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.     

Structural basis for nuclear import complex dissociation by RanGTP

Nuclear protein import is mediated mainly by the transport factor importin-beta that binds cytoplasmic cargo, most often via the importin-alpha adaptor, and then transports it through nuclear pore complexes. This active transport is driven by disassembly of the import complex by nuclear RanGTP. The switch I and II loops of Ran change conformation with nucleotide state, and regulate its interactions with nuclear trafficking components. Importin-beta consists of 19 HEAT repeats that are based on a pair of antiparallel alpha-helices (referred to as the A- and B-helices). The HEAT repeats stack to yield two C-shaped arches, linked together to form a helicoidal molecule that has considerable conformational flexibility. Here we present the structure of full-length yeast importin-beta (Kap95p or karyopherin-beta) complexed with RanGTP, which provides a basis for understanding the crucial cargo-release step of nuclear import. We identify a key interaction site where the RanGTP switch I loop binds to the carboxy-terminal arch of Kap95p. This interaction produces a change in helicoidal pitch that locks Kap95p in a conformation that cannot bind importin-alpha or cargo. We suggest an allosteric mechanism for nuclear import complex disassembly by RanGTP.     

Tom22 is a multifunctional organizer of the mitochondrial preprotein translocase.

Mitochondrial preproteins are imported by a multisubunit translocase of the outer membrane (TOM), including receptor proteins and a general import pore. The central receptor Tom22 binds preproteins through both its cytosolic domain and its intermembrane space domain and is stably associated with the channel protein Tom40 (refs 11-13). Here we report the unexpected observation that a yeast strain can survive without Tom22, although it is strongly reduced in growth and the import of mitochondrial proteins. Tom22 is a multifunctional protein that is required for the higher-level organization of the TOM machinery. In the absence of Tom22, the translocase dissociates into core complexes, representing the basic import units, but lacks a tight control of channel gating. The single membrane anchor of Tom22 is required for a stable interaction between the core complexes, whereas its cytosolic domain serves as docking point for the peripheral receptors Tom20 and Tom70. Thus a preprotein translocase can combine receptor functions with distinct organizing roles in a multidomain protein.     

The molecular architecture of the nuclear pore complex

Nuclear pore complexes (NPCs) are proteinaceous assemblies of approximately 50 MDa that selectively transport cargoes across the nuclear envelope. To determine the molecular architecture of the yeast NPC, we collected a diverse set of biophysical and proteomic data, and developed a method for using these data to localize the NPC's 456 constituent proteins (see the accompanying paper). Our structure reveals that half of the NPC is made up of a core scaffold, which is structurally analogous to vesicle-coating complexes. This scaffold forms an interlaced network that coats the entire curved surface of the nuclear envelope membrane within which the NPC is embedded. The selective barrier for transport is formed by large numbers of proteins with disordered regions that line the inner face of the scaffold. The NPC consists of only a few structural modules that resemble each other in terms of the configuration of their homologous constituents, the most striking of these being a 16-fold repetition of 'columns'. These findings provide clues to the evolutionary origins of the NPC.     

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.     

川东北地区碳酸盐岩储层孔隙度预测方法研究

许多研究都已经证实在碳酸盐岩储层中孔隙结构对声波速度影响很大,因此在孔隙度反演时必须考虑孔隙结构的影响。通过对Gassmann方程的合理简化并引入Eshelby-Walsh干燥岩石椭球包体近似公式;导出了计算岩石孔隙结构参数的公式,并统计分析出岩石孔隙结构与其它弹性参数间的变化规律。利用这种统计规律推出了考虑碳酸盐岩孔隙结构孔隙度计算公式。由于该式计算孔隙度时考虑了纵波速度、横波速度、密度、岩石孔隙结构参数的影响,使求取孔隙度更加合理。最后测井数据试算结果与川东北地区应用实例都证实利用该方法预测碳酸盐岩孔隙度精度高于常规方法。     

Toll-dependent selection of microbial antigens for presentation by dendritic cells

Dendritic cells constitutively sample the tissue microenvironment and phagocytose both microbial and host apoptotic cells. This leads to the induction of immunity against invading pathogens or tolerance to peripheral self antigens, respectively. The outcome of antigen presentation by dendritic cells depends on their activation status, such that Toll-like receptor (TLR)-induced dendritic cell activation makes them immunogenic, whereas steady-state presentation of self antigens leads to tolerance. TLR-inducible expression of co-stimulatory signals is one of the mechanisms of self/non-self discrimination. However, it is unclear whether or how the inducible expression of co-stimulatory signals would distinguish between self antigens and microbial antigens when both are encountered by dendritic cells during infection. Here we describe a new mechanism of antigen selection in dendritic cells for presentation by major histocompatibility complex class II molecules (MHC II) that is based on the origin of the antigen. We show that the efficiency of presenting antigens from phagocytosed cargo is dependent on the presence of TLR ligands within the cargo. Furthermore, we show that the generation of peptide-MHC class II complexes is controlled by TLRs in a strictly phagosome-autonomous manner.     

