119 results on '"Enno Hartmann"'
Search Results
2. Data on draft genomes and transcriptomes from females and males of the flour moth, Ephestia kuehniella
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Axel Künstner, Hauke Busch, Enno Hartmann, and Walther Traut
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Female and male genomes ,Female and male transcriptomes ,Lepidoptera ,de novo assembly ,Heterozygosity ,Computer applications to medicine. Medical informatics ,R858-859.7 ,Science (General) ,Q1-390 - Abstract
We present genomes and pupal transcriptomes of the Mediterranean flour moth, Ephestia kuehniella. The moth is a world-wide storage pest as well as a laboratory species with a considerable background in developmental biology, genetics, and cytogenetics. The sequence data were derived from a highly inbred laboratory strain and, hence, display very little heterozygosity. Female and male genomes and transcriptomes are represented separately in two sets each of raw and assembled sequence data. They are designed as a basis to develop new strategies in pest control, to elucidate the molecular adaptation for its peculiar lifestyle, and for research on sex chromosome structure, sex determination and sex-specific gene activity. For a test, all genes known or suspected to have a role in sex determination were extracted from the data. Raw sequencing data and assemblies are available at European Nucleotide Archive under accession number PRJEB49052.
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- 2022
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3. Cellular Importin-α3 Expression Dynamics in the Lung Regulate Antiviral Response Pathways against Influenza A Virus Infection
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Swantje Thiele, Stephanie Stanelle-Bertram, Sebastian Beck, Nancy Mounogou Kouassi, Martin Zickler, Martin Müller, Berfin Tuku, Patricia Resa-Infante, Debby van Riel, Malik Alawi, Thomas Günther, Franziska Rother, Stefanie Hügel, Susanne Reimering, Alice McHardy, Adam Grundhoff, Wolfram Brune, Albert Osterhaus, Michael Bader, Enno Hartmann, and Gülsah Gabriel
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Biology (General) ,QH301-705.5 - Abstract
Summary: Importin-α adaptor proteins orchestrate dynamic nuclear transport processes involved in cellular homeostasis. Here, we show that importin-α3, one of the main NF-κB transporters, is the most abundantly expressed classical nuclear transport factor in the mammalian respiratory tract. Importin-α3 promoter activity is regulated by TNF-α-induced NF-κB in a concentration-dependent manner. High-level TNF-α-inducing highly pathogenic avian influenza A viruses (HPAIVs) isolated from fatal human cases harboring human-type polymerase signatures (PB2 627K, 701N) significantly downregulate importin-α3 mRNA expression in primary lung cells. Importin-α3 depletion is restored upon back-mutating the HPAIV polymerase into an avian-type signature (PB2 627E, 701D) that can no longer induce high TNF-α levels. Importin-α3-deficient mice show reduced NF-κB-activated antiviral gene expression and increased influenza lethality. Thus, importin-α3 plays a key role in antiviral immunity against influenza. Lifting the bottleneck in importin-α3 availability in the lung might provide a new strategy to combat respiratory virus infections. : Thiele et al. show that importin-α3 is one of the major nuclear transporters of NF-κB in the mammalian lung. High-level TNF-α-inducing HPAIVs inhibit importin-α3 mRNA transcription by interfering with its promoter activity. Thus, HPAIVs may evade antiviral immunity in the respiratory tract by generating a bottleneck in importin-α3 availability. Keywords: lung, influenza, pneumonia, immune sensor, cytokine storm
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- 2020
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4. Reduced DNAJC3 Expression Affects Protein Translocation across the ER Membrane and Attenuates the Down-Modulating Effect of the Translocation Inhibitor Cyclotriazadisulfonamide
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Eva Pauwels, Becky Provinciael, Anita Camps, Enno Hartmann, and Kurt Vermeire
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co-translational translocation ,endoplasmic reticulum ,cyclotriazadisulfonamide ,ER quality control ,DNAJC3 ,signal peptide ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
One of the reported substrates for the endoplasmic reticulum (ER) translocation inhibitor cyclotriazadisulfonamide (CADA) is DNAJC3, a chaperone of the unfolded protein response during ER stress. In this study, we investigated the impact of altered DNAJC3 protein levels on the inhibitory activity of CADA. By comparing WT DNAJC3 with a CADA-resistant DNAJC3 mutant, we observed the enhanced sensitivity of human CD4, PTK7 and ERLEC1 for CADA when DNAJC3 was expressed at high levels. Combined treatment of CADA with a proteasome inhibitor resulted in synergistic inhibition of protein translocation and in the rescue of a small preprotein fraction, which presumably corresponds to the CADA affected protein fraction that is stalled at the Sec61 translocon. We demonstrate that DNAJC3 enhances the protein translation of a reporter protein that is expressed downstream of the CADA-stalled substrate, suggesting that DNAJC3 promotes the clearance of the clogged translocon. We propose a model in which a reduced DNAJC3 level by CADA slows down the clearance of CADA-stalled substrates. This results in higher residual translocation into the ER lumen due to the longer dwelling time of the temporarily stalled substrates in the translocon. Thus, by directly reducing DNAJC3 protein levels, CADA attenuates its net down-modulating effect on its substrates.
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- 2022
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5. Importin α5 Regulates Anxiety through MeCP2 and Sphingosine Kinase 1
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Nicolas Panayotis, Anton Sheinin, Shachar Y. Dagan, Michael M. Tsoory, Franziska Rother, Mayur Vadhvani, Anna Meshcheriakova, Sandip Koley, Letizia Marvaldi, Didi-Andreas Song, Eitan Reuveny, Britta J. Eickholt, Enno Hartmann, Michael Bader, Izhak Michaelevski, and Mike Fainzilber
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Biology (General) ,QH301-705.5 - Abstract
Summary: Importins mediate transport from synapse to soma and from cytoplasm to nucleus, suggesting that perturbation of importin-dependent pathways should have significant neuronal consequences. A behavioral screen on five importin α knockout lines revealed that reduced expression of importin α5 (KPNA1) in hippocampal neurons specifically decreases anxiety in mice. Re-expression of importin α5 in ventral hippocampus of knockout animals increased anxiety behaviors to wild-type levels. Hippocampal neurons lacking importin α5 reveal changes in presynaptic plasticity and modified expression of MeCP2-regulated genes, including sphingosine kinase 1 (Sphk1). Knockout of importin α5, but not importin α3 or α4, reduces MeCP2 nuclear localization in hippocampal neurons. A Sphk1 blocker reverses anxiolysis in the importin α5 knockout mouse, while pharmacological activation of sphingosine signaling has robust anxiolytic effects in wild-type animals. Thus, importin α5 influences sphingosine-sensitive anxiety pathways by regulating MeCP2 nuclear import in hippocampal neurons. : Panayotis et al. found decreased anxiety in importin α5 knockout mice. They report that importin α5 influences sphingosine-sensitive anxiety pathways by regulating MeCP2 nuclear import in hippocampal neurons. Keywords: anxiety, anxiolytic, importin, karyopherin, KPNA1, MeCP2, sphingosine kinase, synapse-nucleus communication
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- 2018
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6. Importin α1 is required for nuclear import of herpes simplex virus proteins and capsid assembly in fibroblasts and neurons.
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Katinka Döhner, Ana Ramos-Nascimento, Dagmara Bialy, Fenja Anderson, Ana Hickford-Martinez, Franziska Rother, Thalea Koithan, Kathrin Rudolph, Anna Buch, Ute Prank, Anne Binz, Stefanie Hügel, Robert Jan Lebbink, Rob C Hoeben, Enno Hartmann, Michael Bader, Rudolf Bauerfeind, and Beate Sodeik
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
Herpesviruses are large DNA viruses which depend on many nuclear functions, and therefore on host transport factors to ensure specific nuclear import of viral and host components. While some import cargoes bind directly to certain transport factors, most recruit importin β1 via importin α. We identified importin α1 in a small targeted siRNA screen to be important for herpes simplex virus (HSV-1) gene expression. Production of infectious virions was delayed in the absence of importin α1, but not in cells lacking importin α3 or importin α4. While nuclear targeting of the incoming capsids, of the HSV-1 transcription activator VP16, and of the viral genomes were not affected, the nuclear import of the HSV-1 proteins ICP4 and ICP0, required for efficient viral transcription, and of ICP8 and pUL42, necessary for DNA replication, were reduced. Furthermore, quantitative electron microscopy showed that fibroblasts lacking importin α1 contained overall fewer nuclear capsids, but an increased proportion of mature nuclear capsids indicating that capsid formation and capsid egress into the cytoplasm were impaired. In neurons, importin α1 was also not required for nuclear targeting of incoming capsids, but for nuclear import of ICP4 and for the formation of nuclear capsid assembly compartments. Our data suggest that importin α1 is specifically required for the nuclear localization of several important HSV1 proteins, capsid assembly, and capsid egress into the cytoplasm, and may become rate limiting in situ upon infection at low multiplicity or in terminally differentiated cells such as neurons.
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- 2018
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7. Signal peptide-binding drug as a selective inhibitor of co-translational protein translocation.
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Kurt Vermeire, Thomas W Bell, Victor Van Puyenbroeck, Anne Giraut, Sam Noppen, Sandra Liekens, Dominique Schols, Enno Hartmann, Kai-Uwe Kalies, and Mark Marsh
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Biology (General) ,QH301-705.5 - Abstract
In eukaryotic cells, surface expression of most type I transmembrane proteins requires translation and simultaneous insertion of the precursor protein into the endoplasmic reticulum (ER) membrane for subsequent routing to the cell surface. This co-translational translocation pathway is initiated when a hydrophobic N-terminal signal peptide (SP) on the nascent protein emerges from the ribosome, binds the cytosolic signal recognition particle (SRP), and targets the ribosome-nascent chain complex to the Sec61 translocon, a universally conserved protein-conducting channel in the ER-membrane. Despite their common function in Sec61 targeting and ER translocation, SPs have diverse but unique primary sequences. Thus, drugs that recognise SPs could be exploited to inhibit translocation of specific proteins into the ER. Here, through flow cytometric analysis the small-molecule macrocycle cyclotriazadisulfonamide (CADA) is identified as a highly selective human CD4 (hCD4) down-modulator. We show that CADA inhibits CD4 biogenesis and that this is due to its ability to inhibit co-translational translocation of CD4 into the lumen of the ER, both in cells as in a cell-free in vitro translation/translocation system. The activity of CADA maps to the cleavable N-terminal SP of hCD4. Moreover, through surface plasmon resonance analysis we were able to show direct binding of CADA to the SP of hCD4 and identify this SP as the target of our drug. Furthermore, CADA locks the SP in the translocon during a post-targeting step, possibly in a folded state, and prevents the translocation of the associated protein into the ER lumen. Instead, the precursor protein is routed to the cytosol for degradation. These findings demonstrate that a synthetic, cell-permeable small-molecule can be developed as a SP-binding drug to selectively inhibit protein translocation and to reversibly regulate the expression of specific target proteins.
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- 2014
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8. Importin α7 is essential for zygotic genome activation and early mouse development.
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Franziska Rother, Tatiana Shmidt, Elena Popova, Alexander Krivokharchenko, Stefanie Hügel, Larissa Vilianovich, Michael Ridders, Katja Tenner, Natalia Alenina, Matthias Köhler, Enno Hartmann, and Michael Bader
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Medicine ,Science - Abstract
Importin α is involved in the nuclear import of proteins. It also contributes to spindle assembly and nuclear membrane formation, however, the underlying mechanisms are poorly understood. Here, we studied the function of importin α7 by gene targeting in mice and show that it is essential for early embryonic development. Embryos lacking importin α7 display a reduced ability for the first cleavage and arrest completely at the two-cell stage. We show that the zygotic genome activation is severely disturbed in these embryos. Our findings indicate that importin α7 is a new member of the small group of maternal effect genes.
