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。     

生物大分子的细胞核质转运

生物大分子通过细胞核孔复合体的转运是真核细胞基因复制、转录和翻译的必要环节 ,也是联系细胞核内外信号转递与参与细胞内核反应 (即细胞增殖、分化、凋亡等核反应 )调控的重要环节。本文主要介绍细胞核孔复合体结构、出入细胞核的转运过程及核转运蛋白与亲核素方面的研究进展 ,细胞核转运过程的深入研究在医药学基础和临床实践都有十分重要的意义。     

植物病毒蛋白核质转运的研究进展

植物病毒编码一些含有核定位信号(nuclear localization signal,NLS)或者核输出信号(nuclear export signal,NES)的核质转运蛋白,这些已被验证的转运蛋白有三种类型:核输入蛋白、核输出蛋白和核质穿梭蛋白。它们通过识别寄主核质转运受体Importinα和Importinβ,介导含有经典核定位信号的蛋白质入核过程,以及寄主蛋白Ran参与,由XPO1介导的富含亮氨酸核输出信号的蛋白质出核过程。植物病毒核质转运蛋白利用寄主的转运机制,进出细胞核发挥相应功能,如介导病毒基因组的核输入和核输出、介导病毒长距离运输及系统侵染、抵抗寄主细胞启动的RNA沉默、调节寄主细胞转录活性、调控病毒的复制及表达和参与病毒症状的形成等。对植物病毒蛋白核质转运的相关研究进展进行综述,着重介绍植物病毒蛋白核质转运类型、核输入和输出信号、转运机制和生物学意义,以及寄主蛋白介导的互作等研究的最新成果。     

Kap与Ran—GTP在核浆转运中的作用

生物大分子（cargo）的核浆转运是由一些转运因子介导的,这些因子包括Kap-α、Kap-β、Ran-GTP及其调节蛋白等。本文阐述了在核浆转运过程中,各因子之间的相互作用及其结构变化,并提出cargo被释放入核的可能机制及一些有待深入研究的问题。     

细胞核质转运及其受体在植物抗病防御反应中的调控作用

植物病害严重威胁全球粮食生产,研究植物对病原菌防御机制和病原菌对寄主作物的侵染过程和分子机制,有助于改良植物种源使其获得持久抗性。近年来, 日渐增多的研究表明, 一些抗病蛋白需要转移到细胞核内才能启动免疫反应,进而发挥抗病防御作用,而细胞核质转运受体是实现这些抗病蛋白核质转运必不可少的“载体”。因此,细胞核质转运及转运...     

KPNB1和Ran蛋白共同介导新城疫病毒基质蛋白的入核转运

【目的】鉴定与新城疫病毒(Newcastle disease virus,NDV)基质蛋白(matrix protein,M)入核相关的细胞蛋白,以阐明NDV M蛋白细胞核定位的分子机制。【方法】从鸡胚成纤维细胞中分别克隆核转运受体蛋白KPNA1–KPNA6和KPNB1基因,将其构建到真核表达载体,并与表达NDV M蛋白的重组真核表达载体分别共转染HEK-293T细胞,通过免疫共沉淀方法鉴定与NDV M蛋白相互作用的核转运受体蛋白。另外,将M蛋白与Ran蛋白突变体或与M蛋白互作的核转运受体蛋白缺失体分别共表达,通过荧光共定位确定M蛋白入核转运相关的细胞蛋白。【结果】构建的重组真核表达载体在HEK-293T细胞中能够正确表达;通过间接免疫荧光观察发现,重组蛋白中除Myc-KPNA2蛋白定位在细胞质外,其它核转运受体蛋白均与M蛋白表现出相同的细胞核定位。免疫共沉淀试验结果表明,M蛋白与KPNA1蛋白和KPNB1蛋白均存在相互作用。进一步通过荧光共定位观察发现,M蛋白与KPNA1蛋白缺失体(DN-KPNA1)共表达不改变M蛋白的细胞核定位,而与KPNB1蛋白缺失体(DN-KPNB1)共表达后导致M蛋白变为细胞质定位,说明M蛋白入核转运需要KPNB1蛋白的参与。另外,将M蛋白与Ran蛋白突变体Ran-Q69L共表达,荧光观察发现M蛋白同样由细胞核定位变为细胞质定位,说明M蛋白入核转运还需要Ran蛋白的辅助。【结论】KPNB1和Ran蛋白共同介导NDV M蛋白的入核转运,其过程是KPNB1蛋白首先和M蛋白发生相互作用并形成复合物,然后通过Ran蛋白的辅助作用完成入核转运。     

