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PubMed1999

SMART: identification and annotation of domains from signalling and extracellular protein sequences.

SMART is a simple modular architecture research tool and database that provides domain identification and annotation on the WWW (http://coot.embl-heidelberg.de/SMART). The tool compares query sequences with its databases of domain sequences and multiple alignments whilst concurrently identifying compositionally biased regions such as signal peptide, transmembrane and coiled coil segments. Annotated and unannotated regions of the sequence can be used as queries in searches of sequence databases. The SMART alignment collection represents more than 250 signalling and extracellular domains. Each alignment is curated to assign appropriate domain boundaries and to ensure its quality. In addition, each domain is annotated extensively with respect to cellular localisation, species distribution, functional class, tertiary structure and functionally important residues.

Amino Acid Sequence↗

PubMed1991

Protein export in prokaryotes and eukaryotes. Theme with variations.

Protein export in prokaryotes as well as in eukaryotes can be defined as protein transport across the plasma membrane. In both types of organisms there are various apparently ATP-dependent transport mechanisms which can be distinguished from one another and which show similarities when the prokaryotic mechanism is compared with the respective eukaryotic mechanism. First, one can distinguish between transport mechanisms which involve so-called signal or leader peptides and those which do not. The latter mechanisms seem to employ ATP-dependent transport systems which belong to the family of oligopeptide permeases and multiple drug resistance proteins. Second, in signal or leader peptide-dependent transport one can distinguish between transport mechanisms which involve ribonucleoparticles and those which employ molecular chaperones. Both mechanisms appear to converge at the level of ATP-dependent translocases.

Animals↗

PubMed2004

The signal peptide of the Junín arenavirus envelope glycoprotein is myristoylated and forms an essential subunit of the mature G1-G2 complex.

Arenaviruses comprise a diverse family of rodent-borne viruses that are responsible for recurring and emerging outbreaks of viral hemorrhagic fevers worldwide. The Junín virus, a member of the New World arenaviruses, is endemic to the pampas grasslands of Argentina and is the etiologic agent of Argentine hemorrhagic fever. In this study, we have analyzed the assembly and function of the Junín virus envelope glycoproteins. The mature envelope glycoprotein complex is proteolytically processed from the GP-C precursor polypeptide and consists of three noncovalently associated subunits, G1, G2, and a stable 58-amino-acid signal peptide. This tripartite organization is found both on virions of the attenuated Candid 1 strain and in cells expressing the pathogenic MC2 strain GP-C gene. Replacement of the Junín virus GP-C signal peptide with that of human CD4 has little effect on glycoprotein assembly while abolishing the ability of the G1-G2 complex to mediate pH-dependent cell-cell fusion. In addition, we demonstrate that the Junín virus GP-C signal peptide subunit is myristoylated at its N-terminal glycine. Alanine substitution for the modified glycine residue in the GP-C signal peptide does not affect formation of the tripartite envelope glycoprotein complex but markedly reduces its membrane fusion activity. In contrast to the classical view that signal peptides act primarily in targeting nascent polypeptides to the endoplasmic reticulum, we suggest that the signal peptide of the arenavirus GP-C may serve additional functions in envelope glycoprotein structure and trafficking.

Amino Acid Sequence↗

PubMed2001

Sonic hedgehog facilitates dopamine differentiation in the presence of a mesencephalic glial cell line.