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- 2011
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9. Structural insights into TRAP association with ribosome-Sec61 complex and translocon inhibition by a CADA derivative
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Eva Pauwels, Neesha R. Shewakramani, Brent De Wijngaert, Anita Camps, Becky Provinciael, Joren Stroobants, Kai-Uwe Kalies, Enno Hartmann, Piet Maes, Kurt Vermeire, and Kalyan Das
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Multidisciplinary - Abstract
During co-translational translocation, the signal peptide of a nascent chain binds Sec61 translocon to initiate protein transport through the ER membrane. Our cryo-EM structure of ribosome-Sec61 shows binding of an ordered heterotetrameric TRranslocon-Associated Protein (TRAP) complex, in which TRAP-γ is anchored at two adjacent positions of 28S rRNA and interacts with ribosomal protein L38 and Sec61α/γ. Four transmembrane helices (TMHs) of TRAP-γ cluster with one C-terminal helix of each α, β, and δ subunits. The seven TMH bundle helps position a crescent-shaped trimeric TRAP–α/β/δ core in the ER lumen, facing the Sec61 channel. Further, our in vitro assay establishes the CADA derivative CK147 as a translocon inhibitor. A structure of ribosome-Sec61-CK147 reveals CK147 binding the channel and interacting with the plug helix from the lumenal side. The CK147-resistance mutations surround the inhibitor. These structures help in understanding the TRAP functions and provide a new Sec61 site for designing translocon inhibitors.Short SummaryCryo-EM structures reveal TRAP binding to ribosome-Sec61 complex, and CK147 inhibiting Sec61 by arresting the plug helix inside the channel.
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- 2023
10. Importin α3 regulates chronic pain pathways in peripheral sensory neurons
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Letizia Marvaldi, Franziska Rother, Shachar Y. Dagan, Didi-Andreas Song, Agostina Di Pizio, Stefanie Alber, Yarden Tzur, Mike Fainzilber, Nicolas Panayotis, Indrek Koppel, Marco Terenzio, Enno Hartmann, Michael Bader, Ida Rishal, Nataliya Okladnikov, and Dalia Gordon
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alpha Karyopherins ,animal structures ,Sensory Receptor Cells ,importins ,Active Transport, Cell Nucleus ,Animals ,Chronic Pain ,Gene Expression Profiling ,Gene Knockdown Techniques ,sulmazole ,Mice ,Mice, Inbred C57BL ,Neuralgia ,Proto-Oncogene Proteins c-fos ,Transcription Factor AP-1 ,Sensory system ,Importin ,Biology ,Inbred C57BL ,environment and public health ,Benzophenones ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Noxious stimulus ,030304 developmental biology ,Karyopherin ,Cell Nucleus ,chemistry.chemical_classification ,0303 health sciences ,Multidisciplinary ,Chronic pain ,Isoxazoles ,medicine.disease ,Active Transport ,chemistry ,embryonic structures ,Neuropathic pain ,Nuclear transport ,Neuroscience ,030217 neurology & neurosurgery ,Nuclear localization sequence - Abstract
How is neuropathic pain regulated in peripheral sensory neurons? Importins are key regulators of nucleocytoplasmic transport. In this study, we found that importin α3 (also known as karyopherin subunit alpha 4) can control pain responsiveness in peripheral sensory neurons in mice. Importin α3 knockout or sensory neuron–specific knockdown in mice reduced responsiveness to diverse noxious stimuli and increased tolerance to neuropathic pain. Importin α3–bound c-Fos and importin α3–deficient neurons were impaired in c-Fos nuclear import. Knockdown or dominant-negative inhibition of c-Fos or c-Jun in sensory neurons reduced neuropathic pain. In silico screens identified drugs that mimic importin α3 deficiency. These drugs attenuated neuropathic pain and reduced c-Fos nuclear localization. Thus, perturbing c-Fos nuclear import by importin α3 in peripheral neurons can promote analgesia.
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- 2020
11. Wohin mit uns?
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Enno Hartmann
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- 2021
12. Kpna6 deficiency causes infertility in male mice by disrupting spermatogenesis
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Hauke Busch, Ilya Chuykin, Franziska Rother, Na Liu, Gabin Sihn, Stefanie Huegel, Michael Bader, Enno Hartmann, and Fatimunnisa Qadri
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Male ,alpha Karyopherins ,animal structures ,Mouse ,Spermiogenesis ,Active Transport, Cell Nucleus ,Importin ,Biology ,environment and public health ,Mice ,Loss of Function Mutation ,Testis ,medicine ,Animals ,Spermatogenesis ,Molecular Biology ,Infertility, Male ,Karyopherin ,chemistry.chemical_classification ,Cell Nucleus ,Reproductive Biology ,Sertoli Cells ,Sertoli cell ,Sperm ,Spermatogonia ,Cell biology ,Androgen receptor ,Mice, Inbred C57BL ,medicine.anatomical_structure ,chemistry ,Cardiovascular and Metabolic Diseases ,Receptors, Androgen ,Male fertility ,embryonic structures ,Nuclear localization sequence ,Developmental Biology ,Research Article - Abstract
Spermatogenesis is driven by an ordered series of events, which rely on trafficking of specific proteins between nucleus and cytoplasm. The karyopherin α family of proteins mediates movement of specific cargo proteins when bound to karyopherin β. Karyopherin α genes have distinct expression patterns in mouse testis, implying they may have unique roles during mammalian spermatogenesis. Here, we use a loss-of-function approach to determine specifically the role of Kpna6 in spermatogenesis and male fertility. We show that ablation of Kpna6 in male mice leads to infertility and has multiple cumulative effects on both germ cells and Sertoli cells. Kpna6-deficient mice exhibit impaired Sertoli cell function, including loss of Sertoli cells and a compromised nuclear localization of the androgen receptor. Furthermore, our data demonstrate devastating defects on spermiogenesis, including incomplete sperm maturation and a massive reduction in sperm number, accompanied by disturbed histone-protamine exchange, differential localization of the transcriptional regulator BRWD1 and altered expression of RFX2 target genes. Our work uncovers an essential role of Kpna6 in spermatogenesis and, hence, in male fertility., Summary: Two different mouse models delineate the morphological and functional impact of Kpna6 on spermatogenesis and Sertoli cell function and show that this protein is crucial for fertility in male mice.
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- 2021
13. Preprotein signature for full susceptibility to the co‐translational translocation inhibitor cyclotriazadisulfonamide
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Eva Pauwels, Kai-Uwe Kalies, Dominique Schols, Kurt Vermeire, Enno Hartmann, Victor Van Puyenbroeck, Thomas W. Bell, and Becky Provinciael
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Signal peptide ,Resistant phenotype ,cyclotriazadisulfonamide ,Chromosomal translocation ,Protein Sorting Signals ,Biology ,Endoplasmic Reticulum ,Biochemistry ,Article ,03 medical and health sciences ,0302 clinical medicine ,Structural Biology ,CADA ,Genetics ,Humans ,signal peptide ,Molecular Biology ,030304 developmental biology ,Protein Synthesis Inhibitors ,chemistry.chemical_classification ,0303 health sciences ,Endoplasmic reticulum ,small molecule inhibitor ,Cell Biology ,Alanine scanning ,Translocon ,CD4 ,In vitro ,Cell biology ,Amino acid ,co-translational translocation ,alanine scanning ,ER ,chemistry ,CD4 Antigens ,Hydrophobic and Hydrophilic Interactions ,Protein Processing, Post-Translational ,030217 neurology & neurosurgery - Abstract
Cyclotriazadisulfonamide (CADA) inhibits the co-translational translocation of human CD4 (huCD4) into the endoplasmic reticulum lumen in a signal peptide (SP)-dependent way. We propose that CADA binds the nascent huCD4 SP in a folded conformation within the translocon resembling a normally transitory state during translocation. Here, we used alanine scanning on the huCD4 SP to identify the signature for full susceptibility to CADA. In accordance with our previous work, we demonstrate that residues in the vicinity of the hydrophobic h-region are critical for sensitivity to CADA. In particular, exchanging Gln-15, Val-17 or Pro-20 in the huCD4 SP for Ala resulted in a resistant phenotype. Together with positively charged residues at the N-terminal portion of the mature protein, these residues mediate full susceptibility to the co-translational translocation inhibitory activity of CADA towards huCD4. In addition, sensitivity to CADA is inversely related to hydrophobicity in the huCD4 SP. In vitro translocation experiments confirmed that the general hydrophobicity of the h-domain and positive charges in the mature protein are key elements that affect both the translocation efficiency of huCD4 and the sensitivity towards CADA. Besides these two general SP parameters that determine the functionality of the signal sequence, unique amino acid pairs (L14/Q15 and L19/P20) in the SP hydrophobic core add specificity to the sensitivity signature for a co-translational translocation inhibitor. ispartof: TRAFFIC vol:21 issue:2 pages:250-264 ispartof: location:England status: published
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- 2019
14. A Proteomic Study on the Membrane Protein Fraction of T Cells Confirms High Substrate Selectivity for the ER Translocation Inhibitor Cyclotriazadisulfonamide
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Becky Provinciael, Eberhard Krause, Claudia Rutz, Dominique Schols, Heike Stephanowitz, Joren Stroobants, Eva Pauwels, Enno Hartmann, Kurt Vermeire, and Ralf Schülein
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Signal peptide ,Proteomics ,CADA, cyclotriazadisulfonamide ,SILAC, stable isotope labeling by amino acids in cell culture ,T-Lymphocytes ,V5, virus 5 ,cyclotriazadisulfonamide ,ERAD, endoplasmic reticulum–associated degradation ,TMD, transmembrane domain ,Endoplasmic-reticulum-associated protein degradation ,DMSO, dimethyl sulfoxide ,Endoplasmic Reticulum ,ER cotranslational translocation ,ERLEC1, endoplasmic reticulum lectin 1 ,SILAC ,SAS, signal anchor sequence ,Cell Line ,Substrate Specificity ,ER, endoplasmic reticulum ,Stable isotope labeling by amino acids in cell culture ,Humans ,signal peptide ,DNAJC3, DnaJ homolog subfamily C member 3 ,PL, prolactin ,eGFP, enhanced GFP ,Integral membrane protein ,SORT, sortilin ,Inactive Tyrosine-Protein Kinase 7 ,Sulfonamides ,huCD4, human CD4 ,Chemistry ,Endoplasmic reticulum ,Research ,Membrane Proteins ,General Medicine ,HEK293T, human embryonic kidney 293T ,BFP, blue fluorescent protein ,CD4 ,Cell biology ,Membrane protein ,Isotope Labeling ,SP, signal peptide ,DNAJC3 ,tGFP, turbo GFP ,PTK7, inactive tyrosine-protein kinase 7 ,small-molecule inhibitor - Abstract
Cyclotriazadisulfonamide (CADA) inhibits the cotranslational translocation of type I integral membrane protein human CD4 (huCD4) across the endoplasmic reticulum in a signal peptide (SP)–dependent way. Previously, sortilin was identified as a secondary substrate for CADA but showed reduced CADA sensitivity as compared with huCD4. Here, we performed a quantitative proteomic study on the crude membrane fraction of human T-cells to analyze how many proteins are sensitive to CADA. To screen for these proteins, we employed stable isotope labeling by amino acids in cell culture technique in combination with quantitative MS on CADA-treated human T-lymphoid SUP-T1 cells expressing high levels of huCD4. In line with our previous reports, our current proteomic analysis (data available via ProteomeXchange with identifier PXD027712) demonstrated that only a very small subset of proteins is depleted by CADA. Our data also confirmed that cellular expression of both huCD4 and sortilin are affected by CADA treatment of SUP-T1 cells. Furthermore, three additional targets for CADA are identified, namely, endoplasmic reticulum lectin 1 (ERLEC1), inactive tyrosine-protein kinase 7 (PTK7), and DnaJ homolog subfamily C member 3 (DNAJC3). Western blot and flow cytometry analysis of ERLEC1, PTK7, and DNAJC3 protein expression validated susceptibility of these substrates to CADA, although with varying degrees of sensitivity. Additional cell-free in vitro translation/translocation data demonstrated that the new substrates for CADA carry cleavable SPs that are targets for the cotranslational translocation inhibition exerted by CADA. Thus, our quantitative proteomic analysis demonstrates that ERLEC1, PTK7, and DNAJC3 are validated additional substrates of CADA; however, huCD4 remains the most sensitive integral membrane protein for the endoplasmic reticulum translocation inhibitor CADA. Furthermore, to our knowledge, CADA is the first compound that specifically interferes with only a very small subset of SPs and does not affect signal anchor sequences., Graphical Abstract, Highlights • About 3007 proteins quantified in SILAC/MS study on CD4+ T-cells treated with CADA. • Three new targets for CADA were identified: ERLEC1, PTK7, and DNAJC3. • All CADA substrates carry cleavable signal peptides for translocation into ER. • huCD4 remains the most sensitive substrate for the ER translocation inhibitor CADA., In Brief In mammalian cells, one-third of all polypeptides enter the secretory pathway via the ER, driven by specific targeting signals in their sequences, such as N-terminal signal peptides. The synthetic macrocycle CADA was identified as a selective ER translocation inhibitor for huCD4 and SORT. Our current SILAC/MS proteomic survey identified and validated only three additional targets for CADA, thus, confirming the high substrate selectivity of CADA, with the strongest effect on huCD4. Therefore, CADA holds great potential as an immunosuppressive drug.
