scholarly journals Amino Acid Substitutions of Coiled-Coil Protein Tpr Abrogate Anchorage to the Nuclear Pore Complex but Not Parallel, In-Register Homodimerization

2001 ◽  
Vol 12 (8) ◽  
pp. 2433-2452 ◽  
Author(s):  
Manuela E. Hase ◽  
Nikolai V. Kuznetsov ◽  
Volker C. Cordes
Keyword(s):  
Electron Microscopy ◽  
Amino Acid ◽  
Nuclear Pore Complex ◽  
Nuclear Periphery ◽  
Hybrid Approach ◽  
Coiled Coil ◽  
Nuclear Pore ◽  
Amino Acid Substitutions ◽  
Cell Fractionation ◽  
Terminal Domain

Tpr is a protein component of nuclear pore complex (NPC)-attached intranuclear filaments. Secondary structure predictions suggest a bipartite structure, with a large N-terminal domain dominated by heptad repeats (HRs) typical for coiled-coil–forming proteins. Proposed functions for Tpr have included roles as a homo- or heteropolymeric architectural element of the nuclear interior. To gain insight into Tpr's ultrastructural properties, we have studied recombinant Tpr segments by circular dichroism spectroscopy, chemical cross-linking, and rotary shadowing electron microscopy. We show that polypeptides of the N-terminal domain homodimerize in vitro and represent α-helical molecules of extended rod-like shape. With the use of a yeast two-hybrid approach, arrangement of the coiled-coil is found to be in parallel and in register. To clarify whether Tpr can self-assemble further into homopolymeric filaments, the full-length protein and deletion mutants were overexpressed in human cells and then analyzed by confocal immunofluorescence microscopy, cell fractionation, and immuno-electron microscopy. Surplus Tpr, which does not bind to the NPC, remains in a soluble state of ∼7.5 S and occasionally forms aggregates of entangled molecules but neither self-assembles into extended linear filaments nor stably binds to other intranuclear structures. Binding to the NPC is shown to depend on the integrity of individual HRs; amino acid substitutions within these HRs abrogate NPC binding and render the protein soluble but do not abolish Tpr's general ability to homodimerize. Possible contributions of Tpr to the structural organization of the nuclear periphery in somatic cells are discussed.

scholarly journals Nuclear pore complex evolution: a trypanosome Mlp analogue functions in chromosomal segregation but lacks transcriptional barrier activity

2014 ◽  
Vol 25 (9) ◽  
pp. 1421-1436 ◽  
Author(s):  
Jennifer M. Holden ◽  
Ludek Koreny ◽  
Samson Obado ◽  
Alexander V. Ratushny ◽  
Wei-Ming Chen ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Mitotic Spindle ◽  
Nuclear Periphery ◽  
Coiled Coil ◽  
Nuclear Lamina ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Dependent Manner ◽  
Major Barrier ◽  
African Trypanosomes

The nuclear pore complex (NPC) has dual roles in nucleocytoplasmic transport and chromatin organization. In many eukaryotes the coiled-coil Mlp/Tpr proteins of the NPC nuclear basket have specific functions in interactions with chromatin and defining specialized regions of active transcription, whereas Mlp2 associates with the mitotic spindle/NPC in a cell cycle–dependent manner. We previously identified two putative Mlp-related proteins in African trypanosomes, TbNup110 and TbNup92, the latter of which associates with the spindle. We now provide evidence for independent ancestry for TbNup92/TbNup110 and Mlp/Tpr proteins. However, TbNup92 is required for correct chromosome segregation, with knockout cells exhibiting microaneuploidy and lowered fidelity of telomere segregation. Further, TbNup92 is intimately associated with the mitotic spindle and spindle anchor site but apparently has minimal roles in control of gene transcription, indicating that TbNup92 lacks major barrier activity. TbNup92 therefore acts as a functional analogue of Mlp/Tpr proteins, and, together with the lamina analogue NUP-1, represents a cohort of novel proteins operating at the nuclear periphery of trypanosomes, uncovering complex evolutionary trajectories for the NPC and nuclear lamina.

scholarly journals A Drosophila Tpr protein homolog is localized both in the extrachromosomal channel network and to nuclear pore complexes

1997 ◽  
Vol 110 (8) ◽  
pp. 927-944 ◽  
Author(s):  
G. Zimowska ◽  
J.P. Aris ◽  
M.R. Paddy
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Periphery ◽  
Coiled Coil ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Channel Network ◽  
Nuclear Interior ◽  
Cytoplasmic Transport ◽  
Complex Protein ◽  
Pore Complexes

