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Results for “Protein Sorting Signals”

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PubMed1992

Proteolysis in protein import and export: signal peptide processing in eu- and prokaryotes.

Numerous proteins in pro- and eukaryotes must cross cellular membranes in order to reach their site of function. Many of these proteins carry signal sequences that are removed by specific signal peptidases during, or shortly after, membrane transport. Signal peptidases have been identified in the rough endoplasmic reticulum, the matrix and inner membrane of mitochondria, the stroma and thylakoid membrane of chloroplasts, the bacterial plasma membrane and the thylakoid membrane of cyanobacteria. The composition of these peptidases varies between one and several subunits. No site-specific inhibitors are known for the majority of these enzymes. Accordingly, signal peptidases recognize structural motifs rather than linear amino acid sequences. Such motifs have become evident by employing extensive site-directed mutagenesis to investigate the anatomy of signal sequences. Analysis of the reaction specificities and the primary sequences of several signal peptidases suggests that the enzymes of the endoplasmic reticulum, the inner mitochondrial membrane and the thylakoid membrane of chloroplasts all have evolved from bacterial progenitors.

Animals↗

PubMed2001

Basolateral sorting of human poliovirus receptor alpha involves an interaction with the mu1B subunit of the clathrin adaptor complex in polarized epithelial cells.

Poliovirus receptor (hPVR/CD155) is a cell surface glycoprotein that belongs to the immunoglobulin superfamily but its natural function remains unknown. Two membrane-bound isoforms, hPVRalpha and hPVRdelta, are known to date, and they differ only in the amino acid sequence of their cytoplasmic domains. To gain an insight into the possible function of the cytoplasmic domains, we examined the localization of introduced hPVRalpha and hPVRdelta in polarized epithelial cells deficient of native hPVRs. Basolateral sorting of hPVRalpha was observed in Madine-Darby canine kidney cells expressing mu1B, but not in LLC-PK1 porcine kidney cells deficient in mu1B. Distribution of hPVRdelta, however, occurred both on the apical and basolateral plasma membranes of these two cell lines. Basolateral sorting of hPVRalpha was also seen in LLC-PK1 cells that expressed an intact exogenous mu1B, but not in the cells that expressed a mutant mu1B lacking binding ability to tyrosine-containing signals. These results indicate that mu1B is involved in the distribution of hPVRalpha to the basolateral membrane. Comparative distribution analysis of hPVRalpha using a series of mutants with truncations and substitutions in the cytoplasmic tail demonstrated that determinant for the basolateral sorting resided in the tyrosine-containing motif of the cytoplasmic tail. Furthermore, yeast two hybrid analysis strongly suggested that the tyrosine motif directly interacted with mu1B protein. Thus, basolateral sorting of hPVRalpha appears to involve the interaction with mu1B through a tyrosine motif existing in the cytoplasmic domain.

Adaptor Protein Complex 1↗

PubMed2001

SRbeta coordinates signal sequence release from SRP with ribosome binding to the translocon.

Protein targeting to the endoplasmic reticulum (ER) membrane is regulated by three GTPases, the 54 kDa subunit of the signal recognition particle (SRP) and the alpha- and beta-subunits of the SRP receptor (SR). Using a soluble form of SR and an XTP-binding mutant of SRbeta, we show that SRbeta is essential for protein translocation across the ER membrane. SRbeta can be cross-linked to a 21 kDa ribosomal protein in its empty and GDP-bound state, but not when GTP is bound. GTP binding to SRbeta is required to induce signal sequence release from SRP. This is achieved by the presence of the translocon, which changes the interaction between the 21 kDa ribosomal protein and SRbeta and thereby allows SRbeta to bind GTP. We conclude that SRbeta coordinates the release of the signal sequence from SRP with the presence of the translocon.

Animals↗

PubMed2001

Native and artificial reticuloplasmins co-accumulate in distinct domains of the endoplasmic reticulum and in post-endoplasmic reticulum compartments.

