Karyopherins and nuclear import

Proteins of the karyopherin alpha and karyopherin beta families play a central role in nucleocytoplasmic transport. Recently, crystal structures of karyopherin alpha and its complexes with nuclear localization signal peptides, a karyopherin beta2-Ran complex and complexes of full-length and fragments of karyopherin beta1 with import substrates, Ran and nucleoporins have been solved. These karyopherin structures provide valuable insights into understanding the molecular mechanism of nuclear import, especially substrate recognition, substrate release by GTPase and interactions with the nuclear pore complex.     

Nucleoporin FG Domains Facilitate mRNP Remodeling at the Cytoplasmic Face of the Nuclear Pore Complex

Directional export of messenger RNA (mRNA) protein particles (mRNPs) through nuclear pore complexes (NPCs) requires multiple factors. In Saccharomyces cerevisiae, the NPC proteins Nup159 and Nup42 are asymmetrically localized to the cytoplasmic face and have distinct functional domains: a phenylalanine-glycine (FG) repeat domain that docks mRNP transport receptors and domains that bind the DEAD-box ATPase Dbp5 and its activating cofactor Gle1, respectively. We speculated that the Nup42 and Nup159 FG domains play a role in positioning mRNPs for the terminal mRNP-remodeling steps carried out by Dbp5. Here we find that deletion (Δ) of both the Nup42 and Nup159 FG domains results in a cold-sensitive poly(A)+ mRNA export defect. The nup42ΔFG nup159ΔFG mutant also has synthetic lethal genetic interactions with dbp5 and gle1 mutants. RNA cross-linking experiments further indicate that the nup42ΔFG nup159ΔFG mutant has a reduced capacity for mRNP remodeling during export. To further analyze the role of these FG domains, we replaced the Nup159 or Nup42 FG domains with FG domains from other Nups. These FG “swaps” demonstrate that only certain FG domains are functional at the NPC cytoplasmic face. Strikingly, fusing the Nup42 FG domain to the carboxy-terminus of Gle1 bypasses the need for the endogenous Nup42 FG domain, highlighting the importance of proximal positioning for these factors. We conclude that the Nup42 and Nup159 FG domains target the mRNP to Gle1 and Dbp5 for mRNP remodeling at the NPC. Moreover, these results provide key evidence that character and context play a direct role in FG domain function and mRNA export.     

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

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.     

蛋白质的入核运送和它在基因表达中的调节作用

蛋白质进入细胞核是由蛋白质分子内部的核定位信号（nuclear localization signal, NLS）引导的.NLS蛋白首先与NLS受体结合,然后在多种胞浆因子及核孔复合物蛋白的作用下穿过核孔、转位入核.蛋白质上存在NLS并不一定总能够引导蛋白质入核.当NLS被修饰或遮掩时,它们便不能被核转运装置所识别.因而,NLS的遮掩被解除之前,蛋白质一直被扣留在胞浆中.以调节转录因子的入核运送来控制转录因子的活性是基因表达调节的一个新概念,也是细胞生长和分化的另一水平的调节.     

核定位信号及其分析策略

真核细胞核膜上的核孔复合体 (nuclear pore complex, NPC) 是细胞核内外进行物质交换的主要通道, 分子量较小的化合物可自由通过NPC或采取被动扩散的方式进入细胞核, 而分子量为50 kD以上的蛋白质则只能通过主动转运进入细胞核. 以这种方式进入细胞核的 蛋白质必须在其氨基酸序列上拥有特殊的核定位信号(nuclear localization signal, NLS)以被相应的核转运蛋白(karyopherins) 识别. 核定位信号具有多样性, 包括经典核定位信号(classical NLS,cNLS), 内输蛋白β2识别的核定位信号（又称PY模体-NLS）和其它类型的NLS. 每一类NLS具有相似的特征, 但并不具有完全保守的氨基酸组成. 不同的NLS, 往往对应着各不相同的核输入机制. 而对同一蛋白质来说, 也可能同时拥有几个功能性的NLS. 研究核定位信号一方面可以帮助揭示新的大分子物质核转运机制, 另一方面也有助于发现一些蛋白质的新功能. 本文对常见NLS的分类进行了总结, 并介绍了两种常用的NLS预测软件及鉴定NLS的一般策略.     

