Structural basis for the assembly of a nuclear export complex

The nuclear import and export of macromolecular cargoes through nuclear pore complexes is mediated primarily by carriers such as importin-beta. Importins carry cargoes into the nucleus, whereas exportins carry cargoes to the cytoplasm. Transport is orchestrated by nuclear RanGTP, which dissociates cargoes from importins, but conversely is required for cargo binding to exportins. Here we present the 2.0 A crystal structure of the nuclear export complex formed by exportin Cse1p complexed with its cargo (Kap60p) and RanGTP, thereby providing a structural framework for understanding nuclear protein export and the different functions of RanGTP in export and import. In the complex, Cse1p coils around both RanGTP and Kap60p, stabilizing the RanGTP-state and clamping the Kap60p importin-beta-binding domain, ensuring that only cargo-free Kap60p is exported. Mutagenesis indicated that conformational changes in exportins couple cargo binding to high affinity for RanGTP, generating a spring-loaded molecule to facilitate disassembly of the export complex following GTP hydrolysis in the cytoplasm.     

Structure of importin-beta bound to the IBB domain of importin-alpha.

Cytosolic proteins bearing a classical nuclear localization signal enter the nucleus bound to a heterodimer of importin-alpha and importin-beta (also called karyopherin-alpha and -beta). The formation of this heterodimer involves the importin-beta-binding (IBB) domain of importin-alpha, a highly basic amino-terminal region of roughly 40 amino-acid residues. Here we report the crystal structure of human importin-beta bound to the IBB domain of importin-alpha, determined at 2.5 A and 2.3 A resolution in two crystal forms. Importin-beta consists of 19 tandemly repeated HEAT motifs and wraps intimately around the IBB domain. The association involves two separate regions of importin-beta, recognizing structurally distinct parts of the IBB domain: an amino-terminal extended moiety and a carboxy-terminal helix. The structure indicates that significant conformational changes occur when importin-beta binds or releases the IBB domain domain and suggests how dissociation of the importin-alpha/beta heterodimer may be achieved upon nuclear entry.     

Structure of the nuclear transport complex karyopherin-beta2-Ran x GppNHp.

Transport factors in the karyopherin-beta (also called importin-beta) family mediate the movement of macromolecules in nuclear-cytoplasmic transport pathways. Karyopherin-beta2 (transportin) binds a cognate import substrate and targets it to the nuclear pore complex. In the nucleus, Ran x GTP binds karyopherin-beta2 and dissociates the substrate. Here we present the 3.0 A structure of the karyopherin-beta2-Ran x GppNHp complex where GppNHp is a non-hydrolysable GTP analogue. Karyopherin-beta2 contains eighteen HEAT repeats arranged into two continuous orthogonal arches. Ran is clamped in the amino-terminal arch and substrate-binding activity is mapped to the carboxy-terminal arch. A large loop in HEAT repeat 7 spans both arches. Interactions of the loop with Ran and the C-terminal arch implicate it in GTPase-mediated dissociation of the import-substrate. Ran x GppNHp in the complex shows extensive structural rearrangement, compared to Ran GDP, in regions contacting karyopherin-beta2. This provides a structural basis for the specificity of the karyopherin-beta family for the GTP-bound state of Ran, as well as a rationale for interactions of the karyopherin-Ran complex with the regulatory proteins ranGAP, ranGEF and ranBP1.     

Analysis of a RanGTP-regulated gradient in mitotic somatic cells

The RanGTPase cycle provides directionality to nucleocytoplasmic transport, regulating interactions between cargoes and nuclear transport receptors of the importin-beta family. The Ran-importin-beta system also functions in mitotic spindle assembly and nuclear pore and nuclear envelope formation. The common principle underlying these diverse functions throughout the cell cycle is thought to be anisotropy of the distribution of RanGTP (the RanGTP gradient), driven by the chromatin-associated guanine nucleotide exchange factor RCC1 (refs 1, 4, 5). However, the existence and function of a RanGTP gradient during mitosis in cells is unclear. Here we examine the Ran-importin-beta system in cells by conventional and fluorescence lifetime microscopy using a biosensor, termed Rango, that increases its fluorescence resonance energy transfer signal when released from importin-beta by RanGTP. Rango is predominantly free in mitotic cells, but is further liberated around mitotic chromatin. In vitro experiments and modelling show that this localized increase of free cargoes corresponds to changes in RanGTP concentration sufficient to stabilize microtubules in extracts. In cells, the Ran-importin-beta-cargo gradient kinetically promotes spindle formation but is largely dispensable once the spindle has been established. Consistent with previous reports, we observe that the Ran system also affects spindle pole formation and chromosome congression in vivo. Our results demonstrate that conserved Ran-regulated pathways are involved in multiple, parallel processes required for spindle function, but that their relative contribution differs in chromatin- versus centrosome/kinetochore-driven spindle assembly systems.     

