Biomedical subjects

D Görlich

Publications and source records attributed to D Görlich.

At least 19 recordsLinked to original sources

PubMed2001

Adenoviral E1A protein nuclear import is preferentially mediated by importin alpha3 in vitro.

Nuclear import of some viral proteins depends on importin alpha proteins. However, no preferences of distinct alpha-importins for any viral protein import have been demonstrated. We used in vitro import assays and observed that all ubiquitously expressed human importin alpha isoforms mediate nuclear translocation of adenoviral E1A. Competition with nucleoplasmin suggests that importin alpha3 is the most efficient import mediator of E1A.

Adenovirus E1A Proteins↗

PubMed2001

Importin 13: a novel mediator of nuclear import and export.

Importin beta-related receptors mediate translocation through nuclear pore complexes. Co-operation with the RanGTPase system allows them to bind and subsequently release their substrates on opposite sides of the nuclear envelope, which in turn ensures a directed nucleocytoplasmic transport. Here we identify a novel family member from higher eukaryotes that functions primarily, but not exclusively, in import. It accounts for nuclear accumulation of the SUMO-1/sentrin-conjugating enzyme hUBC9 and mediates import of the RBM8 (Y14) protein, and is therefore referred to as importin 13 (Imp13). Unexpectedly, Imp13 also shows export activity towards the translation initiation factor eIF1A and is thus a case where a single importin beta-like receptor transports different substrates in opposite directions. However, Imp13 operates differently from typical exportins in that the binding of eIF1A to Imp13 is only regulated indirectly by RanGTP, and the cytoplasmic release of eIF1A from Imp13 is triggered by the loading of import substrates onto Imp13.

Cell Nucleus↗

PubMed2001

Kinetic analysis of translocation through nuclear pore complexes.

The mechanism of facilitated translocation through nuclear pore complexes (NPCs) is only poorly understood. Here, we present a kinetic analysis of the process using various model substrates. We find that the translocation capacity of NPCs is unexpectedly high, with a single NPC allowing a mass flow of nearly 100 MDa/s and rates in the order of 10(3) translocation events per second. Our data further indicate that high affinity interactions between the translocation substrate and NPC components are dispensable for translocation. We propose a 'selective phase model' that could explain how NPCs function as a permeability barrier for inert molecules and yet become selectively permeable for nuclear transport receptors and receptor-cargo complexes.

Active Transport, Cell Nucleus↗

PubMed2001

Caspases mediate nucleoporin cleavage, but not early redistribution of nuclear transport factors and modulation of nuclear permeability in apoptosis.

In eukaryotic cells, both soluble transport factors and components of the nuclear pore complex mediate protein and RNA trafficking between the nucleus and the cytoplasm. Here, we investigated whether caspases, the major execution system in apoptosis, target the nuclear pore or components of the nuclear transport machinery. Four nucleoporins, Nup153, RanBP2, Nup214 and Tpr are cleaved by caspases during apoptosis. In contrast, the nuclear transport factors, Ran, importin alpha and importin beta are not proteolytically processed, but redistribute across the nuclear envelope independently and prior to caspase activation. Also, mRNA accumulates into the nucleus before caspases become active. Microinjection experiments further revealed that early in apoptosis, the nucleus becomes permeable to dextran molecules of 70 kD molecular weight. Redistribution of import factors and mRNA, as well as nuclear permeabilisation, occur prior to caspase-mediated nucleoporin cleavage. Our findings suggest that the apoptotic programme includes modifications in the machinery responsible for nucleocytoplasmic transport, which are independent from caspase-mediated degradation of nuclear proteins.

Active Transport, Cell Nucleus↗

PubMed2001

Signal recognition particle protein 19 is imported into the nucleus by importin 8 (RanBP8) and transportin.

The signal recognition particle (SRP) is a cytoplasmic RNA-protein complex that targets proteins to the rough endoplasmic reticulum. Although SRP functions in the cytoplasm, RNA microinjection and cDNA transfection experiments in animal cells, as well as genetic analyses in yeast, have indicated that SRP assembles in the nucleus. Nonetheless, the mechanisms responsible for nuclear-cytoplasmic transport of SRP RNA and SRP proteins are largely unknown. Here we show that the 19 kDa protein subunit of mammalian SRP, SRP19, was efficiently imported into the nucleus in vitro by two members of the importin beta superfamily of transport receptors, importin 8 and transportin; SRP19 was also imported less efficiently by several other members of the importin beta family. Although transportin is known to import a variety of proteins, SRP19 import is the first function assigned to importin 8. Furthermore, we show that a significant pool of endogenous SRP19 is located in the nucleus, as well as the nucleolus. Our results show that at least one mammalian SRP protein is specifically imported into the nucleus, by members of the importin beta family of transport receptors, and the findings add additional evidence for nuclear assembly of SRP.

