Human Nucleoporins Promote HIV-1 Docking at the Nuclear Pore,Nuclear Import and Integration

The nuclear pore complex (NPC) mediates nucleo-cytoplasmic transport of macromolecules and is an obligatory point of passage and functional bottleneck in the replication of some viruses. The Human Immunodeficiency Virus (HIV) has evolved the required mechanisms for active nuclear import of its genome through the NPC. However the mechanisms by which the NPC allows or even assists HIV translocation are still unknown. We investigated the involvement of four key nucleoporins in HIV-1 docking, translocation, and integration: Nup358/RanBP2, Nup214/CAN, Nup98 and Nup153. Although all induce defects in infectivity when depleted, only Nup153 actually showed any evidence of participating in HIV-1 translocation through the nuclear pore. We show that Nup358/RanBP2 mediates docking of HIV-1 cores on NPC cytoplasmic filaments by interacting with the cores and that the C-terminus of Nup358/RanBP2 comprising a cyclophilin-homology domain contributes to binding. We also show that Nup214/CAN and Nup98 play no role in HIV-1 nuclear import per se: Nup214/CAN plays an indirect role in infectivity read-outs through its effect on mRNA export, while the reduction of expression of Nup98 shows a slight reduction in proviral integration. Our work shows the involvement of nucleoporins in diverse and functionally separable steps of HIV infection and nuclear import.     

核孔复合物的结构、组装及功能

核孔复合物(NPC)是一个巨型分子复合物,相对分子质量约125×106。脊椎动物的NPC由大约30种蛋白质组成,这些蛋白质的序列大多具有FG(苯丙氨酸-甘氨酸)重复序列。NPC锚定于双层核膜上,并且是物质跨核膜运输的惟一通道,它可快速介导小分子物质的被动运输以及大分子物质的主动运输过程。虽然NPC具有较大的相对分子质量和复杂的结构,但它可在细胞分裂过程中分离并重新组装。生物大分子经NPC的跨核膜运输直接影响真核细胞的生长、增殖、分化、发育等多种生命活动。本文重点介绍NPC的结构、组装及其功能特点。     

Crystal Structure of the Human Cytomegalovirus pUL50-pUL53 Core Nuclear Egress Complex Provides Insight into a Unique Assembly Scaffold for Virus-Host Protein Interactions

Nuclear replication of cytomegalovirus relies on elaborate mechanisms of nucleocytoplasmic egress of viral particles. Thus, the role of two essential and conserved viral nuclear egress proteins, pUL50 and pUL53, is pivotal. pUL50 and pUL53 heterodimerize and form a core nuclear egress complex (NEC), which is anchored to the inner nuclear membrane and provides a scaffold for the assembly of a multimeric viral-cellular NEC. Here, we report the crystal structure of the pUL50-pUL53 heterodimer (amino acids 1–175 and 50–292, respectively) at 2.44 Å resolution. Both proteins adopt a globular fold with mixed α and β secondary structure elements. pUL53-specific features include a zinc-binding site and a hook-like N-terminal extension, the latter representing a hallmark element of the pUL50-pUL53 interaction. The hook-like extension (amino acids 59–87) embraces pUL50 and contributes 1510 Ų to the total interface area (1880 Ų). The pUL50 structure overall resembles the recently published NMR structure of the murine cytomegalovirus homolog pM50 but reveals a considerable repositioning of the very C-terminal α-helix of pUL50 upon pUL53 binding. pUL53 shows structural resemblance with the GHKL domain of bacterial sensory histidine kinases. A close examination of the crystal structure indicates partial assembly of pUL50-pUL53 heterodimers to hexameric ring-like structures possibly providing additional scaffolding opportunities for NEC. In combination, the structural information on pUL50-pUL53 considerably improves our understanding of the mechanism of HCMV nuclear egress. It may also accelerate the validation of the NEC as a unique target for developing a novel type of antiviral drug and improved options of broad-spectrum antiherpesviral therapy.     

