Peptide Motifs ◽  
Computational Analyses

ABSTRACT Signal peptides are a cornerstone mechanism for cellular protein localization, yet until now experimental determination of signal peptides has come from only a narrow taxonomic sampling. As a result, the dominant view is that Sec-cleaved signal peptides in prokaryotes are defined by a canonical AxA motif. Although other residues are permitted in the motif, alanine is by far the most common. Here we broadly examine proteomics data to reveal the signal peptide sequences for 32 bacterial and archaeal organisms from nine phyla and demonstrate that this alanine preference is not universal. Discoveries include fundamentally distinct signal peptide motifs from Alphaproteobacteria, Spirochaetes, Thermotogae and Euryarchaeota. In these novel motifs, alanine is no longer the dominant residue but has been replaced in a different way for each taxon. Surprisingly, divergent motifs correlate with a proteome-wide reduction in alanine. Computational analyses of ~1,500 genomes reveal numerous major evolutionary clades which have replaced the canonical signal peptide sequence with novel motifs. IMPORTANCE This article replaces a widely held general model with a more detailed model describing phylogenetically correlated variation in motifs for Sec secretion.

scholarly journals Annotating eukaryotic and toxin-specific signal peptides using Razor

2020 ◽  
Author(s):  
Bikash K. Bhandari ◽  
Paul P. Gardner ◽  
Chun Shen Lim
Keyword(s):  
Signal Peptide ◽  
Web Application ◽  
Protein Transport ◽  
Protein Translocation ◽  
Recombinant Protein Expression ◽  
Disease Diagnosis ◽  
Signal Peptides ◽  
Specific Signal ◽  
Link Type ◽  
Command Line Tool

ABSTRACTMotivationSignal peptides are responsible for protein transport and secretion and are ubiquitous to all forms of life. The annotation of signal peptides is important for understanding protein translocation and toxin secretion, optimising recombinant protein expression, as well as for disease diagnosis and metagenomics.ResultsHere we explore the features of these signal sequences across eukaryotes. We find that different kingdoms have their characteristic distributions of signal peptide residues. Additionally, the signal peptides of secretory toxins have common features across kingdoms. We leverage these subtleties to build Razor, a simple yet powerful tool for annotating signal peptides, which additionally predicts toxin- and fungal-specific signal peptides based on the first 23 N-terminal residues. Finally, we demonstrate the usability of Razor by scanning all reviewed sequences from UniProt. Indeed, Razor is able to identify toxins using their signal peptide sequences only. Strikingly, we discover that many defensive proteins across kingdoms harbour a toxin-like signal peptide; some of these defensive proteins have emerged through convergent evolution, e.g. defensin and defensin-like protein families, and phospholipase families.Availability and implementationRazor is available as a web application (https://tisigner.com/razor) and a command-line tool (https://github.com/Gardner-BinfLab/Razor).

scholarly journals Competitive Microtubule Binding of PEX14 Coordinates Peroxisomal Protein Import and Motility

2020 ◽  
Author(s):  
Maren Reuter ◽  
Hamed Kooshapur ◽  
Jeff-Gordian Suda ◽  
Alexander Neuhaus ◽  
Lena Brühl ◽  
...  
Keyword(s):  
Binding Site ◽  
Mammalian Cells ◽  
Matrix Protein ◽  
Protein Import ◽  
Spatial Proximity ◽  
Binding Motifs ◽  
Docking Protein ◽  
Nanomolar Range ◽  
Import Receptor

AbstractPEX14 functions as peroxisomal docking protein for the import receptor PEX5. For docking, the conserved N-terminal domain of PEX14 (PEX14-NTD) binds amphipathic alpha-helical ligands, typically comprising one or two aromatic residues, of which human PEX5 possesses eight. Here, we show that the PEX14-NTD also binds to microtubular filaments in vitro with a dissociation constant in nanomolar range. PEX14 interacts with two motifs in the C-terminal region of human ß-tubulin. At least one of the binding motifs is in spatial proximity to the binding site of microtubules (MT) for kinesin. Both PEX14 and kinesin can bind to MT simultaneously. Notably, binding of PEX14 to tubulin can be prevented by its association with PEX5. The data suggest that PEX5 competes peroxisome anchoring to MT by occupying the ß-tubulin-binding site of PEX14. The competitive correlation of matrix protein import and motility may facilitate the homogeneous dispersion of peroxisomes in mammalian cells.

scholarly journals Phoenix Enhancer: proteomics data mining using clustered spectra

2019 ◽  
Author(s):  
Mingze Bai ◽  
Chunyuan Qin ◽  
Kunxian Shu ◽  
Johannes Griss ◽  
Yasset Perez-Riverol ◽  
...  
Keyword(s):  
Supplementary Information ◽  
Proteomics Data ◽  
Web Based ◽  
Tandem Mass Spectra ◽  
Link Type ◽  
False Discovery ◽  
Cluster Data ◽  
Public Data ◽  
Visualization Tools ◽  
Individual Dataset

AbstractMotivationSpectrum clustering has been used to enhance proteomics data analysis: some originally unidentified spectra can potentially be identified and individual peptides can be evaluated to find potential mis-identifications by using clusters of identified spectra. The Phoenix Enhancer provides an infrastructure to analyze tandem mass spectra and the corresponding peptides in the context of previously identified public data. Based on PRIDE Cluster data and a newly developed pipeline, four functionalities are provided: i) evaluate the original peptide identifications in an individual dataset, to find low confidence peptide spectrum matches (PSMs) which could correspond to mis-identifications; ii) provide confidence scores for all originally identified PSMs, to help users evaluate their quality (complementary to getting a global false discovery rate); iii) identify potential new PSMs for originally unidentified spectra; and iv) provide a collection of browsing and visualization tools to analyze and export the results. In addition to the web based service, the code is open-source and easy to re-deploy on local computers using Docker containers.AvailabilityThe service of Phoenix Enhancer is available at http://enhancer.ncpsb.org. All source code is freely available in GitHub (https://github.com/phoenix-cluster/) and can be deployed in the Cloud and HPC [email protected] informationSupplementary data are available online.

The Role of Intracellular Membranes in Protein Processing

1981 ◽  
Vol 39 ◽  
pp. 514-515
Author(s):  
Dennis Shields ◽  
Thomas G. Warren ◽  
Sara E. Roth ◽  
Reza F. Green
Keyword(s):  
Endoplasmic Reticulum ◽  
Signal Peptide ◽  
Messenger Rna ◽  
Polypeptide Chain ◽  
Secretory Protein ◽  
Free Protein ◽  
Amino Terminal ◽  
Free Translation ◽  
Microsomal Membranes

Most polypeptides destined for secretion are synthesized on polyribosomes bound to the membrane of the endoplasmic reticulum (E.R.), in contrast, cytosolic proteins are made on free ribosomes. When the messenger RNA (mRNA) for a secretory protein is translated in a cell-free protein synthesizing system, the product is usually larger than the mature protein by about 3,000 daltons. Numerous studies have demonstrated that the higher molecular weight of the cell-free translation product can be attributed to an amino terminal extension of about 20-30 amino acids termed the “signal peptide”. This signal peptide is thought to mediate binding of ribosomes bearing the nascent polypeptide chain to the membrane of the endoplasmic reticulum. Upon interaction with the E.R., the polypeptide chain is translocated across the membrane usually resulting in proteolytic removal of the signal peptide and segregation of the “processed” polypeptide into the ER. cisternae. This series of reactions can be followed in vitro by supplementing the cell-free protein synthesizing system with heterologous microsomal membranes which have been stripped of their endogenous ribosomes.

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