Active Transport, Cell Nucleus↗

PubMed2000

Different structural and kinetic requirements for the interaction of Ran with the Ran-binding domains from RanBP2 and importin-beta.

The cytoplasmic disassembly of Ran.GTP.importin and Ran.GTP.exportin. cargo complexes is an essential step in the corresponding nuclear import and export cycles. It has previously been shown that such disassembly can be mediated by RanBP1 in the presence of RanGAP. The nuclear pore complex protein RanBP2 (Nup358) contains four Ran-binding domains (RanBDi) that might function like RanBP1. We used biophysical assays based on fluorescence-labeled probes and on surface plasmon resonance to investigate the dynamic interplay of Ran in its GDP- and GTP-complexed states with RanBDis and with importin-beta. We show that RanBP1 and the four RanBDis from RanBP2 have comparable affinities for Ran.GTP (10(8)-10(9) M(-1)). Deletion of Ran's C-terminal (211)DEDDDL(216) sequence weakens the interaction of Ran.GTP with RanBPis approximately 2000-fold, but accelerates the association of Ran.GTP with importin-beta 10-fold. Importin-beta binds Ran.GTP with a moderate rate, but attains a high affinity for Ran (K(D) = 140 pM) via an extremely low dissociation rate of 10(-5) s(-)(1). Association with Ran is accelerated 3-fold in the presence of RanBP1, which presumably prevents steric hindrance caused by the Ran C-terminus. In addition, we show that the RanBDis of RanBP2 are full equivalents of RanBP1 in that they also costimulate RanGAP-catalyzed GTP hydrolysis in Ran and relieve the GTPase block in a Ran.GTP.transportin complex. Our data suggest that the C-terminus of Ran functions like a loose tether in Ran.GTP complexes of importins or exportins that exit the nucleus. This flag is then recognized by the multiple RanBDis at or near the nuclear pore complex, allowing efficient disassembly of these Ran.GTP complexes.

Amino Acid Motifs↗

PubMed2000

Exportin 4: a mediator of a novel nuclear export pathway in higher eukaryotes.

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.

Amino Acid Sequence↗

PubMed2000

Acetylation of importin-alpha nuclear import factors by CBP/p300.

Histone acetylases were originally identified because of their ability to acetylate histone substrates [1] [2] [3]. Acetylases can also target other proteins such as transcription factors [4] [5] [6] [7]. We asked whether the acetylase CREB-binding protein (CBP) could acetylate proteins not directly involved in transcription. A large panel of proteins, involved in a variety of cellular processes, were tested as substrates for recombinant CBP. This screen identified two proteins involved in nuclear import, Rch1 (human importin-alpha) and importin-alpha7, as targets for CBP. The acetylation site within Rch1 was mapped to a single residue, Lys22. By comparing the context of Lys22 with the sequences of other known substrates of CBP and the closely related acetylase p300, we identified G/SK (in the single-letter amino acid code) as a consensus acetylation motif. Mutagenesis of the glycine, as well as the lysine, severely impaired Rch1 acetylation, supporting the view that GK is part of a recognition motif for acetylation by CBP/p300. Using an antibody raised against an acetylated Rch1 peptide, we show that Rch1 was acetylated at Lys22 in vivo and that CBP or p300 could mediate this reaction. Lys22 lies within the binding site for a second nuclear import factor, importin-beta. Acetylation of Lys22 promoted interaction with importin-beta in vitro. Collectively, these results demonstrate that acetylation is not unique to proteins involved in transcription. Acetylation may regulate a variety of biological processes, including nuclear import.

Acetylation↗

PubMed2000

The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates.

