Mediators of Nuclear Protein Import Target Karyophilic Proteins to Pore Complexes of Cytoplasmic Annulate Lamellae

Nuclear pore complexes are constitutive structures of the nuclear envelope in eukaryotic cells and represent the sites where transport of molecules between nucleus and cytoplasm takes place. However, pore complexes of similar structure, but with largely unknown functional properties, are long known to occur also in certain cytoplasmic cisternae that have been termed annulate lamellae (AL). To analyze the capability of the AL pore complex to interact with the soluble mediators of nuclear protein import and their karyophilic protein substrates, we have performed a microinjection study in stage VI oocytes ofXenopus laevis.In these cells AL are especially abundant and can easily be identified by light and electron microscopy. Following injection into the cytoplasm, fluorochrome-labeled mediators of two different nuclear import pathways, importin β and transportin, not only associate with the nuclear envelope but also with AL. Likewise, nuclear localization signals (NLS) of the basic and M9 type, but not nuclear export signals, confer targeting and transient binding of fluorochrome-labeled proteins to cytoplasmic AL. Mutation or deletion of the NLS signals prevents these interactions. Furthermore, binding to AL is abolished by dominant negative inhibitors of nuclear protein import. Microinjections of gold-coupled NLS-bearing proteins reveal specific gold decoration at distinct sites within the AL pore complex. These include such at the peripheral pore complex-attached fibrils and at the central “transporter” and closely resemble those of “transport intermediates” found in electron microscopic studies of the nuclear pore complex (NPC). These data demonstrate that AL can represent distinct sites within the cytoplasm of transient accumulation of nuclear proteins and that the AL pore complex shares functional binding properties with the NPC.     

Karyopherins: from nuclear-transport mediators to nuclear-function regulators

The karyopherin beta (or importin beta) family comprises soluble transport factors that mediate the movement of proteins and RNAs between the nucleus and cytoplasm. Recent studies have extended the role of karyopherins to regulating assembly of the nuclear pore complex (NPC), assembly of the nuclear envelope, mitosis and replication. New data also address how karyopherins specifically recognize and transport many distinct cargoes and traverse the NPC. These data raise the possibility that, although there might be a universal mechanism for nuclear transport, specific interactions between karyopherins and components of the NPC might function to regulate differentially the ability of the different karyopherins to cross the NPC.     

蛋白质入核转运的机制和研究进展

细胞核膜是由外膜和内膜组成的磷脂双分子层结构,同时镶嵌一些核孔复合体（NPC）.核孔复合体是胞浆和胞核之间主动和被动转运的生理屏障.核内功能蛋白在胞浆内合成后通过核孔复合体进入胞核,这个过程除了需要NPC上核孔蛋白、胞浆内核转运受体和RanGTP等蛋白的参与外, 货物蛋白本身的结构特征在其入核转运过程中亦发挥重要作用.本文着重就蛋白入核转运的机制及近年来取得的相关进展进行综述.     

Real-Time Imaging of Nuclear Permeation by EGFP in Single Intact Cells

The NPC is the portal for the exchange of proteins, mRNA, and ions between nucleus and cytoplasm. Many small molecules (<10 kDa) permeate the nucleus by simple diffusion through the pore, but molecules larger than 70 kDa require ATP and a nuclear localization sequence for their transport. In isolated Xenopus oocyte nuclei, diffusion of intermediate-sized molecules appears to be regulated by the NPC, dependent upon [Ca²⁺] in the nuclear envelope. We have applied real-time imaging and fluorescence recovery after photobleaching to examine the nuclear pore permeability of 27-kDa EGFP in single intact cells. We found that EGFP diffused bidirectionally via the NPC across the nuclear envelope. Although diffusion is slowed ~100-fold at the nuclear envelope boundary compared to diffusion within the nucleus or cytoplasm, this delay is expected for the reduced cross-sectional area of the NPCs. We found no evidence for significant nuclear pore gating or block of EGFP diffusion by depletion of perinuclear Ca²⁺ stores, as assayed by a nuclear cisterna-targeted Ca²⁺ indicator. We also found that EGFP exchange was not altered significantly during the cell cycle.     

Yeast and vertebrate nuclear-pore complexes: evolutionary conserved, yet divergent macromolecular assemblies

Summary The nuclear-pore complex (NPC), which consists of ca. 50 proteins called nucleoporins, is a huge macromolecular structure that spans the nuclear envelope and is an obligatory passage for molecules in transit between the cytoplasm and the nucleus. In the last years, major progress has allowed the characterization of the so-called soluble phase of nucleocytoplasmic transport, that involves transport substrates, import and export receptors of which some belong to the karyopherin- family, and the small GTPase Ran and its modulators. In addition, the knowledge of the NPC architecture, the identification of its constituents, and the determination of the hierarchy of interactions within the pore should help to understand how nucleoporins are assembled, and how they give rise to a functional NPC through interactions with specific transport factors. In this review, we will focus on recent insights into the stationary phase of nucleocytoplasmic transport (i.e., the NPCs) that have been gained from exploiting the benefits of several organisms, such asXenopus laevis oocytes, mammalian cell lines, and the yeastSaccharomyces cerevisiae.Abbreviations FXFG phenylalanine X phenylalanine glycine - GLFG glycine leucine phenylalanine glycine - NPC nuclear-pore complex - RLNE rat liver nuclear envelope - WGA wheat germ agglutinin     

