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ABSTRACT: Nuclear pore complexes (NPCs) mediate the exchange of macromolecules between the cytoplasm and the nucleoplasm. Soluble nuclear transport receptors bind signal-dependent cargos to form transport complexes that diffuse through the NPC and are then disassembled. Although transport receptors enable the NPC's permeability barrier to be overcome, directionality is established by complex assembly and disassembly. Here, we delineate the choreography of importin-α/CAS complex assembly and disassembly in permeabilized cells, using single-molecule fluorescence resonance energy transfer and particle tracking. Monitoring interaction sequences in intact NPCs ensures spatiotemporal preservation of structures and interactions critical for activity in vivo. We show that key interactions between components are reversible, multiple outcomes are often possible, and the assembly and disassembly of complexes are precisely controlled to occur at the appropriate place and time. Importin-α mutants that impair interactions during nuclear import were used together with cytoplasmic Ran GTPase-activating factors to demonstrate that importin-α/CAS complexes form in the nuclear basket region, at the termination of protein import, and disassembly of importin-α/CAS complexes after export occurs in the cytoplasmic filament region of the NPC. Mathematical models derived from our data emphasize the intimate connection between transport and the coordinated assembly and disassembly of importin-α/CAS complexes for generating productive transport cycles.
Proceedings of the National Academy of Sciences 04/2013; · 9.68 Impact Factor
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ABSTRACT: The PCI fold is based on a stack of α-helices topped with a winged-helix domain and is found in a range of proteins that form central parts of large complexes such as the proteasome lid, the COP9 signalosome, elongation factor eIF3, and the TREX-2 complex. Recent structural determinations have given intriguing insight into how these folds function both to facilitate the generation of larger proteinaceous assembles and also to interact functionally with nucleic acids.
Bioarchitecture. 07/2012; 2(4).
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Christoph Brockmann,
Sharon Soucek,
Sonja I Kuhlmann,
Katherine Mills-Lujan,
Seth M Kelly,
Ji-Chun Yang,
Nahid Iglesias,
Francoise Stutz,
Anita H Corbett,
David Neuhaus, Murray Stewart
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ABSTRACT: Polyadenylation regulation and efficient nuclear export of mature mRNPs both require the polyadenosine-RNA-binding protein, Nab2, which contains seven CCCH Zn fingers. We describe here the solution structure of fingers 5-7, which are necessary and sufficient for high-affinity polyadenosine-RNA binding, and identify key residues involved. These Zn fingers form a single structural unit. Structural coherence is lost in the RNA-binding compromised Nab2-C437S mutant, which also suppresses the rat8-2 allele of RNA helicase Dbp5. Structure-guided Nab2 variants indicate that dbp5(rat8-2) suppression is more closely linked to hyperadenylation and suppression of mutant alleles of the nuclear RNA export adaptor, Yra1, than to affinity for polyadenosine-RNA. These results indicate that, in addition to modulating polyA tail length, Nab2 has an unanticipated function associated with generating export-competent mRNPs, and that changes within fingers 5-7 lead to suboptimal assembly of mRNP export complexes that are more easily disassembled by Dbp5 upon reaching the cytoplasm.
Structure 05/2012; 20(6):1007-18. · 6.35 Impact Factor
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ABSTRACT: Most of the individual components of the nuclear elements of the gene expression pathway have been identified and high-resolution structural information is becoming available for many of them. Information is also starting to become available on the larger complexes they form and is beginning to give clues about how the dynamics of their interactions generate function. Although the translocation of export-competent messenger ribonucleoprotein particles (mRNPs) through the nuclear pore transport channel that is mediated by interactions with nuclear pore proteins (nucleoporins) is relatively well understood, the precise molecular mechanisms underlying the assembly of export-competent mRNPs in the nucleus and their Dbp5-mediated disassembly in the cytoplasm is less well defined. Considerable information has been obtained on the structure of Dbp5 in its different nucleotide-bound states and in complex with Gle1 or Nup159/NUP214. Although the precise manner by which the Dbp5 ATPase cycle is coupled to mRNP remodelling remains to be established, current models capture many key details of this process. The formation of export-competent mRNPs in the nucleus remains an elusive component of this pathway and the precise nature of the remodelling that generates these mRNPs as well as detailed understanding of the molecular mechanisms by which this step is integrated with the transcriptional, splicing and polyadenylation machinery by the TREX and TREX-2 complexes remain obscure. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.
