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Results for “Protein Sorting Signals”

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At least 703 records · Page 39Linked to original sources

PubMed2001

Aggrecan domains expected to traffic through the exocytic pathway are misdirected to the nucleus.

In this article, we report the misdirected targeting of expressed aggrecan domains. Aggrecan, the chondroitin sulfate (CS) proteoglycan of cartilage, normally progresses through the exocytic pathway. Proteins expressed from constructs containing the putative aggrecan signal sequence (i.e., the first 23 N-terminal amino acids), specified globular (G) domains G1 and/or G3, and a segment of the CS domain were detected in the endoplasmic reticulum (ER) and Golgi complex. Although proteins expressed from constructs containing the putative signal and G3, but lacking G1, were detected to a limited extent in the secretory pathway, they primarily accumulated in nuclei. Discrete nuclear inclusions were seen when G3 was expressed. Immunoelectron microscopic characterization of the inclusions suggested the association of nuclear G3 with other proteins. When signal-free G3 constructs and those with G3 immediately following the N-terminal signal were expressed, abundant dispersed accumulations filled the nucleoplasm. The data suggest first, that signal-free and signal-containing G3 proteins enter the nucleus from the cytosol, and second, that the entry of signal-containing G3 proteins into the ER lumen is inefficient. Hsp25, Hsp70, and ubiquitin were colocalized with nuclear G3, indicating the involvement of chaperones and the degradative machinery in the formation and/or attempted disposal of the abnormal nuclear inclusions. Overall, the results focus attention on (1) intracellular protein trafficking at the ER membrane and the nuclear envelope and (2) chaperone interactions and mechanisms leading to abnormal protein deposition in the nucleus.

Aggrecans↗

PubMed2002

Diverse trafficking patterns due to multiple traffic motifs in G protein-activated inwardly rectifying potassium channels from brain and heart.

G protein-activated inwardly rectifying potassium channels (Kir3, GIRK) provide an important mechanism for neurotransmitter regulation of membrane excitability. GIRK channels are tetramers containing various combinations of Kir3 subunits (Kir3.1--Kir3.4). We find that different combinations of Kir3 subunits exhibit a surprisingly complex spectrum of trafficking phenotypes. Kir3.2 and Kir3.4, but not Kir3.1, contain ER export signals that are important for plasma membrane expression of Kir3.1/Kir3.2 and Kir3.1/Kir3.4 heterotetramers, the GIRK channels found in the brain and the heart, respectively. Additional motifs in Kir3.2 and Kir3.4 control the trafficking between endosome and plasma membrane. In contrast, the Kir3.3 subunit potently inhibits plasma membrane expression by diverting the heterotetrameric channels to lysosomes. Such rich trafficking behaviors provide a mechanism for dynamic regulation of GIRK channel density in the plasma membrane.

Amino Acid Motifs↗

PubMed1998

Role of neurofibromin in modulation of expression of the tyrosinase-related protein 2 gene.

Tyrosinase-related protein 2 (TRP-2)/DOPAchrome tautomerase is an enzyme involved in melanin biosynthesis and plays an important role in cytoprotection by preventing the production of a toxic melanin precursor, 5,6-dihydroxyindole. Neurofibromin is the protein product of a gene linked to neurofibromatosis type 1 (NF1), which is characterized by multiple neurofibromas and abnormalities in skin pigmentation. To explore the pathogenesis of NF1, we studied the role of neurofibromin in the regulation of TRP-2 gene expression. By means of transient cotransfection assays, we show that the expression of a reporter gene under the control of the TRP-2 gene promoter was increased by a neurofibromin-dependent signal through the 71-bp region (positions -415 to -345). A Lys-to-Glu substitution at position 1425 in neurofibromin abrogated this activating function. A dominant negative Ki-ras inhibitor mimics neurofibromin's function, and additively increases TRP-2 promoter activity when coexpressed with neurofibromin. Therefore, we suggest that neurofibromin is involved in the regulation of TRP-2 gene expression. Moreover, we found a single case of a glioblastoma multiforme that expresses TRP-2 mRNA but not tyrosinase mRNA, suggesting that TRP-2 may function in human neural tissues under certain conditions.

Animals↗

PubMed2005

Importin-alpha promotes passage through the nuclear pore complex of human immunodeficiency virus type 1 Vpr.