核定位信号及其在病毒感染机制中的研究进展

核定位信号介导的蛋白入核是细胞内信号传递网络中核内外物质信息交流的重要一环,绝大多数病毒蛋白进入细胞核均需要核质转运受体识别和结合入核蛋白携带的核定位信号序列。病毒蛋白的入核转运机制在病毒感染过程中起着至关重要的作用,对于病毒的复制、毒力具有重要意义,针对该机制的研究有利于新的抗病毒靶点的发现。本文对核定位信号的分类信息进行了总结,并对不同的核质转运受体及其介导的入核机制进行了比较分析,概述了病毒入核蛋白及其核定位信号在病毒感染机制中的研究发现。     

质膜转运蛋白及其与植物耐盐性关系研究进展

植物细胞质膜有两种主要功能：⑴溶质运输（进出细胞）,溶质运输主要由转运蛋白完成;⑵信号传导,即接收信号并引发细胞生理生化响应。盐分过多对植物的伤害主要是离子毒害。质膜转运蛋白活性环境变化能做现迅速响应。本文简要叙述了植物细胞质膜转运蛋白类型、分子特性、生理功能及其活性调节。介绍了植物细胞质膜Ｈ＾＋－ＡＴＰａｓｅ、质膜氧化还原系统、质膜离子载体和离子通道对盐胁迫的响应及其这些响应与植物耐盐性之间的关     

细胞核质转运受体Importin β家族与转运调控

核质转运是真核细胞的基本生命活动之一。Importinβ家族的蛋白质成员作为核质转运的受体,负责细胞内大部分蛋白质和核酸等生物大分子的跨核膜运输。同时,细胞通过多种方式对核质转运的过程进行精确调控,使底物能够在正确的时间与空间发挥功能,保证细胞增殖与分化的正常进行。核质转运的失调,则使得底物不能正常执行功能,导致个体发育的异常与疾病的发生。     

质膜转运蛋白及其与植物耐盐性关系研究进展

植物细胞质膜有两种主要功能：(1)溶质运输(进出细胞),溶质运输主要由转运蛋白完成;(2)信号传导,即接收信号并引发细胞生理生化响应。盐分过多对植物的伤害主要是离子毒害。质膜转运蛋白活性对环境变化能做出迅速响应。本文简要叙述了植物细胞质膜转运蛋白类型、分子特性、生理功能及其活性调节。介绍了植物细胞质膜H+_ATPase、质膜氧化还原系统、质膜离子载体和离子通道对盐胁迫的响应及其这些响应与植物耐盐性之间的关系。     

Purification of the vertebrate nuclear pore complex by biochemical criteria

The nuclear pore is a large and complex biological machine, mediating all signal-directed transport between the nucleus and the cytoplasm. The vertebrate pore has a mass of ∼120 million daltons or 30 times the size of a ribosome. The large size of the pore, coupled to its tight integration in the nuclear lamina, has hampered the isolation of pore complexes from vertebrate sources. We have now developed a strategy for the purification of nuclear pores from in vitro assembled annulate lamellae (AL), a cytoplasmic mimic of the nuclear envelope that lacks a lamina, nuclear matrix, and chromatin-associated proteins. We find that purified pore complexes from annulate lamellae contain every nuclear pore protein tested. In addition, immunoblotting reveals the presence of soluble transport receptors and factors known to play important roles in the transport of macromolecules through the pore. While transport factors such as Ran and NTF2 show only transient interaction with the pores, a number of soluble transport receptors, including importin β, show a tight association with the purified pores. In summary, we report that we have purified the vertebrate pore by biochemical criteria; silver staining reveals ∼40–50 distinct protein bands.     

Should I stay or should I go? Nucleocytoplasmic trafficking in plant innate immunity

Communication between the cytoplasm and the nucleus is a fundamental feature of eukaryotic cells. Bidirectional transport of macromolecules across the nuclear envelope is typically mediated by receptors and occurs exclusively through nuclear pore complexes (NPCs). The components and molecular mechanisms regulating nucleocytoplasmic trafficking and signalling processes are well studied in animals and yeast but are poorly understood in plants. Current work shows that components of the NPC and the nuclear import and export machinery play essential roles in plant innate immunity. Translocation of defence regulators and Resistance (R) proteins between the cytoplasm and the nucleus are recently uncovered aspects of plant defence responses against pathogens. Future studies will reveal more details on the spatial and temporal dynamics and regulation of this process.     

NTF2 mediates nuclear import of Ran.

