scholarly journals Nuclear Export Signal-Interacting Protein Forms Complexes with Lamin A/C-Nups To Mediate the CRM1-Independent Nuclear Export of Large Hepatitis Delta Antigen

2012 ◽  
Vol 87 (3) ◽  
pp. 1596-1604 ◽  
Author(s):  
Cheng Huang ◽  
Jia-Yin Jiang ◽  
Shin C. Chang ◽  
Yeou-Guang Tsay ◽  
Mei-Ru Chen ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Chromosome Region ◽  
Nuclear Export Signal ◽  
Lamin A ◽  
Hepatitis Delta ◽  
Interacting Protein ◽  
Delta Antigen ◽  
C Terminus ◽  
Hepatitis Delta Antigen ◽  
Export Signal

ABSTRACTNuclear export is an important process that not only regulates the functions of cellular factors but also facilitates the assembly of viral nucleoprotein complexes. Chromosome region maintenance 1 (CRM1) that mediates the transport of proteins bearing the classical leucine-rich nuclear export signal (NES) is the best-characterized nuclear export receptor. Recently, several CRM1-independent nuclear export pathways were also identified. The nuclear export of the large form of hepatitis delta antigen (HDAg-L), a nucleocapsid protein of hepatitis delta virus (HDV), which contains a CRM1-independent proline-rich NES, is mediated by the host NES-interacting protein (NESI). The mechanism of the NESI protein in mediating nuclear export is still unknown. In this study, NESI was characterized as a highly glycosylated membrane protein. It interacted and colocalized well in the nuclear envelope with lamin A/C and nucleoporins. Importantly, HDAg-L could be coimmunoprecipitated with lamin A/C and nucleoporins. In addition, binding of the cargo HDAg-L to the C terminus of NESI was detected for the wild-type protein but not for the nuclear export-defective HDAg-L carrying a P205A mutation [HDAg-L(P205A)]. Knockdown of lamin A/C effectively reduced the nuclear export of HDAg-L and the assembly of HDV. These data indicate that by forming complexes with lamin A/C and nucleoporins, NESI facilitates the CRM1-independent nuclear export of HDAg-L.

scholarly journals Novel Nuclear Export Signal-Interacting Protein, NESI, Critical for the Assembly of Hepatitis Delta Virus

2005 ◽  
Vol 79 (13) ◽  
pp. 8113-8120 ◽  
Author(s):  
Yun-Hsin Wang ◽  
Shin C. Chang ◽  
Cheng Huang ◽  
Ya-Ping Li ◽  
Chia-Huei Lee ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Hepatitis Delta Virus ◽  
Critical Role ◽  
Specific Interaction ◽  
Nuclear Export Signal ◽  
Actin Binding ◽  
Hepatitis Delta ◽  
Interacting Protein ◽  
Export Signal ◽  
Delta Virus

ABSTRACT The process of host factor-mediated nucleocytoplasmic transport is critical for diverse cellular events in eukaryotes and the life cycle of viruses. We have previously identified a chromosome region maintenance 1-independent nuclear export signal (NES) at the C terminus of the large form of hepatitis delta antigen (HDAg), designated NES(HDAg-L) that is required for the assembly of hepatitis delta virus (HDV) (C.-H. Lee et al., J. Biol. Chem. 276:8142-8148, 2001). To look for interacting proteins of the NES(HDAg-L), yeast two-hybrid screening was applied using the GAL4-binding domain fused to the NES(HDAg-L) as bait. Among the positive clones, one encodes a protein, designated NESI [NES(HDAg-L) interacting protein] that specifically interacted with the wild-type NES(HDAg-L) but not with the export/package-defective HDAg-L mutant, NES*(HDAg-L), in which Pro-205 has been replaced by Ala. Northern blot analysis revealed NESI as the gene product of a 1.9-kb endogenous mRNA transcript that is present predominantly in human liver tissue. NESI consists of 467 amino acid residues and bears a putative actin-binding site and a bipartite nuclear localization signal. Specific interaction between HDAg-L and NESI was further confirmed by coimmunoprecipitation and immunofluorescence staining. Overexpression of antisense NESI RNAs inhibited the expression of NESI and abolished HDAg-L-mediated nuclear export and assembly of HDV genomic RNA. These data indicate a critical role of NESI in the assembly of HDV through interaction with HDAg-L.

