[30] Analysis of Ran/TC4 function in nuclear protein import

Melchior, Frauke; Sweet, Deborah; Gerace, Larry

doi:10.1016/s0076-6879(95)57032-2

Cited by 60 publications

(44 citation statements)

References 21 publications

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“…GST-Ran and His-SPN1 were expressed in BL21(DE3) upon induction with 500 mM isopropyl b-D-1-thiogalactopyranoside and purified according to standard protocols. GST-SPN1 (Strasser et al, 2004), RanBP1 (Kehlenbach et al, 2001), RanGAP (Mahajan et al, 1997), CRM1-His (Guan et al, 2000), Ran and RanQ69L (Melchior et al, 1995), MBP-Nup88 (aa 500-741) and MBP-Nup214 (aa1859-2090) (Hutten and Kehlenbach, 2006) were purified as described before. His-Nup214 was expressed in BL21(DE3) and purified as described above for MBP-NLP1 using Ni-NTA agarose beads and 300 mM imidazole for protein elution.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

The nucleoporin-like protein NLP1 (hCG1) promotes CRM1-dependent nuclear protein export

Waldmann

Spillner

Kehlenbach

2012

Journal of Cell Science

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SummaryTranslocation of transport complexes across the nuclear envelope is mediated by nucleoporins, proteins of the nuclear pore complex that contain phenylalanine-glycine (FG) repeats as a characteristic binding motif for transport receptors. CRM1 (exportin 1), the major export receptor, forms trimeric complexes with RanGTP and proteins containing nuclear export sequences (NESs). We analyzed the role of the nucleoporin-like protein 1, NLP1 (also known as hCG1 and NUPL2) in CRM1-dependent nuclear transport. NLP1, which contains many FG repeats, localizes to the nuclear envelope and could also be mobile within the nucleus. It promotes the formation of complexes containing CRM1 and RanGTP, with or without NES-containing cargo proteins, that can be dissociated by RanBP1 and/or the cytoplasmic nucleoporin Nup214. The FG repeats of NLP1 do not play a major role in CRM1 binding. Overexpression of NLP1 promotes CRM1-dependent export of certain cargos, whereas its depletion by small interfering RNAs leads to reduced export rates. Thus, NLP1 functions as an accessory factor in CRM1-dependent nuclear protein export.

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Section: Protein Expression and Purificationmentioning

confidence: 99%

The nucleoporin-like protein NLP1 (hCG1) promotes CRM1-dependent nuclear protein export

Waldmann

Spillner

Kehlenbach

2012

Journal of Cell Science

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“…GST-M9M was expressed in BL21(-DE3) cells and natively purified by single-step affinity chromatography using glutathione Sepharose beads (Amersham Biosciences), according to the instructions of the manufacturer. Importin-β (Chi and Adam, 1997), importin-5 (Jäkel and Görlich, 1998), importin-7 (Wohlwend et al, 2007, importin-9 (Mühlhäusser et al, 2001), importin-13 (Mingot et al, 2001), transportin (Baake et al, 2001), Ran and RanQ69L (Melchior et al, 1995), and GST-Rev (Arnold et al, 2006a) were expressed as described previously. These proteins were dialysed against transport buffer (TPB; 20 mM HEPES-KOH, pH 7.3, 110 mM KOAc, 2 mM Mg(OAc) 2 , 1 mM EGTA, 2 mM DTT, and 1 μg/ml each of aprotinin, leupeptin and pepstatin), frozen in liquid nitrogen and stored at -80°C.…”

Section: Protein Purificationmentioning

confidence: 99%

The nuclear pore component Nup358 promotes transportin-dependent nuclear import

Hutten

Wälde

Spillner

et al. 2009

Journal of Cell Science

125

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Nup358 (also known as RanBP2), a component of the cytoplasmic filaments of the nuclear pore complex, has been implicated in various nucleocytoplasmic transport pathways. Here, we identify Nup358 as an important factor for transportin-mediated nuclear import. Depletion of Nup358 resulted in a strong inhibition of nuclear import of the human immunodeficiency virus type 1 (HIV-1) Rev protein. HIV-1 Rev is an RNA-binding protein that is required for CRM1 (also known as exportin 1)-dependent nuclear export of unspliced or partially spliced viral RNA. We show that transportin is the major nuclear import receptor for HIV-1 Rev in HeLa cells. Overexpression of transportin strongly promoted nuclear import of HIV-1 Rev in Nup358-depleted cells, indicating that the import receptor becomes rate-limiting under these conditions. Importantly, the import rate of other transportindependent proteins was also significantly reduced in Nup358-depleted cells. Our data therefore suggest a general role for Nup358 in transportin-mediated nuclear import. Supplementary material available online at

