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Results for “Protein Sorting Signals”

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PubMed1997

Cloning of a new chicken trkC extracellular isoform and its mRNA expression in E9 sensory and autonomic ganglia.

Neuronal development and maintenance are regulated by trophic interactions with the target tissues and the innervating nerve. The neurotrophin family of polypeptide growth factors, consisting of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5), are produced in limited amounts in target areas. They bind to tyrosine receptor kinases of the trk family, including trkA, trkB and trkC, which mediate intracellular signalling in the responding neurons. There are reports of different isoforms of trkA, trkB and trkC having different signalling capacities. This study reports a novel deletion of the first cysteine-rich domain in the extracellular part of chicken trkC. We describe the mRNA expression of this isoform compared to non-deleted forms in E9 peripheral ganglia studied by reversetranscriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. We also compare the mRNA expression pattern of two existing signal peptide sequences and the distribution of trkC mRNA detected by the use of a kinase specific probe. The results show that the novel isoform is expressed in peripheral sensory and autonomic ganglia. Moreover both signal peptide forms are detected in these ganglia by RT-PCR. In addition, in situ hybridization results showed a weak mRNA expression of the novel isoform in the E9 dorsal root ganglion (DRG) but not in Remak's ganglion. The two existing signal peptides are equally expressed in the DRG and Remak's ganglion, at labelling densities comparable to those for the full-length catalytic form of trkC.

Amino Acid Sequence↗

PubMed2004

A signal-arrest-release sequence mediates export and control of the phage P1 endolysin.

The Lyz endolysin of bacteriophage P1 was found to cause lysis of the host without a holin. Induction of a plasmid-cloned lyz resulted in lysis, and the lytic event could be triggered prematurely by treatments that dissipate the proton-motive force. Instead of requiring a holin, export was mediated by an N-terminal transmembrane domain (TMD) and required host sec function. Exported Lyz of identical SDS/PAGE mobility was found in both the membrane and periplasmic compartments, indicating that periplasmic Lyz was not generated by the proteolytic cleavage of the membrane-associated form. In gene fusion experiments, the Lyz TMD directed PhoA to both the membrane and periplasmic compartments, whereas the TMD of the integral membrane protein FtsI restricts Lyz to the membrane. Thus, the N-terminal domain of Lyz is both necessary and sufficient not only for export of this endolysin to the membrane but also for its release into the periplasm. The unusual N-terminal domain, rich in residues that are weakly hydrophobic, thus functions as a signal-arrest-release sequence, which first acts as a normal signal-arrest domain to direct the endolysin to the periplasm in membrane-tethered form and then allows it to be released as a soluble active enzyme in the periplasm. Examination of the protein sequences of related bacteriophage endolysins suggests that the presence of an N-terminal signal-arrest-release sequence is not unique to Lyz. These observations are discussed in relation to the role of holins in the control of host lysis by bacteriophage encoding a secretory endolysin.

Amino Acid Sequence↗

PubMed2003

High-level expression of human glycosyltransferases in insect cells as biochemically active form.

cDNAs, encoding human beta1,4-galactosyltransferase (hGalT I, EC 2.4.1.22), human Galbeta1,3(4)-GlcNAc alpha2,3-sialyltransferase (hST3GalIII, EC 2.4.99), and human Galbeta1,4-GlcNAc alpha2,6-sialyltransferase (hST6Gal I, EC 2.4.99.1), were cloned from human cell lines. In order to express these glycosyltransferases as secreted form in insect cells, cDNAs were inserted into a novel baculovirus transfer vector equipped with the mouse IgM signal peptide and IgG binding domain of the Staphylococcus aureus protein A as an N-terminal fusion partner. About 14 mg hGalT I, 8 mg hST3GalIII, and 6.4 mg hST6Gal I were purified from 1 liter of recombinant baculovirus infected insect cell culture media. The specific activities of recombinant hGalT I and hST6Gal I were determined as 0.65 and 1.6 U/mg protein, respectively. These results indicated that the recombinant hGalT I and hST6Gal I retained enzyme activities at similar level to those of the authentic one although they were fused with the IgG binding domain at the N-terminus. Taken together, the mouse IgM signal peptide and IgG binding domain of the protein A could be efficiently used as an N-terminus fusion partner for the over-expression of heterologous proteins in insect cells.

Amino Acid Sequence↗

PubMed2004

Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 beta-lactamase as a new fluorescence-based reporter.