Snapshots of nuclear pore complexes in action captured by cryo-electron tomography

Nuclear pore complexes reside in the nuclear envelope of eukaryotic cells and mediate the nucleocytoplasmic exchange of macromolecules. Traffic is regulated by mobile transport receptors that target their cargo to the central translocation channel, where phenylalanine-glycine-rich repeats serve as binding sites. The structural analysis of the nuclear pore is a formidable challenge given its size, its location in a membranous environment and its dynamic nature. Here we have used cryo-electron tomography to study the structure of nuclear pore complexes in their functional environment, that is, in intact nuclei of Dictyostelium discoideum. A new image-processing strategy compensating for deviations of the asymmetric units (protomers) from a perfect eight-fold symmetry enabled us to refine the structure and to identify new features. Furthermore, the superposition of a large number of tomograms taken in the presence of cargo, which was rendered visible by gold nanoparticles, has yielded a map outlining the trajectories of import cargo. Finally, we have performed single-molecule Monte Carlo simulations of nuclear import to interpret the experimentally observed cargo distribution in the light of existing models for nuclear import.     

Structure of a Ran-binding domain complexed with Ran bound to a GTP analogue: implications for nuclear transport

The protein Ran is a small GTP-binding protein that binds to two types of effector inside the cell: Ran-binding proteins, which have a role in terminating export processes from the nucleus to the cytoplasm, and importin-beta-like molecules that bind cargo proteins during nuclear transport. The Ran-binding domain is a conserved sequence motif found in several proteins that participate in these transport processes. The Ran-binding protein RanBP2 contains four of these domains and constitutes a large part of the cytoplasmic fibrils that extend from the nuclear-pore complex. The structure of Ran bound to a non-hydrolysable GTP analogue (Ran x GppNHp) in complex with the first Ran-binding domain (RanBD1) of human RanBP2 reveals not only that RanBD1 has a pleckstrin-homology domain fold, but also that the switch-I region of Ran x GppNHp resembles the canonical Ras GppNHp structure and that the carboxy terminus of Ran is wrapped around RanBD1, contacting a basic patch on RanBD1 through its acidic end. This molecular 'embrace' enables RanBDs to sequester the Ran carboxy terminus, triggering the dissociation of Ran x GTP from importin-beta-related transport factors and facilitating GTP hydrolysis by the GTPase-activating protein ranGAP. Such a mechanism represents a new type of switch mechanism and regulatory protein-protein interaction for a Ras-related protein.     

Structural basis of family-wide Rab GTPase recognition by rabenosyn-5

Rab GTPases regulate all stages of membrane trafficking, including vesicle budding, cargo sorting, transport, tethering and fusion. In the inactive (GDP-bound) conformation, accessory factors facilitate the targeting of Rab GTPases to intracellular compartments. After nucleotide exchange to the active (GTP-bound) conformation, Rab GTPases interact with functionally diverse effectors including lipid kinases, motor proteins and tethering complexes. How effectors distinguish between homologous Rab GTPases represents an unresolved problem with respect to the specificity of vesicular trafficking. Using a structural proteomic approach, we have determined the specificity and structural basis underlying the interaction of the multivalent effector rabenosyn-5 with the Rab family. The results demonstrate that even the structurally similar effector domains in rabenosyn-5 can achieve highly selective recognition of distinct subsets of Rab GTPases exclusively through interactions with the switch and interswitch regions. The observed specificity is determined at a family-wide level by structural diversity in the active conformation, which governs the spatial disposition of critical conserved recognition determinants, and by a small number of both positive and negative sequence determinants that allow further discrimination between Rab GTPases with similar switch conformations.     