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- 2021
15. Importin α7 deficiency causes infertility in male mice by disrupting spermatogenesis
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Michael Bader, Franziska Rother, Stefanie Huegel, Na Liu, Enno Hartmann, Hauke Busch, Gabin Sihn, Fatimunnisa Qadri, and Ilya Chuykin
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chemistry.chemical_classification ,animal structures ,Spermiogenesis ,Importin ,Biology ,Sertoli cell ,environment and public health ,Cell biology ,Androgen receptor ,medicine.anatomical_structure ,chemistry ,Cytoplasm ,medicine ,Spermatogenesis ,Nuclear localization sequence ,Karyopherin - Abstract
Spermatogenesis is driven by an ordered series of events, which rely on trafficking of specific proteins between nucleus and cytoplasm. The importin α family of proteins mediates movement of specific cargo proteins when bound to importin β. Importin α genes have distinct expression patterns in mouse testis, implying they may have unique roles during mammalian spermatogenesis. Here we use a loss-of-function approach to specifically determine the role of importin α7 in spermatogenesis and male fertility. We show that ablation of importin α7 in male mice leads to infertility and has multiple cumulative effects on both germ cells and Sertoli cells. Importin α7-deficient mice exhibit an impaired Sertoli cell function, including loss of Sertoli cells and a compromised nuclear transport of the androgen receptor. Furthermore, our data demonstrate devastating defects in spermiogenesis that are accompanied by disturbed histone-protamine-exchange, absence of the transcriptional regulator Brwd1 and altered expression of Rfx2 target genes, resulting in incomplete sperm maturation and massive loss of sperms. Our work uncovers the essential role of importin α7 in spermatogenesis and hence in male fertility.
- Published
- 2020
16. Cellular Importin-α3 Expression Dynamics in the Lung Regulate Antiviral Response Pathways against Influenza A Virus Infection
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Gülsah Gabriel, Martin Zickler, Nancy Mounogou Kouassi, Stefanie Hügel, Franziska Rother, Susanne Reimering, Sebastian Beck, Enno Hartmann, Michael Bader, Swantje Thiele, Stephanie Stanelle-Bertram, Patricia Resa-Infante, Wolfram Brune, Martin Müller, Debby van Riel, Albert D. M. E. Osterhaus, Berfin Tuku, Alice C. McHardy, Thomas Günther, Adam Grundhoff, Malik Alawi, BRICS, Braunschweiger Zentrum für Systembiologie, Rebenring 56,38106 Braunschweig, Germany., and Virology
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0301 basic medicine ,Cellular homeostasis ,medicine.disease_cause ,environment and public health ,Mice ,0302 clinical medicine ,Chlorocebus aethiops ,Influenza A virus ,immune sensor ,lcsh:QH301-705.5 ,Polymerase ,Mice, Knockout ,biology ,Signal transducing adaptor protein ,Middle Aged ,Cell biology ,embryonic structures ,cytokine storm ,Respiratory virus ,Female ,influenza ,alpha Karyopherins ,animal structures ,Down-Regulation ,Importin ,Article ,General Biochemistry, Genetics and Molecular Biology ,lung ,03 medical and health sciences ,Orthomyxoviridae Infections ,Cell Line, Tumor ,Influenza, Human ,medicine ,Animals ,Humans ,pneumonia ,RNA, Messenger ,Vero Cells ,medicine.disease ,Influenza A virus subtype H5N1 ,Mice, Inbred C57BL ,HEK293 Cells ,030104 developmental biology ,lcsh:Biology (General) ,A549 Cells ,Cardiovascular and Metabolic Diseases ,biology.protein ,Cytokine storm ,030217 neurology & neurosurgery - Abstract
Summary Importin-α adaptor proteins orchestrate dynamic nuclear transport processes involved in cellular homeostasis. Here, we show that importin-α3, one of the main NF-κB transporters, is the most abundantly expressed classical nuclear transport factor in the mammalian respiratory tract. Importin-α3 promoter activity is regulated by TNF-α-induced NF-κB in a concentration-dependent manner. High-level TNF-α-inducing highly pathogenic avian influenza A viruses (HPAIVs) isolated from fatal human cases harboring human-type polymerase signatures (PB2 627K, 701N) significantly downregulate importin-α3 mRNA expression in primary lung cells. Importin-α3 depletion is restored upon back-mutating the HPAIV polymerase into an avian-type signature (PB2 627E, 701D) that can no longer induce high TNF-α levels. Importin-α3-deficient mice show reduced NF-κB-activated antiviral gene expression and increased influenza lethality. Thus, importin-α3 plays a key role in antiviral immunity against influenza. Lifting the bottleneck in importin-α3 availability in the lung might provide a new strategy to combat respiratory virus infections., Graphical Abstract, Highlights • Importin-α3 is the most abundantly expressed isoform in the mammalian lung • Importin-α3 is highly conserved across species • Importin-α3 is one of the major nuclear transporters of NF-κB • Importin-α3 acts as an immune sensor of influenza A virus infections, Thiele et al. show that importin-α3 is one of the major nuclear transporters of NF-κB in the mammalian lung. High-level TNF-α-inducing HPAIVs inhibit importin-α3 mRNA transcription by interfering with its promoter activity. Thus, HPAIVs may evade antiviral immunity in the respiratory tract by generating a bottleneck in importin-α3 availability.
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- 2020
17. Mediators of protein topogenesis of Hypoxia Inducible Factors (HIF) and Prolyl-Hydroxylases (PHD)
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Friederike, Pientka, Amrei, Steinhoff, Enno, Hartmann, Matthias, Köhler, and Reinhard, Depping
- Published
- 2009
18. Author response for 'Pre‐protein signature for full susceptibility to the co‐translational translocation inhibitor cyclotriazadisulfonamide (CADA)'
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Eva Pauwels, Enno Hartmann, Dominique Schols, Victor Van Puyenbroeck, Becky Provinciael, Kurt Vermeire, Kai-Uwe Kalies, and Thomas W. Bell
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Chemistry ,Protein signature ,Chromosomal translocation ,Cell biology - Published
- 2019
19. Importin α5 regulates anxiety through MeCP2 and sphingosine kinase 1
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Britta J. Eickholt, Sandip Koley, Enno Hartmann, Anton Sheinin, Michael Bader, Michael Tsoory, Nicolas Panayotis, Izhak Michaelevski, Franziska Rother, Shachar Y. Dagan, Anna Meshcheriakova, Mayur Vadhvani, Letizia Marvaldi, Eitan Reuveny, Mike Fainzilber, and Didi-Andreas Song
- Subjects
0301 basic medicine ,Transcription, Genetic ,KPNA1 ,Methyl-CpG-Binding Protein 2 ,Sphingosine kinase ,Anxiety ,Hippocampal formation ,Hippocampus ,environment and public health ,anxiety ,anxiolytic ,importin ,karyopherin ,MeCP2 ,sphingosine kinase ,synapse-nucleus communication ,Animals ,Anti-Anxiety Agents ,Behavior, Animal ,Carbolines ,Mice, Knockout ,Neurons ,Phenotype ,Phosphotransferases (Alcohol Group Acceptor) ,Synapses ,alpha Karyopherins ,Mice ,chemistry.chemical_compound ,lcsh:QH301-705.5 ,Karyopherin ,chemistry.chemical_classification ,biology ,Cell biology ,Sphingosine kinase 1 ,Knockout mouse ,embryonic structures ,Transcription ,animal structures ,Knockout ,Importin ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Genetic ,Behavior ,Sphingosine ,Animal ,030104 developmental biology ,chemistry ,lcsh:Biology (General) ,Cardiovascular and Metabolic Diseases ,biology.protein ,Nuclear localization sequence - Abstract
Summary Importins mediate transport from synapse to soma and from cytoplasm to nucleus, suggesting that perturbation of importin-dependent pathways should have significant neuronal consequences. A behavioral screen on five importin α knockout lines revealed that reduced expression of importin α5 (KPNA1) in hippocampal neurons specifically decreases anxiety in mice. Re-expression of importin α5 in ventral hippocampus of knockout animals increased anxiety behaviors to wild-type levels. Hippocampal neurons lacking importin α5 reveal changes in presynaptic plasticity and modified expression of MeCP2-regulated genes, including sphingosine kinase 1 (Sphk1). Knockout of importin α5, but not importin α3 or α4, reduces MeCP2 nuclear localization in hippocampal neurons. A Sphk1 blocker reverses anxiolysis in the importin α5 knockout mouse, while pharmacological activation of sphingosine signaling has robust anxiolytic effects in wild-type animals. Thus, importin α5 influences sphingosine-sensitive anxiety pathways by regulating MeCP2 nuclear import in hippocampal neurons., Graphical Abstract, Highlights • Reduced expression of importin α5 in hippocampal neurons decreases anxiety • Importin α5 is required for nuclear localization of MeCP2 in hippocampal neurons • Importin α5 knockout increases expression of Sphk1, an MeCP2-regulated gene • Pharmacological modulation of Sphk1 and the S1P receptor affects anxiety, Panayotis et al. found decreased anxiety in importin α5 knockout mice. They report that importin α5 influences sphingosine-sensitive anxiety pathways by regulating MeCP2 nuclear import in hippocampal neurons.
- Published
- 2018
20. A hyperactive mutant of interferon-regulatory factor 4
- Author
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Anne Brüstle, Michael Lohoff, Alekya Porapu, Hartmann Raifer, Bärbel Camara, Chol Ho Kang, Maria Bieringer, Daniel Staudenraus, Enno Hartmann, Olaf Pinkenburg, Elfadil Abass, and Lisa Menke
- Subjects
0301 basic medicine ,Immunology ,Mutant ,Biology ,medicine.disease_cause ,03 medical and health sciences ,0302 clinical medicine ,T-Lymphocyte Subsets ,medicine ,Immunology and Allergy ,Animals ,Humans ,Transcription factor ,chemistry.chemical_classification ,Transcriptional activity ,Mutation ,Molecular biology ,Amino acid ,030104 developmental biology ,chemistry ,T cell differentiation ,Interferon Regulatory Factors ,CD8 ,030215 immunology ,IRF4 - Abstract
We found that deletion of the final 30 amino acids of transcription factor IRF4's (interferon-regulatory factor) C-terminus creates hyperactive IRF4. When introduced into IRF4-deficient CD4+ or CD8+ T cells, more type 17 differentiation was found compared to WT IRF4. Interestingly, Th9 differentiation and Th2-linked IL-13 production were much less altered.
- Published
- 2018
21. TRAP assists membrane protein topogenesis at the mammalian ER membrane
- Author
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Martin Spiess, Tina Junne, Nicole Sommer, Kai-Uwe Kalies, and Enno Hartmann
- Subjects
Small interfering RNA ,Vesicle-associated membrane protein 8 ,Receptors, Peptide ,Molecular Sequence Data ,Receptors, Cytoplasmic and Nuclear ,Membrane protein topogenesis ,Biology ,Endoplasmic Reticulum ,03 medical and health sciences ,RNA interference ,0302 clinical medicine ,Animals ,Humans ,Amino Acid Sequence ,Gene Silencing ,Molecular Biology ,030304 developmental biology ,Mammals ,0303 health sciences ,Membrane Glycoproteins ,Translocon associated proteins ,Membrane transport protein ,Endoplasmic reticulum ,Calcium-Binding Proteins ,Membrane Proteins ,Intracellular Membranes ,Cell Biology ,Translocon ,SEC61 Translocon ,Cell biology ,Protein Transport ,Membrane ,Membrane protein ,Gene Knockdown Techniques ,Multiprotein Complexes ,Sec61 complex ,biology.protein ,Mutant Proteins ,Hydrophobic and Hydrophilic Interactions ,030217 neurology & neurosurgery ,HeLa Cells - Abstract
Membrane protein insertion and topogenesis generally occur at the Sec61 translocon in the endoplasmic reticulum membrane. During this process, membrane spanning segments may adopt two distinct orientations with either their N- or C-terminus translocated into the ER lumen. While different topogenic determinants in membrane proteins, such as flanking charges, polypeptide folding, and hydrophobicity, have been identified, it is not well understood how the translocon and/or associated components decode them. Here we present evidence that the translocon-associated protein (TRAP) complex is involved in membrane protein topogenesis in vivo. Small interfering RNA (siRNA)-mediated silencing of the TRAP complex in HeLa cells enhanced the topology effect of mutating the flanking charges of a signal-anchor, but not of increasing signal hydrophobicity. The results suggest a role of the TRAP complex in moderating the ‘positive-inside’ rule.