Viral protein R (Vpr) of human immunodeficiency virus type 1 has potent karyophilic properties, but details of the mechanism by which it enters the nucleus remain to be clarified. We reported previously that two regions, located between residues 17 and 34 (alphaH1) and between residues 46 and 74 (alphaH2), are indispensable for the nuclear localization of Vpr. Here, we reveal that a chimeric protein composed of the nuclear localization signal of Vpr, glutathione S-transferase, and green fluorescent protein was localized at the nuclear envelope and then entered the nucleus upon addition of importin-alpha. An in vitro transport assay using a series of derivatives of importin-alpha demonstrated that the carboxyl terminus was required for this nuclear import process. We also showed that Vpr interacts with importin-alpha through alphaH1 and alphaH2; only the interaction via alphaH1 is indispensable for the nuclear entry of Vpr. These observations indicate that importin-alpha functions as a mediator for the nuclear entry of Vpr.

Cell Nucleus↗

PubMed2005

Functional periplasmic secretion of organophosphorous hydrolase using the twin-arginine translocation pathway in Escherichia coli.

Recombinant Escherichia coli systems expressing organophosphorous hydrolase (OPH) have been proposed for biotransformation of toxic organophosphate compounds. However, whole cell biocatalyst systems are critically disadvantaged due to substrate diffusion limitations. To enhance whole cell biocatalytic efficiency, we engineered E. coli, for the first time, to secrete metal ion cofactor-requiring OPH into the periplasmic space using the twin-arginine translocation (Tat) pathway. In particular, the twin-arginine signal sequence of E. coli trimethylamine N-oxide (TMAO) reductase (TorA) was employed. Even though total OPH activity in the cell lysate fraction was lower in the periplasmic-secreting strain than in the control cytosolic-expressing strain, whole cell OPH activity was approximately 2.8-fold higher due to successful translocation of OPH into the periplasmic space. In addition, whole cell OPH activity in the periplasmic-secreting strain was far more stable than that in the cytosolic-expressing strain. Therefore, Tat-driven periplasmic-secreting E. coli can be successfully employed as efficient whole cell biocatalysts.

Arginine↗

PubMed2003

DePIE: Designing Primers for Protein Interaction Experiments.

Several primer prediction and analysis programs have been developed for diverse applications. However, none of these existing programs can be directly used for the design of primers in protein interaction experiments, since proteins may have transmembrane domains (TMDs) and/or a signal peptide that must be excluded from experiments. Furthermore, it is frequently the case that a short restriction sequences must be added to each primer in order to clone PCR products into a given destination vectors for expression. DePIE, a web-based primer design tool, was developed to address these deficiencies. The program takes as input NCBI protein accession numbers and returns primer information including nucleotide sequences, thermodynamic melting temperature of the nucleotide sequences and the target positions. DePIE is implemented in JAVA, PERL and PHP and has proven to be very efficient in designing primers for our interaction experiments. DePIE services can be accessed at the web site: http://biocore.unl.edu/primer/primerPI.html.

DNA Primers↗

PubMed2007

Export pathway selectivity of Escherichia coli twin arginine translocation signal peptides.

The Escherichia coli genome encodes at least 29 putative signal peptides containing a twin arginine motif characteristic of proteins exported via the twin arginine translocation (Tat) pathway. Fusions of the putative Tat signal peptides plus six to eight amino acids of the mature proteins to three reporter proteins (short-lived green fluorescent protein, maltose-binding protein (MBP), and alkaline phosphatase) and also data from the cell localization of epitope-tagged full-length proteins were employed to determine the ability of the 29 signal peptides to direct export through the Tat pathway, through the general secretory pathway (Sec), or through both. 27/29 putative signal peptides could export one or more reporter proteins through Tat. Of these, 11 signal peptides displayed Tat specificity in that they could not direct the export of Sec-only reporter proteins. The rest (16/27) were promiscuous and were capable of directing export of the appropriate reporter either via Tat (green fluorescent protein, MBP) or via Sec (PhoA, MBP). Mutations that conferred a >or=+1 charge to the N terminus of the mature protein abolished or drastically reduced routing through the Sec pathway without affecting the ability to export via the Tat pathway. These experiments demonstrate that the charge of the mature protein N terminus affects export promiscuity, independent of the effect of the folding state of the mature protein.