Messenger RNAs are exported from the nucleus as large ribonucleoprotein complexes (mRNPs). To date, proteins implicated in this process include TAP/Mex67p and RAE1/Gle2p and are distinct from the nuclear transport receptors of the beta-related, Ran-binding protein family. Mex67p is essential for mRNA export in yeast. Its vertebrate homolog TAP has been implicated in the export of cellular mRNAs and of simian type D viral RNAs bearing the constitutive transport element (CTE). Here we show that TAP is predominantly localized in the nucleoplasm and at both the nucleoplasmic and cytoplasmic faces of the nuclear pore complex (NPC). TAP interacts with multiple components of the NPC including the nucleoporins CAN, Nup98, Nup153, p62, and with three major NPC subcomplexes. The nucleoporin-binding domain of TAP comprises residues 508-619. In HeLa cells, this domain is necessary and sufficient to target GFP-TAP fusions to the nuclear rim. Moreover, the isolated domain strongly competes multiple export pathways in vivo, probably by blocking binding sites on the NPC that are shared with other transport receptors. Microinjection experiments implicate this domain in the export of specific CTE-containing RNAs. Finally, we show that TAP interacts with transportin and with two proteins implicated in the export of cellular mRNAs: RAE1/hGle2 and E1B-AP5. The interaction of TAP with nucleoporins, its direct binding to the CTE RNA, and its association with two mRNP binding proteins suggest that TAP is an RNA export mediator that may bridge the interaction between specific RNP export substrates and the NPC.

Amino Acid Sequence↗

PubMed2000

The Ketel gene encodes a Drosophila homologue of importin-beta.

The Drosophila melanogaster Ketel gene was identified via the Ketel(D) dominant female sterile mutations and their ketel(r) revertant alleles that are recessive zygotic lethals. The maternally acting Ketel(D) mutations inhibit cleavage nuclei formation. We cloned the Ketel gene on the basis of a common breakpoint in 38E1. 2-3 in four ketel(r) alleles. The Ketel(+) transgenes rescue ketel(r)-associated zygotic lethality and slightly reduce Ketel(D)-associated dominant female sterility. Ketel is a single copy gene. It is transcribed to a single 3.6-kb mRNA, predicted to encode the 97-kD Ketel protein. The 884-amino-acid sequence of Ketel is 60% identical and 78% similar to that of human importin-beta, the nuclear import receptor for proteins with a classical NLS. Indeed, Ketel supports import of appropriately designed substrates into nuclei of digitonin-permeabilized HeLa cells. As shown by a polyclonal anti-Ketel antibody, nurse cells synthesize and transfer Ketel protein into the oocyte cytoplasm from stage 11 of oogenesis. In cleavage embryos the Ketel protein is cytoplasmic. The Ketel gene appears to be ubiquitously expressed in embryonic cells. Western blot analysis revealed that the Ketel gene is not expressed in several larval cell types of late third instar larvae.

Amino Acid Sequence↗

PubMed1999

Interaction between NTF2 and xFxFG-containing nucleoporins is required to mediate nuclear import of RanGDP.

Nuclear transport factor 2 (NTF2) is a small, homodimeric protein that binds to both RanGDP and xFxFG repeat-containing nucleoporins, such as yeast Nsp1p and vertebrate p62. NTF2 is required for efficient nuclear protein import and has been shown to mediate the nuclear import of RanGDP. We have used the crystal structures of rat NTF2 and its complex with RanGDP to design a mutant, W7A-NTF2, in which the affinity for xFxFG-repeat nucleoporins is reduced while wild-type binding to RanGDP is retained. The 2.5 A resolution crystal structure of W7A-NTF2 is virtually superimposable upon the wild-type protein structure, indicating that the mutation had not introduced a more general conformational change. Therefore, our data suggest that the exposed side-chain of residue 7 is crucial to the interaction between NTF2 and xFxFG repeat-containing nucleoporins. Consistent with its reduced affinity for xFxFG nucleoporins, fluorescently labelled W7A-NTF2 binds less strongly to the nuclear envelope of permeabilized cultured cells than wild-type NTF2 and, when microinjected into Xenopus oocytes, colloidal gold coated with W7A-NTF2 binds less strongly to the central channel of nuclear pore complexes than wild-type NTF2-coated gold. Significantly, W7A-NTF2 only weakly stimulated the nuclear import of fluorescein-labelled RanGDP, providing direct evidence that an interaction between NTF2 and xFxFG repeat-containing nucleoporins is required to mediate the nuclear import of RanGDP.

Animals↗

PubMed1999

Nuclear import of RPA in Xenopus egg extracts requires a novel protein XRIPalpha but not importin alpha.