The Nuclear Pore Complex and Nuclear Transport

Internal membrane bound structures sequester all genetic material in eukaryotic cells. The most prominent of these structures is the nucleus, which is bounded by a double membrane termed the nuclear envelope (NE). Though this NE separates the nucleoplasm and genetic material within the nucleus from the surrounding cytoplasm, it is studded throughout with portals called nuclear pore complexes (NPCs). The NPC is a highly selective, bidirectional transporter for a tremendous range of protein and ribonucleoprotein cargoes. All the while the NPC must prevent the passage of nonspecific macromolecules, yet allow the free diffusion of water, sugars, and ions. These many types of nuclear transport are regulated at multiple stages, and the NPC carries binding sites for many of the proteins that modulate and modify the cargoes as they pass across the NE. Assembly, maintenance, and repair of the NPC must somehow occur while maintaining the integrity of the NE. Finally, the NPC appears to be an anchor for localization of many nuclear processes, including gene activation and cell cycle regulation. All these requirements demonstrate the complex design of the NPC and the integral role it plays in key cellular processes.Taxonomically speaking, all life on earth falls into one of two fundamental groups, the prokaryotes and the eukaryotes. The prokaryotes, the first group to evolve, are single cell organisms bounded by a single membrane. About 1.5 billion years later, a series of evolutionary innovations led to the emergence of eukaryotes. Eukaryotes have multiple inner membrane structures that allow for compartmentalization within the cell, and therefore differentiation of the cell and regulation within it. Ultimately, the greater cellular complexity of eukaryotes allowed them to adopt a multicellular lifestyle, as seen in the plants, fungi and animals of today (reviewed in Field and Dacks 2009).Internal membrane bound structures sequester all genetic material in eukaryotic cells. The most prominent of these structures, which gives the eukaryotes their Greek-rooted name, is the nucleus—the central “kernel” (gr. “karyo-”) of the cell. The nucleus is bounded by a double membrane termed the nuclear envelope (NE), which separates the nucleoplasm and genetic material from the surrounding cytoplasm. However the genetic material in the nucleus is not totally isolated from the rest of the cell. Studded throughout the NE are portals called nuclear pore complexes (NPCs). The NPC is a highly selective, bidirectional transporter for a tremendous range of cargoes. Going into the nucleus, these cargoes include inner nuclear membrane proteins and all the proteins in the nucleoplasm. Going out are RNA-associated proteins that are assembled into ribosomal subunits or messenger ribonucleoproteins (mRNPs). Once transported, the NPC must ensure these cargos are retained in their respective nuclear and cytoplasmic compartments. All the while the NPC must prevent the passage of nonspecific macromolecules, yet allow the free diffusion of water, sugars, and ions. These many types of nuclear transport are regulated at multiple stages, providing a powerful extra level of cellular control that is not necessary in prokaryotes. Assembly, maintenance, and repair of the NPC must somehow occur while maintaining the integrity of the NE. Finally, the NPC appears to be an anchor for localization of many nuclear processes, including gene activation and cell cycle regulation (reviewed in Ahmed and Brickner 2007; Hetzer and Wente 2009). All these requirements demonstrate the complex design of the NPC and the integral role it plays in key cellular processes.     

核孔复合体的动态组装

核孔是介导所有大分子入核出核的唯一通道。在整个生命活动中,核孔复合体的组成蛋白总是处于动态变化中。核孔复合体的动态组装改变了核质转运状态,并最终改变了细胞的功能。     

Nuclear Pore Complex Structure in Birds

The nuclear envelope consists of two parallel membranes enclosing an aqueous lumen. In places there are pores in both membranes at which the two membranes are joined. Within these pores reside the nuclear pore complexes. The current structural models of the nuclear pore complex have been derived from a number of studies using different electron microscopical techniques. Recently, using surface imaging techniques such as field emission in-lens scanning electron microscopy, novel structures have been identified, particularly at the periphery of the structure, most notably the nucleoplasmic basket. One limitation of the current models is that they are based almost entirely on nuclear envelopes isolated from amphibian oocytes and a pressing question is whether this structure is the same in other organisms and tissues. Here we have studied the structure of nuclear envelopes isolated from bird oocytes. We show that the overall structure is remarkably conserved. In particular, recently discovered peripheral structures appear very similar. We see variations in basket conformation but believe that this is related to the functional states of individual pore complexes.     

A Novel Fluorescence-based Genetic Strategy Identifies Mutants of Saccharomyces cerevisiae Defective for Nuclear Pore Complex Assembly

Nuclear pore complexes (NPCs) are large proteinaceous portals for exchanging macromolecules between the nucleus and the cytoplasm. Revealing how this transport apparatus is assembled will be critical for understanding the nuclear transport mechanism. To address this issue and to identify factors that regulate NPC formation and dynamics, a novel fluorescence-based strategy was used. This approach is based on the functional tagging of NPC proteins with the green fluorescent protein (GFP), and the hypothesis that NPC assembly mutants will have distinct GFP-NPC signals as compared with wild-type (wt) cells. By fluorescence-activated cell sorting for cells with low GFP signal from a population of mutagenized cells expressing GFP-Nup49p, three complementation groups were identified: two correspond to mutant nup120 and gle2 alleles that result in clusters of NPCs. Interestingly, a third group was a novel temperature-sensitive allele of nup57. The lowered GFP-Nup49p incorporation in the nup57-E17 cells resulted in a decreased fluorescence level, which was due in part to a sharply diminished interaction between the carboxy-terminal truncated nup57p^E17 and wt Nup49p. Interestingly, the nup57-E17 mutant also affected the incorporation of a specific subset of other nucleoporins into the NPC. Decreased levels of NPC-associated Nsp1p and Nup116p were observed. In contrast, the localizations of Nic96p, Nup82p, Nup159p, Nup145p, and Pom152p were not markedly diminished. Coincidentally, nuclear import capacity was inhibited. Taken together, the identification of such mutants with specific perturbations of NPC structure validates this fluorescence-based strategy as a powerful approach for providing insight into the mechanism of NPC biogenesis.     