- Published
- 2013
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22. Single rRNA Helices Bind Independently to the Protein-Conducting Channel SecYEG
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Christina Behrens, Enno Hartmann, and Kai-Uwe Kalies
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0303 health sciences ,Sec61 ,030302 biochemistry & molecular biology ,5.8S ribosomal RNA ,Cell Biology ,Ribosomal RNA ,Biology ,Biochemistry ,Ribosome ,Conserved sequence ,Cell biology ,03 medical and health sciences ,Structural Biology ,Ribosomal protein ,23S ribosomal RNA ,Genetics ,Binding site ,Molecular Biology ,030304 developmental biology - Abstract
Ribosomes tightly interact with protein-conducting channels in the plasma membrane of bacteria (SecYEG) and in the endoplasmic reticulum of eukaryotes (Sec61 complex). This interaction is mediated by multiple junctions and is highly conserved during evolution. Although it is well known that both ribosomal proteins and ribosomal RNA (rRNA) are involved in the ribosome–channel interaction, detailed analyses on how these components contribute to this binding are lacking. Here, we demonstrate that the evolutionary conservation of ribosome binding is solely mediated by rRNA. Moreover, we show that in vitro transcribed 23 S rRNA binds with similar characteristics to protein translocation channels as native 23 S rRNA or 50 S ribosomal subunits. This indicates that base modifications, which exist in native rRNA, do not crucially influence the binding. In two of the ribosome-channel junctions (c1 and c2), exclusively rRNA helices are involved. Using in vitro transcribed rRNA mutants, we now provide evidence that large parts of the rRNA can be deleted without altering its binding properties, as long as the rRNA helices of the c1 and c2 junctions remain intact. We demonstrate that the connection sites c1 and c2 generate high-affinity binding sites that act independently of each other. This could explain why membrane-bound ribosomes have an extremely low off-rate.
- Published
- 2013
23. Prediction of Apoptosis Protein Locations with Genetic Algorithms and Support Vector Machines Through a New Mode of Pseudo Amino Acid Composition
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Ganesan Pugalenthi, Kai Uwe Kalies, Ponnuthurai Nagaratnam Suganthan, Steffen Möller, Enno Hartmann, Krishna Kumar Kandaswamy, and Thomas Martinetz
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Programmed cell death ,Drug discovery ,Sorting ,General Medicine ,Computational biology ,Biology ,Bioinformatics ,Biochemistry ,Support vector machine ,Protein Transport ,Artificial Intelligence ,Structural Biology ,Genetic algorithm ,Apoptosis Regulatory Proteins ,Protein secondary structure ,Pseudo amino acid composition ,Algorithms ,Tissue homeostasis - Abstract
Apoptosis is an essential process for controlling tissue homeostasis by regulating a physiological balance between cell proliferation and cell death. The subcellular locations of proteins performing the cell death are determined by mostly independent cellular mechanisms. The regular bioinformatics tools to predict the subcellular locations of such apoptotic proteins do often fail. This work proposes a model for the sorting of proteins that are involved in apoptosis, allowing us to both the prediction of their subcellular locations as well as the molecular properties that contributed to it. We report a novel hybrid Genetic Algorithm (GA)/Support Vector Machine (SVM) approach to predict apoptotic protein sequences using 119 sequence derived properties like frequency of amino acid groups, secondary structure, and physicochemical properties. GA is used for selecting a near-optimal subset of informative features that is most relevant for the classification. Jackknife cross-validation is applied to test the predictive capability of the proposed method on 317 apoptosis proteins. Our method achieved 85.80% accuracy using all 119 features and 89.91% accuracy for 25 features selected by GA. Our models were examined by a test dataset of 98 apoptosis proteins and obtained an overall accuracy of 90.34%. The results show that the proposed approach is promising; it is able to select small subsets of features and still improves the classification accuracy. Our model can contribute to the understanding of programmed cell death and drug discovery. The software and dataset are available at http://www.inb.uni-luebeck.de/tools-demos/apoptosis/GASVM.
- Published
- 2010
24. SPRED: A machine learning approach for the identification of classical and non-classical secretory proteins in mammalian genomes
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Kai-Uwe Kalies, Ganesan Pugalenthi, Krishna Kumar Kandaswamy, Thomas Martinetz, Ponnuthurai Nagaratnam Suganthan, Steffen Möller, and Enno Hartmann
- Subjects
Signal peptide ,Proteome ,Genome, Human ,Biophysics ,Proteins ,Cell Biology ,Biology ,Biochemistry ,Cell biology ,Secretory protein ,Artificial Intelligence ,Sequence Analysis, Protein ,Protein methods ,Animals ,Humans ,Secretion ,Molecular Biology ,Peptide sequence ,Secretory pathway ,Galectin - Abstract
Eukaryotic protein secretion generally occurs via the classical secretory pathway that traverses the ER and Golgi apparatus. Secreted proteins usually contain a signal sequence with all the essential information required to target them for secretion. However, some proteins like fibroblast growth factors (FGF-1, FGF-2), interleukins (IL-1 alpha, IL-1 beta), galectins and thioredoxin are exported by an alternative pathway. This is known as leaderless or non-classical secretion and works without a signal sequence. Most computational methods for the identification of secretory proteins use the signal peptide as indicator and are therefore not able to identify substrates of non-classical secretion. In this work, we report a random forest method, SPRED, to identify secretory proteins from protein sequences irrespective of N-terminal signal peptides, thus allowing also correct classification of non-classical secretory proteins. Training was performed on a dataset containing 600 extracellular proteins and 600 cytoplasmic and/or nuclear proteins. The algorithm was tested on 180 extracellular proteins and 1380 cytoplasmic and/or nuclear proteins. We obtained 85.92% accuracy from training and 82.18% accuracy from testing. Since SPRED does not use N-terminal signals, it can detect non-classical secreted proteins by filtering those secreted proteins with an N-terminal signal by using SignalP. SPRED predicted 15 out of 19 experimentally verified non-classical secretory proteins. By scanning the entire human proteome we identified 566 protein sequences potentially undergoing non-classical secretion. The dataset and standalone version of the SPRED software is available at http://www.inb.uni-luebeck.de/tools-demos/spred/spred.
- Published
- 2010
25. A single Sec61-complex functions as a protein-conducting channel
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Enno Hartmann, Kai-Uwe Kalies, and Vivica Stokes
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Sec61 ,Proteases ,Transcription, Genetic ,medicine.medical_treatment ,Peptide Chain Elongation, Translational ,Chromosomal translocation ,Biology ,Ribosome ,Ion Channels ,Protein-conducting channel ,chemistry.chemical_compound ,Dogs ,Cotranslational protein translocation ,medicine ,Animals ,Pancreas ,Molecular Biology ,Protease ,Endoplasmic reticulum ,Membrane Proteins ,Cell Biology ,Protein Transport ,ER-membrane ,chemistry ,Membrane protein ,Biochemistry ,Ribosome binding ,Puromycin ,Protein Biosynthesis ,Gene Targeting ,Biophysics ,Endoplasmic Reticulum, Rough ,Ribosomes ,SEC Translocation Channels - Abstract
During cotranslational translocation of proteins into the endoplasmic reticulum (ER) translating ribosomes bind to Sec61-complexes. Presently two models exist how these membrane protein complexes might form protein-conducting channels. While electron microscopic data suggest that a ring-like structure consisting of four Sec61-complexes build the channel, the recently solved crystal structure of a homologous bacterial protein complex led to the speculation that the actual tunnel is formed by just one individual Sec61-complex. Using protease protection assays together with quantitative immunoblotting we directly examined the structure of mammalian protein-conducting channels. We found that in native ER-membranes one single Sec61α-molecule is preferentially protected by a membrane bound ribosome, both, in the presence and absence of nascent polypeptides. In addition we present evidence that the nascent polypeptide destabilizes the ring-like translocation apparatus formed by four Sec61-complexes. Moreover, we found that after solubilization of ER-membranes a single Sec61-complex is sufficient to protect the nascent polypeptide chain against added proteases. Finally, we could show that this single Sec61-complex allows the movement of the nascent chain, when it has been released from the ribosome by puromycin treatment. Collectively, our data suggest that the active protein-conducting channel in the ER is formed by a single Sec61-complex.
- Published
- 2008
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26. Importin-α7 Is Involved in the Formation of Ebola Virus Inclusion Bodies but Is Not Essential for Pathogenicity in Mice
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Hideki Ebihara, Heinz Feldmann, Meike Fischer, Enno Hartmann, Friederike Feldmann, Thomas Hoenen, Swantje Thiele, Michael Bader, Rudolph Reimer, and Gülsah Gabriel
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DNA Replication ,alpha Karyopherins ,animal structures ,Alpha (ethology) ,Importin ,Biology ,medicine.disease_cause ,Virus Replication ,environment and public health ,Inclusion bodies ,Cell Line ,Inclusion Bodies, Viral ,Mice ,Viral Proteins ,Interferon ,Chlorocebus aethiops ,medicine ,Immunology and Allergy ,Animals ,Ebola and Marburg Viruses-Research, Outbreak Management, Epidemiology and Ecology ,Vero Cells ,Ebolavirus ,Ebola virus ,Virulence ,Alpha Karyopherins ,Hemorrhagic Fever, Ebola ,Virology ,Mice, Inbred C57BL ,Infectious Diseases ,Viral replication ,embryonic structures ,medicine.drug - Abstract
Ebola virus (EBOV) protein 24 antagonizes the host interferon (IFN) response by hijacking select nuclear importin-{alpha} isoforms. Thereby, it blocks STAT1-mediated IFN-{alpha}/{beta} and IFN-{gamma} synthesis. However, owing to the lack of importin-{alpha} knockout animal models in the past, their role in EBOV pathogenesis remained largely unknown. Here, we demonstrate that importin-{alpha}7 is involved in the formation of EBOV inclusion bodies and replication. However, deletion of the gene encoding importin-{alpha}7 was not sufficient to increase survival rates among mice infected with EBOV.
- Published
- 2015
27. Arf1p, Chs5p and the ChAPs are required for export of specialized cargo from the Golgi
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Mark Trautwein, Jörn Dengjel, Christina Schindler, Anne Spang, Enno Hartmann, and Robert Gauss
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Saccharomyces cerevisiae Proteins ,Molecular Sequence Data ,Golgi Apparatus ,Chitin ,Saccharomyces cerevisiae ,Chromatography, Affinity ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cell membrane ,symbols.namesake ,Chaps ,medicine ,Small GTPase ,Amino Acid Sequence ,Molecular Biology ,Sequence Deletion ,Chitin Synthase ,Sequence Homology, Amino Acid ,General Immunology and Microbiology ,biology ,General Neuroscience ,Cell Membrane ,Chitin synthase ,Golgi apparatus ,Transport protein ,Cell biology ,Vesicular transport protein ,Adaptor Proteins, Vesicular Transport ,Protein Transport ,medicine.anatomical_structure ,Microscopy, Fluorescence ,Biochemistry ,Exomer complex ,biology.protein ,symbols ,ADP-Ribosylation Factor 1 ,Carrier Proteins ,trans-Golgi Network - Abstract
In Saccharomyces cerevisiae, the synthesis of chitin is temporally and spatially regulated through the transport of Chs3p (chitin synthase III) to the plasma membrane in the bud neck region. Traffic of Chs3p from the trans-Golgi network (TGN)/early endosome to the plasma membrane requires the function of Chs5p and Chs6p. Chs6p belongs to a family of four proteins that we have named ChAPs for Chs5p-Arf1p-binding Proteins. Here, we show that all ChAPs physically interact not only with Chs5p but also with the small GTPase Arf1p. A short sequence at the C-terminus of the ChAPs is required for protein function and the ability to bind to Chs5p. Simultaneous disruption of two members, Deltabud7 and Deltabch1, phenocopies a Deltachs6 or Deltachs5 deletion with respect to Chs3p transport. Moreover, the ChAPs interact with each other and can form complexes. In addition, they are all at least partially localized to the TGN in a Chs5p-dependent manner. Most importantly, several ChAPs can interact physically with Chs3p. We propose that the ChAPs facilitate export of cargo out of the Golgi.