Replication protein A (RPA) is a eukaryotic single-stranded (ss) DNA-binding protein that is essential for general DNA metabolism. RPA consists of three subunits (70, 33 and 14 kDa). We have identified by two-hybrid screening a novel Xenopus protein called XRIPalpha that interacts with the ssDNA-binding domain of the largest subunit of RPA. XRIPalpha homologues are found in human and in Drosophila but not in yeast. XRIPalpha is complexed with RPA in Xenopus egg extracts together with another 90 kDa protein that was identified as importin beta. We have demonstrated that XRIPalpha, but not importin alpha, is required for nuclear import of RPA. Immunodepletion of XRIPalpha from the egg extracts blocks nuclear import of RPA but not that of nucleoplasmin, a classical import substrate. RPA import can be restored by addition of recombinant XRIPalpha. Conversely, depletion of importin alpha blocks import of nucleoplasmin but not that of RPA. GST-XRIPalpha pull-down assay shows that XRIPalpha interacts directly with recombinant importin beta as well as with RPA in vitro. Finally, RPA import can be reconstituted from the recombinant proteins. We propose that XRIPalpha plays the role of importin alpha in the RPA import scheme: XRIPalpha serves as an adaptor to link RPA to importin beta.

Amino Acid Sequence↗

PubMed1999

Structural view of the Ran-Importin beta interaction at 2.3 A resolution.

Transport receptors of the Importin beta family shuttle between the nucleus and cytoplasm and mediate transport of macromolecules through nuclear pore complexes. They interact specifically with the GTP-binding protein Ran, which in turn regulates their interaction with cargo. Here, we report the three-dimensional structure of a complex between Ran bound to the nonhydrolyzable GTP analog GppNHp and a 462-residue fragment from Importin beta. The structure of Importin beta shows 10 tandem repeats resembling HEAT and Armadillo motifs. They form an irregular crescent, the concave site of which forms the interface with Ran-triphosphate. The importin-binding site of Ran does not overlap with that of the Ran-binding domain of RanBP2.

Amino Acid Sequence↗

PubMed1999

The importin beta/importin 7 heterodimer is a functional nuclear import receptor for histone H1.

Import of proteins into the nucleus proceeds through nuclear pore complexes and is largely mediated by nuclear transport receptors of the importin beta family that use direct RanGTP-binding to regulate the interaction with their cargoes. We investigated nuclear import of the linker histone H1 and found that two receptors, importin beta (Impbeta) and importin 7 (Imp7, RanBP7), play a critical role in this process. Individually, the two import receptors bind H1 weakly, but binding is strong for the Impbeta/Imp7 heterodimer. Consistent with this, import of H1 into nuclei of permeabilized mammalian cells requires exogenous Impbeta together with Imp7. Import by the Imp7/Impbeta heterodimer is strictly Ran dependent, the Ran-requiring step most likely being the disassembly of the cargo-receptor complex following translocation into the nucleus. Disassembly is brought about by direct binding of RanGTP to Impbeta and Imp7, whereby the two Ran-binding sites act synergistically. However, whereas an Impbeta/RanGTP interaction appears essential for H1 import, Ran-binding to Imp7 is dispensable. Thus, Imp7 can function in two modes. Its Ran-binding site is essential when operating as an autonomous import receptor, i.e. independently of Impbeta. Within the Impbeta/Imp7 heterodimer, however, Imp7 plays a more passive role than Impbeta and resembles an import adapter.

Allosteric Regulation↗

PubMed1999

CRM1-mediated recycling of snurportin 1 to the cytoplasm.

Importin beta is a major mediator of import into the cell nucleus. Importin beta binds cargo molecules either directly or via two types of adapter molecules, importin alpha, for import of proteins with a classical nuclear localization signal (NLS), or snurportin 1, for import of m3G-capped U snRNPs. Both adapters have an NH2-terminal importin beta-binding domain for binding to, and import by, importin beta, and both need to be returned to the cytoplasm after having delivered their cargoes to the nucleus. We have shown previously that CAS mediates export of importin alpha. Here we show that snurportin 1 is exported by CRM1, the receptor for leucine-rich nuclear export signals (NESs). However, the interaction of CRM1 with snurportin 1 differs from that with previously characterized NESs. First, CRM1 binds snurportin 1 50-fold stronger than the Rev protein and 5,000-fold stronger than the minimum Rev activation domain. Second, snurportin 1 interacts with CRM1 not through a short peptide but rather via a large domain that allows regulation of affinity. Strikingly, snurportin 1 has a low affinity for CRM1 when bound to its m3G-capped import substrate, and a high affinity when substrate-free. This mechanism appears crucial for productive import cycles as it can ensure that CRM1 only exports snurportin 1 that has already released its import substrate in the nucleus.

Animals↗

PubMed1999

Import of DNA into mammalian nuclei by proteins originating from a plant pathogenic bacterium.