scholarly journals Structural determinants of nuclear export signal orientation in binding to exportin CRM1

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Ho Yee Joyce Fung ◽  
Szu-Chin Fu ◽  
Chad A Brautigam ◽  
Yuh Min Chook
Keyword(s):  
Crystal Structures ◽  
Nuclear Export ◽  
Chromosome Region ◽  
Nuclear Export Signal ◽  
Opposite Orientation ◽  
Structural Determinants ◽  
Large Expansion ◽  
Export Signal

The Chromosome Region of Maintenance 1 (CRM1) protein mediates nuclear export of hundreds of proteins through recognition of their nuclear export signals (NESs), which are highly variable in sequence and structure. The plasticity of the CRM1-NES interaction is not well understood, as there are many NES sequences that seem incompatible with structures of the NES-bound CRM1 groove. Crystal structures of CRM1 bound to two different NESs with unusual sequences showed the NES peptides binding the CRM1 groove in the opposite orientation (minus) to that of previously studied NESs (plus). Comparison of minus and plus NESs identified structural and sequence determinants for NES orientation. The binding of NESs to CRM1 in both orientations results in a large expansion in NES consensus patterns and therefore a corresponding expansion of potential NESs in the proteome.

scholarly journals Calcium-calmodulin kinase I cooperatively regulates nucleocytoplasmic shuttling of CCTα by accessing a nuclear export signal

2012 ◽  
Vol 23 (14) ◽  
pp. 2755-2769 ◽  
Author(s):  
Marianna Agassandian ◽  
Bill B. Chen ◽  
Roopa Pulijala ◽  
Leah Kaercher ◽  
Jennifer R. Glasser ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Membrane Phospholipids ◽  
Nuclear Entry ◽  
Nuclear Trafficking ◽  
C Terminus ◽  
Calmodulin Kinase ◽  
Calcium Stimulation ◽  
Export Signal ◽  
In Cells

We identified a new calmodulin kinase I (CaMKI) substrate, cytidyltransferase (CCTα), a crucial enzyme required for maintenance of cell membranes. CCTα becomes activated with translocation from the cytoplasm to the nuclear membrane, resulting in increased membrane phospholipids. Calcium-activated CCTα nuclear import is mediated by binding of its C-terminus to 14-3-3 ζ, a regulator of nuclear trafficking. Here CaMK1 phosphorylates residues within this C-terminus that signals association of CCTα with 14-3-3 ζ to initiate calcium-induced nuclear entry. CaMKI docks within the CCTα membrane-binding domain (residues 290–299), a sequence that displays similarities to a canonical nuclear export signal (NES) that also binds CRM1/exportin 1. Expression of a CFP-CCTα mutant lacking residues 290–299 in cells results in cytosolically retained enzyme. CRM1/exportin 1 was required for CCTα nuclear export, and its overexpression in cells was partially sufficient to trigger CCTα nuclear export despite calcium stimulation. An isolated CFP-290-299 peptide remained in the nucleus in the presence of leptomycin B but was able to target to the cytoplasm with farnesol. Thus CaMKI vies with CRM1/exportin 1 for access to a NES, and assembly of a CaMKI–14-3-3 ζ–CCTα complex is a key effector mechanism that drives nuclear CCTα translocation.

scholarly journals Clathrin-Mediated Post-Golgi Membrane Trafficking in the Morphogenesis of Hepatitis Delta Virus