Amino Acid Sequence↗

PubMed2001

Molecular determinants of metabotropic glutamate receptor 1B trafficking.

The metabotropic glutamate receptor mGluR1 undergoes alternative splicing to generate isoforms differing in C-terminal sequence. The mechanism by which these isoforms give different functional responses to agonists in vitro is so far unclear. Using the native mGluR1 and CD2-mGluR1 chimeric molecules, as well as their C-terminal truncations and mutants, we identified an endoplasmic reticulum (ER) retention signal Arg-Arg-Lys-Lys within the C-terminal sequence of mGluR1b. Its presence results in a much reduced cell surface expression of the receptor and chimeric molecules in cell lines and their restricted trafficking in neurones. This motif is also present in the C-terminus of mGluR1a, but its effect is overcome by a region of the mGluR1a-specific C-terminal sequence (amino acids 975-1098). Our results indicate that these splice variants of mGluR1 utilize different targeting pathways and suggest that this may be a general phenomenon in the metabotropic glutamate receptor gene family.

Amino Acid Sequence↗

PubMed1997

A role for the M9 transport signal of hnRNP A1 in mRNA nuclear export.

Among the nuclear proteins associated with mRNAs before their export to the cytoplasm are the abundant heterogeneous nuclear (hn) RNPs. Several of these contain the M9 signal that, in the case of hnRNP A1, has been shown to be sufficient to signal both nuclear export and nuclear import in cultured somatic cells. Kinetic competition experiments are used here to demonstrate that M9-directed nuclear import in Xenopus oocytes is a saturable process. Saturating levels of M9 have, however, no effect on the import of either U snRNPs or proteins carrying a classical basic NLS. Previous work demonstrated the existence of nuclear export factors specific for particular classes of RNA. Injection of hnRNP A1 but not of a mutant protein lacking the M9 domain inhibited export of mRNA but not of other classes of RNA. This suggests that hnRNP A1 or other proteins containing an M9 domain play a role in mRNA export from the nucleus. However, the requirement for M9 function in mRNA export is not identical to that in hnRNP A1 protein transport.

Animals↗

PubMed1999

The nuclear envelope serves as an intermediary between the ER and Golgi complex in the intracellular parasite Toxoplasma gondii.

Morphological examination of the highly polarized protozoan parasite Toxoplasma gondii suggests that secretory traffic in this organism progresses from the endoplasmic reticulum to the Golgi apparatus using the nuclear envelope as an intermediate compartment. While the endoplasmic reticulum is predominantly located near the basal end of the parasite, the Golgi is invariably adjacent to the apical end of the nucleus, and the space between the Golgi and nuclear envelope is filled with numerous coatomer-coated vesicles. Staining with antiserum raised against recombinant T. gondii beta-COP confirms its association with the apical juxtanuclear region. Perturbation of protein secretion using brefeldin A, microtubule inhibitors or dithiothreitol disrupts the Golgi, causing swelling of the nuclear envelope, particularly at its basal end. Prolonged drug treatment leads to gross distention of the endoplasmic reticulum, filling the basal end of the parasite. Cloning and sequencing of the T. gondii homolog of the chaperonin protein BiP identifies the carboxy-terminal amino acid sequence HDEL as this organism's endoplasmic reticulum-retention signal. Appending the HDEL motif to a recombinant secretory protein (a chimera between the parasite's major surface protein fusion, P30, and the Green Fluorescent Protein) causes this secretory reporter to be retained intracellularly. P30-GFP-HDEL fluorescence was most intense within the nuclear envelope, particularly at the apical end. These data support a model of secretion in which protein traffic from the endoplasmic reticulum to Golgi occurs via the apical end of the nuclear envelope.

Animals↗

PubMed2003

Primary structure of echotoxin 2, an actinoporin-like hemolytic toxin from the salivary gland of the marine gastropod Monoplex echo.