Binding Site Distribution of Nuclear Transport Receptors and Transport Complexes in Single Nuclear Pore Complexes

Transport through the nuclear pore complex (NPC) involves a large channel and an abundance of binding sites for nuclear transport receptors (NTRs). However, the mechanistically important distribution of NTR-binding sites along the channel is vividly debated. In this study, we visualized binding site distributions directly by two complementary optical super-resolution methods, single-molecule microscopy and 4Pi microscopy. First, we analyzed the distribution of RanGDP because this important nuclear transport substrate has two types of binding sites at the NPC, direct and indirect, NTR-mediated sites. We found that the direct binding sites had a maximum at approximately −30 nm with regard to the NPC center, whereas the indirect transport-relevant binding sites peaked at approximately −10 nm. The 20 nm-shift could be only resolved by 4Pi microscopy because of a two to threefold improved localization precision as compared with single-molecule microscopy. Then we analyzed the distribution of the NTR Kapβ1 and a Kapβ1-based transport complex and found them to have also binding maxima at approximately −10 nm. These observations support transport models in which NTR binding sites are distributed all along the transport channel and argue against models in which the cytoplasmic entrance of the channel is surrounded by a large cloud of binding sites.     

Nucleocytoplasmic transport of proteins

In eukaryotic cells, the movement of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC)--a large protein complex spanning the nuclear envelope. The nuclear transport of proteins is usually mediated by a family of transport receptors known as karyopherins. Karyopherins bind to their cargoes via recognition of nuclear localization signal (NLS) for nuclear import or nuclear export signal (NES) for export to form a transport complex. Its transport through NPC is facilitated by transient interactions between the karyopherins and NPC components. The interactions of karyopherins with their cargoes are regulated by GTPase Ran. In the current review, we describe the NPC structure, NLS, and NES, as well as the model of classic Ran-dependent transport, with special emphasis on existing alternative mechanisms; we also propose a classification of the basic mechanisms of protein transport regulation.     

Centromere Protein B Null Mice are Mitotically and Meiotically Normal but Have Lower Body and Testis Weights

CENP-B is a constitutive centromere DNA-binding protein that is conserved in a number of mammalian species and in yeast. Despite this conservation, earlier cytological and indirect experimental studies have provided conflicting evidence concerning the role of this protein in mitosis. The requirement of this protein in meiosis has also not previously been described. To resolve these uncertainties, we used targeted disruption of the Cenpb gene in mouse to study the functional significance of this protein in mitosis and meiosis. Male and female Cenpb null mice have normal body weights at birth and at weaning, but these subsequently lag behind those of the heterozygous and wild-type animals. The weight and sperm content of the testes of Cenpb null mice are also significantly decreased. Otherwise, the animals appear developmentally and reproductively normal. Cytogenetic fluorescence-activated cell sorting and histological analyses of somatic and germline tissues revealed no abnormality. These results indicate that Cenpb is not essential for mitosis or meiosis, although the observed weight reduction raises the possibility that Cenpb deficiency may subtly affect some aspects of centromere assembly and function, and result in reduced rate of cell cycle progression, efficiency of microtubule capture, and/or chromosome movement. A model for a functional redundancy of this protein is presented.     

Quantitative Analysis of Membrane Protein Transport Across the Nuclear Pore Complex

Nuclear transport of the Saccharomyces cerevisiae membrane proteins Src1/Heh1 and Heh2 across the NPC is facilitated by a long intrinsically disordered linker between the nuclear localization signal (NLS) and the transmembrane domain. The import of reporter proteins derived from Heh2 is dependent on the FG‐Nups in the central channel, and the linker can position the transport factor‐bound NLS in the vicinity of the FG‐Nups in the central channel, while the transmembrane segment resides in the pore membrane. Here, we present a quantitative analysis of karyopherin‐mediated import and passive efflux of reporter proteins derived from Heh2, including data on the mobility of the reporter proteins in different membrane compartments. We show that membrane proteins with extralumenal domains up to 174 kDa, terminal to the linker and NLS, passively leak out of the nucleus via the NPC, albeit at a slow rate. We propose that also during passive efflux, the unfolded linker facilitates the passage of extralumenal domains through the central channel of the NPC .     

昆虫中的核输出现象

核质转运是真核细胞生命活动重要的组成部分,细胞核内蛋白的平衡及转录出的RNA出核成熟过程都依赖于核输出,核输出与真核生物的生命活动密切相关;病原物在侵染昆虫时也会利用宿主的核输出机制干扰宿主的免疫反应或劫持宿主的核输出蛋白以利于病毒的组装及复制.本文主要介绍真核细胞中核输出的基本机制、蚊子和果蝇等模式昆虫中核输出通路;...     