We compared the subcellular distribution of native and artificial reticuloplasmins in endosperm, callus, and leaf tissues of transgenic rice (Oryza sativa) to determine the distribution of these proteins among endoplasmic reticulum (ER) and post-ER compartments. The native reticuloplasmin was calreticulin. The artificial reticuloplasmin was a recombinant single-chain antibody (scFv), expressed with an N-terminal signal peptide and the C-terminal KDEL sequence for retrieval to the ER (scFvT84.66-KDEL). We found that both molecules were distributed in the same manner. In endosperm, each accumulated in ER-derived prolamine protein bodies, but also in glutelin protein storage vacuoles, even though glutelins are known to pass through the Golgi apparatus en route to these organelles. This finding may suggest that similar mechanisms are involved in the sorting of reticuloplasmins and rice seed storage proteins. However, the presence of reticuloplasmins in protein storage vacuoles could also be due to simple dispersal into these compartments during protein storage vacuole biogenesis, before glutelin deposition. In callus and leaf mesophyll cells, both reticuloplasmins accumulated in ribosome-coated vesicles probably derived directly from the rough ER.

Calcium-Binding Proteins↗

PubMed2002

Expression and purification of the chloroplast putative nitrogen sensor, PII, of Arabidopsis thaliana.

The bacterial PII protein was discovered over 30 years ago and is known to be a key player in orchestrating the coordination of nitrogen metabolism with changes in carbon flux. Bacterial PII is regulated by covalent modification and binding to effector molecules in response to the nitrogen/carbon status of the cell and appropriately coordinates the activity of glutamine synthetase and the transcription of a nitrogen sensitive regulon. Recently, a PII protein was identified in higher plants and the protein was found to be localized to the chloroplast. The Arabidopsis thaliana putative nitrogen sensor protein, PII, was cloned and overexpressed with a C-terminal 6-histidine tag. The full-length protein, which included the chloroplast transit peptide, was overexpressed in Escherichia coli, but was very susceptible to proteolytic degradation. Removal of the transit peptide yielded a highly pure, stable recombinant protein whose identity was established as PII by matrix assisted laser desorption ionization-time of flight mass spectrometry. Polyclonal antibodies generated against the recombinant protein effectively immunoprecipitated PII from an A. thaliana extract and the protein was confirmed to be 17 kDa in mass. The availability of milligram amounts of PII will allow a complete biophysical characterization of the protein and antibodies should aid in the identification of PII interacting proteins and the establishment of the higher plant PII signal transduction cascade.

Amino Acid Sequence↗

PubMed1988

Protein translocation across membranes.

Many newly synthesized proteins must be translocated across a membrane to reach their final destinations. Translocation requires a signal on the protein itself, a loose conformation of the protein, energy, and receptor-like components in the cytosol and on the target membrane.

Animals↗

PubMed1999

PHYSEAN: PHYsical SEquence ANalysis for the identification of protein domains on the basis of physical and chemical properties of amino acids.

MOTIVATION: PHYSEAN predicts protein classes with highly variable sequences on the basis of their physical, chemical and biological characteristics such as diverse hydrophobicity, structural propensity and steric properties. These characteristics, calculated from multiple positions in a sequence, may be conserved even between sequences that fail to produce alignments at any acceptable level of statistical significance. PHYSEAN complements methods that require sequence alignments (BLAST, FASTA, dynamic programming) by adding less residue- and position-specific physicochemical information on the protein or the domain. RESULTS: We predict proteins or their domains like signal peptides using physical, chemical, geometric, and biological properties of the 20 amino acids. This comprehensive set of properties may cover the diagnostic functional and structural aspects of a domain or a protein class. We automatically select and weight a subset of properties so as to discriminate between, e.g., signal peptides and amino-termini of cytosolic proteins with the lowest number of incorrect predictions. This optimal selection of properties and their weights significantly decreases the number of incorrect predictions as compared to any single property or any combination of unweighted properties. Weights have been optimized by high-performance linear programming models that systematically find the optimal solution from among an astronomic number of property/weight combinations. PHYSEAN's performance is demonstrated by highly accurate predictions of signal peptides (the vehicles for protein transport across membranes) and their cleavage sites. The results indicate reliable predictions are possible even in the lack of sequence conservation using an automated physical and chemical analysis of proteins.