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“…Imp␤_N-396, Imp␤_304-C were expressed and purified as described previously (15). Ran/TC4 was expressed in E. coli BL21 (DE3) RP in 2YT medium with 0.5 mM isopropyl 1-thio-␤-D-galactopyranoside at 18°C over night and purified as described (16). The nucleotide exchange of GDP against GMPPNP, a nonhydrolyzable analog of GTP, was performed with the alkaline phosphatase method (17).…”

Section: Recombinant Protein Expression and Protein Purification-mentioning

confidence: 99%

Thermodynamic Analysis of H1 Nuclear Import

Wohlwend

Strasser

Dickmanns

et al. 2007

Journal of Biological Chemistry

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The nuclear import of H1 linker histones is mediated by a heterodimer of transport receptors, known as importin␤ and importin7. Interestingly, both importins separately interact with H1, but only as a dimer they facilitate the translocation through the nuclear pore. We identified the H1 binding site of importin7, comprising two extended acidic loops near the C terminus of importin7. The analysis of the H1 import complex assembly by means of isothermal titration calorimetry revealed that the formation of a receptor heterodimer in vitro is an enthalpy-driven process, whereas subsequent binding of H1 to the heterodimer is entropy-driven. Furthermore, we show that the importin␤ binding domain of importin7 plays a key role in the activation of importin7 by importin␤. This process is allosterically regulated by importin␤ and accounts for a specific tuning of the activity of the importin␤⅐importin7 heterodimer. The results presented here provide new insights into cellular strategies to even energy balances in nuclear import and point toward a general regulation of importin␤-related nuclear import processes.The nuclear transport machinery represents one of the major transport systems in eukaryotic cells connecting the nucleus and the cytosol by bridging the nuclear envelope. Nuclear pore complexes (NPCs) 2 are embedded in the nuclear envelope and provide the channels for nuclear transport. NPCs have a dual function; whereas solutes, ions and small macromolecules (20 -40 kDa) are allowed to diffuse passively through the nuclear pore, larger macromolecules or such, whose transfer has to be tightly controlled, are transported by specific, soluble transport receptors in a signal-dependent manner (for review, see Refs. 1-4). The majority of known transport signals are specifically recognized by members of the importin␤/karyopherin␤ protein superfamily shuttling between nucleus and cytosol, also known as importins and exportins. They are composed of a number of helix-turn-helix motifs termed HEAT tandem repeats, which pack side by side in an almost parallel fashion, forming elongated molecules with a superhelical twist (5-8). Besides one or more substrate binding sites, these proteins additionally comprise a binding site for the small GTPase Ran (9). Ran represents the central mediator of directionality of nucleocytoplasmic trafficking. Its low intrinsic GTPase activity allows for a rigid control of GTP hydrolysis. The cytosolic GTPase-activating protein RanGAP1 acts as activator of Ran; the additional factors RanBP1 and RanBP2 act as GTPase enhancers and further stimulate Ran's GTP hydrolysis rate. Due to the nucleocytoplasmic compartmentalization of the regulatory factors, with RanGAP in the cytosol and RanGEF in the nucleus, a steep gradient of RanGTP is established across the nuclear envelope with high concentrations in the nucleus and low ones in the cytosol. The RanGTP gradient across the nuclear envelope is thought to provoke a unidirectional translocation of cargoes through the NPC; in terms of nuclear import, bindi...

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[30] Analysis of Ran/TC4 function in nuclear protein import

Cited by 60 publications

References 21 publications

The nucleoporin-like protein NLP1 (hCG1) promotes CRM1-dependent nuclear protein export

The nucleoporin-like protein NLP1 (hCG1) promotes CRM1-dependent nuclear protein export

The nuclear pore component Nup358 promotes transportin-dependent nuclear import

Thermodynamic Analysis of H1 Nuclear Import

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