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) strains are human and animal pathogens that inject effector proteins into host cells via a type III secretion system (TTSS). Cif is an effector protein which induces host cell cycle arrest and reorganization of the actin cytoskeleton. Cif is encoded by a lambdoid prophage present in most of the EPEC and EHEC strains. In this study, we analyzed the domain that targets Cif to the TTSS by using a new reporter system based on a translational fusion of the effector proteins with mature TEM-1 beta-lactamase. Translocation was detected directly in living host cells by using the fluorescent beta-lactamase substrate CCF2/AM. We show that the first 16 amino acids (aa) of Cif were necessary and sufficient to mediate translocation into the host cells. Similarly, the first 20 aa of the effector proteins Map, EspF, and Tir, which are encoded in the same region as the TTSS, mediated secretion and translocation in a type III-dependent but chaperone-independent manner. A truncated form of Cif lacking its first 20 aa was no longer secreted and translocated, but fusion with the first 20 aa of Tir, Map, or EspF restored both secretion and translocation. In addition, the chimeric proteins were fully able to trigger host cell cycle arrest and stress fiber formation. In conclusion, our results demonstrate that Cif is composed of a C-terminal effector domain and an exchangeable N-terminal translocation signal and that the TEM-1 reporter system is a convenient tool for the study of the translocation of toxins or effector proteins into host cells.

Actins↗

PubMed2000

A new determinant of endoplasmic reticulum localization is contained in the juxtamembrane region of the ectodomain of hepatitis C virus glycoprotein E1.

Hepatitis C virus glycoproteins E1 and E2 do not reach the plasma membrane of the cell but accumulate intracellularly, mostly in the endoplasmic reticulum. Previous studies based on transient expression assays have shown that the transmembrane domains of both glycoproteins are sufficient to localize reporter proteins in the endoplasmic reticulum and that other localization signals may be contained in the ectodomain of E1 protein. To identify such signals we generated chimeric proteins between E1 and two reporter proteins, the human CD8 glycoprotein and the human alkaline phosphatase, and analyzed their subcellular localization in stable as well as transient transfectants. Our results showed that (i) an independent localization determinant for the endoplasmic reticulum is present in the juxtamembrane region of the ectodomain of E1 protein and (ii) the localization dictated by this determinant is either due to direct retention or to a recycling mechanism from the intermediate compartment/cis-Golgi complex region, which is clearly different from those previously described for other retrieval signals. These results show for the first time in mammalian cells that the localization in the endoplasmic reticulum of transmembrane protein can be determined by specific targeting signals acting in the lumen of the compartment.

Amino Acid Sequence↗

PubMed1996

Nucleocytoplasmic transport of proteins and control of cellular function.

Nucleocytoplasmic transport of proteins occurs through pores embedded in the nuclear envelope. Recent studies have defined the cytoplasmic factors required for signal-mediated import of nuclear proteins. Considerable progress has been made in understanding the mechanism of nuclear export of proteins by the identification of specific signal sequences needed for export. Regulatory molecules have been shown to adopt novel mechanisms to control their entry into the nucleus and thereby regulate their functions.

Amino Acid Sequence↗

PubMed2003

Genetic analysis of pathway specificity during posttranslational protein translocation across the Escherichia coli plasma membrane.

In Escherichia coli, the SecB/SecA branch of the Sec pathway and the twin-arginine translocation (Tat) pathway represent two alternative possibilities for posttranslational translocation of proteins across the cytoplasmic membrane. Maintenance of pathway specificity was analyzed using a model precursor consisting of the mature part of the SecB-dependent maltose-binding protein (MalE) fused to the signal peptide of the Tat-dependent TorA protein. The TorA signal peptide selectively and specifically directed MalE into the Tat pathway. The characterization of a spontaneous TorA signal peptide mutant (TorA*), in which the two arginine residues in the c-region had been replaced by one leucine residue, showed that the TorA*-MalE mutant precursor had acquired the ability for efficiently using the SecB/SecA pathway. Despite the lack of the "Sec avoidance signal," the mutant precursor was still capable of using the Tat pathway, provided that the kinetically favored Sec pathway was blocked. These results show that the h-region of the TorA signal peptide is, in principle, sufficiently hydrophobic for Sec-dependent protein translocation, and therefore, the positively charged amino acid residues in the c-region represent a major determinant for Tat pathway specificity. Tat-dependent export of TorA-MalE was significantly slower in the presence of SecB than in its absence, showing that SecB can bind to this precursor despite the presence of the Sec avoidance signal in the c-region of the TorA signal peptide, strongly suggesting that the function of the Sec avoidance signal is not the prevention of SecB binding; rather, it must be exerted at a later step in the Sec pathway.