Import of DNA into mammalian nuclei is generally inefficient. Therefore, one of the current challenges in human gene therapy is the development of efficient DNA delivery systems. Here we tested whether bacterial proteins could be used to target DNA to mammalian cells. Agrobacterium tumefaciens, a plant pathogen, efficiently transfers DNA as a nucleoprotein complex to plant cells. Agrobacterium-mediated T-DNA transfer to plant cells is the only known example for interkingdom DNA transfer and is widely used for plant transformation. Agrobacterium virulence proteins VirD2 and VirE2 perform important functions in this process. We reconstituted complexes consisting of the bacterial virulence proteins VirD2, VirE2, and single-stranded DNA (ssDNA) in vitro. These complexes were tested for import into HeLa cell nuclei. Import of ssDNA required both VirD2 and VirE2 proteins. A VirD2 mutant lacking its C-terminal nuclear localization signal was deficient in import of the ssDNA-protein complexes into nuclei. Import of VirD2-ssDNA-VirE2 complexes was fast and efficient, and was shown to depended on importin alpha, Ran, and an energy source. We report here that the bacterium-derived and plant-adapted protein-DNA complex, made in vitro, can be efficiently imported into mammalian nuclei following the classical importin-dependent nuclear import pathway. This demonstrates the potential of our approach to enhance gene transfer to animal cells.

Agrobacterium tumefaciens↗

PubMed1999

The translocation of transportin-cargo complexes through nuclear pores is independent of both Ran and energy.

Active transport between nucleus and cytoplasm proceeds through nuclear pore complexes (NPCs) and is mediated largely by shuttling transport receptors that use direct RanGTP binding to coordinate loading and unloading of cargo [1] [2] [3] [4]. Import receptors such as importin beta or transportin bind their substrates at low RanGTP levels in the cytoplasm and release them upon encountering RanGTP in the nucleus, where a high RanGTP concentration is predicted. This substrate release is, in the case of import by the importin alpha/beta heterodimer, coupled directly to importin beta release from the NPCs. If the importin beta -RanGTP interaction is prevented, import intermediates arrest at the nuclear side of the NPCs [5] [6]. This arrest makes it difficult to probe directly the Ran and energy requirements of the actual translocation from the cytoplasmic to the nuclear side of the NPC, which immediately precedes substrate release. Here, we have shown that in the case of transportin, dissociation of transportin-substrate complexes is uncoupled from transportin release from NPCs. This allowed us to dissect the requirements of translocation through the NPC, substrate release and transportin recycling. Surprisingly, translocation of transportin-substrate complexes into the nucleus requires neither Ran nor nucleoside triphosphates (NTPs). It is only nuclear RanGTP, not GTP hydrolysis, that is needed for dissociation of transportin-substrate complexes and for re-export of transportin to the cytoplasm. GTP hydrolysis is apparently required only to restore the import competence of the re-exported transportin and, thus, for multiple rounds of transportin-dependent import. In addition, we provide evidence that at least one type of substrate can also complete NPC passage mediated by importin beta independently of Ran and energy.

Guanosine Triphosphate↗

PubMed1999

Coordination of tRNA nuclear export with processing of tRNA.

Eukaryotic tRNAs are synthesized in the nucleus and need to be exported to the cytoplasm where they function in translation. tRNA export is mediated by exportin-t, which binds tRNA directly and with high affinity. tRNAs are initially synthesized as precursor molecules. Maturation to functional tRNA takes place in the nucleus, precedes export, and includes trimming of the 5' and 3' ends, posttranscriptional addition of the 3' CCA end, nucleoside modifications, and in some cases splicing. Here we address the question of how tRNA maturation is coordinated with export and thus how cytoplasmic accumulation of inactive maturation intermediates is avoided. This could, in principle, be achieved by nuclear retention of immature tRNA or by selective export of the fully mature form. We show that exportin-t has a strong preference for tRNA with correctly processed 5' and 3' ends and nucleoside modification. tRNA recognition by exportin-t can thus be considered as a quality control mechanism for these maturation steps prior to tRNA export. Surprisingly however, exportin-t can efficiently bind unspliced tRNA and intron-containing tRNA is exported when the rate of splicing is slow. During characterization of the exportin-t/tRNA interaction we found that exportin-t recognizes features in the tRNA that are conserved between prokaryotic and eukaryotic tRNAs. Our data suggest that correct tRNA shape, the 5' and 3' terminal ends, and the TpsiC loop are critical for exportin-t binding.

Animals↗