The aim of this study was to establish a cellular system to investigate the requirement for cell surface and diffusible molecules in the differentiation of fetal mesencephalic cells toward the dopamine lineage. Toward this end, we immortalized rat embryonic day 14 (E14) mesencephalon with a regulatable retroviral vector encoding v-myc. The stably transduced cells were pooled and designated as VME14 cells. VME14 cells proliferated rapidly, stopped proliferating, extended processes, and expressed GFAP after suppression of the v-myc expression with tetracycline, suggesting that VME14 cells differentiated into glial cells. Dissociated cells derived from the E11 rat mesencephalon gave rise to only a small number of tyrosine hydroxylase (TH)-positive neurons. However, when grown on a monolayer of the differentiated VME14 cells, a significantly higher number of cells differentiated into TH-positive neurons. VME14 cells were transduced with the secreted N-terminal cleavage product of the Sonic hedgehog gene (SHH-N), an inducer of mesencephalic dopaminergic neurons. This monoclonal, SHH-N-overexpressing cell line further enhanced dopaminergic differentiation of E11 rat mesencephalon cells. Thus, SHH-N and signals derived from fetal mesencephalic glia act cooperatively to facilitate dopaminergic differentiation. These fetal mesencephalon-derived cell lines will provide tools for the study of signals involved in dopaminergic differentiation.

Animals↗

PubMed1994

Mitochondrial Mas70p signal anchor sequence. Mutations in the transmembrane domain that disrupt dimerization but not targeting or membrane insertion.

Mas70p is an integral membrane protein in Saccharomyces cerevisiae that is targeted and inserted into the mitochondrial outer membrane in an N(in)-Ccyto orientation by its NH2-terminal 29-amino acid signal anchor sequence. Recently, we demonstrated that the signal anchor was capable of mediating homo-oligomerization of a fusion protein, pOMD29, in the outer membrane in vitro (Millar, D. G., and Shore, G. C. (1992) J. Biol. Chem. 268, 18403-18406). Consistent with this finding, we show here that a synthetic peptide corresponding to the Mas70p signal anchor is capable of independent membrane insertion and dimerization with pOMD29. To further map the oligomerization domain in the signal anchor sequence, a deletion mutant of pOMD29 that lacks amino acids 2-10 was constructed. This protein, pOMD29 delta 2-10, efficiently participated in dimer formation following import, indicating that dimerization was mediated by the putative membrane spanning segment (amino acids 11-29). This segment is predicted to form an alpha-helix that has an alanine-rich face and contains multiple copies of a pentapeptide dimerization motif that is widespread among members of the receptor tyrosine kinase family. Substitution of the alanine residues in one of these copies with isoleucine, producing a potentially bulkier contact surface, resulted in a protein which was targeted and inserted into the outer membrane but failed to assemble into dimers. Taken together, these results identify a structural feature of the signal anchor transmembrane domain that is important for oligomerization but is not required for targeting and membrane insertion.

Amino Acid Sequence↗

PubMed2006

Common infection strategies of pathogenic eukaryotes.

Pathogenic eukaryotes belong to several distinct phylogenetic lineages and have evolved the ability to colonize a range of hosts, including animals and plants. Pathogenic lifestyles have evolved repeatedly in eukaryotes, indicating that unique molecular processes are involved in host infection. However, evidence is now emerging that divergent eukaryotic pathogens might share common mechanisms of pathogenicity. The results from recent studies demonstrate that Plasmodium falciparum and Phytophthora infestans use equivalent host-targeting signals to deliver virulence adhesins and avirulence gene products into human and plant cells, respectively. Remodelling of host cells by different eukaryotic pathogens might therefore share some common features.

Amino Acid Motifs↗

PubMed2005

The signal peptide of the rat corticotropin-releasing factor receptor 1 promotes receptor expression but is not essential for establishing a functional receptor.

Approximately 5-10% of the GPCRs (G-protein-coupled receptors) contain N-terminal signal peptides that are cleaved off during receptor insertion into the ER (endoplasmic reticulum) membrane by the signal peptidases of the ER. The reason as to why only a subset of GPCRs requires these additional signal peptides is not known. We have recently shown that the signal peptide of the human ET(B)-R (endothelin B receptor) does not influence receptor expression but is necessary for the translocation of the receptor's N-tail across the ER membrane and thus for the establishment of a functional receptor [Köchl, Alken, Rutz, Krause, Oksche, Rosenthal and Schülein (2002) J. Biol. Chem. 277, 16131-16138]. In the present study, we show that the signal peptide of the rat CRF-R1 (corticotropin-releasing factor receptor 1) has a different function: a mutant of the CRF-R1 lacking the signal peptide was functional and displayed wild-type properties with respect to ligand binding and activation of adenylate cyclase. However, immunoblot analysis and confocal laser scanning microscopy revealed that the mutant receptor was expressed at 10-fold lower levels than the wild-type receptor. Northern-blot and in vitro transcription translation analyses precluded the possibility that the reduced receptor expression is due to decreased transcription or translation levels. Thus the signal peptide of the CRF-R1 promotes an early step of receptor biogenesis, such as targeting of the nascent chain to the ER membrane and/or the gating of the protein-conducting translocon of the ER membrane.