Algorithms↗

PubMed1991

Protein secretion in bacteria.

Most secretory proteins are synthesized as precursors with an amino-terminal signal peptide. Genetic identification of proteins essential for signal peptide dependent translocation to the Escherichia coli periplasm has led to the biochemical dissection of the secretion pathway. Additional mechanisms exist in Gram-negative bacteria for protein secretion to the extracellular environment.

Bacterial Proteins↗

PubMed2002

Support vector machine prediction of signal peptide cleavage site using a new class of kernels for strings.

A new class of kernels for strings is introduced. These kernels can be used by any kernel-based data analysis method, including support vector machines (SVM). They are derived from probabilistic models to integrate biologically relevant information. We show how to compute the kernels corresponding to several classical probabilistic models, and illustrate their use by building a SVM for the problem of predicting the cleavage site of signal peptides from the amino-acid sequence of a protein. At a given rate of false positive this method retrieves up to 47% more true positives than the classical weight matrix method.

Algorithms↗

PubMedDate not supplied

On remaining cytoplasmic.

The published literature contains a number of examples of normally non-cytoplasmic proteins whose transport out of the cytoplasm is not completely abolished by drastic alterations to their routing signals (signal sequences, etc). Furthermore, there are numerous examples of cytoplasmic proteins that can be routed to and across plasma or organelle membranes by fusing them to routing signals. These 2 sets of observations lead to a re-evaluation of the reliability and accuracy of protein routing and to consideration of the consequences of the errors which might occur.

Biological Transport↗

PubMed2000

Oligomerization of pro-opiomelanocortin is independent of pH, calcium and the sorting signal for the regulated secretory pathway.

Studies indicate that pro-opiomelanocortin (POMC) is sorted to the regulated secretory pathway by binding to a sorting receptor identified as membrane-bound carboxypeptidase E (CPE) [Cool et al. (1997) Cell 88, 73-83]. The efficiency of this sorting mechanism could be enhanced if POMC molecules were to self-associate to form oligomers, prior or subsequent to binding to CPE. Using cross-linking and gel filtration techniques, we demonstrated that POMC forms oligomers at both neutral and acidic pHs and calcium was not necessary. delta N-POMC, which lacks the N-terminal sorting signal for the regulated secretory pathway, also formed similar oligomers, indicating that the sorting and oligomerization domains are different.

Animals↗

PubMed2000

Large-scale predictions of secretory proteins from mammalian genomic and EST sequences.

Machine learning techniques have improved predictions of secretory proteins from protein, genomic and expressed sequence tag (EST) sequences. Artificial neural networks, physical sequence analysis using high-performance optimization, and hidden Markov models identify extremely variable signal peptides (the vehicles of protein transport across the endoplasmic reticulum membrane), transmembrane segments, and specific extracellular and intracellular domains as indicators of possible roles in the intercellular and intracellular chemical signaling pathways. The major role of peptide hormones, blood coagulation factors, carcinogenesis agents, and other secretory proteins in orchestrating multicellular life indicates pharmacological potential in the cure of major diseases and numerous biotechnological applications.

Animals↗

PubMed1997

Characterization of a novel tyrosine phosphorylated 100-kDa protein that binds to SHP-2 and phosphatidylinositol 3'-kinase in myeloid cells.