Importin beta family transport receptors shuttle between the nucleus and the cytoplasm and mediate transport of macromolecules through nuclear pore complexes (NPCs). The interactions between these receptors and their cargoes are regulated by binding RanGTP; all receptors probably exit the nucleus complexed with RanGTP, and so should deplete RanGTP continuously from the nucleus. We describe here the development of an in vitro system to study how nuclear Ran is replenished. Nuclear import of Ran does not rely on simple diffusion as Ran's small size would permit, but instead is stimulated by soluble transport factors. This facilitated import is specific for cytoplasmic RanGDP and employs nuclear transport factor 2 (NTF2) as the actual carrier. NTF2 binds RanGDP initially to NPCs and probably also mediates translocation of the NTF2-RanGDP complex to the nuclear side of the NPCs. A direct NTF2-RanGDP interaction is crucial for this process, since point mutations that disturb the RanGDP-NTF2 interaction also interfere with Ran import. The subsequent nuclear accumulation of Ran also requires GTP, but not GTP hydrolysis. The release of Ran from NTF2 into the nucleus, and thus the directionality of Ran import, probably involves nucleotide exchange to generate RanGTP, for which NTF2 has no detectable affinity, followed by binding of the RanGTP to an importin beta family transport receptor.     

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.     

Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import

Nuclear import and export signals on macromolecules mediate directional, receptor-driven transport through the nuclear pore complex (NPC) by a process that is suggested to involve the sequential binding of transport complexes to different nucleoporins. The directionality of transport appears to be partly determined by the nucleocytoplasmic compartmentalization of components of the Ran GTPase system. We have analyzed whether the asymmetric localization of discrete nucleoporins can also contribute to transport directionality. To this end, we have used quantitative solid phase binding analysis to determine the affinity of an importin beta cargo complex for Nup358, the Nup62 complex, and Nup153, which are in the cytoplasmic, central, and nucleoplasmic regions of the NPC, respectively. These nucleoporins are proposed to provide progressively more distal binding sites for importin beta during import. Our results indicate that the importin beta transport complex binds to nucleoporins with progressively increasing affinity as the complex moves from Nup358 to the Nup62 complex and to Nup153. Antibody inhibition studies support the possibility that importin beta moves from Nup358 to Nup153 via the Nup62 complex during import. These results indicate that nucleoporins themselves, as well as the nucleocytoplasmic compartmentalization of the Ran system, are likely to play an important role in conferring directionality to nuclear protein import.     

The mRNA export pathway in plants

A double lipid bilayer separating the nucleus from the cytoplasm, termed the nuclear envelope, is a defining feature of eukaryotes. Nucleocytoplasmic transport of macromolecules through the nuclear pores enables fine-tuned regulation of biologic processes. All mature mRNAs are delivered to the cytoplasm from the nucleus via an mRNA export pathway. Much work has been done in yeast and animals to study the machinery of mRNA export. However, until recently, research on plant mRNA export has been quite limited. Genetic, bioinformatic, and biochemical investigations have expanded our understanding of the mRNA export process in plants. Here, we review recent progress that has been made elucidating the components of the mRNA export pathway in plants. MOS3 (MODIFIER OF SNC1, 3) /AtNup96 and AtNup160 are both components of the highly conserved Nup107–160 nucleoporin complex and were shown to play key roles in mRNA export. MOS11 (MODIFIER OF SNC1, 11), which is homologous to the RNA helicase enhancer CIP29 in human, was recently found to be involved in the same pathway as MOS3. A DEAD Box RNA helicase, LOS4 (low expression of osmotically responsive genes 4) was also found to play a role in the mRNA export process, putatively by carrying mRNA molecules through the nuclear envelope. Recently, a protein complex homologous to the yeast TREX-2 complex was also found to play important roles in mRNA export in plants. It appears that most players in the mRNA export pathway are highly conserved among plants, yeast and animals.     

Nuclear export of yeast signal recognition particle lacking Srp54p by the Xpo1p/Crm1p NES-dependent pathway

BACKGROUND: The movement of macromolecules through the nuclear pores requires energy and transport receptors that bind both cargo and nuclear pores. Different molecules/complexes often require different transport receptors. The signal recognition particle (SRP) is a conserved cytosolic ribonucleoprotein that targets proteins to the endoplasmic reticulum. Previous studies have shown that the export of SRP RNA from the nucleus requires trans-acting factors and that SRP may be at least partly assembled in the nucleus, but little else is known about how it is assembled and exported into the cytoplasm. RESULTS: Of the six proteins that constitute the yeast SRP, we found that all except Srp54p were imported into the nucleus. Four of these had nucleolar pools. The same four proteins are required for stability of the yeast SRP RNA scR1, suggesting that they assemble with the RNA in the nucleus to form a central core SRP. This core SRP was a competent export substrate. Of the remaining components, Sec65p entered the nucleus and was assembled onto the core particle there, whereas Srp54p was solely cytoplasmic. The export of SRP from the nucleus required the transport receptor Xpo1p/Crm1p and Yrb2p, both components of the pathway that exports leucine-rich nuclear export signal (NES)-containing proteins from the nucleus. CONCLUSIONS: The SRP is assembled in the nucleus into a complex lacking only Srp54p. It is then exported through the NES pathway into the cytoplasm where Srp54p binds to it. This transport route for a ribonucleoprotein complex is so far unique in yeast.