2009 ◽  
Vol 83 (23) ◽  
pp. 12314-12324 ◽  
Author(s):  
Cheng Huang ◽  
Shin C. Chang ◽  
Hui-Chin Yang ◽  
Chung-Liang Chien ◽  
Ming-Fu Chang
Keyword(s):  
Membrane Trafficking ◽  
Nuclear Export ◽  
Hepatitis Delta Virus ◽  
Intracellular Transport ◽  
Nuclear Export Signal ◽  
Single Amino Acid ◽  
Dominant Negative Mutant ◽  
Hepatitis Delta ◽  
C Terminus ◽  
Delta Virus

ABSTRACT Clathrin is involved in the endocytosis and exocytosis of cellular proteins and the process of virus infection. We have previously demonstrated that large hepatitis delta antigen (HDAg-L) functions as a clathrin adaptor, but the detailed mechanisms of clathrin involvement in the morphogenesis of hepatitis delta virus (HDV) are not clear. In this study, we found that clathrin heavy chain (CHC) is a key determinant in the morphogenesis of HDV. HDAg-L with a single amino acid substitution at the clathrin box retained nuclear export activity but failed to interact with CHC and to assemble into virus-like particles. Downregulation of CHC function by a dominant-negative mutant or by short hairpin RNA reduced the efficiency of HDV assembly, but not the secretion of hepatitis B virus subviral particles. In addition, the coexistence of a cell-permeable peptide derived from the C terminus of HDAg-L significantly interfered with the intracellular transport of HDAg-L. HDAg-L, small HBsAg, and CHC were found to colocalize with the trans-Golgi network and were highly enriched on clathrin-coated vesicles. Furthermore, genotype II HDV, which assembles less efficiently than genotype I HDV does, has a putative clathrin box in its HDAg-L but interacted only weakly with CHC. The assembly efficiency of the various HDV genotypes correlates well with the CHC-binding activity of their HDAg-Ls and coincides with the severity of disease outcome. Thus, the clathrin box and the nuclear export signal at the C terminus of HDAg-L are potential new molecular targets for HDV therapy.

scholarly journals Facilitated Nucleocytoplasmic Shuttling of the Ran Binding Protein RanBP1

2000 ◽  
Vol 20 (10) ◽  
pp. 3510-3521 ◽  
Author(s):  
Kendra Plafker ◽  
Ian G. Macara
Keyword(s):  
Nuclear Export ◽  
Binding Protein ◽  
Nuclear Export Signal ◽  
Nuclear Accumulation ◽  
Leptomycin B ◽  
Simple Diffusion ◽  
Permeabilized Cells ◽  
C Terminus ◽  
Transport Receptors ◽  
Export Signal

ABSTRACT The Ran binding protein RanBP1 is localized to the cytosol of interphase cells. A leucine-rich nuclear export signal (NES) near the C terminus of RanBP1 is essential to maintain this distribution. We now show that RanBP1 accumulates in nuclei of cells treated with the export inhibitor, leptomycin B, and collapse of the nucleocytoplasmic Ran:GTP gradient leads to equilibration of RanBP1 across the nuclear envelope. Low temperature prevents nuclear accumulation of RanBP1, suggesting that import does not occur via simple diffusion. Glutathione S-transferase (GST)–RanBP1(1-161), which lacks the NES, accumulates in the nucleus after cytoplasmic microinjection. In permeabilized cells, nuclear accumulation of GST-RanBP1(1-161) requires nuclear Ran:GTP but is not inhibited by a dominant interfering G19V mutant of Ran. Nuclear accumulation is enhanced by addition of exogenous karyopherins/importins or RCC1, both of which also enhance nuclear Ran accumulation. Import correlates with Ran concentration. Remarkably, an E37K mutant of RanBP1 does not import into the nuclei under any conditions tested despite the fact that it can form a ternary complex with Ran and importin β. These data indicate that RanBP1 translocates through the pores by an active, nonclassical mechanism and requires Ran:GTP for nuclear accumulation. Shuttling of RanBP1 may function to clear nuclear pores of Ran:GTP, to prevent premature release of import cargo from transport receptors.