核孔复合体的结构组装及其生理功能

核孔复合体(nuclear pore complex,NPC)介导了大分子物质在细胞质与细胞核之间的穿梭运动。NPC定位于核膜,形成一个疏水通道,使得蛋白质等大分子物质与转运受体结合,进行跨膜转运。这种经核孔复合体进行的跨核膜转运在细胞增殖、细胞分化以及个体发育等生命活动中发挥了重要的生理功能。     

Energetics of Transport through the Nuclear Pore Complex

Molecular transport across the nuclear envelope in eukaryotic cells is solely controlled by the nuclear pore complex (NPC). The NPC provides two types of nucleocytoplasmic transport: passive diffusion of small molecules and active chaperon-mediated translocation of large molecules. It has been shown that the interaction between intrinsically disordered proteins that line the central channel of the NPC and the transporting cargoes is the determining factor, but the exact mechanism of transport is yet unknown. Here, we use coarse-grained molecular dynamics simulations to quantify the energy barrier that has to be overcome for molecules to pass through the NPC. We focus on two aspects of transport. First, the passive transport of model cargo molecules with different sizes is studied and the size selectivity feature of the NPC is investigated. Our results show that the transport probability of cargoes is significantly reduced when they are larger than ∼5 nm in diameter. Secondly, we show that incorporating hydrophobic binding spots on the surface of the cargo effectively decreases the energy barrier of the pore. Finally, a simple transport model is proposed which characterizes the energy barrier of the NPC as a function of diameter and hydrophobicity of the transporting particles.     

Structure of an Asymmetric Ternary Protein Complex Provides Insight for Membrane Interaction

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Highlights? S100A10, annexin A2, and AHNAK are involved in membrane repair ? The AHNAK binding region forms a coiled structure ? Both S100A10 and annexin A2 make contacts to AHNAK and are necessary for binding ? The asymmetric architecture provides a model for AHNAK translocation to the membrane     

FUS-NLS/Transportin 1 Complex Structure Provides Insights into the Nuclear Targeting Mechanism of FUS and the Implications in ALS

The C-terminal nuclear localization sequence of FUsed in Sarcoma (FUS-NLS) is critical for its nuclear import mediated by transportin (Trn1). Familial amyotrophic lateral sclerosis (ALS) related mutations are clustered in FUS-NLS. We report here the structural, biochemical and cell biological characterization of the FUS-NLS and its clinical implications. The crystal structure of the FUS-NLS/Trn1 complex shows extensive contacts between the two proteins and a unique α-helical structure in the FUS-NLS. The binding affinity between Trn1 and FUS-NLS (wide-type and 12 ALS-associated mutants) was determined. As compared to the wide-type FUS-NLS (K_D = 1.7 nM), each ALS-associated mutation caused a decreased affinity and the range of this reduction varied widely from 1.4-fold over 700-fold. The affinity of the mutants correlated with the extent of impaired nuclear localization, and more importantly, with the duration of disease progression in ALS patients. This study provides a comprehensive understanding of the nuclear targeting mechanism of FUS and illustrates the significance of FUS-NLS in ALS.     

The Combination of X-Ray Crystallography and Cryo-Electron Microscopy Provides Insight into the Overall Architecture of the Dodecameric Rvb1/Rvb2 Complex

The Rvb1/Rvb2 complex is an essential component of many cellular pathways. The Rvb1/Rvb2 complex forms a dodecameric assembly where six copies of each subunit form two heterohexameric rings. However, due to conformational variability, the way the two rings pack together is still not fully understood. Here, we present the crystal structure and two cryo-electron microscopy reconstructions of the dodecameric, full-length Rvb1/Rvb2 complex, all showing that the interaction between the two heterohexameric rings is mediated through the Rvb1/Rvb2-specific domain II. Two conformations of the Rvb1/Rvb2 dodecamer are present in solution: a stretched conformation also present in the crystal, and a compact conformation. Novel asymmetric features observed in the reconstruction of the compact conformation provide additional insight into the plasticity of the Rvb1/Rvb2 complex.     