- Published
- 2006
28. Signal peptide-binding drug as a selective inhibitor of co-translational protein translocation
- Author
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Victor Van Puyenbroeck, Mark Marsh, Thomas W. Bell, Dominique Schols, Sam Noppen, Enno Hartmann, Sandra Liekens, Kai-Uwe Kalies, Anne Giraut, Kurt Vermeire, and Hegde, Ramanujan
- Subjects
Signal peptide ,Sec61 ,Transmembrane Receptors ,Protein Conformation ,QH301-705.5 ,Molecular Sequence Data ,Down-Regulation ,Protein Synthesis ,Protein Sorting Signals ,Biology ,Biochemistry ,Cell-Free Protein Synthesis ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,0302 clinical medicine ,Protein structure ,Humans ,Amino Acid Sequence ,Biology (General) ,Integral membrane protein ,030304 developmental biology ,Protein Synthesis Inhibitors ,Sulfonamides ,0303 health sciences ,Signal recognition particle ,General Immunology and Microbiology ,General Neuroscience ,Endoplasmic reticulum ,Biology and Life Sciences ,Proteins ,Translocon ,3. Good health ,Transport protein ,Cell biology ,Protein Transport ,Protein Biosynthesis ,CD4 Antigens ,General Agricultural and Biological Sciences ,Hydrophobic and Hydrophilic Interactions ,030217 neurology & neurosurgery ,Research Article - Abstract
A small chemical drug CADA specifically binds to the signal peptide of the membrane pre-protein CD4, disturbing its synthesis, impeding the routing to and expression on the cell surface., In eukaryotic cells, surface expression of most type I transmembrane proteins requires translation and simultaneous insertion of the precursor protein into the endoplasmic reticulum (ER) membrane for subsequent routing to the cell surface. This co-translational translocation pathway is initiated when a hydrophobic N-terminal signal peptide (SP) on the nascent protein emerges from the ribosome, binds the cytosolic signal recognition particle (SRP), and targets the ribosome-nascent chain complex to the Sec61 translocon, a universally conserved protein-conducting channel in the ER-membrane. Despite their common function in Sec61 targeting and ER translocation, SPs have diverse but unique primary sequences. Thus, drugs that recognise SPs could be exploited to inhibit translocation of specific proteins into the ER. Here, through flow cytometric analysis the small-molecule macrocycle cyclotriazadisulfonamide (CADA) is identified as a highly selective human CD4 (hCD4) down-modulator. We show that CADA inhibits CD4 biogenesis and that this is due to its ability to inhibit co-translational translocation of CD4 into the lumen of the ER, both in cells as in a cell-free in vitro translation/translocation system. The activity of CADA maps to the cleavable N-terminal SP of hCD4. Moreover, through surface plasmon resonance analysis we were able to show direct binding of CADA to the SP of hCD4 and identify this SP as the target of our drug. Furthermore, CADA locks the SP in the translocon during a post-targeting step, possibly in a folded state, and prevents the translocation of the associated protein into the ER lumen. Instead, the precursor protein is routed to the cytosol for degradation. These findings demonstrate that a synthetic, cell-permeable small-molecule can be developed as a SP-binding drug to selectively inhibit protein translocation and to reversibly regulate the expression of specific target proteins., Author Summary All cells are highly crowded with proteins that, once synthesized, have to reach their proper subcellular location in order to maintain the cellular homeostasis. Approximately 30% of the proteome needs to be sorted from the cytosol and inserted into, or transported through, biological membranes. For proteins sorted via the secretory pathway, an important step is the translocation into a cellular compartment called the endoplasmic reticulum (ER). The cell uses an elegant way to discriminate proteins that need to be translocated into the ER from those that have to reside in the cytosol by scanning for the presence of an N-terminal ER-entry tag. Although these tags, called signal peptides, have a common structure, they each contain a unique hydrophobic peptide sequence. In this work, we describe how a small chemical drug, CADA, can bind to one specific signal peptide present in the human CD4 pre-protein. We show that by influencing the signal peptide orientation in the translocation channel located in the ER membrane, CADA prevents CD4 translocation into the ER lumen. As a consequence, the CD4 protein is not properly synthesized and routed to the cell surface, resulting in a clear reduction in the amount of surface CD4, a membrane protein found on immune cells, and implicated in HIV-infection and other diseases. We believe that other drugs can be designed to selectively regulate, in a similar way, ER translocation of specific target proteins.
- Published
- 2014
29. In Vivo Analysis of Importin α Proteins Reveals Cellular Proliferation Inhibition and Substrate Specificity
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Thomas Sommer, Christina Quensel, Beate Friedrich, Enno Hartmann, and Matthias Köhler
- Subjects
alpha Karyopherins ,Time Factors ,animal structures ,Blotting, Western ,Down-Regulation ,Apoptosis ,Cell Cycle Proteins ,Cell Separation ,Importin ,Biology ,Transfection ,environment and public health ,Substrate Specificity ,Cytosol ,medicine ,Importin-alpha ,Guanine Nucleotide Exchange Factors ,Humans ,Cycloheximide ,RNA, Small Interfering ,Cell Growth and Development ,Molecular Biology ,Cell Proliferation ,Cell Nucleus ,Protein Synthesis Inhibitors ,Dose-Response Relationship, Drug ,urogenital system ,Nuclear Proteins ,Alpha Karyopherins ,Cell Biology ,Flow Cytometry ,beta Karyopherins ,Recombinant Proteins ,Cell biology ,Cell nucleus ,medicine.anatomical_structure ,Microscopy, Fluorescence ,Biochemistry ,embryonic structures ,Ran ,RNA Interference ,Beta Karyopherins ,Guanine nucleotide exchange factor ,Nuclear transport ,HeLa Cells - Abstract
The "classical" nuclear import pathway depends on importins alpha and beta. Humans have only one importin beta, while six alpha importins have been described. Whether or not distinct alpha importins are essential for specific import pathways in living human cells is unclear. We used RNA interference technology to specifically down-regulate the expression of ubiquitously expressed human alpha importins in HeLa cells. Down-regulation of importins alpha3, alpha5, alpha7, and beta strongly inhibited HeLa cell proliferation, while down-regulation of importins alpha1 and alpha4 had only a minor effect or no effect. Nucleoplasmin import was not prevented by down-regulation of any alpha importin, indicating that the importin alpha/beta pathway was generally not affected. In contrast, importin alpha3 or alpha5 down-regulation specifically inhibited the nuclear import of the Ran guanine nucleotide exchange factor, RCC1. Coinjection of recombinant alpha importins and RCC1 into down-regulated cells demonstrated that these transport defects were specifically caused by the limited availability of importin alpha3 in both cases. Thus, importin alpha3 is the only alpha importin responsible for the classical nuclear import of RCC1 in living cells.
- Published
- 2004
30. Exportin 6: a novel nuclear export receptor that is specific for profilin{middle dot}actin complexes
- Author
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Dirk Görlich, Enno Hartmann, and Theis Stüven
- Subjects
General Immunology and Microbiology ,General Neuroscience ,Arp2/3 complex ,Actin remodeling ,macromolecular substances ,Importin ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Cell biology ,Profilin ,Profilin-1 ,biology.protein ,Actin-binding protein ,MDia1 ,Nuclear export signal ,Molecular Biology - Abstract
Active macromolecular transport between the nucleus and cytoplasm proceeds through nuclear pore complexes and is mostly mediated by transport receptors of the importin β-superfamily. Here we identify exportin 6 (Exp6) as a novel family member from higher eukaryotes and show that it mediates nuclear export of profilin·actin complexes. Exp6 appears to contact primarily actin, but the interaction is greatly enhanced by the presence of profilin. Profilin thus functions not only as the nucleotide exchange factor for actin, but can also be regarded as a cofactor of actin export and hence as a suppressor of actin polymerization in the nucleus. Even though human and Drosophila Exp6 share only ∼20% identical amino acid residues, their function in profilin·actin export is conserved. A knock-down of Drosophila Exp6 by RNA interference abolishes nuclear exclusion of actin and results in the appearance of nuclear actin paracrystals. In contrast to a previous report, we found no indications of a major and direct role for CRM1 in actin export from mammalian or insect nuclei.
- Published
- 2003
31. The enigma of ribonuclease P evolution
- Author
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Enno Hartmann and Roland K. Hartmann
- Subjects
Genetics ,Bacteria ,biology ,RNase P ,Escherichia coli Proteins ,Protein subunit ,biology.organism_classification ,Archaea ,RNase PH ,Ribonuclease P ,Evolution, Molecular ,RNase MRP ,Bacterial Proteins ,Molecular evolution ,biology.protein ,Animals ,Humans ,Nucleic Acid Conformation ,RNA ,RNA, Catalytic ,RNase H ,Phylogeny ,Ribonucleoprotein - Abstract
The 5'-end maturation of tRNAs is catalyzed by the ribonucleoprotein enzyme ribonuclease P (RNase P) in all organisms. Here we provide, for the first time, a comprehensive overview on the representation of individual RNase P protein homologs within the Eukarya and Archaea. Most eukaryotes have homologs for all four protein subunits (Pop4, Rpp1, Pop5 and Rpr2) present in the majority of Archaea. Pop4 is the only RNase P protein subunit identifiable in all Eukarya and Archaea with available genome sequences. Remarkably, there is no structural homology between bacterial and archaeal-eukaryotic RNase P proteins. The simplest interpretation is that RNase P has an 'RNA-alone' origin and progenitors of Bacteria and Archaea diverged very early in evolution and then pursued completely different strategies in the recruitment of protein subunits during the transition from the 'RNA-alone' to the 'RNA-protein' state of the enzyme.
- Published
- 2003
32. SMNrp is an essential pre-mRNA splicing factor required for the formation of the mature spliceosome
- Author
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Stanislav Fakan, Enno Hartmann, Stefan Hannus, Tonie Luise Baars, Bernhard Laggerbauer, Gunter Meister, Oliver Plöttner, and Utz Fischer
- Subjects
RNA Splicing Factors ,Spliceosome ,Transcription, Genetic ,Ribonucleoprotein, U4-U6 Small Nuclear ,RNA Splicing ,Xenopus ,Exonic splicing enhancer ,Nerve Tissue Proteins ,Prp24 ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,03 medical and health sciences ,Splicing factor ,0302 clinical medicine ,Minor spliceosome ,RNA Precursors ,Animals ,Humans ,Cyclic AMP Response Element-Binding Protein ,Molecular Biology ,030304 developmental biology ,Cell Nucleus ,Genetics ,0303 health sciences ,General Immunology and Microbiology ,General Neuroscience ,RNA-Binding Proteins ,SMN Complex Proteins ,Ribonucleoprotein, U2 Small Nuclear ,Ribonucleoproteins, Small Nuclear ,Recombinant Proteins ,Protein Structure, Tertiary ,Cell biology ,Polypyrimidine tract ,Mutation ,RNA splicing ,Spliceosomes ,030217 neurology & neurosurgery ,Protein Binding - Abstract
SMNrp, also termed SPF30, has recently been identified in spliceosomes assembled in vitro. We have functionally characterized this protein and show that it is an essential splicing factor. We show that SMNrp is a 17S U2 snRNP-associated protein that appears in the pre-spliceosome (complex A) and the mature spliceosome (complex B) during splicing. Immunodepletion of SMNrp from nuclear extract inhibits the first step of pre-mRNA splicing by preventing the formation of complex B. Re-addition of recombinant SMNrp to immunodepleted extract reconstitutes both spliceosome formation and splicing. Mutations in two domains of SMNrp, although similarly deleterious for splicing, differed in their consequences on U2 snRNP binding, suggesting that SMNrp may also engage in interactions with splicing factors other than the U2 snRNP. In agreement with this, we present evidence for an additional interaction between SMNrp and the [U4/U6⋅U5] tri-snRNP. A candidate that may mediate this interaction, namely the U4/U6-90 kDa protein, has been identified. We suggest that SMNrp, as a U2 snRNP-associated protein, facilitates the recruitment of the [U4/U6⋅U5] tri-snRNP to the pre-spliceosome.