Biochimica et Biophysica Acta 03/2012; 1819(6):578-92. · 4.66 Impact Factor
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ABSTRACT: Export of messenger RNA (mRNA) from the nucleus to the cytoplasm is a critical step in the gene expression pathway of eukaryotic cells. Here, we report the functional and structural characterization of the mammalian TREX-2 complex and show how it links transcription/processing with nuclear mRNA export. Mammalian TREX-2 is based on a germinal-centre associated nuclear protein (GANP) scaffold to which ENY2, PCID2 and centrins bind and depletion of any of these components inhibits mRNA export. The crystal structure of the GANP:ENY2 complex shows that two ENY2 chains interact directly with GANP, but they have different orientations from those observed on yeast Sac3. GANP is required to recruit ENY2 to nuclear pore complexes (NPCs), but ENY2 is not necessary to recruit GANP, which requires both its CID and MCM3AP domains, together with nucleoporin Nup153. GANP and ENY2 associate with RNA polymerase II and inhibition of mRNA processing redistributes GANP from NPCs into nuclear foci indicating that mammalian TREX-2 is associated with transcription. Thus, we implicate TREX-2 as an integral component of the mammalian mRNA export machinery where it links transcription and nuclear export by facilitating the transfer of mature mRNPs from the nuclear interior to NPCs.
Nucleic Acids Research 02/2012; 40(10):4562-73. · 8.03 Impact Factor
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ABSTRACT: Nematode sperm offer a unique perspective for investigating amoeboid cell motility. These cells display the hallmark features of amoeboid movement but power their locomotion with a cytoskeleton composed of major sperm protein (MSP) filaments in place of the familiar actin cytoskeleton found in other crawling cells. Thus, properties of sperm can be compared to those of actin-rich cells to identify the shared features that are essential to motility. Sperm are simple cells in which cytoskeletal dynamics are tightly coupled to protrusion of the leading edge and retraction of the cell body. These features have facilitated reconstitution of both protrusion and retraction in cell-free extracts and enabled identification of accessory components in the motility apparatus as well as elucidation of the mechanical basis of movement. Six MSP accessory proteins have been isolated including four components of the sperm cytoskeleton and two enzymes that play key roles in regulating cytoskeletal dynamics and locomotion. Analysis of this versatile in vitro motility system has identified motor-independent mechanisms for protrusion and retraction that are based on changes in filament-packing density. These changes result in expansion and contraction of the MSP-filament network that generate the forces for movement. We discuss how the mechanisms of motility that operate in nematode sperm may contribute generally to the movement of crawling cells.
International review of cell and molecular biology 01/2012; 297:265-93. · 4.48 Impact Factor
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ABSTRACT: The conserved TREX-2 transcription-export complex integrates transcription and processing of many actively transcribed nascent mRNAs with the recruitment of export factors at nuclear pores and also contributes to transcriptional memory and genomic stability. We report the crystal structure of the Sac3-Thp1-Sem1 segment of Saccharomyces cerevisiae TREX-2 that interfaces with the gene expression machinery. Sac3-Thp1-Sem1 forms a previously uncharacterized PCI-domain complex characterized by the juxtaposition of Sac3 and Thp1 winged helix domains, forming a platform that mediates nucleic acid binding. Our structure-guided mutations support the idea that the Thp1-Sac3 interaction is an essential requirement for mRNA binding and for the coupling of transcription and processing to mRNP assembly and export. These results provide insight into how newly synthesized transcripts are efficiently transferred from TREX-2 to the principal mRNA export factor, and they reveal how Sem1 stabilizes PCI domain-containing proteins and promotes complex assembly.