CONSTRICTOR: Constraint Modification Provides Insight into Design of Biochemical Networks

Advances in computational methods that allow for exploration of the combinatorial mutation space are needed to realize the potential of synthetic biology based strain engineering efforts. Here, we present Constrictor, a computational framework that uses flux balance analysis (FBA) to analyze inhibitory effects of genetic mutations on the performance of biochemical networks. Constrictor identifies engineering interventions by classifying the reactions in the metabolic model depending on the extent to which their flux must be decreased to achieve the overproduction target. The optimal inhibition of various reaction pathways is determined by restricting the flux through targeted reactions below the steady state levels of a baseline strain. Constrictor generates unique in silico strains, each representing an “expression state”, or a combination of gene expression levels required to achieve the overproduction target. The Constrictor framework is demonstrated by studying overproduction of ethylene in Escherichia coli network models iAF1260 and iJO1366 through the addition of the heterologous ethylene-forming enzyme from Pseudomonas syringae. Targeting individual reactions as well as combinations of reactions reveals in silico mutants that are predicted to have as high as 25% greater theoretical ethylene yields than the baseline strain during simulated exponential growth. Altering the degree of restriction reveals a large distribution of ethylene yields, while analysis of the expression states that return lower yields provides insight into system bottlenecks. Finally, we demonstrate the ability of Constrictor to scan networks and provide targets for a range of possible products. Constrictor is an adaptable technique that can be used to generate and analyze disparate populations of in silico mutants, select gene expression levels and provide non-intuitive strategies for metabolic engineering.     

Scyl1 Facilitates Nuclear tRNA Export in Mammalian Cells by Acting at the Nuclear Pore Complex

Scyl1 is an evolutionarily conserved N-terminal protein kinase-like domain protein that plays a role in COP1-mediated retrograde protein trafficking in mammalian cells. Furthermore, loss of Scyl1 function has been shown to result in neurodegenerative disorders in mice. Here, we report that Scyl1 is also a cytoplasmic component of the mammalian nuclear tRNA export machinery. Like exportin-t, overexpression of Scyl1 restored export of a nuclear export-defective serine amber suppressor tRNA mutant in COS-7 cells. Scyl1 binds tRNA saturably, and associates with the nuclear pore complex by interacting, in part, with Nup98. Scyl1 copurifies with the nuclear tRNA export receptors exportin-t and exportin-5, the RanGTPase, and the eukaryotic elongation factor eEF-1A, which transports aminoacyl-tRNAs to the ribosomes. Scyl1 interacts directly with exportin-t and RanGTP but not with eEF-1A or RanGDP in vitro. Moreover, exportin-t containing tRNA, Scyl1, and RanGTP form a quaternary complex in vitro. Biochemical characterization also suggests that the nuclear aminoacylation-dependent pathway is primarily responsible for tRNA export in mammalian cells. These findings together suggest that Scyl1 participates in the nuclear aminoacylation-dependent tRNA export pathway and may unload aminoacyl-tRNAs from the nuclear tRNA export receptor at the cytoplasmic side of the nuclear pore complex and channels them to eEF-1A.     

Structural and Functional Analysis of SoPIP2;1 Mutants Adds Insight into Plant Aquaporin Gating

Plant plasma membrane aquaporins facilitate water flux into and out of plant cells, thus coupling their cellular function to basic aspects of plant physiology. Posttranslational modifications of conserved phosphorylation sites, changes in cytoplasmic pH and the binding of Ca²⁺ can regulate water transport activity by gating the plasma membrane aquaporins. A structural mechanism unifying these diverse biochemical signals has emerged for the spinach aquaporin SoPIP2;1, although several questions concerning the opening mechanism remain. Here, we describe the X-ray structures of the S115E and S274E single SoPIP2;1 mutants and the corresponding double mutant. Phosphorylation of these serines is believed to increase water transport activity of SoPIP2;1 by opening the channel. However, all mutants crystallised in a closed conformation, as confirmed by water transport assays, implying that neither substitution fully mimics the phosphorylated state. Nevertheless, a half-turn extension of transmembrane helix 1 occurs upon the substitution of Ser115, which draws the C^α atom of Glu31 10 Å away from its wild-type conformation, thereby disrupting the divalent cation binding site involved in the gating mechanism. Mutation of Ser274 disorders the C-terminus but no other significant conformational changes are observed. Inspection of the hydrogen-bond interactions within loop D suggested that the phosphorylation of Ser188 may also produce an open channel, and this was supported by an increased water transport activity for the S188E mutant and molecular dynamics simulations. These findings add additional insight into the general mechanism of plant aquaporin gating.