- Published
- 2001
33. Identification of Two Novel RanGTP-binding Proteins Belonging to the Importin β Superfamily
- Author
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Maria Carmo-Fonseca, Dirk Görlich, Siegfried Prehn, Ulrike Kutay, N. Treichel, F. R. Bischoff, Regine Kraft, A. Calado, and Enno Hartmann
- Subjects
Molecular Sequence Data ,Importin ,Karyopherins ,Biology ,Biochemistry ,DNA-binding protein ,03 medical and health sciences ,0302 clinical medicine ,Affinity chromatography ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Nuclear pore ,Receptor ,Molecular Biology ,DNA Primers ,030304 developmental biology ,0303 health sciences ,Base Sequence ,Sequence Homology, Amino Acid ,Nuclear Proteins ,Cell Biology ,Cell biology ,ran GTP-Binding Protein ,medicine.anatomical_structure ,Ran ,Nuclear transport ,Nucleus ,030217 neurology & neurosurgery ,HeLa Cells - Abstract
Nucleo-cytoplasmic transport comprises a large number of distinct pathways, many of which are defined by members of the importin beta superfamily of nuclear transport receptors. These transport receptors all directly interact with RanGTP to modulate the compartment-specific binding of their transport substrates. To identify new members of the importin beta family, we used affinity chromatography on immobilized RanGTP and isolated Ran-binding protein (RanBP) 16 from HeLa cell extracts. RanBP16 and its close human homologue, RanBP17, are distant members of the importin beta family. Like the other members of the transport receptor superfamily, RanBP16 interacts with the nuclear pore complex and is able to enter the nucleus independent of energy and additional nuclear transport receptors.
- Published
- 2000
34. Interactions between Spc2p and Other Components of the Endoplasmic Reticulum Translocation Sites of the YeastSaccharomyces cerevisiae
- Author
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H E Meyer, Wolfram Antonin, and Enno Hartmann
- Subjects
Signal peptide ,Saccharomyces cerevisiae Proteins ,Saccharomyces cerevisiae ,Chromosomal translocation ,Biology ,Endoplasmic Reticulum ,Biochemistry ,Fungal Proteins ,03 medical and health sciences ,0302 clinical medicine ,Molecular Biology ,DNA Primers ,030304 developmental biology ,0303 health sciences ,Base Sequence ,Endoplasmic reticulum ,STIM1 ,Cell Biology ,biology.organism_classification ,Yeast ,3. Good health ,Cell biology ,Membrane protein ,Mutagenesis ,Microsome ,030217 neurology & neurosurgery ,Protein Binding - Abstract
In yeast, the endoplasmic reticulum membrane proteins Sec11p and Spc3p are essential for the cleavage of signal peptides of nascent polypeptide chains during their passage through translocation sites. Genetic and biochemical experiments demonstrate that Sec11p and Spc3p are tightly associated with two other proteins, Spc1p and Spc2p, whose functions are largely unknown. Using anti-Spc2p antibodies, we show here that this heterotetrameric complex associates with Sbh1p and Sbh2p, the beta-subunits of the Sec61p complex and the Ssh1p complex, respectively. Depletion of Spc2p decreased the enzymatic activity of the SPC in vitro, led to a loss of Spc1p, and led to a down-regulation of the amount of Sec11p and Spc3p in the endoplasmic reticulum. Moreover, the deletion of Spc2p also decreased the expression level of Sbh2p. These data implicate that Spc2p not only enhances the enzymatic activity of the SPC but also facilitates the interactions between different components of the translocation site.
- Published
- 2000
35. Exportin 4: a mediator of a novel nuclear export pathway in higher eukaryotes
- Author
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Petra Schwarzmaier, Gerd Lipowsky, Enno Hartmann, Susanne Kostka, Regine Kraft, Ulrike Kutay, F. Ralf Bischoff, and Dirk Görlich
- Subjects
Cytoplasm ,DNA, Complementary ,Time Factors ,Molecular Sequence Data ,Importin ,Karyopherins ,Biology ,environment and public health ,Chromatography, Affinity ,General Biochemistry, Genetics and Molecular Biology ,GTP Phosphohydrolases ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Peptide Initiation Factors ,Animals ,Humans ,Amino Acid Sequence ,Cloning, Molecular ,Nuclear pore ,Nuclear protein ,Nuclear export signal ,Molecular Biology ,030304 developmental biology ,Cell Nucleus ,Hypusine ,0303 health sciences ,Dose-Response Relationship, Drug ,General Immunology and Microbiology ,Lysine ,General Neuroscience ,Nuclear Proteins ,RNA-Binding Proteins ,Articles ,Protein Structure, Tertiary ,Kinetics ,ran GTP-Binding Protein ,Microscopy, Fluorescence ,Biochemistry ,chemistry ,030220 oncology & carcinogenesis ,Ran ,RNA ,Nucleoporin ,Nuclear transport ,Carrier Proteins ,HeLa Cells ,Protein Binding - Abstract
Transport receptors of the importin beta superfamily account for many of the nuclear import and export events in eukaryotic cells. They mediate translocation through nuclear pore complexes, shuttle between nucleus and cytoplasm and co-operate with the RanGTPase system to regulate their interactions with cargo molecules in a compartment-specific manner. We used affinity chromatography on immobilized RanGTP to isolate further candidate nuclear transport receptors and thereby identified exportin 4 as the most distant member of the importin beta family so far. Exportin 4 appears to be conserved amongst higher eukaryotes, but lacks obvious orthologues in yeast. It mediates nuclear export of eIF-5A (eukaryotic translation initiation factor 5A) and possibly that of other cargoes. The export signal in eIF-5A appears to be complex and to involve the hypusine modification that is unique to eIF-5A. We discuss possible cellular roles for nuclear export of eIF-5A.
- Published
- 2000
36. Stress-Associated Endoplasmic Reticulum Protein 1 (Serp1)/Ribosome-Associated Membrane Protein 4 (Ramp4) Stabilizes Membrane Proteins during Stress and Facilitates Subsequent Glycosylation
- Author
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Masaya Tohyama, Atsushi Yamaguchi, David M. Stern, Satoshi Ogawa, Enno Hartmann, and Osamu Hori
- Subjects
Male ,Protein Denaturation ,Vesicle-associated membrane protein 8 ,Glycosylation ,Calnexin ,Molecular Sequence Data ,Protein Renaturation ,Biology ,Endoplasmic Reticulum ,Brain Ischemia ,Cell Line ,refolding ,Rats, Sprague-Dawley ,03 medical and health sciences ,0302 clinical medicine ,Animals ,Homeostasis ,Humans ,Amino Acid Sequence ,RNA, Messenger ,Cloning, Molecular ,Integral membrane protein ,translocon ,Glycoproteins ,030304 developmental biology ,0303 health sciences ,Sequence Homology, Amino Acid ,hypoxia ,Endoplasmic reticulum ,Calcium-Binding Proteins ,Peripheral membrane protein ,Membrane Proteins ,aggregation/degradation ,Cell Biology ,Translocon ,Membrane contact site ,Cell Hypoxia ,Rats ,Cell biology ,Membrane protein ,Astrocytes ,endoplasmic reticulum stress ,Unfolded protein response ,Original Article ,SEC Translocation Channels ,030217 neurology & neurosurgery ,Molecular Chaperones ,Protein Binding - Abstract
Application of differential display to cultured rat astrocytes subjected to hypoxia allowed cloning of a novel cDNA, termed stress-associated endoplasmic reticulum protein 1 (SERP1). Expression of SERP1 was enhanced in vitro by hypoxia and/or reoxygenation or other forms of stress, causing accumulation of unfolded proteins in endoplasmic reticulum (ER) stress, and in vivo by middle cerebral artery occlusion in rats. The SERP1 cDNA encodes a 66–amino acid polypeptide which was found to be identical to ribosome-associated membrane protein 4 (RAMP4) and bearing 29% identity to yeast suppressor of SecY 6 protein (YSY6p), suggesting participation in pathways controlling membrane protein biogenesis at ER. In cultured 293 cells subjected to ER stress, overexpression of SERP1/RAMP4 suppressed aggregation and/or degradation of newly synthesized integral membrane proteins, and subsequently, facilitated their glycosylation when the stress was removed. SERP1/RAMP4 interacted with Sec61α and Sec61β, which are subunits of translocon, and a molecular chaperon calnexin. Furthermore, Sec61α and Sec61β, but not SERP1/RAMP4, were found to associate with newly synthesized integral membrane proteins under stress. These results suggest that stabilization of membrane proteins in response to stress involves the concerted action of a rescue unit in the ER membrane comprised of SERP1/RAMP4, other components of translocon, and molecular chaperons in ER.
- Published
- 1999
37. Control of glycosylation of MHC class II-associated invariant chain by translocon-associated RAMP4
- Author
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Bernhard Dobberstein, Michael Hofmann, Enno Hartmann, Tom A. Rapoport, Bruno Martoglio, Christina Hölscher, Katja Schröder, and Siegfried Prehn
- Subjects
DNA, Complementary ,Glycosylation ,Time Factors ,CD74 ,Molecular Sequence Data ,Models, Biological ,General Biochemistry, Genetics and Molecular Biology ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,MHC class I ,Animals ,Humans ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,030304 developmental biology ,0303 health sciences ,MHC class II ,Sequence Homology, Amino Acid ,General Immunology and Microbiology ,biology ,General Neuroscience ,Endoplasmic reticulum ,030302 biochemistry & molecular biology ,Histocompatibility Antigens Class II ,Membrane Proteins ,Translocon ,Precipitin Tests ,Rats ,Antigens, Differentiation, B-Lymphocyte ,chemistry ,Membrane protein ,Biochemistry ,Mutagenesis ,Protein Biosynthesis ,biology.protein ,Ribosomes ,Protein Binding ,Research Article - Abstract
Protein translocation across the membrane of the endoplasmic reticulum (ER) proceeds through a proteinaceous translocation machinery, the translocon. To identify components that may regulate translocation by interacting with nascent polypeptides in the translocon, we used site-specific photo-crosslinking. We found that a region C-terminal of the two N-glycosylation sites of the MHC class II-associated invariant chain (Ii) interacts specifically with the ribosome-associated membrane protein 4 (RAMP4). RAMP4 is a small, tail-anchored protein of 66 amino acid residues that is homologous to the yeast YSY6 protein. YSY6 suppresses a secretion defect of a secY mutant in Escherichia coli. The interaction of RAMP4 with Ii occurred when nascent Ii chains reached a length of 170 amino acid residues and persisted until Ii chain completion, suggesting translocational pausing. Site-directed mutagenesis revealed that the region of Ii interacting with RAMP4 contains essential hydrophobic amino acid residues. Exchange of these residues for serines led to a reduced interaction with RAMP4 and inefficient N-glycosylation. We propose that RAMP4 controls modification of Ii and possibly also of other secretory and membrane proteins containing specific RAMP4-interacting sequences. Efficient or variable glycosylation of Ii may contribute to its capacity to modulate antigen presentation by MHC class II molecules.
- Published
- 1999
38. Nuclear Protein Transport Pathways
- Author
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Hermann Haller, Enno Hartmann, and Matthias Köhler
- Subjects
Cytoplasm ,Nuclear Envelope ,Physiology ,Biological Transport, Active ,Importin ,Mitochondrial membrane transport protein ,Ribosomal protein ,Genetics ,medicine ,Animals ,Humans ,Nuclear protein ,Cell Nucleus ,biology ,Nuclear Proteins ,General Medicine ,Transport protein ,Cell biology ,Cell nucleus ,ran GTP-Binding Protein ,medicine.anatomical_structure ,Biochemistry ,Nephrology ,biology.protein ,Nucleoporin ,Nuclear transport - Abstract
Nuclear proteins like transcription factors and ribosomal proteins are synthesized in the cytoplasm and have to be transported into the nucleus to fulfill their functions. The transport of proteins >20–60 kD through the nuclear pore complex (NPC) into the nucleus is an active, energy-requiring process. Transport substrates are recognized by their transport proteins via certain signals. The best-characterized protein import pathway is the ‘classical’ nuclear localization signal-dependent pathway with importin α and β carrying the substrate to the NPC. The transport of the importin-substrate complex into the nucleus is regulated by the small GTPase Ran/TC4. During the last years more than ten proteins have been discovered which have already been proven or are very likely to be nuclear transport factors of distinct import pathways: members of the importin α protein family are very similar and transport in complex with importin β nuclear localization signal-bearing proteins into the nucleus. Members of the Ran-binding protein family show some weak similarity to importin β. Sharing a common domain at the amino terminus, they are able to bind RanGTP, a prerequisite for their function as nuclear import or export factors for distinct proteins or RNAs. However, Ran/TC4 seems to play a key regulatory role in all nuclear transport pathways described so far, although the molecular mechanism of the translocation step through the NPC is still unclear.