Amino Acid Sequence↗

PubMed2006

Identification of a novel gene family involved in osmotic stress response in Caenorhabditis elegans.

Organisms exposed to the damaging effects of high osmolarity accumulate solutes to increase cytoplasmic osmolarity. Yeast accumulates glycerol in response to osmotic stress, activated primarily by MAP kinase Hog1 signaling. A pathway regulated by protein kinase C (PKC1) also responds to changes in osmolarity and cell wall integrity. C. elegans accumulates glycerol when exposed to high osmolarity, but the molecular pathways responsible for this are not well understood. We report the identification of two genes, osm-7 and osm-11, which are related members of a novel gene family. Mutations in either gene lead to high internal levels of glycerol and cause an osmotic resistance phenotype (Osr). These mutants also have an altered defecation rhythm (Dec). Mutations in cuticle collagen genes dpy-2, dpy-7, and dpy-10 cause a similar Osr Dec phenotype. osm-7 is expressed in the hypodermis and may be secreted. We hypothesize that osm-7 and osm-11 interact with the cuticle, and disruption of the cuticle causes activation of signaling pathways that increase glycerol production. The phenotypes of osm-7 are not suppressed by mutations in MAP kinase or PKC pathways, suggesting that C. elegans uses signaling pathways different from yeast to mount a response to osmotic stress.

Amino Acid Sequence↗

PubMed2006

The N-terminal hydrophobic sequence of autotaxin (ENPP2) functions as a signal peptide.

Autotaxin, also known as ENPP2, was originally isolated from the culture medium of melanoma cells as a cell-motility promoting protein. It regulates cell growth, motility, and angiogenesis through the production of lysophosphatidic acid and sphingosine 1-phosphate. Because autotaxin shows overall structural similarity to the well-characterized PC-1, it has been assumed to be a type II transmembrane protein that is expressed on the cell surface and is released into the extracellular space after proteolytic cleavage. We found, however, that while autotaxin was efficiently secreted into the extracellular space both in vitro and in vivo, it was expressed neither on the surfaces of autotaxin-transfected cells nor on those of the autotaxin-expressing choroid plexus epithelium cells. N-terminal sequencing of the secreted autotaxin revealed that it was cleaved at two N-terminal sites that match the consensus sequences for cleavage by a signal peptidase and furin. In addition, when translated in vitro, autotaxin was co-translationally translocated into microsome membranes, and its N-terminal 3-kDa fragment corresponding to a signal sequence was cleaved. These data demonstrate that the N-terminal hydrophobic sequence of autotaxin functions as a signal peptide, not as a transmembrane segment, and thus autotaxin is synthesized as a secreted protein.

Amino Acid Sequence↗

PubMed1997

Tromp1, a putative rare outer membrane protein, is anchored by an uncleaved signal sequence to the Treponema pallidum cytoplasmic membrane.

Treponema pallidum rare outer membrane protein 1 (Tromp1) has extensive sequence homology with substrate-binding proteins of ATP-binding cassette transporters. Because such proteins typically are periplasmic or cytoplasmic membrane associated, experiments were conducted to clarify Tromp1's physicochemical properties and cellular location in T. pallidum. Comparison of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities of (i) native Tromp1 and Tromp1 synthesized by coupled in vitro transcription-translation and (ii) native Tromp1 and recombinant Tromp1 lacking the N-terminal signal sequence revealed that the native protein is not processed. Other studies demonstrated that recombinant Tromp1 lacks three basic porin-like properties: (i) the ability to form aqueous channels in liposomes which permit the influx of small hydrophilic solutes, (ii) an extensive beta-sheet secondary structure, and (iii) amphiphilicity. Subsurface localization of native Tromp1 was demonstrated by immunofluorescence analysis of treponemes encapsulated in gel microdroplets, while opsonization assays failed to detect surface-exposed Tromp1. Incubation of motile treponemes with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazarine, a photoactivatable, lipophilic probe, also did not result in the detection of Tromp1 within the outer membranes of intact treponemes but, instead, resulted in the labeling of a basic 30.5-kDa presumptive outer membrane protein. Finally, analysis of fractionated treponemes revealed that native Tromp1 is associated predominantly with cell cylinders. These findings comprise a body of evidence that Tromp1 actually is anchored by an uncleaved signal sequence to the periplasmic face of the T. pallidum cytoplasmic membrane, where it likely subserves a transport-related function.