Here we report structural, molecular, and biochemical characterizations of Bx34, a Drosophila melanogaster nuclear coiled-coil protein which is localized to extrachromosomal and extranucleolar spaces in the nuclear interior and which is homologous to the mammalian nuclear pore complex protein Tpr. In the nuclear interior, Bx34 is excluded from chromosomes and the nucleolus and generally localizes to regions between these structures and the nuclear periphery. This distribution matches the ‘extrachromosomal channel network’ described previously. In the nuclear periphery, Bx34 localizes on or near nuclear pore complexes. Biochemically, Bx34 isolates exclusively with the nuclear matrix fraction. The Bx34 cDNA sequence predicts a large protein (262 kDa) with two distinct structural domains. The Bx34 N-terminal 70% (180 kDa) is predicted to form an extended region of coiled-coil, while the C-terminal 30% (82 kDa) is predicted to be unstructured and acidic. Bx34 shows moderate sequence identity over its entire length to the mammalian nuclear pore complex protein ‘Tpr’ (28% amino acid identity and 50% similarity). Furthermore, several of the sequence motifs and biochemical similarities between Bx34 and Tpr are sufficiently striking that it is likely that Bx34 and Tpr are functionally related. The Bx34 gene exists in a single copy in region 48C of chromosome 2R. The localization of coiled-coil Bx34 to both the nuclear interior and nuclear pore complexes and its sequence similarity to a known nuclear pore complex protein leads to speculations about a role for Bx34 in nucleo-cytoplasmic transport which we can test using molecular genetic approaches.

scholarly journals Structure of the Cytoplasmic Ring of the Xenopus laevis Nuclear Pore Complex

2020 ◽  
Author(s):  
Gaoxingyu Huang ◽  
Yanqing Zhang ◽  
Xuechen Zhu ◽  
Chao Zeng ◽  
Qifan Wang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Xenopus Laevis ◽  
Nuclear Pore Complex ◽  
Single Particle ◽  
Coiled Coil ◽  
Structural Features ◽  
Nuclear Pore ◽  
Structural Elements ◽  
Cryo Electron Microscopy ◽  
Secondary Structural Elements

Nuclear pore complex (NPC) exhibits structural plasticity and has only been characterized at local resolutions of up to 15 Å for the cytoplasmic ring (CR). Here we present a single-particle cryo-electron microscopy (cryo-EM) structure of the CR from Xenopus laevis NPC at average resolutions of 5.5-7.9 Å, with local resolutions reaching 4.5 Å. Improved resolutions allow identification and placement of secondary structural elements in the majority of the CR components. The two Y complexes in each CR subunit interact with each other and associate with those from flanking subunits, forming a circular scaffold. Within each CR subunit, the Nup358-containing region wraps around the stems of both Y complexes, likely stabilizing the scaffold. Nup205 connects the short arms of the two Y complexes and associates with the stem of a neighbouring Y complex. The Nup214-containing region uses an extended coiled-coil to link Nup85 of the two Y complexes and protrudes into the axial pore of the NPC. These previously uncharacterized structural features reveal insights into NPC assembly.

scholarly journals P granules extend the nuclear pore complex environment in the C. elegans germ line

2011 ◽  
Vol 192 (6) ◽  
pp. 939-948 ◽  
Author(s):  
Dustin L. Updike ◽  
Stephanie J. Hachey ◽  
Jeremy Kreher ◽  
Susan Strome
Keyword(s):  
Nuclear Pore Complex ◽  
Germ Plasm ◽  
Germ Line ◽  
Hydrophobic Interactions ◽  
Nuclear Periphery ◽  
Size Exclusion ◽  
Nuclear Pore ◽  
Complex Environment ◽  
P Granules ◽  
C Elegans

The immortal and totipotent properties of the germ line depend on determinants within the germ plasm. A common characteristic of germ plasm across phyla is the presence of germ granules, including P granules in Caenorhabditis elegans, which are typically associated with the nuclear periphery. In C. elegans, nuclear pore complex (NPC)–like FG repeat domains are found in the VASA-related P-granule proteins GLH-1, GLH-2, and GLH-4 and other P-granule components. We demonstrate that P granules, like NPCs, are held together by weak hydrophobic interactions and establish a size-exclusion barrier. Our analysis of intestine-expressed proteins revealed that GLH-1 and its FG domain are not sufficient to form granules, but require factors like PGL-1 to nucleate the localized concentration of GLH proteins. GLH-1 is necessary but not sufficient for the perinuclear location of granules in the intestine. Our results suggest that P granules extend the NPC environment in the germ line and provide insights into the roles of the PGL and GLH family proteins.

scholarly journals Nuclear Import of Adenovirus DNA Involves Direct Interaction of Hexon with an N-Terminal Domain of the Nucleoporin Nup214

2014 ◽  
Vol 89 (3) ◽  
pp. 1719-1730 ◽  
Author(s):  
Aurélia Cassany ◽  
Jessica Ragues ◽  
Tinglu Guan ◽  
Dominique Bégu ◽  
Harald Wodrich ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Nuclear Import ◽  
Viral Genome ◽  
Molecular Basis ◽  
Nuclear Pore ◽  
Viral Dna ◽  
Terminal Domain ◽  
Cytoplasmic Face ◽  
The Impact