Fms is a tyrosine kinase-containing receptor for macrophage colony-stimulating factor (M-CSF) that regulates survival, growth, and differentiation of cells along the monocyte/macrophage lineage. M-CSF stimulation of murine myeloid FDC-P1 cells expressing Fms resulted in the tyrosine phosphorylation of a number of signal transduction proteins, including an unidentified 100-kDa protein. This 100-kDa protein associated with the tyrosine phosphatase SHP-2 but not with the related phosphatase SHP-1. The kinetics of tyrosine phosphorylation of p100 and SHP-2 suggest that p100 may be a direct substrate of SHP-2. p100 bound directly to the SH2 domains of both SHP-2 and the p85 subunit of phosphatidylinositol 3'-kinase. The 100-kDa protein did not appear to bind directly to Fms, Ship, Cbl, Shc, or Grb2, although all of these proteins were coimmunoprecipitated with p85 after M-CSF stimulation. Association of p100 with SHP-2 and p85 did not require the major autophosphorylation sites on Fms nor binding of p85 to Fms. A tyrosine phosphorylated protein of 100 kDa also coprecipitated with SHP-2 from several other myeloid cell lines after M-CSF stimulation but was not seen in immunoprecipitates from Rat2 fibroblasts expressing Fms. Stimulation of FDC-P1 cells with additional cytokines also resulted in coprecipitation of a 100-kDa protein with SHP-2. p100 may therefore be a common component of the signaling pathways of cytokine receptors in myeloid cells.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Secretion and circular dichroism analysis of the C-terminal signal peptides of HlyA and LktA.

The secretion of the 107 kDa hemolysin A (HlyA) from Escherichia coli is mediated by membrane proteins hemolysin B (HlyB) and hemolysin D (HlyD). The signal for transport has been mapped to the C-terminal 60 amino acids of the HylA molecule. We have shown previously that the C-terminal 70 amino acids of leukotoxin (LktA) from Pasteurella hemolytica can substitute functionally for the HlyA signal sequence. This 70 amino acid peptide contains little primary sequence similarity to the HlyA signal sequence, and we have hypothesized that these signal sequences assume a similar higher-order structure which is recognized by the HlyB/D transporter. In the present study, we have expressed and purified small peptides containing the C-terminal 61 amino acids of HlyA and the C-terminal 70 amino acids of LktA. We show that these signal peptides are sufficient for secretion from E. coli in a HlyB/D dependent manner. Circular dichroism analyses show that both molecules exhibit common biophysical properties. In aqueous solution, they appear to be mainly unstructured, but in a membrane mimetic environment they assume a helical secondary structure. The conformational change observed for both peptides going from an aqueous to a membrane mimetic environment may be an important feature of these signal sequences necessary for their recognition and transport.

Bacterial Proteins↗

PubMed1994

Identification of the nucleolar targeting signal of human angiogenin.

Angiogenin is endocytosed by subconfluent endothelial cells, translocated to the nucleus and accumulates in the nucleolus. It also localizes into the nucleolus of digitonin-permeabilized endothelial cells. The peptide RRRGL corresponding to residues 31-35 of human angiogenin specifically targets non-nuclear carrier proteins such as albumin, an anti-human nucleolus monoclonal antibody and R33A angiogenin to the nucleolus of permeabilized endothelial cells. Proteins conjugated with a "mutant" peptide, RRAGL, are not imported. Fluorescein isothiocyanate-conjugated RRRGL is also rapidly imported into the nucleus and localized to the nucleolus, whereas the "mutant" peptide is not. Residue R33 is essential for nuclear translocation and R31 and R32 appear to modulate this process. Thus, 31RRRGL35 is a nuclear localization signal responsible for the nucleolar targeting of human angiogenin.

Amino Acid Sequence↗

PubMed2000

Requirement for the COOH-terminal pro-sequence in the translocation of aqualysin I across the cytoplasmic membrane in Escherichia coli.