Azirines↗

PubMed1998

Cloning and sequencing of cDNA encoding the LS-12 antifreeze protein in the longhorn sculpin, Myoxocephalus octodecimspinosis.

cDNA coding for an antifreeze protein (LS-12) in the longhorn sculpin, Myoxocephalus octodecimspinosis, was prepared from liver mRNA using reverse transcriptase-polymerase chain reaction coupled with 3' and 5' RACE procedures. This cDNA contains 609 base pairs, including a 384-bp open reading frame which codes for a 128-residue LS-12 precursor protein. The predicted amino acid sequence of the mature LS-12 corresponds exactly to the amino acid sequence obtained from Edman degradation [G. Deng, D.W. Andrews, R.A. Laursen, FEBS Lett., 402, 1997, pp. 17-20]. The 20 residues preceding mature LS-12 are predicted to be a signal sequence, which is presumably cleaved off before the mature, 108-residue protein is secreted into the circulatory system. This is the first report of a cDNA sequence from M. octodecimspinosis.

Amino Acid Sequence↗

PubMed2001

Using subsite coupling to predict signal peptides.

Given a nascent protein sequence, how can one predict its signal peptide or "Zipcode" sequence? This is a first important problem for scientists to use signal peptides as a vehicle to find new drugs or to reprogram cells for gene therapy. Based on a model that takes into account the coupling effect among some key subsites, the so-called [-3, -1, +1] coupling model, a new prediction algorithm is developed. The overall rate of correct prediction for 1939 secretory proteins and 1440 non-secretary proteins was over 92%. It has not escaped our attention that the new method may also serve as a useful tool for helping investigate further many unclear details regarding the molecular mechanism of the ZIP code protein-sorting system in cells.

Algorithms↗

PubMed1994

Mammalian and Escherichia coli signal recognition particles.

Recent evidence from both biochemical and genetic studies indicates that protein targeting to the prokaryotic cytoplasmic membrane and the eukaryotic endoplasmic reticulum membrane may have more in common than previously thought. A ribonucleoprotein particle was identified in Escherichia coli that consists of at least one protein (P48 or Ffh) and one RNA molecule (4.5S RNA), both of which exhibit strong sequence similarity with constituents of the mammalian signal recognition particle (SRP). Like the mammalian SRP, the E. coli SRP binds specifically to the signal sequence of presecretory proteins. Depletion of either P48 or 4.5S RNA affects translation and results in the accumulation of precursors of several secreted proteins. This review discusses the recent studies and speculates on the position of the SRP in the complex network of protein interactions involved in translation and membrane targeting in E. coli.

Animals↗

PubMed2002

The Erv41p-Erv46p complex: multiple export signals are required in trans for COPII-dependent transport from the ER.

Erv41p and Erv46p form an integral membrane protein complex that cycles between the endoplasmic reticulum (ER) and Golgi. Both proteins contain a large lumenal domain and short N- and C-terminal tail sequences exposed to the cytosol. The coat protein complex II (COPII) packages the Erv41p-Erv46p complex into ER-derived vesicles for delivery to the Golgi. We determined signals in the Erv41p-Erv46p complex that are required for COPII-dependent export from the ER. Mutants lacking the Erv41p or Erv46p C-terminus accumulated in the ER and were not packaged efficiently into vesicles. We identified an isoleucine-leucine sequence in the Erv41p tail that was required for COPII binding and inclusion of the complex into vesicles. This signal was sufficient for COPII binding but not for ER export. The Erv46p tail contains a phenylalanine-tyrosine sequence required together with the isoleucine-leucine signal in Erv41p for export of the complex. Surprisingly, Erv41p- Erv46p tail-swapped chimeras were not exported from the ER, indicating that signals in both the Erv41p and the Erv46p tail sequences are required in a specific orientation for efficient packaging of the Erv41p-Erv46p complex.

Amino Acid Motifs↗

PubMed2002

Characterization of signal that directs C-tail-anchored proteins to mammalian mitochondrial outer membrane.