纳米-滑溜水在致密砂岩中的滞留及其对微观孔隙结构的影响

纳米材料已被证明可以提高非常规油气采收率,但其在储层孔隙中的吸附与滞留机理尚未明确。本文以大庆油田上白垩统青山口组致密砂岩为研究对象,采用低温液氮吸附、润湿角测定、扫描电镜、核磁共振及离心实验方法,研究了纳米-滑溜水压裂液在孔隙中的吸附与滞留,以及其对微观孔隙结构参数的影响。结果表明,纳米滑溜水压裂液处理后,扫描电镜观察到纳米颗粒在孔隙中滞留,岩石润湿角降低30.28%~58.17%;孔隙结构由平板孔向墨水瓶孔过渡,比表面积及吸附量显著增加;微孔占比减小20%~25%,过渡孔占比增大21%~26%,总孔体积增大;分形维数变小更接近2,孔隙结构变简单。纳米颗粒在储层孔隙中的吸附与滞留,导致微观孔隙结构发生变化。实验结果与认识对纳米-滑溜水压裂液在致密砂岩储层中的应用具有重要意义。     

TMP21 is a presenilin complex component that modulates gamma-secretase but not epsilon-secretase activity

The presenilin proteins (PS1 and PS2) and their interacting partners nicastrin, aph-1 (refs 4, 5) and pen-2 (ref. 5) form a series of high-molecular-mass, membrane-bound protein complexes that are necessary for gamma-secretase and epsilon-secretase cleavage of selected type 1 transmembrane proteins, including the amyloid precursor protein, Notch and cadherins. Modest cleavage activity can be generated by reconstituting these four proteins in yeast and Spodoptera frugiperda (sf9) cells. However, a critical but unanswered question about the biology of the presenilin complexes is how their activity is modulated in terms of substrate specificity and/or relative activities at the gamma and epsilon sites. A corollary to this question is whether additional proteins in the presenilin complexes might subsume these putative regulatory functions. The hypothesis that additional proteins might exist in the presenilin complexes is supported by the fact that enzymatically active complexes have a mass that is much greater than predicted for a 1:1:1:1 stoichiometric complex (at least 650 kDa observed, compared with about 220 kDa predicted). To address these questions we undertook a search for presenilin-interacting proteins that differentially affected gamma- and epsilon-site cleavage events. Here we report that TMP21, a member of the p24 cargo protein family, is a component of presenilin complexes and differentially regulates gamma-secretase cleavage without affecting epsilon-secretase activity.     

半栽培与栽培大豆输导组织的比较研究

使用光学显微技术 ,对半栽培大豆和栽培大豆进行了输导组织的形态研究。观察结果表明 :半栽培大豆都是典型的散口材 ,栽培大豆在结构上要比半栽培大豆进化 ,对环境的适应能力比半栽培大豆强。通过不同时期取材连续切片观察证明半栽培大豆的输导组织的管孔呈管孔链排列 ,端壁倾斜 ,射线宽度较窄 ,分布不密集 ;而栽培大豆的管孔呈管孔团排列 ,端壁平坦 ,射线宽度较半栽培大豆宽 ,且分布密集 ,这些结构的不同使得栽培大豆的输导能力更强 ,输导组织更进化     

低渗透油藏二元复合驱微观机制研究

 选取不同渗透率级别的低渗和特低渗储层岩心进行水驱、二元驱油实验,研究不同驱替方式、驱替压力梯度对驱油效率的影响.实验结果表明,对于低渗透油藏,二元驱油效率高于水驱,随着压力梯度的增大,驱油效率增加,并且渗透率越低,增加幅度越明显.通过核磁共振T₂谱与恒速压汞微观孔喉测试相结合,分析岩心束缚水状态含油饱和度分布及残余油分布规律,发现较高压力梯度下二元驱替后,低渗透岩心中细微孔隙残余油较少,而高渗透岩心中较大孔隙残余油较多.     

特低渗透储层岩石渗透率应力敏感新机制

 地层岩石多孔介质的渗透率应力敏感性一直是石油工业与岩土工程建设等领域持续研究的一个热点课题.在低渗透岩石渗透率应力敏感性实验研究的基础上,提出了毛管束-孔隙网络渗流模型的多孔介质渗透率应力敏感新机制,该模型充分考虑了多孔介质孔隙之间相互连通的复杂性、渗流迂曲度以及不同类型和大小的孔隙对多孔介质渗透率贡献率的差异.有效应力作用下,低渗岩石中作为主要渗流通道的较大孔喉首先被压缩变小,流体渗流阻力和孔隙迂曲度均同时增大,这是导致有效应力加载初期岩石渗透率急剧减小的主要原因.同时渗透率越小的岩心,其中所发育的较大孔喉越少,该部分孔喉闭合后对岩心渗透率的影响越大,因此渗透率越小的岩石应力敏感性越强.与相关学者的研究成果对比表明,本文提出的新模型能够更好地解释低渗透岩石应力敏感性较强的内在原因.     