Nature Structural & Molecular Biology 01/2012; 19(3):328-36. · 12.71 Impact Factor
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ABSTRACT: Crawling movement in eukaryotic cells requires coordination of leading-edge protrusion with cell body retraction [1-3]. Protrusion is driven by actin polymerization along the leading edge [4]. The mechanism of retraction is less clear; myosin contractility may be involved in some cells [5] but is not essential in others [6-9]. In Ascaris sperm, protrusion and retraction are powered by the major sperm protein (MSP) motility system instead of the conventional actin apparatus [10, 11]. These cells lack motor proteins [12] and so are well suited to explore motor-independent mechanisms of retraction. We reconstituted protrusion and retraction simultaneously in MSP filament meshworks, called fibers, that assemble behind plasma membrane-derived vesicles. Retraction is triggered by depolymerization of complete filaments in the rear of the fiber [13]. The surviving filaments reorganize to maintain their packing density. By packing fewer filaments into a smaller volume, the depolymerizing network shrinks and thereby generates sufficient force to move an attached load. Our work provides direct evidence for motor-independent retraction in the reconstituted MSP motility system of nematode sperm. This mechanism could also apply to actin-based cells and may explain reports of cells that crawl even when their myosin activity is compromised.
Current biology: CB 10/2011; 21(20):1727-31. · 10.99 Impact Factor
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ABSTRACT: The toroid-shaped nuclear protein export factor CRM1 is constructed from 21 tandem HEAT repeats, each of which contains an inner (B) and outer (A) α-helix joined by loops. Proteins targeted for export have a nuclear export signal (NES) that binds between the A-helices of HEAT repeats 11 and 12 on the outer surface of CRM1. RanGTP binding increases the affinity of CRM1 for NESs. In the absence of RanGTP, the CRM1 C-terminal helix, together with the HEAT repeat 9 loop, modulates its affinity for NESs. Here we show that there is an electrostatic interaction between acidic residues at the extreme distal tip of the C-terminal helix and basic residues on the HEAT repeat 12 B-helix that lies on the inner surface of CRM1 beneath the NES binding site. Small angle x-ray scattering indicates that the increased affinity for NESs generated by mutations in the C-terminal helix is not associated with large scale changes in CRM1 conformation, consistent with the modulation of NES affinity being mediated by a local change in CRM1 near the NES binding site. These data also suggest that in the absence of RanGTP, the C-terminal helix lies across the CRM1 toroid in a position similar to that seen in the CRM1-Snurportin crystal structure. By creating local changes that stabilize the NES binding site in its closed conformation and thereby reducing the affinity of CRM1 for NESs, the C-terminal helix and HEAT 9 loop facilitate release of NES-containing cargo in the cytoplasm and also inhibit their return to the nucleus.
Journal of Biological Chemistry 06/2011; 286(33):29325-35. · 4.77 Impact Factor
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Murray Stewart
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ABSTRACT: The nuclear export of mRNA, in which Mex67-Mtr2 mediates movement of mature transcripts through nuclear pores, represents the culmination of the nuclear portion of the gene expression pathway. Nuclear export is closely integrated with transcription and processing, and is based on forming a messenger ribonucleoprotein (mRNP) export complex in the nucleus that is able to diffuse back and forth through the pores. Directionality is imposed by remodelling of the mRNP in the cytoplasm, thereby removing key transport-related proteins and preventing its return to the nucleus. The nuclear and cytoplasmic steps of this pathway, in which Mex67-Mtr2 and Nab2 are added and removed, are crucial, and both involve remodelling of the mRNP, which is mediated by DEAD-box helicases together with adaptor and accessory proteins. Recent structural and cell biology results provide key information that should enable development of a detailed understanding of this central cellular process at a molecular level.
Trends in Biochemical Sciences 11/2010; 35(11):609-17. · 10.85 Impact Factor
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ABSTRACT: The structure of solenoid proteins facilitates a higher degree of flexibility than most folded proteins. In importin-β, a nuclear import factor built from 19 tandem HEAT repeats, flexibility plays a crucial role in allowing interactions with a range of different partners. We present a comprehensive analysis of importin-β flexibility based on a number of different approaches. We determined the crystal structure of unliganded Saccharomyces cerevisiae importin-β (Kap95) to allow a quantitative comparison with importin-β bound to different partners. Complementary mutagenesis, small angle X-ray scattering and molecular dynamics studies suggest that the protein samples several conformations in solution. The analyses suggest the flexibility of the solenoid is generated by cumulative small movements along its length. Importin-β illustrates how solenoid proteins can orchestrate protein interactions in many cellular pathways.