Amino Acid Sequence↗

PubMed1992

Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space.

Cytochrome b2 reaches the intermembrane space of mitochondria by transport into the matrix followed by export across the inner membrane. While in the matrix, the protein interacts with hsp60, which arrests its folding prior to export. The bacterial-type export sequence in pre-cytochrome b2 functions by inhibiting the ATP-dependent release of the protein from hsp60. Release for export apparently requires, in addition to ATP, the interaction of the signal sequence with a component of the export machinery in the inner membrane. Export can occur before import is complete provided that a critical length of the polypeptide chain has been translocated into the matrix. Thus, hsp60 combines two activities: catalysis of folding of proteins destined for the matrix, and maintaining proteins in an unfolded state to facilitate their channeling between the machineries for import and export across the inner membrane. Anti-folding signals such as the hydrophobic export sequence in cytochrome b2 may act as switches between these two activities.

Base Sequence↗

PubMed1990

Targeting of proteins into the eukaryotic secretory pathway: signal peptide structure/function relationships.

Much progress has been made in recent years regarding the mechanisms of targeting of secretory proteins to, and across, the endoplasmic reticulum (ER) membrane. Many of the cellular components involved in mediating translocation across this bilayer have been identified and characterized. Polypeptide domains of secretory proteins, termed signal peptides, have been shown to be necessary, and in most cases sufficient, for entry of preproteins into the lumen of the ER. These NH2-terminal segments appear to serve multiple roles in targeting and translocation. The structural features which mediate their multiple functions are currently the subject of intense study.

Animals↗

PubMed1994

Mechanisms of limb patterning.

The development of the vertebrate limb requires the coordinated action of multiple signals to achieve the proper arrangement of adult tissues. Recently, several molecules have been identified which play central roles in patterning of the limb bud. Sonic hedgehog, a homolog of the Drosophila segment polarity gene hedgehog, is likely to regulate anterior/posterior pattern formation. FGF-2 and FGF-4, members of the fibroblast growth factor family, have been shown to provide important signals for limb bud outgrowth and to indirectly regulate proximal/distal patterning. Some candidate effectors of the activity of Sonic hedgehog and of FGFs are known, including members of the clustered Hox genes.

Animals↗

PubMed1997

Cloning of a mouse smoothened cDNA and expression patterns of hedgehog signalling molecules during chondrogenesis and cartilage differentiation in clonal mouse EC cells, ATDC5.

Hedgehog (hh) family proteins appear to use the conserved targets in their signalling pathway including Patched (Ptc), Smoothened (Smo), and Gli. Although Indian hedgehog (Ihh) plays an important role in endochondral bone formation, the involvement of hh signalling molecules in skeletogenesis is unknown. We cloned a mouse (m) Smo cDNA and studied the expression patterns of Ihh, Ptc, Smo, and Gli mRNAs in mouse chondrogenic EC cells, ATDC5. The deduced amino acid sequence of mSmo consisted of 793 amino acids and was 98 and 93% homologous to the rat (r) Smo and human (h) Smo, respectively. In ATDC5 cells, the expression of Ihh mRNA paralleled that of type X collagen mRNA. Smo, Ptc, and Gli mRNAs were constitutively expressed throughout chondrogenesis and the subsequent cartilage differentiation processes except for the transient decrease in Ptc mRNA at the cellular condensation stage. Our data suggest that hh signalling molecules may be involved in chondrogenesis and cartilage differentiation in ATDC5 cells.