川东南盆缘复杂构造区龙马溪组页岩孔隙结构及分形特征

川东南盆缘复杂构造区龙马溪组页岩总有机碳(total organic carbon, TOC)含量高、热演化成熟度高,但构造条件复杂,其微观孔隙结构特征及分形特征相关研究较少且与四川盆内页岩存在差异,亟需进一步深入研究。为更好地表征页岩孔隙结构非均质性及其对页岩气富集的影响,综合运用核磁共振、高压压汞及扫描电镜等技术,定量表征复杂构造区页岩微观孔隙结构特征。基于分形理论,利用高压压汞、核磁共振方法获得不同尺度孔隙的分形维数,并探讨分形维数与孔喉结构参数、TOC含量、矿物组分含量的关系及其地质意义。结果表明：川东南盆缘复杂构造区页岩主要发育有机孔、粒间孔和微裂缝。孔隙结构具有多重分形特征,不同尺度孔喉分形维数存在差异,大孔喉复杂程度高于小孔喉,孔隙总分形维数为2.470 2～2.819 1,均值为2.625 6,反映复杂构造区页岩发育更为复杂的孔隙结构,为页岩气提供大量吸附点位,对页岩气聚集具有积极作用。TOC含量和石英含量等因素的共同影响,造成研究区页岩的强非均质性和复杂的孔隙结构特征。与四川盆内页岩相比,研究区页岩孔径分布较广、分形维数偏低。综合分析页岩孔隙结构及分形特征可为昭通示范...     

特低渗储层孔隙结构对CO2驱特征影响

由于强烈的成岩作用,特低渗储层孔隙结构特征复杂,核磁共振实验结果表明,渗透率相近的岩心具有不同的孔隙结构,实验特低渗岩心孔隙结构特征表现为双峰分散型、双峰偏细歪度型及双峰偏粗歪度型;通过核磁共振方法研究了孔隙结构对水驱及后续CO2驱特征的影响,结果表明,不同孔隙结构岩心CO2驱提高采收率机理不同,对于双峰偏细歪度型岩心,CO2驱主要驱替小孔隙中的富集原油,提高了微观波及效率;对于双峰偏粗歪度岩心,CO2驱主要驱替大、中孔隙中的残余油滴和油膜,提高了驱油效率,实验结果为CO2驱提高采收率机理研究及油藏优选提供了参考。     

比表面积及孔径对花岗岩力学性质影响

为研究花岗岩内部结构对其物理力学性质的影响,利用氮吸附原理的比表面积及孔径分析仪测得两种花岗岩的比表面积和孔径分布情况,对比分析了两种花岗岩的密度、孔隙率等物理性质及单轴压缩强度,发现由比表面积和孔径表征的岩石孔隙结构更能合理的解释其宏观力学行为。试验结果表明:比表面积较大的岩样强度较低。比表面积较大的岩样加剧了岩样在加载过程中微裂隙的发育,使试样的力学性能降低;孔体积较大的岩样的强度较低。岩样内部孔隙的存在加剧了内部微裂隙的发育。随着轴向压力的不断加载,这些孔隙被压缩,有的成为了微裂隙发育的通道、有的成为新裂隙产生的起源。这些微裂隙不断延伸、扩张、贯通最终使得试样在压力的作用下破坏;利用排水法测得的孔隙率不能完整的反映岩样内部孔隙的发育状态,而通过氮吸附静态容量法测得的孔体积能够较为完整的反映岩样中孔隙的发育状态。比表面积及孔径分析测试更为准确地得到岩石内部的孔隙结构,测试结果对花岗岩的宏观力学行为做出了合理解释。     

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.     

Karyopherin-mediated import of integral inner nuclear membrane proteins

Targeting of newly synthesized integral membrane proteins to the appropriate cellular compartment is specified by discrete sequence elements, many of which have been well characterized. An understanding of the signals required to direct integral membrane proteins to the inner nuclear membrane (INM) remains a notable exception. Here we show that integral INM proteins possess basic sequence motifs that resemble 'classical' nuclear localization signals. These sequences can mediate direct binding to karyopherin-alpha and are essential for the passage of integral membrane proteins to the INM. Furthermore, karyopherin-alpha, karyopherin-beta1 and the Ran GTPase cycle are required for INM targeting, underscoring parallels between mechanisms governing the targeting of integral INM proteins and soluble nuclear transport. We also provide evidence that specific nuclear pore complex proteins contribute to this process, suggesting a role for signal-mediated alterations in the nuclear pore complex to allow for passage of INM proteins along the pore membrane.