Structure 09/2010; 18(9):1171-83. · 6.35 Impact Factor
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ABSTRACT: Following transcription, mRNA is processed, packaged into messenger ribonucleoprotein (mRNP) particles, and transported through nuclear pores (NPCs) to the cytoplasm. At the NPC cytoplasmic face, Dbp5 mediates mRNP remodeling and mRNA export factor dissociation, releasing transcripts for translation. In Saccharomyces cerevisiae, the conserved poly(A) RNA-binding protein, Nab2, facilitates NPC targeting of transcripts and also modulates poly(A) tail length. Dbp5 removes Nab2 from mRNPs at the cytoplasmic face of the pore and, importantly, a Nab2 RNA-binding mutant suppresses the thermosensitive rat8-2 (dbp5) mutant. GFD1 is a multicopy suppressor of rat8-2 (dbp5), and Gfd1 interacts physically with both Dbp5 and the Nab2 N-terminal domain (Nab2-N). Here, we present a structural and functional analysis of the Gfd1/Nab2-N interaction. Crystallography, supported by solution NMR, shows that Gfd1 residues 126-150 form an alpha-helix when bound to Nab2-N. Engineered Nab2-N and Gfd1 mutants that inhibit this interaction in vitro were used to probe its function in vivo using the genetic interaction between GFD1 and NAB2. Although GFD1 is not essential for viability, its deletion severely impairs growth of rat8-2 (dbp5) cells. Moreover, although Gfd1 overexpression suppresses rat8-2 (dbp5), Gfd1 mutants that do not bind Nab2 only partially suppress rat8-2 (dbp5). Furthermore, rat8-2 (dbp5) cells that express nab2-Y34A, in which binding to Gfd1 is impaired, show a synthetic growth phenotype and nuclear accumulation of poly(A) RNA. These data support the importance of the Gfd1/Nab2 interaction for Dbp5 activity and provide further molecular details of the interactions that facilitate Dbp5-mediated mRNP remodeling in the terminal step of mRNA export.
Journal of Biological Chemistry 05/2010; 285(27):20704-15. · 4.77 Impact Factor
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ABSTRACT: Several aspects of mitotic spindle assembly are orchestrated by the Ran GTPase through its modulation of the interaction between spindle assembly factors and importin-alpha. One such factor is TPX2 that promotes microtubule assembly in the vicinity of chromosomes. TPX2 is inhibited when bound to importin-alpha, which occurs when the latter is bound to importin-beta. The importin-alpha:beta interaction is disrupted by the high RanGTP concentration near the chromosomes, releasing TPX2. In more distal regions, where Ran is predominantly GDP-bound, TPX2 remains bound to importin-alpha and so is inhibited. Here we use a combination of structural and biochemical methods to define the basis for TPX2 binding to importin-alpha. A 2.2 A resolution crystal structure shows that the primary nuclear localization signal ((284)KRKH(287)) of TPX2, which has been shown to be crucial for inhibition, binds to the minor NLS-binding site on importin-alpha. This atypical interaction pattern was confirmed using complementary binding studies that employed importin-alpha variants in which binding to either the major or minor NLS-binding site was impaired, together with competition assays using the SV40 monopartite NLS that binds primarily to the major site. The different way in which TPX2 binds to importin-alpha could account for much of the selectivity necessary during mitosis because this would reduce the competition for binding to importin-alpha from other NLS-containing proteins.
Journal of Biological Chemistry 03/2010; 285(23):17628-35. · 4.77 Impact Factor
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ABSTRACT: Sus1 is a central component of the yeast gene gating machinery, the process by which actively transcribing genes such as GAL1 become associated with nuclear pore complexes. Sus1 is a component of both the SAGA transcriptional co-activator complex
and the TREX-2 complex that binds to nuclear pore complexes. TREX-2 contains two Sus1 chains that have an articulated helical
hairpin fold, enabling them to wrap around an extended α-helix in Sac3, following a helical hydrophobic stripe. In SAGA, Sus1
binds to Sgf11 and has been proposed to provide a link between SAGA and TREX-2. We present here the crystal structure of the
complex between Sus1 and the N-terminal region of Sgf11 that forms an extended α-helix around which Sus1 wraps in a manner
that shares some similarities with the Sus1-Sac3 interface in TREX-2. However, the Sus1-binding site on Sgf11 is somewhat
shorter than on Sac3 and is based on a narrower hydrophobic stripe. Engineered mutants that disrupt the Sgf11-Sus1 interaction
in vitro confirm the importance of the hydrophobic helical stripe in molecular recognition. Helix α1 of the Sus1-articulated hairpin
does not bind directly to Sgf11 and adopts a wide range of conformations within and between crystal forms, consistent with
the presence of a flexible hinge and also with results from previous extensive mutagenesis studies (Klöckner, C., Schneider,
M., Lutz, S., Jani, D., Kressler, D., Stewart, M., Hurt, E., and Köhler, A. (2009) J. Biol. Chem. 284, 12049–12056). A single Sus1 molecule cannot bind Sgf11 and Sac3 simultaneously and this, combined with the structure
of the Sus1-Sgf11 complex, indicates that Sus1 forms separate subcomplexes within SAGA and TREX-2.
