Export of Importin α from the Nucleus Is Mediated by a Specific Nuclear Transport Factor

Kutay, Ulrike; Bischoff, F. Ralf; Kostka, Susanne; Kraft, Regine; Görlich, Dirk

doi:10.1016/s0092-8674(00)80372-4

Cited by 597 publications

(654 citation statements)

References 71 publications

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“…The cDNA of human importin-a5 (provided by Matthias Köhler; Reha-Klinik Damp, Damp) (20) was amplified by PCR and cloned into the EcoRI and XhoI sites of pASK-IBA3plus (IBA, Göttingen). A plasmid encoding Histagged human importin-b was provided by Dr. Dirk Görlich, MPI für Biophysikalische Chemie, Göttingen (21).…”

Section: Plasmidsmentioning

confidence: 99%

Microinjected antibodies interfere with protein nucleocytoplasmic shuttling by distinct molecular mechanisms

et al. 2008

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The observation that some antibodies can enter the nucleus after their microinjection into the cytoplasm established the principle of protein nucleocytoplasmic shuttling. Here, we introduce the concept of stationary antibodies for studying nuclear transport, particularly of native proteins. Contrary to the aforementioned translocating immunoglobulins, stationary antibodies do not cross the nuclear envelope. They are distinguished by their ability to trigger the nucleocytoplasmic redistribution of their antigen. What determines these apparently contradictory outcomes has not been explored. We studied a stationary STAT1 antibody and a translocating importin-b antibody. The stationary phenotype resulted from the inhibition of carrier-independent transport. This was not due to crosslinking or precipitation of antigen, because the antigen-antibody complex remained highly mobile. Rather, decoration with stationary antibody precluded actual nuclear pore passage of antigen. In addition, both antibodies inhibited the carrier-dependent translocation via importin-a, but by diverse mechanisms. The translocating antibody blocked the association with importin-a, whereas the stationary antibody prevented the phosphorylation of its antigen, and thus functioned upstream of the importin-a binding step. We identified a stationary antibody to green-fluorescent protein (GFP) and probed the translocation of GFP fusions of STAT1, thyroid hormone receptor and histones, demonstrating general application of this approach. Our results provide an experimental rationale for the use of antibodies as unique tools for dissecting protein nuclear translocation. As the microinjection of stationary antibodies extends to analyses of native proteins, this method can complement and validate results obtained with fluorescent-labeled derivatives. ' 2008 International Society for Advancement of Cytometry Key terms antibody; native protein; microinjection; nucleocytoplasmic shuttling; import; export; GFP TRANSPORT of proteins and macromolecules between the nucleus and the cytoplasm of eukaryotic cells is an important mechanism for cytoplasmic regulation of nuclear activities. Nucleocytoplasmic exchange occurs through specialized channels called ''nuclear pore complexes'' (NPCs), which perforate the double layer of the nuclear envelope (1). The NPCs function as selectivity filters allowing passive diffusion of molecules up to $40 kDa, whereas components of higher molecular weight are usually restricted from autonomous exchange (2,3). A well-characterized pathway for nuclear transport of proteins relies on the GTPase Ran and the transport factors (also called carriers or karyopherins) importin-a and importin-b or CRM1 for import or export, respectively (4,5). In addition, certain large proteins can enter the nucleus, independent of metabolic energy and transport factors via direct interactions with constituents of the nuclear pore (6), and an increasing number of proteins, e.g. STAT1 and importin-b are known to use both pathways consecutively or i...

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Section: Plasmidsmentioning

confidence: 99%

Microinjected antibodies interfere with protein nucleocytoplasmic shuttling by distinct molecular mechanisms