ABSTRACTIn this study, we characterized the molecular basis for binding of adenovirus (AdV) to the cytoplasmic face of the nuclear pore complex (NPC), a key step during delivery of the viral genome into the nucleus. We used RNA interference (RNAi) to deplete cells of either Nup214 or Nup358, the two major Phe-Gly (FG) repeat nucleoporins localized on the cytoplasmic side of the NPC, and evaluated the impact on hexon binding and AdV infection. The accumulation of purified hexon trimers or partially disassembled AdV at the nuclear envelope (NE) was observed in digitonin-permeabilized cells in the absence of cytosolic factors. Bothin vitrohexon binding andin vivonuclear import of the AdV genome were strongly reduced in Nup214-depleted cells but still occurred in Nup358-depleted cells, suggesting that Nup214 is a major binding site of AdV during infection. The expression of an NPC-targeted N-terminal domain of Nup214 in Nup214-depleted cells restored the binding of hexon at the NE and the nuclear import of protein VII (pVII), indicating that this region is sufficient to allow AdV binding. We further narrowed the binding site to a 137-amino-acid segment in the N-terminal domain of Nup214. Together, our results have identified a specific region within the N terminus of Nup214 that acts as a direct NPC binding site for AdV.IMPORTANCEAdVs, which have the largest genome of nonenveloped DNA viruses, are being extensively explored for use in gene therapy, especially in alternative treatments for cancers that are refractory to traditional therapies. In this study, we characterized the molecular basis for binding of AdV to the cytoplasmic face of the NPC, a key step for delivery of the viral genome into the nucleus. Our data indicate that a 137-amino-acid region of the nucleoporin Nup214 is a binding site for the major AdV capsid protein, hexon, and that this interaction is required for viral DNA import. These findings provide additional insight on how AdV exploits the nuclear transport machinery for infection. The results could promote the development of new strategies for gene transfer and enhance understanding of the nuclear import of other viral DNA genomes, such as those of papillomavirus or hepatitis B virus that induce specific cancers.

scholarly journals Amino acid substitutions in human growth hormone affect coiled-coil content and receptor binding

2021 ◽  
Author(s):  
Andrei Rajkovic ◽  
Sandesh Kanchugal ◽  
Eldar Abdurakhmanov ◽  
Rebecca Howard ◽  
Astrid Gräslund ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Amino Acid ◽  
Binding Site ◽  
Human Growth Hormone ◽  
Receptor Binding ◽  
Coiled Coil ◽  
Human Growth ◽  
Zinc Binding ◽  
Amino Acid Substitutions ◽  
Great Relevance

The interaction between human Growth Hormone (hGH) and hGH Receptor (hGHR) has great relevance to human diseases such as acromegaly and cancer. HGH has been extensively engineered by other workers to improve binding and other properties. We used a computational screen to select substitutions at single hGH positions within the hGHR-binding site. We find that, while many successfully slow down dissociation of the hGH-hGHR complex once bound, they also slow down the association of hGH to hGHR. We are particularly interested in E174 which belongs to the hGH zinc-binding triad, and which spans coiled-coil helices and obeys the coiled-coil heptad pattern. Surprisingly, substituting E174 with A leads to substantial increase in an experimental measure of coiled-coil content. E174A is known to increase affinity of hGH against hGHR; here we show that this is simply because the off-rate is slowed down more than the on-rate, in line with what has been found for other affinity-improving mutations. For E174Y (and mutations at other sites) the slowdown in on-rate was greater, leading to decreased affinity. The results point to a link between coiled-coiling, zinc binding, and hGHR-binding affinity in hGH, and also suggest rules for choosing affinity-increasing substitutions.

scholarly journals Functional Analysis of Tpr: Identification of Nuclear Pore Complex Association and Nuclear Localization Domains and a Role in mRNA Export

1998 ◽  
Vol 143 (7) ◽  
pp. 1801-1812 ◽  
Author(s):  
Peter Bangs ◽  
Brian Burke ◽  
Christine Powers ◽  
Roger Craig ◽  
Aruna Purohit ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Pore Complex ◽  
Ectopic Expression ◽  
Coiled Coil ◽  
Full Length ◽  
Nuclear Localization Sequence ◽  
Nuclear Pore ◽  
Nuclear Interior ◽  
Mammalian Cell Lines ◽  
Localization Sequence

Tpr is a 270-kD coiled-coil protein localized to intranuclear filaments of the nuclear pore complex (NPC). The mechanism by which Tpr contributes to the structure and function of the nuclear pore is currently unknown. To gain insight into Tpr function, we expressed the full-length protein and several subdomains in mammalian cell lines and examined their effects on nuclear pore function. Through this analysis, we identified an NH2-terminal domain that was sufficient for association with the nucleoplasmic aspect of the NPC. In addition, we unexpectedly found that the acidic COOH terminus was efficiently transported into the nuclear interior, an event that was apparently mediated by a putative nuclear localization sequence. Ectopic expression of the full-length Tpr caused a dramatic accumulation of poly(A)+ RNA within the nucleus. Similar results were observed with domains that localized to the NPC and the nuclear interior. In contrast, expression of these proteins did not appear to affect nuclear import. These data are consistent with a model in which Tpr is tethered to intranuclear filaments of the NPC by its coiled coil domain leaving the acidic COOH terminus free to interact with soluble transport factors and mediate export of macromolecules from the nucleus.

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