Journal of Biological Chemistry 02/2010; 285(6):3850-3856. · 4.77 Impact Factor
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ABSTRACT: Sus1 is a central component of the yeast gene gating machinery, the process by which actively transcribing genes such as GAL1 become associated with nuclear pore complexes. Sus1 is a component of both the SAGA transcriptional co-activator complex and the TREX-2 complex that binds to nuclear pore complexes. TREX-2 contains two Sus1 chains that have an articulated helical hairpin fold, enabling them to wrap around an extended alpha-helix in Sac3, following a helical hydrophobic stripe. In SAGA, Sus1 binds to Sgf11 and has been proposed to provide a link between SAGA and TREX-2. We present here the crystal structure of the complex between Sus1 and the N-terminal region of Sgf11 that forms an extended alpha-helix around which Sus1 wraps in a manner that shares some similarities with the Sus1-Sac3 interface in TREX-2. However, the Sus1-binding site on Sgf11 is somewhat shorter than on Sac3 and is based on a narrower hydrophobic stripe. Engineered mutants that disrupt the Sgf11-Sus1 interaction in vitro confirm the importance of the hydrophobic helical stripe in molecular recognition. Helix alpha1 of the Sus1-articulated hairpin does not bind directly to Sgf11 and adopts a wide range of conformations within and between crystal forms, consistent with the presence of a flexible hinge and also with results from previous extensive mutagenesis studies (Klöckner, C., Schneider, M., Lutz, S., Jani, D., Kressler, D., Stewart, M., Hurt, E., and Köhler, A. (2009) J. Biol. Chem. 284, 12049-12056). A single Sus1 molecule cannot bind Sgf11 and Sac3 simultaneously and this, combined with the structure of the Sus1-Sgf11 complex, indicates that Sus1 forms separate subcomplexes within SAGA and TREX-2.
Journal of Biological Chemistry 12/2009; 285(6):3850-6. · 4.77 Impact Factor
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Murray Stewart
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ABSTRACT: In eukaryotic organisms, RNAs are generated in the cell nucleus but many perform their functions in the cytoplasm and so need
to be exported through pores in the nuclear envelope. Because many RNAs must be processed or modified in the nucleus, it is
important that only the fully mature RNA is exported. Stringent molecular recognition is required to identify the class of
RNA and ensure that its processing has been completed. On page 1275 of this issue, Okada et al. provide remarkable insight into this export process for pre-microRNAs (1), which—after further digestion in the cytoplasm—function to regulate specific mRNAs. Unexpectedly, pre-microRNAs are recognized
for export in a quite different way to that recently reported by Cook et al. for transfer RNA (tRNA) (2).
Science 11/2009; 326(5957):1195-6. · 31.20 Impact Factor
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ABSTRACT: The crawling movement of nematode sperm requires coordination of leading edge protrusion with cell body retraction, both of which are powered by modulation of a cytoskeleton based on major sperm protein (MSP) filaments. We used a cell-free in vitro motility system in which both protrusion and retraction can be reconstituted, to identify two proteins involved in cell body retraction. Pharmacological and depletion-add back assays showed that retraction was triggered by a putative protein phosphatase 2A (PP2A, a Ser/Thr phosphatase activated by tyrosine dephosphorylation). Immunofluorescence showed that PP2A was present in the cell body and was concentrated at the base of the lamellipod where the force for retraction is generated. PP2A targeted MSP fiber protein 3 (MFP3), a protein unique to nematode sperm that binds to the MSP filaments in the motility apparatus. Dephosphorylation of MFP3 caused its release from the cytoskeleton and generated filament disassembly. Our results suggest that interaction between PP2A and MFP3 leads to local disassembly of the MSP cytoskeleton at the base of the lamellipod in sperm that in turn pulls the trailing cell body forward.