RanGTP mediates nuclear pore complex assembly

In metazoa, the nuclear envelope breaks down and reforms during each cell cycle. Nuclear pore complexes (NPCs), which serve as channels for transport between the nucleus and cytoplasm, assemble into the reforming nuclear envelope in a sequential process involving association of a subset of NPC proteins, nucleoporins, with chromatin followed by the formation of a closed nuclear envelope fenestrated by NPCs. How chromatin recruitment of nucleoporins and NPC assembly are regulated is unknown. Here we demonstrate that RanGTP production is required to dissociate nucleoporins Nup107, Nup153 and Nup358 from Importin beta, to target them to chromatin and to induce association between separate NPC subcomplexes. Additionally, either an excess of RanGTP or removal of Importin beta induces formation of NPC-containing membrane structures--annulate lamellae--both in vitro in the absence of chromatin and in vivo. Annulate lamellae formation is strongly and specifically inhibited by an excess of Importin beta. The data demonstrate that RanGTP triggers distinct steps of NPC assembly, and suggest a mechanism for the spatial restriction of NPC assembly to the surface of chromatin.     

TRAPPI tethers COPII vesicles by binding the coat subunit Sec23

The budding of endoplasmic reticulum (ER)-derived vesicles is dependent on the COPII coat complex. Coat assembly is initiated when Sar1-GTP recruits the cargo adaptor complex, Sec23/Sec24, by binding to its GTPase-activating protein (GAP) Sec23 (ref. 2). This leads to the capture of transmembrane cargo by Sec24 (refs 3, 4) before the coat is polymerized by the Sec13/Sec31 complex. The initial interaction of a vesicle with its target membrane is mediated by tethers. We report here that in yeast and mammalian cells the tethering complex TRAPPI (ref. 7) binds to the coat subunit Sec23. This event requires the Bet3 subunit. In vitro studies demonstrate that the interaction between Sec23 and Bet3 targets TRAPPI to COPII vesicles to mediate vesicle tethering. We propose that the binding of TRAPPI to Sec23 marks a coated vesicle for fusion with another COPII vesicle or the Golgi apparatus. An implication of these findings is that the intracellular destination of a transport vesicle may be determined in part by its coat and its associated cargo.     

Identification of specific binding proteins for a nuclear location sequence

The nuclear envelope is a selective barrier against the movement of macromolecules between the nucleus and cytoplasm. Nuclear proteins larger than relative molecular mass 20,000-40,000 are probably actively transported across the envelope through the nuclear pore complex and are directed by specific nuclear location sequences (NLS) in the proteins. NLS mediate the nuclear import of isolated nuclear proteins after microinjection into whole cells and the nuclear accumulation of chimaeric proteins or of non-nuclear proteins conjugated to synthetic peptides. The best-characterized NLS is the simian virus 40 large T-antigen sequence. We have identified two proteins of rat liver by chemical cross-linking that interact with a synthetic peptide containing this sequence: this interaction is specific for a functional NLS, is saturable, and high affinity. The binding proteins are present in a post-mitochondrial supernatant, in nuclei and in a nuclear envelope fraction, which is consistent with a role in the transport of nuclear proteins from the cytoplasm to the nucleus.     

Regulated degradation of a class V myosin receptor directs movement of the yeast vacuole

Normal cellular function requires that organelles be positioned in specific locations. The direction in which molecular motors move organelles is based in part on the polarity of microtubules and actin filaments. However, this alone does not determine the intracellular destination of organelles. For example, the yeast class V myosin, Myo2p, moves several organelles to distinct locations during the cell cycle. Thus the movement of each type of Myo2p cargo must be regulated uniquely. Here we report a regulatory mechanism that specifically provides directionality to vacuole movement. The vacuole-specific Myo2p receptor, Vac17p, has a key function in this process. Vac17p binds simultaneously to Myo2p and to Vac8p, a vacuolar membrane protein. The transport complex, Myo2p-Vac17p-Vac8p, moves the vacuole to the bud, and is then disrupted through the degradation of Vac17p. The vacuole is ultimately deposited near the centre of the bud. Removal of a PEST sequence (a potential signal for rapid protein degradation) within Vac17p causes its stabilization and the subsequent 'backward' movement of vacuoles, which mis-targets them to the neck between the mother cell and the bud. Thus the regulated disruption of this transport complex places the vacuole in its proper location. This may be a general mechanism whereby organelles are deposited at their terminal destination.     