Aqualysin I from Thermus aquaticus YT-1 is an extracellular subtilisin-type serine protease. The protease is synthesized as a distinct precursor composed of four functional domains: an N-terminal signal sequence, an N-terminal pro-sequence, a protease domain, and a C-terminal pro-sequence. The N-terminal pro-sequence is essential for the production of active aqualysin I while the C-terminal pro-sequence is required for extracellular secretion of aqualysin I. In an E. coli expression system, the function of C-terminal pro-sequence in the translocation of aqualysin I across the cytoplasmic membrane was investigated. More than 60-70% of the total activity was detected in the cytoplasmic fraction in the deletion mutations of the C-terminal pro-sequence while less than 30% was found in this fraction in wild type. In addition, in vitro processing of aqualysin I precursors with these mutations to a mature form promptly occurred and the folding into active aqualysin I was rapid. These results suggest that the C-terminal pro-sequence, probably in conjunction with the signal sequence, facilitates the translocation of the precursor across the cytoplasmic membrane by preventing the precursor from taking on an active conformation.

Cell Membrane↗

PubMed2000

Aberrant signal peptide cleavage of collagen X in Schmid metaphyseal chondrodysplasia. Implications for the molecular basis of the disease.

Schmid metaphyseal chondrodysplasia results from mutations in the collagen X (COL10A1) gene. With the exception of two cases, the known mutations are clustered in the C-terminal nonhelical (NC1) domain of the collagen X. In vitro and cell culture studies have shown that the NC1 mutations result in impaired collagen X trimer assembly and secretion. In the two other cases, missense mutations that alter Gly(18) at the -1 position of the putative signal peptide cleavage site were identified (Ikegawa, S., Nakamura, K., Nagano, A., Haga, N., and Nakamura, Y. (1997) Hum. Mutat. 9, 131-135). To study their impact on collagen X biosynthesis using in vitro cell-free translation in the presence of microsomes, and cell transfection assays, these two mutations were created in COL10A1 by site-directed mutagenesis. The data suggest that translocation of the mutant pre-alpha1(X) chains into the microsomes is not affected, but cleavage of the signal peptide is inhibited, and the mutant chains remain anchored to the membrane of microsomes. Cell-free translation and transfection studies in cells showed that the mutant chains associate into trimers but cannot form a triple helix. The combined effect of both the lack of signal peptide cleavage and helical configuration is impaired secretion. Thus, despite the different nature of the NC1 and signal peptide mutations in collagen X, both result in impaired collagen X secretion, probably followed by intracellular retention and degradation of mutant chains, and causing the Schmid metaphyseal chondrodysplasia phenotype.

Animals↗

PubMed1997

Spectroscopic studies of the C-terminal secretion signal of the Serratia marcescens haem acquisition protein (HasA) in various membrane-mimetic environments.

The structure of a peptide comprising the last 56 C-terminal residues of the Serratia marcescens haem acquisition protein (HasA) secreted by an ATP-binding cassette exporter was examined by 1H-NMR, circular dichroic and fluorescence spectroscopies. The peptide, which contains the secretion signal of HasA, is efficiently secreted by the HasA transporter. It is largely unstructured and flexible in aqueous buffer solution, but its helical content increases upon addition of trifluoroethanol, detergents and lipids. By circular dichroism, a stable helical conformation is observed between 20% and 70% (by vol.) trifluoroethanol. The 1H-NMR spectrum was analysed at these two trifluoroethanol concentrations; residues 7-15, 21-30 and 40-50 were shown to form relatively stable helices. In the presence of neutral detergent, alpha-helix is induced to a similar extent upon micelle formation; in this case, fluorescence data indicate that at least the N-terminus of the peptide interacts with the micelle. In the presence of negatively charged detergent, alpha-helix is induced before micelle formation and the N-terminus of the peptide seems not to be involved in this interaction. In the presence of negatively charged liposomes, the peptide interacts with the vesicle, again inducing a helical conformation. However, the helical content remains lower than upon addition of trifluoroethanol or neutral micelles. These results are compared to those previously obtained with the secretion signal of one of the Erwinia chrysanthemi metalloproteases which are transported efficiently by the HasA transporter. Both signals exhibit similar conformational features, despite their low sequence similarity.

ATP-Binding Cassette Transporters↗