Echotoxins are 25 kDa proteins with both hemolytic and lethal activities, previously purified from the salivary gland of the marine gastropod Monoplex echo. In this study, a cDNA encoding echotoxin 2 was cloned by RT-PCR, 3'-RACE and 5'-RACE, based on its partial amino acid sequence. The full-length echotoxin 2 cDNA (1000 bp) obtained contains an open reading frame (825 bp) coding for a precursor protein of 274 amino acid residues. Mature echotoxin 2 composed of 226 amino acid residues is assumed to be produced by post-translational removal of N-terminal 23 residues (predicted as a signal peptide) and C-terminal 25 residues from the precursor protein. Very interestingly, a homology search revealed that echotoxin 2 is analogous to actinoporins, 20 kDa pore-forming hemolysins reported from various sea anemones. In addition to the similarities in biological activity, molecular size and basicity between echotoxin 2 and actinoporins, two prominent structural features, an N-terminal amphiphilic alpha-helix and an aromatic patch comprising Trp and Tyr residues, both of which are important for the pore-forming activity of actinoporins, are also recognized in echotoxin 2. However, echotoxin 2 is distinguishable from actinoporins in having Cys residues and lacking an RGD motif.

Amino Acid Sequence↗

PubMedDate not supplied

Differential oligomerization of endoplasmic reticulum-retained connexin43/connexin32 chimeras.

To examine early events in connexin oligomerization, we made connexin constructs containing a C-terminal di-lysine based endoplasmic reticulum (ER) retention/retrieval signal (HKKSL). Previously, we found that both Cx32-HKKSL and Cx43-HKKSL were retained in the ER. However, Cx32-HKKSL oligomerized into hexameric hemichannels, but Cx43-HKKSL was retained as an apparent monomer. To define elements that prevent Cx43-HKKSL oligomerization in the ER, we made a series of HKKSL-tagged Cx43/Cx32 chimeras. When expressed by HeLa cells, some chimeras were retained in the ER as apparent monomers, whereas others oligomerized in the ER. To date, the second and third transmembrane domains and the cytoplasmic loop domain provide the minimal sufficient Cx43 element to inhibit ER oligomerization.

Animals↗

PubMed2004

Identification of Francisella tularensis genes encoding exported membrane-associated proteins using TnphoA mutagenesis of a genomic library.

Francisella tularensis, the causative agent of tularemia, is a highly infectious pathogen of humans and animals, yet little is known about the surface proteins of this organism that mediate mechanisms of pathogenicity. lambdaTnphoA was used to generate random alkaline phosphatase gene fusions in a F. tularensis subsp. tularensis (strain Schu S4) genomic library to identify genes encoding exported extracytoplasmic proteins. Eleven genes encoding membrane-associated proteins were identified by this method and their respective signal peptides were characterized. Three of the genes encoded conserved 'housekeeping' enzymes, while the other eight genes were unique to F. tularensis, encoding proteins with molecular masses ranging from 11 to 78kDa as deduced from the amino acid sequences. Two genes putatively encoded lipoproteins based on the presence of characteristic signal peptidase II cleavage sites. Four selected proteins were found associated with outer membranes from Schu S4 and LVS strains by Western blotting. Indirect immunofluorescence of strain Schu S4 cells also showed evidence of protein localization to the outer membrane. Protein database searches produced significant alignments with proteins from other bacteria involved in carbohydrate transport, lipid metabolism, and cell envelope biogenesis, thereby providing clues for putative functions. These findings demonstrated that TnphoA mutagenesis can be used in conjunction with F. tularensis genome sequence data to provide a foundation for studies to identify and define cellular surface protein virulence factors of this pathogen.

Alkaline Phosphatase↗

PubMed1988

Efficient production of native, biologically active human cystatin C by Escherichia coli.

A cDNA encoding the mature human cysteine proteinase inhibitor cystatin C was fused to the coding sequence for the Escherichia coli outer membrane protein A signal peptide, and the recombinant gene was expressed in E. coli under the control of the lambda PR promoter, an optimized Shine-Dalgarno sequence and the lambda cI 857 repressor. When induced at 42 degrees C, such cells expressed large amounts of recombinant cystatin C. The recombinant protein was isolated in high yield and characterized. All physicochemical properties investigated, including the positions of disulfide bonds, indicated that the E. coli derived cystatin C was identical to cystatin C isolated from human biological fluids, except that the proline residue in position three was not hydroxylated. The recombinant protein displayed full biological activity against papain, cathepsin B and dipeptidyl peptidase I.

Amino Acid Sequence↗

PubMed1995

Exportation of mouse vas deferens protein, a protein without a signal peptide, from mouse vas deferens epithelium: a model of apocrine secretion.