et al. 2008

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“…The preparation of the HeLa extract was as described (Kutay et al+ 1997)+ The expression and purification of recombinant human transportin and RanQ69L have also been described (Izaurralde et al+, 1997b)+ Recombinant zzPABP2 was immobilized on IgG-Sepharose 4B at a concentration of 1 mg/mL of resin+ All binding experiments were performed in 50 mM HEPES, pH 7+6, 150 mM NaCl, 75 mM potassium acetate and 5 mM magnesium acetate+ For each sample, 20 mL of affinity matrix were incubated with either 500 mL of HeLa extract or 200 mL of transportin lysate in a total volume of 1+5 mL for 4 h at 4 8C+ Where indicated RanQ69L was added to 2 mM+ After binding, beads were washed four times with 50 mM HEPES, pH 7+6, 200 mM NaCl, 25 mM potassium acetate, and 5 mM magnesium acetate+ Elution was with 50 mM Tris-HCl, pH 7+5, 1 M MgCl 2 + Samples were precipitated with 90% isopropanol and dissolved in SDS sample buffer+ on cloning experiments+ We are also grateful to Dr+ A+ Nemeth for generously providing PABP2 mutants; Dr+ D+ Görlich and Dr+ G+ Dreyfuss for antibodies directed against importin b, transportin, and hnRNP proteins; Dr+ O+ Bensaude for luciferase plasmid; and Dr+ G+ Dreyfuss for pcDNA-myc-NPc plasmid+ This study was supported by grants from Fundaçãao para a Ciência e Tecnologia/PRAXIS XXI and from the European Union (A+C+ and M+C+-F+); Deutsche Forschungsgemeinschaft (U+K+ and E+W+) and Fonds der Chemischen Industrie (E+W+)+ A+C+ is a fellow from the Gulbenkian Ph+D+ Program+…”

Section: Binding Assaysmentioning

confidence: 99%

Deciphering the cellular pathway for transport of poly(A)-binding protein II

Calado

Kutay

Kühn³

et al. 2000

RNA

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Poly(A)-binding protein II (PABP2)is an abundant nuclear protein that binds with high affinity to nascent poly(A) tails, stimulating their extension and controlling their length. In the cytoplasm, a distinct protein (PABP1) binds to poly(A) tails and participates in mRNA translation and stability. How cytoplasmic PABP1 substitutes for nuclear PABP2 is still unknown. Here we report that PABP2 shuttles back and forth between nucleus and cytoplasm by a carrier-mediated mechanism. A potential novel type of nuclear localization signal exists at the C-terminus of the protein, a domain that is highly enriched in methylated arginines. PABP2 binds directly to transportin in a RanGTP-sensitive manner, suggesting an involvement of this transport receptor in mediating import of the protein into the nucleus. Although PABP2 is small enough to diffuse passively through the nuclear pores, protein fusion experiments reveal the existence of a facilitated export pathway. Accordingly, no transport of PABP2 to the cytoplasm occurs at 4 8C. In contrast, export of PABP2 continues in the absence of transcription, indicating that transport to the cytoplasm is independent of mRNA traffic. Thus, rather than leaving the nucleus as a passive passenger of mRNAs, the data suggest that PABP2 interacts with the nuclear export machinery and may therefore contribute to mRNA transport.

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“…For example, importin 13 (IPO13) has also been shown to have export capacity for the translation initiation factor elF1A (Mingot et al , 2001), and also exportin 4 (XPO4) has a bidirectional transport capability (Gontan et al , 2009). A specialized member of the importin β family is the cellular apoptosis susceptibility (CAS/XPO2) protein that is a recycling factor for importin αs (Kutay et al , 1997). NTRs of the importin α family consist of 10 armadillo (ARM) repeats and an importin β binding (IBB) domain.…”

Section: Introductionmentioning

confidence: 99%

Landscape of nuclear transport receptor cargo specificity

Mackmull

Klaus

Heinze

et al. 2017

Molecular Systems Biology

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Nuclear transport receptors (NTRs) recognize localization signals of cargos to facilitate their passage across the central channel of nuclear pore complexes (NPCs). About 30 different NTRs constitute different transport pathways in humans and bind to a multitude of different cargos. The exact cargo spectrum of the majority of NTRs, their specificity and even the extent to which active nucleocytoplasmic transport contributes to protein localization remains understudied because of the transient nature of these interactions and the wide dynamic range of cargo concentrations. To systematically map cargo–NTR relationships in situ, we used proximity ligation coupled to mass spectrometry (BioID). We systematically fused the engineered biotin ligase BirA* to 16 NTRs. We estimate that a considerable fraction of the human proteome is subject to active nuclear transport. We quantified the specificity and redundancy in NTR interactions and identified transport pathways for cargos. We extended the BioID method by the direct identification of biotinylation sites. This approach enabled us to identify interaction interfaces and to discriminate direct versus piggyback transport mechanisms. Data are available via ProteomeXchange with identifier PXD007976.

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Export of Importin α from the Nucleus Is Mediated by a Specific Nuclear Transport Factor

Cited by 597 publications

References 71 publications

Microinjected antibodies interfere with protein nucleocytoplasmic shuttling by distinct molecular mechanisms

Microinjected antibodies interfere with protein nucleocytoplasmic shuttling by distinct molecular mechanisms

Deciphering the cellular pathway for transport of poly(A)-binding protein II

Landscape of nuclear transport receptor cargo specificity

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