We analyzed the signal that directs the outer membrane protein with the C-terminal transmembrane segment (TMS) to mammalian mitochondria by using yeast Tom5 as a model and green fluorescent protein as a reporter. Deletions or mutations were systematically introduced into the TMS or the flanking regions and their intracellular localization in COS-7 cells was examined using confocal microscopy and cell fractionation. 1) Three basic amino acid residues within the C-terminal five-residue segment (C-segment) contained the information required for mitochondrial-targeting. Reduction of the net positive charge in this segment decreased mitochondrial specificity, and the mutants were distributed throughout the intracellular membranes. 2) Elongation of the TMS interfered with the function of the C-segment and the mutants were delivered to the intracellular membranes. 3) Separation of the TMS and C-segment by linker insertion severely impaired mitochondrial targeting function, leading to mislocalization to the cytoplasm. 4) Mutations or small deletions in the region of the TMS flanking the C-segment also impaired the mitochondrial targeting. Therefore, the moderate length of the TMS, the positive charges in the C-segment, and the distance between or context of the TMS and C-segment are critical for the targeting signal. The structural characteristics of the signal thus defined were also confirmed with mammalian C-tail-anchored protein OMP25.

Amino Acid Sequence↗

PubMed2004

Export from the endoplasmic reticulum of assembled N-methyl-d-aspartic acid receptors is controlled by a motif in the c terminus of the NR2 subunit.

Functional N-methyl-d-aspartic acid (NMDA) receptors are formed from the assembly of NR1 and NR2 subunits. When expressed alone, the major NR1 splice variant and the NR2 subunits are retained in the endoplasmic reticulum (ER), reflecting a quality control mechanism found in many complex multisubunit proteins to ensure that only fully assembled and properly folded complexes reach the cell surface. Recent studies have identified an RRR motif in the C terminus of the NR1 subunit, which controls the ER retention of the unassembled subunit. Here we investigated the mechanisms controlling the ER retention of the NR2 subunit and the export of the assembled complex from the ER. We found that Tac chimeras of the C terminus of the NR2B subunit show that an ER retention signal is also present in the NR2B subunit. In assembled complexes, ER retention signals on the individual subunits must be overcome to allow the complex to leave the ER. One common mechanism involves mutual masking of the signals on the individual subunits. Our data do not support such a mechanism for regulating the release of assembled NMDA receptors from the ER. We found that the motif, HLFY, immediately following transmembrane domain 4 of the NR2 subunit, is required for the assembled complex to exit from the ER. Mutation of this motif allowed the assembly of NR1 and NR2 subunits into a complex that was functional, based on MK-801 binding, but it is retained in the ER. These results are consistent with HLFY functioning as a signal that is necessary for the release of the assembled functional NMDA receptor complex from the ER.

Amino Acid Motifs↗

PubMed2002

Topological changes in the transmembrane domains of hepatitis C virus envelope glycoproteins.

Hepatitis C virus proteins are synthesized as a polyprotein cleaved by a signal peptidase and viral proteases. The behaviour of internal signal sequences at the C-terminus of the transmembrane domains of hepatitis C virus envelope proteins E1 and E2 is essential for the topology of downstream polypeptides. We determined the topology of these transmembrane domains before and after signal sequence cleavage by tagging E1 and E2 with epitopes and by analysing their accessibility in selectively permeabilized cells. We showed that, after cleavage by signal peptidase in the endoplasmic reticulum, the C-terminal orientation of these transmembrane domains changed from luminal to cytosolic. The dynamic behaviour of these transmembrane domains is unique and it is linked to their multifunctionality. By reorienting their C-terminus toward the cytosol and being part of a transmembrane domain, the signal sequences at the C-terminus of E1 and E2 contribute to new functions: (i) membrane anchoring; (ii) E1E2 heterodimerization; and (iii) endoplasmic reticulum retention.

Amino Acid Sequence↗

PubMed2006

Signal recognition particle-dependent inner membrane targeting of the PulG Pseudopilin component of a type II secretion system.

The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner membrane targeting and insertion in Escherichia coli derivatives defective in different protein targeting and export factors. The beta-galactosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the dsbA, dsbB, or all sec genes tested except secB were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePulG* was detected in strains carrying mutations in secA, secY, secE, and secD genes, including some novel alleles of secY and secD. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the Sec-dependent export of periplasmic MalE and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane Sec translocase by SRP.

Amino Acid Sequence↗