Amino Acid Sequence↗

PubMed2000

Characterization of the signal that directs Tom20 to the mitochondrial outer membrane.

Tom20 is a major receptor of the mitochondrial preprotein translocation system and is bound to the outer membrane through the NH(2)-terminal transmembrane domain (TMD) in an Nin-Ccyt orientation. We analyzed the mitochondria-targeting signal of rat Tom20 (rTom20) in COS-7 cells, using green fluorescent protein (GFP) as the reporter by systematically introducing deletions or mutations into the TMD or the flanking regions. Moderate TMD hydrophobicity and a net positive charge within five residues of the COOH-terminal flanking region were both critical for mitochondria targeting. Constructs without net positive charges within the flanking region, as well as those with high TMD hydrophobicity, were targeted to the ER-Golgi compartments. Intracellular localization of rTom20-GFP fusions, determined by fluorescence microscopy, was further verified by cell fractionation. The signal recognition particle (SRP)-induced translation arrest and photo-cross-linking demonstrated that SRP recognized the TMD of rTom20-GFP, but with reduced affinity, while the positive charge at the COOH-terminal flanking segment inhibited the translation arrest. The mitochondria-targeting signal identified in vivo also functioned in the in vitro system. We conclude that NH(2)-terminal TMD with a moderate hydrophobicity and a net positive charge in the COOH-terminal flanking region function as the mitochondria-targeting signal of the outer membrane proteins, evading SRP-dependent ER targeting.

Amino Acid Sequence↗

PubMed2002

Oligomeric properties and signal peptide binding by Escherichia coli Tat protein transport complexes.

The Escherichia coli Tat apparatus is a protein translocation system that serves to export folded proteins across the inner membrane. The integral membrane proteins TatA, TatB and TatC are essential components of this pathway. Substrate proteins are directed to the Tat apparatus by specialized N-terminal signal peptides bearing a consensus twin-arginine sequence motif. Here we have systematically examined the Tat complexes that can be purified from overproducing strains. Our data suggest that the TatA, TatB and TatC proteins are found in at least two major types of high molecular mass complex in detergent solution, one consisting predominantly of TatA but with a small quantity of TatB, and the other based on a TatBC unit but also containing some TatA protein. The latter complex is shown to be capable of binding a Tat signal peptide. Using an alternative purification strategy we show that it is possible to isolate a TatABC complex containing a high molar excess of the TatA component.

Arginine↗

PubMed1994

Structure of the signal sequences for two mitochondrial matrix proteins that are not proteolytically processed upon import.

The N-terminal sequences of rhodanese and 3-oxoacyl-CoA thiolase, two mitochondrial matrix proteins that are not proteolytically processed upon import, have been studied by NMR and CD spectroscopy. In aqueous trifluoroethanol, in the presence of micelles, and in the presence of small unilamellar vesicles (SUVs), these peptides form alpha-helical structures beginning near the N-terminus and extending, continuously, for at least three helical turns. This result is consistent with a previous finding that a mutant rat liver mitochondrial aldehyde dehydrogenase signal sequence we designed, which formed a continuous alpha-helix, could successfully direct protein import but was not proteolytically processed (Thornton, K., Wang, Y., Weiner, H., & Gorenstein, D. G. (1993) J. Biol. Chem. 268, 19906-19914). From these three examples, a model is developed which suggests that a mitochondrial signal sequence that has an N-terminal alpha-helix longer than 11 residues can take on the necessary conformation to be imported but cannot adopt the necessary conformation to be processed.

Acetyl-CoA C-Acetyltransferase↗

PubMed2000

Adaptive encoding neural networks for the recognition of human signal peptide cleavage sites.