The interaction between importin-α and Nup153 promotes importin-α/β-mediated nuclear import

Nuclear transport is mediated by transport factors, including the importin β family members. The directionality of nuclear transport is governed by the asymmetrical distribution of the small GTPase Ran. Of note, importin α/β-mediated import of classical nuclear localization signal (cNLS)--containing cargo is more efficient than other Ran-dependent import pathways that do not require importin α. In this study, we characterized the role of importin α in nuclear transport by examining import efficiencies of cNLS-cargo/importin α/β complexes. We first depleted digitonin-permeabilized semi-intact cells of endogenous importin α and used the cells to show that the interaction between importin α and Nup153--a component of the nuclear pore complex (NPC)--is essential for efficient import of importin β-binding domain containing substrates, but not other cargoes that directly bind to importin β. Moreover, we found that the binding of importin α to Nup153 facilitates cNLS-mediated import, and demonstrated that importin α in import complexes and cargo-free importin α prebound to Nup153 promote efficient import of cNLS-containing proteins. This is the first in vitro study showing that in conjunction with Nup153, importin α contributes to directionally biased exit of cNLS-containing cargo to the nuclear side of NPCs.     

进入细胞核之门：核移位机制研究进展

细胞中DNA复制和RNA生物形成发生在细胞核,而蛋白质的合成场所位于细胞质,这些生命活动的整合依赖于功能蛋白等在两个亚空间尺度的选择性穿梭.这是一个信号介导的过程,需要能量和可溶性因子如穿梭载体的参与.通过介绍功能蛋白受控核移位机制研究进展,拓宽了其潜在的医学应用,该领域的深入研究,将有力推动抗病毒治疗和基因治疗载体的设计研究.     

Cryo-electron Microscopy Reveals the Structure of the Nuclear Pore Complex

The nuclear pore complex (NPC) is a giant protein assembly that penetrates the double layers of the nuclear membrane. The overall structure of the NPC has approximately eightfold symmetry and is formed by approximately 30 nucleoporins. The great size and complexity of the NPC have hindered the study of its structure for many years until recent breakthroughs were achieved by integrating the latest high-resolution cryo-electron microscopy (cryo-EM), the emerging artificial intelligence-based modeling and all other available structural information from crystallography and mass spectrometry. Here, we review our latest knowledge of the NPC architecture and the history of its structural study from in vitro to in situ with progressively improved resolutions by cryo-EM, with a particular focus on the latest subnanometer-resolution structural studies. The future directions for structural studies of NPCs are also discussed.     

The Nucleocytoplasmic Transport of Viral Proteins

Molecules can enter the nucleus by passive diffusion or active transport mechanisms, depending on their size. Small molecules up to size of 50-60 kDa or less than 10 nm in diameter can diffuse passively through the nuclear pore complex (NPC), while most proteins are transported by energy driven transport mechanisms. Active transport of viral proteins is mediated by nuclear localization signals (NLS), which were first identified in Simian Virus 40 large T antigen and had subsequently been identified in a lar...     

Recombinant Nup153 Incorporates in Vivo into Xenopus Oocyte Nuclear Pore Complexes

Nup153 is a molecular constituent of the nuclear basket of the nuclear pore complex (NPC) that plays a critical role in nuclear export of RNAs and proteins. In an effort to map this nucleoporin more precisely within the nuclear basket we have developed an experimental approach for localizing Nup153 expressed and incorporated in vivo into Xenopus oocyte NPCs. This approach involves the microinjection into the cytoplasm of Xenopus oocytes of in vitro synthesized mRNA from a vector encoding an epitope-tagged cDNA. Here we present results obtained by Western blots, fluorescence microscopy, and immuno-electron microscopy, which clearly document that the heterologous protein is properly expressed, targeted, and incorporated into preexisting Xenopus NPCs. This new approach for localizing nucleoporins within the structure of the NPC overcomes limitations of previous techniques and allows for greater specificity and resolution than have been possible with previous methods.     

Nuclear import of the ran exchange factor, RCC1, is mediated by at least two distinct mechanisms

RCC1, the only known guanine-nucleotide exchange factor for the Ran GTPase, is an approximately 45-kD nuclear protein that can bind chromatin. An important question concerns how RCC1 traverses the nuclear envelope. We now show that nuclear RCC1 is not exported readily in interphase cells and that the import of RCC1 into the nucleoplasm is extremely rapid. Import can proceed by at least two distinct mechanisms. The first is a classic import pathway mediated by basic residues within the NH(2)-terminal domain (NTD) of RCC1. This pathway is dependent upon both a preexisting Ran gradient and energy, and preferentially uses the importin-alpha3 isoform of importin-alpha. The second pathway is not mediated by the NTD of RCC1. This novel pathway does not require importin-alpha or importin-beta or the addition of any other soluble factor in vitro; however, this pathway is saturable and sensitive only to a subset of inhibitors of classical import pathways. Furthermore, the nuclear import of RCC1 does not require a preexisting Ran gradient or energy. We speculate that this second import pathway evolved to ensure that RCC1 never accumulates in the cytoplasm.