- Published
- 1999
39. Interaction of Kar2p and Sls1p Is Required for Efficient Co-translational Translocation of Secreted Proteins in the YeastYarrowia lipolytica
- Author
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Jean-Marie Beckerich, Mehdi Kabani, Claude Gaillardin, Enno Hartmann, Anita Boisramé, Institut National de la Recherche Agronomique (INRA), and Georg-August-University [Göttingen]
- Subjects
polypeptide ,Mutant ,Endoplasmic Reticulum ,Biochemistry ,Ribosome ,mutagenèse ,0303 health sciences ,education.field_of_study ,biology ,030302 biochemistry & molecular biology ,Temperature ,[SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM] ,Phenotype ,ribosome ,protéine ,translocation chromosomique ,Protein Binding ,Saccharomyces cerevisiae Proteins ,Molecular Sequence Data ,Population ,Saccharomyces cerevisiae ,Fungal Proteins ,Mitochondrial Proteins ,03 medical and health sciences ,Microsomes ,HSP70 Heat-Shock Proteins ,Amino Acid Sequence ,réticulum endoplasmique ,education ,levure ,Molecular Biology ,030304 developmental biology ,Sequence Homology, Amino Acid ,Endoplasmic reticulum ,Genetic Complementation Test ,Membrane Proteins ,Biological Transport ,Yarrowia ,Cell Biology ,biology.organism_classification ,Yeast ,Secretory protein ,Protein Biosynthesis ,Mutation ,Saccharomycetales ,Carrier Proteins ,yarrowia lipolytica ,Ribosomes ,SEC Translocation Channels - Abstract
The yeast Yarrowia lipolytica is a model organism for in vivo study of the signal recognition particle-dependent targeting pathway. In this report, we defined solubilization conditions and set up a fractionation procedure of Y. lipolytica microsomes to determine the amounts of Sec61p-containing translocation pores linked to ribosomes. In contrast to Saccharomyces cerevisiae, from 70 to 80% of Sec61p associates with ribosomes in this yeast. The chaperone protein Kar2p and the Sls1p product, a resident protein of the endoplasmic reticulum lumen, partially fractionate with this Sec61p population. Moreover, Sls1p can be co-immunoprecipitated with Kar2p, and the two polypeptides are shown to directly interact in the yeast two-hybrid system. A site-directed mutagenesis was performed on the SLS1 coding sequence that allowed us to define a functional domain in Sls1p. Indeed, co-translational translocation of a reporter protein is affected when one of these mutant proteins is expressed. Moreover, this protein has lost its capacity to interact with Kar2p, and the two lumenal polypeptides might thus cooperate to promote secretory protein co-translational translocation.
- Published
- 1998
40. The β Subunit of the Sec61 Complex Facilitates Cotranslational Protein Transport and Interacts with the Signal Peptidase during Translocation
- Author
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Tom A. Rapoport, Kai-Uwe Kalies, and Enno Hartmann
- Subjects
Models, Molecular ,Sec61 ,Glycosylation ,Saccharomyces cerevisiae Proteins ,Transcription, Genetic ,Macromolecular Substances ,Protein Conformation ,Proteolipids ,Protein subunit ,Peptide Chain Elongation, Translational ,Chromosomal translocation ,Saccharomyces cerevisiae ,Biology ,Endoplasmic Reticulum ,Ribosome ,03 medical and health sciences ,0302 clinical medicine ,Microsomes ,Cloning, Molecular ,Protein Precursors ,030304 developmental biology ,0303 health sciences ,Signal peptidase ,Binding Sites ,Serine Endopeptidases ,Membrane Proteins ,Membrane Transport Proteins ,Biological Transport ,Intracellular Membranes ,Articles ,Cell Biology ,Translocon ,Prolactin ,Transport protein ,Cell biology ,Kinetics ,Protein Biosynthesis ,Liposomes ,Signal peptidase complex ,Protein Processing, Post-Translational ,Ribosomes ,SEC Translocation Channels ,030217 neurology & neurosurgery - Abstract
The Sec61 complex is the central component of the protein translocation apparatus of the ER membrane. We have addressed the role of the β subunit (Sec61β) during cotranslational protein translocation. With a reconstituted system, we show that a Sec61 complex lacking Sec61β is essentially inactive when elongation and membrane targeting of a nascent chain occur at the same time. The translocation process is perturbed at a step where the nascent chain would be inserted into the translocation channel. However, if sufficient time is given for the interaction of the nascent polypeptide with the mutant Sec61 complex, translocation is almost normal. Thus Sec61β kinetically facilitates cotranslational translocation, but is not essential for it.Using chemical cross-linking we show that Sec61β not only interacts with subunits of the Sec61 complex but also with the 25-kD subunit of the signal peptidase complex (SPC25), thus demonstrating for the first time a tight interaction between the SPC and the Sec61 complex. Interestingly, the cross-links between Sec61β and SPC25 and between Sec61β and Sec61α depend on the presence of membrane-bound ribosomes, suggesting that these interactions are induced when translocation is initiated. We propose that the SPC is transiently recruited to the translocation site, thus enhancing its activity.
- Published
- 1998
41. Identification of a tRNA-Specific Nuclear Export Receptor
- Author
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Petra Schwarzmaier, Enno Hartmann, Ulrike Kutay, Dirk Görlich, Gerd Lipowsky, F. R. Bischoff, and Elisa Izaurralde
- Subjects
Cytoplasm ,Nucleocytoplasmic Transport Proteins ,RNA, Transfer, Leu ,Xenopus ,Molecular Sequence Data ,Importin ,Biology ,Xenopus Proteins ,RNA, Transfer ,GTP-Binding Proteins ,Animals ,Humans ,Amino Acid Sequence ,RNA, Messenger ,Nuclear protein ,Nuclear pore ,Cloning, Molecular ,Nuclear export signal ,Molecular Biology ,RNA, Transfer, Ser ,Cell Nucleus ,GTPase-Activating Proteins ,Nuclear Proteins ,Cell Biology ,RNA, Transfer, Amino Acid-Specific ,beta Karyopherins ,Cell biology ,ran GTP-Binding Protein ,Biochemistry ,Oocytes ,Beta Karyopherins ,Nucleoporin ,Carrier Proteins ,Nuclear localization sequence ,HeLa Cells ,Protein Binding - Abstract
In eukaryotes, tRNAs are synthesized in the nucleus and after several maturation steps exported to the cytoplasm. Here, we identify exportin-t as a specific mediator of tRNA export. It is a RanGTP-binding, importin beta-related factor with predominantly nuclear localization. It shuttles rapidly between nucleus and cytoplasm and interacts with nuclear pore complexes. Exportin-t binds tRNA directly and with high affinity. Its cellular concentration in Xenopus oocytes was found to be rate-limiting for export of all tRNAs tested, as judged by microinjection experiments. RanGTP regulates the substrate-exportin-t interaction such that tRNA can be preferentially bound in the nucleus and released in the cytoplasm.
- Published
- 1998
- Full Text
- View/download PDF
42. Cloning of two novel human importin-α subunits and analysis of the expression pattern of the importin-α protein family
- Author
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Enno Hartmann, Achim Leutz, Matthias Köhler, Hermann Haller, Siegfried Prehn, and Stéphane Ansieau
- Subjects
DNA, Complementary ,animal structures ,Protein family ,Immunoblotting ,Molecular Sequence Data ,Biophysics ,Gene Expression ,Importin ,Karyopherins ,Biology ,environment and public health ,Biochemistry ,Jurkat Cells ,Importin-α ,Structural Biology ,Tumor Cells, Cultured ,Genetics ,Importin-alpha ,Animals ,Humans ,Tissue Distribution ,Amino Acid Sequence ,RNA, Messenger ,Cloning, Molecular ,Molecular Biology ,Gene ,Cloning ,Base Sequence ,Sequence Homology, Amino Acid ,Protein primary structure ,Nuclear Proteins ,Alpha Karyopherins ,Cell Biology ,Cell biology ,Nuclear signaling ,Nuclear transport ,embryonic structures ,Protein expression ,Rabbits ,Nuclear localization sequence ,HeLa Cells - Abstract
The import of many proteins into the nucleus is mediated by the importin-alpha/beta heterodimer. While only one importin-beta gene has been found, several forms of importin-alpha have been described. In addition to the three human importin-alphas already identified, we report here the primary structure of two new human importin-alpha proteins. The five known human importin-alpha subunits can be classified into three subfamilies that appear conserved in higher eukaryotic organisms. We show by immunoblotting that the different importin-alpha subfamilies are expressed in a variety of human tissues and mammalian cell lines.
- Published
- 1997
43. Protein Translocation in the Three Domains of Life: Variations on a Theme
- Author
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Enno Hartmann, Mechthild Pohlschroder, Jon Beckwith, and Will A. Prinz
- Subjects
Biochemistry, Genetics and Molecular Biology(all) ,Archaeal Proteins ,Proteins ,Biological Transport ,Environmental ethics ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Fungal Proteins ,Scholarship ,Bacterial Proteins ,Three-domain system ,Protein translocation ,Classics ,Theme (narrative) - Abstract
We thank Carol Bult, Jorg Nolling, and Sorel Fitz-Gibbon for comparing sequences of known translocation components with genome sequences of M. jannaschii, M. thermoautotrophicum, and P. aerofilum, respectively. We are grateful to Dana Boyd and other members of the Beckwith laboratory for helpful discussions and comments on the manuscript. We thank Tom Rapoport and William Wickner for critical reading of the manuscript. This work was supported by a National Institute of General Medical Sciences grant (to J. B.) and a Deutsche Forschungsgemeinschaft research scholarship (to M. P.).
- Published
- 1997
44. The Homologue of Mammalian SPC12 Is Important for Efficient Signal Peptidase Activity in
- Author
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Neil E. Green, Chris Mullins, Hong Fang, Steffen Panzner, and Enno Hartmann
- Subjects
Signal peptide ,Signal peptidase ,Endoplasmic reticulum ,Saccharomyces cerevisiae ,Cell Biology ,Protein degradation ,Biology ,biology.organism_classification ,Biochemistry ,Yeast ,Signal peptidase complex ,Molecular Biology ,Signal peptide peptidase - Abstract
The multisubunit signal peptidase catalyzes the cleavage of signal peptides and the degradation of some membrane proteins within the endoplasmic reticulum (ER). The only subunit of this enzyme functionally examined to date, yeast Sec11p, is related to signal peptidase I from bacteria. Since bacterial signal peptidase is capable of processing both prokaryotic and eukaryotic signal sequences as a monomer, it is unclear why the analogous enzyme in the ER contains proteins unrelated to signal peptidase I. To address this issue, the gene encoding Spc1p, the yeast homologue to mammalian SPC12, is isolated from the yeast Saccharomyces cerevisiae. Spc1p co-purifies and genetically interacts with Sec11p, but unlike Sec11p, Spc1p is not required for cell growth or the proteolytic processing of tested proteins in yeast. This indicates that only a subset of the ER signal peptidase subunits is required for signal peptidase and protein degradation activities in vivo. Through both genetic and biochemical criteria, Spc1p appears, however, to be important for efficient signal peptidase activity.