MOTIVATION: Data representation and encoding are essential for classification of protein sequences with artificial neural networks (ANN). Biophysical properties are appropriate for low dimensional encoding of protein sequence data. However, in general there is no a priori knowledge of the relevant properties for extraction of representative features. RESULTS: An adaptive encoding artificial neural network (ACN) for recognition of sequence patterns is described. In this approach parameters for sequence encoding are optimized within the same process as the weight vectors by an evolutionary algorithm. The method is applied to the prediction of signal peptide cleavage sites in human secretory proteins and compared with an established predictor for signal peptides. CONCLUSION: Knowledge of physico-chemical properties is not necessary for training an ACN. The advantage is a low dimensional data representation leading to computational efficiency, easy evaluation of the detected features, and high prediction accuracy. AVAILABILITY: A cleavage site prediction server is located at the Humboldt University http://itb.biologie.hu-berlin.de/ approximately jo/sig-cleave/ACNpredictor.cgi CONTACT: jo@itb.hu-berlin.de; berndj@zedat.fu-berlin.de

Binding Sites↗

PubMed1992

A novel calcium-binding protein from Euglena gracilis. Characterisation of a cDNA encoding a 74-kDa acidic-repeat protein targeted across the endoplasmic reticulum.

We have isolated a novel cDNA from Euglena gracilis that encodes a protein composed of 24.9% aspartate with an estimated pI of 3.56, and a deduced molecular mass of 73,542 Da. The first 20 or so amino acids are hydrophobic and resemble a signal sequence. The rest of the polypeptide is composed of a 23-amino-acid repeat. There are 30 repeats, of which 23 are full length. Part of the consensus sequence derived from the repeats has some similarity to the loop of the EF-hand type calcium-binding motif. Evidence is presented that a fusion protein of this novel protein with beta-galactosidase can bind calcium. Northern blotting indicates a single transcript of 2.3 kb (the same size as the cDNA). In-vitro translation of the cDNA gives a protein that migrates on SDS/PAGE with an apparent molecular mass of 120-125 kDa. The protein is processed into a smaller, protease-protected form (110-120 kDa) when translated in the presence of canine pancreatic microsomal vesicles. This suggests that the protein is targeted across the endoplasmic reticulum membrane in vivo, and is the first report of a signal sequence from E. gracilis. We propose that the cDNA obtained encodes a novel calcium-binding protein that is either secreted or resident in the endomembrane system of E. gracilis, and call it the acidic-repeat protein.

Amino Acid Sequence↗

PubMed2001

Functional signal peptides bind a soluble N-terminal fragment of SecA and inhibit its ATPase activity.

The selective recognition of pre-secretory proteins by SecA is essential to the process of protein export from Escherichia coli, yet very little is known about the requirements for recognition and the mode of binding of precursors to SecA. The major reason for this is the lack of a soluble system suitable for biophysical study of the SecA-precursor complex. Complicating the development of such a system is the likelihood that SecA interacts with the precursor in a high affinity, productive manner only when it is activated by binding to membrane and SecYEG. A critical aspect of the precursor/SecA interaction is that it is regulated by various SecA ligands (nucleotide, lipid, SecYEG) to facilitate the release of the precursor, most likely in a stepwise fashion, for translocation. Several recent reports show that functions of SecA can be studied using separated domains. Using this approach, we have isolated a proteolytically generated N-terminal fragment of SecA, which is stably folded, has high ATPase activity, and represents an activated version of SecA. We report here that this fragment, termed SecA64, binds signal peptides with significantly higher affinity than does SecA. Moreover, the ATPase activity of SecA64 is inhibited by signal peptides to an extent that correlates with the ability of these signal peptides to inhibit either SecA translocation ATPase or in vitro protein translocation, arguing that the interaction with SecA64 is functionally significant. Thus, SecA64 offers a soluble, well defined system to study the mode of recognition of signal peptides by SecA and the regulation of signal peptide release.

Adenosine Triphosphatases↗

PubMed1985

Ribosome-SRP-signal sequence interactions. The relay helix hypothesis.

The role of the signal sequence in protein export is reviewed, and some difficulties inherent in the conventional picture of how it interacts with other components of the export machinery are pointed out. An alternative model is suggested, which seems to account better for some of the critical experimental findings made so far.

Amino Acid Sequence↗