scholarly journals Characterization of the Interaction between the Interferon-Induced Protein P56 and the Int6 Protein Encoded by a Locus of Insertion of the Mouse Mammary Tumor Virus

2000 ◽  
Vol 74 (4) ◽  
pp. 1892-1899 ◽  
Author(s):  
Jinjiao Guo ◽  
Ganes C. Sen
Keyword(s):  
Nuclear Export ◽  
Nuclear Protein ◽  
Nuclear Export Signal ◽  
Structural Basis ◽  
Specific Domain ◽  
Interacting Protein ◽  
Amino Terminal ◽  
Tumor Virus ◽  
Export Signal ◽  
Two Hybrid

ABSTRACT For determining cellular functions of the interferon-inducible human cytoplasmic protein P56, we undertook a Saccharomyces cerevisiae two-hybrid screen that identified Int6 as a P56-interacting protein. That the interaction also occurs in human cells was confirmed by coimmunoprecipitation and the observed cytoplasmic displacement of nuclear Int6 upon coexpression of P56. Because Int6 has been claimed to be both a cytoplasmic and a nuclear protein, we investigated the structural basis of this discrepancy. By mutational analyses, we showed that the Int6 protein contains a bipartite nuclear localization signal and a nuclear export signal at the far end of the amino terminus. The 20 amino-terminal residues of Int6, when they were attached to a different nuclear protein, were sufficient to translocate that protein to the cytoplasm. Within this region, replacement of any of the three leucine residues with alanine destroyed the function of the export signal. The specific domain of P56 that is required for its interaction with Int6 was mapped using the yeast two-hybrid assay and a mammalian coimmunoprecipitation assay. Both assays demonstrated that the C-terminal region of P56 containing three specific tetratricopeptide motifs is required for this interaction. In contrast, removal of an internal domain of P56 enhanced the interaction, as quantified by the two-hybrid assay.

scholarly journals The Nuclear Export Receptor Xpo1p Forms Distinct Complexes with NES Transport Substrates and the Yeast Ran Binding Protein 1 (Yrb1p)

2001 ◽  
Vol 12 (3) ◽  
pp. 539-549 ◽  
Author(s):  
Patrick Maurer ◽  
Michael Redd ◽  
Jens Solsbacher ◽  
F. Ralf Bischoff ◽  
Markus Greiner ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Binding Protein ◽  
Nuclear Export Signal ◽  
Nuclear Pore ◽  
Nuclear Targeting ◽  
Gtp Hydrolysis ◽  
C Terminus ◽  
Transport Receptors ◽  
Nuclear Export Receptor ◽  
Export Signal

Xpo1p (Crm1p) is the nuclear export receptor for proteins containing a leucine-rich nuclear export signal (NES). Xpo1p, the NES-containing protein, and GTP-bound Ran form a complex in the nucleus that translocates across the nuclear pore. We have identified Yrb1p as the major Xpo1p-binding protein in Saccharomyces cerevisiae extracts in the presence of GTP-bound Gsp1p (yeast Ran). Yrb1p is cytoplasmic at steady-state but shuttles continuously between the cytoplasm and the nucleus. Nuclear import of Yrb1p is mediated by two separate nuclear targeting signals. Export from the nucleus requires Xpo1p, but Yrb1p does not contain a leucine-rich NES. Instead, the interaction of Yrb1p with Xpo1p is mediated by Gsp1p-GTP. This novel type of export complex requires the acidic C-terminus of Gsp1p, which is dispensable for the binding to importin β-like transport receptors. A similar complex with Xpo1p and Gsp1p-GTP can be formed by Yrb2p, a relative of Yrb1p predominantly located in the nucleus. Yrb1p also functions as a disassembly factor for NES/Xpo1p/Gsp1p-GTP complexes by displacing the NES protein from Xpo1p/Gsp1p. This Yrb1p/Xpo1p/Gsp1p complex is then completely dissociated after GTP hydrolysis catalyzed by the cytoplasmic GTPase activating protein Rna1p.

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