Molecular biology of the cell 06/2009; 20(14):3200-8. · 5.98 Impact Factor
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ABSTRACT: The ubiquitin-associated (UBA) domain of the principal Saccharomyces cerevisiae mRNA nuclear export factor, Mex67, can bind both nuclear pore protein (nucleoporin) FG repeats and Hpr1, a component of the TREX.THO complex that functions to link transcription and export. Using fluorescence resonance energy transfer-based assays, we show here that Hpr1 and the FG repeats interact with overlapping binding sites on the Mex67 UBA domain. We present the solution structure of the Mex67 UBA domain (UBA-Mex67) complexed with a FXFG nucleoporin peptide and define residues engaged in the interaction and those involved in the FXFG-induced conformational change. We show by NMR titration that the binding of Hpr1 produces analogous changes in chemical shifts in similar regions of the UBA domain. Together the data presented here indicate that both Hpr1 and FXFG nucleoporins may bind in a similar way to the UBA-Mex67 domain. However, whereas binding of Hpr1 allows UBA-Mex67 to interact with tetra-ubiquitin, the complex between UBA-Mex67 and FXFG is unable to bind mono- or tetra-ubiquitin, suggesting that both substrate binding and also the nature of the substrate may influence the affinity of the UBA-Mex67 domain for ubiquitin.
Journal of Biological Chemistry 05/2009; 284(26):17575-83. · 4.77 Impact Factor
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ABSTRACT: The yeast Sac3:Cdc31:Sus1:Thp1 (TREX-2) complex facilitates the repositioning and association of actively transcribing genes with nuclear pores (NPCs)-"gene gating"-that is central to integrating transcription, processing, and mRNA nuclear export. We present here the crystal structure of Sus1 and Cdc31 bound to a central region of Sac3 (the CID domain) that is crucial for its function. Sac3(CID) forms a long, gently undulating alpha helix around which one Cdc31 and two Sus1 chains are wrapped. Sus1 has an articulated helical hairpin fold that facilitates its wrapping around Sac3. In vivo studies using engineered mutations that selectively disrupted binding of individual chains to Sac3 indicated that Sus1 and Cdc31 function synergistically to promote NPC association of TREX-2 and mRNA nuclear export. These data indicate Sac3(CID) provides a scaffold within TREX-2 to integrate interactions between protein complexes to facilitate the coupling of transcription and mRNA export during gene expression.
Molecular cell 04/2009; 33(6):727-37. · 14.61 Impact Factor
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ABSTRACT: Sus1 is an evolutionary conserved protein that functions both in transcription and mRNA export and has been proposed to contribute to coupling these processes in yeast. Sus1 mediates its different roles as a component of both the histone H2B deubiquitinating module (Sus1-Sgf11-Ubp8-Sgf73) of the SAGA (Spt-Ada-Gcn5 acetyltransferase) transcriptional co-activator and the mRNA export complex, TREX-2 (Sus1-Sac3-Thp1-Cdc31). We have dissected the different functions of Sus1 with respect to its partitioning in transcription and export complexes using a mutational approach. Here we show that the sus1-10 (E18A, S19A, and G20A) and sus1-12 (V73A and D75A) alleles of Sus1 can be dissociated from TREX-2 while leaving its interaction with SAGA largely intact. Conversely, the binding to both TREX-2 and SAGA was impaired in the sus1-11 allele (G37A and W38A), in which two highly conserved residues were mutated. In vitro experiments demonstrated that dissociation of mutant Sus1 from its partners is caused by a reduced affinity toward the TREX-2 subunit, Sac3, and the SAGA factor, Sgf11, respectively. Consistent with the biochemical data, these sus1 mutant alleles showed differential genetic relationships with SAGA and mRNA export mutants. In vivo, all three sus1 mutants were impaired in targeting TREX-2 (i.e. Sac3) to the nuclear pore complexes and exhibited nuclear mRNA export defects. This study has implications for how Sus1, in combination with distinct interaction partners, can regulate diverse aspects of gene expression.
Journal of Biological Chemistry 03/2009; 284(18):12049-56. · 4.77 Impact Factor