Lipid packing sensed by ArfGAP1 couples COPI coat disassembly to membrane bilayer curvature

Protein coats deform flat lipid membranes into buds and capture membrane proteins to form transport vesicles. The assembly/disassembly cycle of the COPI coat on Golgi membranes is coupled to the GTP/GDP cycle of the small G protein Arf1. At the heart of this coupling is the specific interaction of membrane-bound Arf1-GTP with coatomer, a complex of seven proteins that forms the building unit of the COPI coat. Although COPI coat disassembly requires the catalysis of GTP hydrolysis in Arf1 by a specific GTPase-activating protein (ArfGAP1), the precise timing of this reaction during COPI vesicle formation is not known. Using time-resolved assays for COPI dynamics on liposomes of controlled size, we show that the rate of ArfGAP1-catalysed GTP hydrolysis in Arf1 and the rate of COPI disassembly increase over two orders of magnitude as the curvature of the lipid bilayer increases and approaches that of a typical transport vesicle. This leads to a model for COPI dynamics in which GTP hydrolysis in Arf1 is organized temporally and spatially according to the changes in lipid packing induced by the coat.     

ESCRT-III recognition by VPS4 ATPases

The ESCRT (endosomal sorting complex required for transport) pathway is required for terminal membrane fission events in several important biological processes, including endosomal intraluminal vesicle formation, HIV budding and cytokinesis. VPS4 ATPases perform a key function in this pathway by recognizing membrane-associated ESCRT-III assemblies and catalysing their disassembly, possibly in conjunction with membrane fission. Here we show that the microtubule interacting and transport (MIT) domains of human VPS4A and VPS4B bind conserved sequence motifs located at the carboxy termini of the CHMP1-3 class of ESCRT-III proteins. Structures of VPS4A MIT-CHMP1A and VPS4B MIT-CHMP2B complexes reveal that the C-terminal CHMP motif forms an amphipathic helix that binds in a groove between the last two helices of the tetratricopeptide-like repeat (TPR) of the VPS4 MIT domain, but in the opposite orientation to that of a canonical TPR interaction. Distinct pockets in the MIT domain bind three conserved leucine residues of the CHMP motif, and mutations that inhibit these interactions block VPS4 recruitment, impair endosomal protein sorting and relieve dominant-negative VPS4 inhibition of HIV budding. Thus, our studies reveal how the VPS4 ATPases recognize their CHMP substrates to facilitate the membrane fission events required for the release of viruses, endosomal vesicles and daughter cells.     

Splicing factor Sub2p is required for nuclear mRNA export through its interaction with Yra1p.

The yeast nuclear protein Yra1p is an essential export factor for mRNA. Yra1p interacts directly with the mRNA transport factor Mex67p/Mtr2p, which is associated with the nuclear pore. Here, we report a genetic interaction between YRA1 and SUB2, the gene for a DEAD box helicase involved in splicing. Mutation of SUB2 as well as its overexpression leads to a defect in mRNA export. Moreover, Yra1p and Sub2p bind directly to each other both in vivo and in vitro. Significantly, Sub2p and Mex67p/Mtr2p bind to the same domains of Yra1p, and the proteins compete for binding to Yra1p. Together, these data indicate that the spliceosomal component Sub2p is also important in mRNA export and may function to recruit Yra1p to the mRNA. Sub2p may then be displaced from Yra1p by the binding of Mex67p/Mtr2p, which participates in the export of mRNA through the nuclear pores.     

The microtubule aster formation and its role in nuclear envelope assembly around the sperm chromatin in<Emphasis Type="Italic">Xenopus</Emphasis> egg extracts

Nuclear envelope separates cell genome from cyto-plasm in eucaryotic cells and plays a pivotal role in the cell life. The nuclear envelope is composed of two jointed membranes, the inner membrane and the out membrane, embedded the nuclear pore complexes. The out membrane is continuous with the endoplasmic reticulum (ER). The ARTICLES inner membrane faces to and connects with the chromatin through the nuclear lamina, an intermediate filamentous network thought to play a structural role for…