Mouse vas deferens protein (MVDP) is a major androgen-dependent protein of deferential fluid. It is specifically expressed in the epithelium of the mouse vas deferens. Its amino acid sequence as deduced from the nucleotidic sequence of its cDNA does not possess a signal sequence characteristic of secretory proteins. In vitro, transcription of MVDP cDNA followed by translation of mRNA in the rabbit reticulocyte system, in the absence or the presence of microsomes, demonstrated that there was no internalization of MVDP into microsomes that could protect it from degradation by proteinase K; this confirmed the absence of signal sequence. Moreover, MVDP has its NH2-terminus blocked. To understand how MVDP can be exported, its ultrastructural distribution and secretion process were analyzed by means of electron microscopy. Immunolocalization of MVDP revealed that it was distributed in the whole cytoplasm; it was never detected in the lumen of endoplasmic reticulum, Golgi apparatus, or vesicles but was abundant in apical protrusions and in the fluid, where it was associated with cellular material undergoing degradation. These data clearly demonstrated that exportation of MVDP into the luminal fluid does not occur in the classical manner for secretory proteins but rather involves an apocrine secretion process.

Aldehyde Reductase↗

PubMed2004

Molecular characterization of guinea - pig (Cavia porcellus) CD8alpha and CD8beta cDNA.

CD8 is expressed on cytotoxic T cells (CTL) and functions as a coreceptor for recognition of major histocompatibility complex (MHC) class I peptide complexes by the T-cell receptor (TCR). The CD8 molecule consists of two subunits (alpha and beta) and exists either as a heterodimer (alphabeta) or a homodimer (alphaalpha). We report the cloning of full-length cDNAs of guinea pig CD8alpha and CD8beta. The deduced amino acid sequence of CD8alpha and CD8beta reveals characteristic structural features including a signal peptide, an immunoglobulin (Ig) variable-like region, hinge region, transmembrane, and cytoplasmic domains. In addition to the full-length cDNA, a splice variant of CD8beta cDNA was observed, suggesting splicing events as reported for human CD8beta. The sequence homology of guinea pig CD8 indicates greater homology to human, canine, and feline counterparts than to rodent CD8. As the guinea pig serves as an ideal non-primate animal model to several human infectious diseases, such as syphilis, tuberculosis, and chlamydial genital and ocular infection, the CD8 sequence information provides a necessary molecular tool for studying the cell-mediated immune response.

Alternative Splicing↗

PubMed2006

High-throughput trapping of secretory pathway genes in mouse embryonic stem cells.

High-throughput gene trapping is a random approach for inducing insertional mutations across the mouse genome. This approach uses gene trap vectors that simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a DNA tag for the rapid identification of the disrupted gene. Gene trapping has been used by both public and private institutions to produce libraries of embryonic stem (ES) cells harboring mutations in single genes. Presently, approximately 66% of the protein coding genes in the mouse genome have been disrupted by gene trap insertions. Among these, however, genes encoding signal peptides or transmembrane domains (secretory genes) are underrepresented because they are not susceptible to conventional trapping methods. Here, we describe a high-throughput gene trapping strategy that effectively targets secretory genes. We used this strategy to assemble a library of ES cells harboring mutations in 716 unique secretory genes, of which 61% were not trapped by conventional trapping, indicating that the two strategies are complementary. The trapped ES cell lines, which can be ordered from the International Gene Trap Consortium (http://www.genetrap.org), are freely available to the scientific community.

Animals↗

PubMed2005

Twin-arginine signal peptide attributes effective display of CD147 to filamentous phage.

A novel phagemid (pTat8) was constructed in this study to improve the quality of a molecule displayed on filamentous phage. The twin-arginine translocation (Tat) pathway was chosen for transporting and integrating a CD147 molecule into a phage particle via gpVIII. The parent vector pComb8-CD147Ex was modified by substituting a Sec signal sequence (PelB) with a twin-arginine signal sequence from trimethylamine N-oxide reductase (TorA). The characteristics of the CD147 displayed on the phage particle were evaluated by Sandwich ELISA and Western immunoblotting. A Tat-dependent leader was found to be superior to the Sec leader for the phage display of CD147. Our findings further support the involvement of an Escherichia coli Tat translocase in mediating the integration of a hydrophobic transmembrane protein into the inner membrane. This modified phagemid will be useful in phage display technique when the correctly folded structure is required (i.e., antibody libraries and ligand-receptor tracing).

Basigin↗