- Published
- 1996
45. A second trimeric complex containing homologs of the Sec61p complex functions in protein transport across the ER membrane of S. cerevisiae
- Author
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Thomas Sommer, Siegfried Prehn, Steffen Panzner, K. Finke, Tom A. Rapoport, Kathrin Plath, and Enno Hartmann
- Subjects
Saccharomyces cerevisiae Proteins ,Protein Conformation ,Protein subunit ,Molecular Sequence Data ,Biological Transport, Active ,Saccharomyces cerevisiae ,Endoplasmic Reticulum ,General Biochemistry, Genetics and Molecular Biology ,Fungal Proteins ,Dogs ,Heterotrimeric G protein ,Animals ,Amino Acid Sequence ,DNA, Fungal ,Molecular Biology ,Endoplasmic reticulum membrane ,Base Sequence ,Sequence Homology, Amino Acid ,General Immunology and Microbiology ,biology ,Membrane transport protein ,General Neuroscience ,Endoplasmic reticulum ,Membrane Proteins ,Membrane Transport Proteins ,Intracellular Membranes ,Transport protein ,Cell biology ,Mutation ,Cyclin-dependent kinase complex ,biology.protein ,Protein Processing, Post-Translational ,SEC Translocation Channels ,Research Article - Abstract
Yeast microsomes contain a heptameric Sec complex involved in post-translational protein transport that is composed of a heterotrimeric Sec61p complex and a tetrameric Sec62-Sec63 complex. The trimeric Sec61p complex also exists as a separate entity that probably functions in co-translational protein transport, like its homolog in mammals. We have now discovered in the yeast endoplasmic reticulum membrane a second, structurally related trimeric complex, named Ssh1p complex. It consists of Ssh1p1 (Sec sixty-one homolog 1), a rather distant relative of Sec61p, of Sbh2p, a homolog of the Sbh1p subunit of the Sec61p complex, and of Sss1p, a component common to both trimeric complexes. In contrast to Sec61p, Ssh1p is not essential for cell viability but it is required for normal growth rates. Sbh1p and Sbh2p individually are also not essential, but cells lacking both proteins are impaired in their growth at elevated temperatures and accumulate precursors of secretory proteins; microsomes isolated from these cells also exhibit a reduced rate of post-translational protein transport. Like the Sec61p complex, the Ssh1p complex interacts with membrane-bound ribosomes, but it does not associate with the Sec62-Sec63p complex to form a heptameric Sec complex. We therefore propose that it functions exclusively in the co-translational pathway of protein transport.
- Published
- 1996
46. Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope
- Author
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Enno Hartmann, Dirk Görlich, Ronald A. Laskey, Regine Kraft, Susanne Kostka, Siegfried Prehn, and Colin Dingwall
- Subjects
alpha Karyopherins ,animal structures ,Nuclear Envelope ,Xenopus ,Molecular Sequence Data ,Receptors, Cytoplasmic and Nuclear ,Importin ,Biology ,Karyopherins ,Protein Sorting Signals ,environment and public health ,General Biochemistry, Genetics and Molecular Biology ,Cytosol ,Importin-alpha ,Animals ,Humans ,Amino Acid Sequence ,Nuclear pore ,Nuclear protein ,Cloning, Molecular ,Karyopherin ,chemistry.chemical_classification ,Sequence Homology, Amino Acid ,Agricultural and Biological Sciences(all) ,Biochemistry, Genetics and Molecular Biology(all) ,Nuclear Proteins ,Alpha Karyopherins ,Biological Transport ,Biological Evolution ,Biochemistry ,chemistry ,embryonic structures ,Nuclear transport ,General Agricultural and Biological Sciences ,Carrier Proteins ,Nuclear localization sequence ,HeLa Cells ,Protein Binding - Abstract
Background: Selective protein import into the cell nucleus occurs in two steps: binding to the nuclear envelope, followed by energy-dependent transit through the nuclear pore complex. A 60 kD protein, importin, is essential for the first nuclear import step, and the small G protein Ran/TC4 is essential for the second. We have previously purified the 60 kD importin protein (importin 60) as a single polypeptide. Results We have identified importin 90, a 90 kD second subunit that dissociates from importin 60 during affinity chromatography on nickel (II)–nitrolotriacetic acid–Sepharose, a technique that was originally used to purify importin 60. Partial amino-acid sequencing of Xenopus importin 90 allowed us to clone and sequence its human homologue; the amino-acid sequence of importin 90 is strikingly conserved between the two species. We have also identified a homologous budding yeast sequence from a database entry. Importin 90 potentiates the effects of importin 60 on nuclear protein import, indicating that the importin complex is the physiological unit responsible for import. To assess whether nuclear localization sequences are recognized by cytosolic receptor proteins, a biotin-tagged conjugate of nuclear localization signals linked to bovine serum albumin was allowed to form complexes with cytosolic proteins in Xenopus egg extracts; the complexes were then retrieved with streptavidin–agarose. The pattern of bound proteins was surprisingly simple and showed only two predominant bands: those of the importin complex. We also expressed the human homologue of importin 60, Rch1p, and found that it was able to replace its Xenopus counterpart in a functional assay. We discuss the relationship of importin 60 and importin 90 to other nuclear import factors. Conclusion Importin consists of a 60 and a 90 kD subunit. Together, they constitute a cytosolic receptor for nuclear localization signals that enables import substrates to bind to the nuclear envelope.
- Published
- 1995
- Full Text
- View/download PDF
47. Transport route for synaptobrevin via a novel pathway of insertion into the endoplasmic reticulum membrane
- Author
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Enno Hartmann, G Ahnert-Hilger, Tom A. Rapoport, Ulrike Kutay, and Bertram Wiedenmann
- Subjects
Sec61 ,Synaptobrevin ,Proteolipids ,Molecular Sequence Data ,Nerve Tissue Proteins ,Biology ,Endoplasmic Reticulum ,medicine.disease_cause ,PC12 Cells ,General Biochemistry, Genetics and Molecular Biology ,R-SNARE Proteins ,Adenosine Triphosphate ,Endopeptidases ,Protein targeting ,medicine ,Animals ,Amino Acid Sequence ,Molecular Biology ,Integral membrane protein ,Secretory pathway ,General Immunology and Microbiology ,General Neuroscience ,Endoplasmic reticulum ,Peripheral membrane protein ,Membrane Proteins ,Biological Transport ,Intracellular Membranes ,Membrane contact site ,Rats ,Cell biology ,Protein Processing, Post-Translational ,Research Article - Abstract
Synaptobrevin/vesicle-associated membrane protein is one of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. It is proposed to provide specificity for the targeting and fusion of vesicles with the plasma membrane. It belongs to a class of membrane proteins which lack a signal sequence and contain a single hydrophobic segment close to their C-terminus, leaving most of the polypeptide chain in the cytoplasm (tail-anchored). We show that in neuroendocrine PC12 cells, synaptobrevin is not directly incorporated into the target organelle, synaptic-like vesicles. Rather, it is first inserted into the endoplasmic reticulum (ER) membrane and is then transported via the Golgi apparatus. Its insertion into the ER membrane in vitro occurs post-translationally, is dependent on ATP and results in a trans-membrane orientation of the hydrophobic tail. Membrane integration requires ER protein(s) different from the translocation components needed for proteins with signal sequences, thus suggesting a novel mechanism of insertion.
- Published
- 1995
48. Single rRNA helices bind independently to the protein-conducting channel SecYEG
- Author
-
Christina, Behrens, Enno, Hartmann, and Kai-Uwe, Kalies
- Subjects
RNA Folding ,Binding Sites ,Base Sequence ,Escherichia coli Proteins ,Molecular Sequence Data ,Membrane Proteins ,Molecular Docking Simulation ,Dogs ,RNA, Ribosomal ,Mutation ,Ribosome Subunits ,Animals ,Amino Acid Sequence ,SEC Translocation Channels ,Protein Binding - Abstract
Ribosomes tightly interact with protein-conducting channels in the plasma membrane of bacteria (SecYEG) and in the endoplasmic reticulum of eukaryotes (Sec61 complex). This interaction is mediated by multiple junctions and is highly conserved during evolution. Although it is well known that both ribosomal proteins and ribosomal RNA (rRNA) are involved in the ribosome-channel interaction, detailed analyses on how these components contribute to this binding are lacking. Here, we demonstrate that the evolutionary conservation of ribosome binding is solely mediated by rRNA. Moreover, we show that in vitro transcribed 23 S rRNA binds with similar characteristics to protein translocation channels as native 23 S rRNA or 50 S ribosomal subunits. This indicates that base modifications, which exist in native rRNA, do not crucially influence the binding. In two of the ribosome-channel junctions (c1 and c2), exclusively rRNA helices are involved. Using in vitro transcribed rRNA mutants, we now provide evidence that large parts of the rRNA can be deleted without altering its binding properties, as long as the rRNA helices of the c1 and c2 junctions remain intact. We demonstrate that the connection sites c1 and c2 generate high-affinity binding sites that act independently of each other. This could explain why membrane-bound ribosomes have an extremely low off-rate.
- Published
- 2012
49. EcmPred: prediction of extracellular matrix proteins based on random forest with maximum relevance minimum redundancy feature selection
- Author
-
Kai-Uwe Kalies, Ganesan Pugalenthi, Krishna Kumar Kandaswamy, Thomas Martinetz, and Enno Hartmann
- Subjects
Statistics and Probability ,InterPro ,Proteome ,Computational biology ,Matrix (biology) ,Biology ,Bioinformatics ,General Biochemistry, Genetics and Molecular Biology ,Extracellular matrix ,Artificial Intelligence ,Extracellular ,Human proteome project ,Minimum redundancy feature selection ,Humans ,Databases, Protein ,chemistry.chemical_classification ,Extracellular Matrix Proteins ,General Immunology and Microbiology ,Applied Mathematics ,Computational Biology ,General Medicine ,Random forest ,chemistry ,ROC Curve ,Modeling and Simulation ,General Agricultural and Biological Sciences ,Glycoprotein ,Algorithms - Abstract
The extracellular matrix (ECM) is a major component of tissues of multicellular organisms. It consists of secreted macromolecules, mainly polysaccharides and glycoproteins. Malfunctions of ECM proteins lead to severe disorders such as marfan syndrome, osteogenesis imperfecta, numerous chondrodysplasias, and skin diseases. In this work, we report a random forest approach, EcmPred, for the prediction of ECM proteins from protein sequences. EcmPred was trained on a dataset containing 300 ECM and 300 non-ECM and tested on a dataset containing 145 ECM and 4187 non-ECM proteins. EcmPred achieved 83% accuracy on the training and 77% on the test dataset. EcmPred predicted 15 out of 20 experimentally verified ECM proteins. By scanning the entire human proteome, we predicted novel ECM proteins validated with gene ontology and InterPro. The dataset and standalone version of the EcmPred software is available at http://www.inb.uni-luebeck.de/tools-demos/Extracellular_matrix_proteins/EcmPred.
- Published
- 2012
50. Isolation of a protein that is essential for the first step of nuclear protein import
- Author
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Ronald A. Laskey, Dirk Görlich, Siegfried Prehn, and Enno Hartmann
- Subjects
alpha Karyopherins ,DNA, Complementary ,Nuclear Envelope ,Xenopus ,Molecular Sequence Data ,Importin ,Karyopherins ,Biology ,environment and public health ,General Biochemistry, Genetics and Molecular Biology ,Adenosine Triphosphate ,Cytosol ,Sequence Homology, Nucleic Acid ,Importin-alpha ,Animals ,Humans ,Amino Acid Sequence ,Cloning, Molecular ,Nuclear pore ,Nuclear protein ,Ovum ,Karyopherin ,Cell Nucleus ,chemistry.chemical_classification ,Base Sequence ,Sequence Homology, Amino Acid ,Histocytochemistry ,Nuclear Proteins ,Biological Transport ,Alpha Karyopherins ,Sequence Analysis, DNA ,Recombinant Proteins ,Cell Compartmentation ,chemistry ,Biochemistry ,embryonic structures ,Ran ,Beta Karyopherins ,HeLa Cells ,Protein Binding - Abstract
We have purified a cytosolic protein from Xenopus eggs that is essential for selective protein import into the cell nucleus. The purified protein, named importin, promotes signal-dependent binding of karyophilic proteins to the nuclear envelope. We have cloned, sequenced, and expressed a corresponding cDNA. Importin shows 44% sequence identity with SRP1p, a protein associated with the yeast nuclear pore complex. Complete, signal-dependent import into HeLa nuclei can be reconstituted by combining importin purified from Xenopus eggs or expressed in E. coli with Ran/TC4. Evidence for additional stimulatory factors is provided.
- Published
- 1994
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