Dynamic nuclear pore complexes: life on the edge

The exchange of molecules between the nucleus and cytoplasm is mediated through nuclear pore complexes (NPCs) embedded in the nuclear envelope. Altering the interactions between transport receptors and their cargo has been shown to be a major regulatory mechanism to control traffic through NPCs. New evidence now suggests that NPC proteins play active roles in translocation, and that transport is also controlled by dynamic changes in NPC composition and architecture. This view of ever-changing NPCs necessitates the re-evaluation of current models of nuclear transport and how this process is regulated.     

Nucleocytoplasmic trafficking of proteins: With or without Ran?

Proteins and RNAs move between the nucleus and cytoplasm by translocation through nuclear pore complexes in the nuclear envelope. To do this, they require specific targeting signals, energy, and a cellular apparatus that catalyzes their transport. Several of the factors involved in nucleocytoplasmic trafficking of proteins have been identified and characterized in some detail. The emerging picture for nuclear transport proposes a central role for the small GTPase Ran and proteins with which it interacts. In particular, asymmetric distribution of these proteins between nucleus and cytoplasm appears to be responsible for the vectorial nature of nucleocytoplasmic transport. Here, we summarize the role of Ran and Ran-binding proteins in nuclear trafficking of proteins with classical nuclear localisation signals. We also discuss examples of the growing number of alternative pathways that are involved in transport of proteins across the nuclear envelope. BioEssays 21:579–589, 1999. © 1999 John Wiley & Sons, Inc.     

The role of the integral membrane nucleoporins Ndc1p and Pom152p in nuclear pore complex assembly and function

The nuclear pore complex (NPC) is a large channel that spans the two lipid bilayers of the nuclear envelope and mediates transport events between the cytoplasm and the nucleus. Only a few NPC components are transmembrane proteins, and the role of these proteins in NPC function and assembly remains poorly understood. We investigate the function of the three integral membrane nucleoporins, which are Ndc1p, Pom152p, and Pom34p, in NPC assembly and transport in Saccharomyces cerevisiae. We find that Ndc1p is important for the correct localization of nuclear transport cargoes and of components of the NPC. However, the role of Ndc1p in NPC assembly is partially redundant with Pom152p, as cells lacking both of these proteins show enhanced NPC disruption. Electron microscopy studies reveal that the absence of Ndc1p and Pom152p results in aberrant pores that have enlarged diameters and lack proteinaceous material, leading to an increased diffusion between the cytoplasm and the nucleus.     

The yeast nuclear pore complex and transport through it

Exchange of macromolecules between the nucleus and cytoplasm is a key regulatory event in the expression of a cell's genome. This exchange requires a dedicated transport system: (1) nuclear pore complexes (NPCs), embedded in the nuclear envelope and composed of proteins termed nucleoporins (or "Nups"), and (2) nuclear transport factors that recognize the cargoes to be transported and ferry them across the NPCs. This transport is regulated at multiple levels, and the NPC itself also plays a key regulatory role in gene expression by influencing nuclear architecture and acting as a point of control for various nuclear processes. Here we summarize how the yeast Saccharomyces has been used extensively as a model system to understand the fundamental and highly conserved features of this transport system, revealing the structure and function of the NPC; the NPC's role in the regulation of gene expression; and the interactions of transport factors with their cargoes, regulatory factors, and specific nucleoporins.     

Nuclear protein accumulation by facilitated transport and intranuclear binding

Nuclear proteins are transported from the cytoplasm into the nucleus via nuclear envelope pore complexes (NPCs). At the molecular level, the mechanisms responsible for this transport remain obscure. However, it is known that, for many proteins, the process requires ATP and proceeds against formidable nucleocytoplasmic concentration gradients. Therefore, the NPC is often thought of as an active transport site. In this article, Philip Paine presents the alternative hypothesis that, on current evidence, protein translocation across the nuclear envelope and accumulation in the nucleus can equally well be explained by facilitated transport through the NPC and subsequent intranuclear binding.     

New steps toward the nucleocytoplasmic traffic of macromolecules

The nucleocytoplasmic transport of functional molecules is mediated bidirectionally through the nuclear pore complex (NPC), which spans the double membranes of the nuclear envelope. It has recently been shown that signaling between the nucleus and the cytoplasm plays a key role in coordinating the cellular processes such as the cell cycle and cell differentiation (Yoneda, 2000). As the result of recent extensive analysis, significant progress has been made in our understanding of the fundamental mechanism of nuclear transport of proteins and RNAs and numerous transport factors have now been identified. In this special issue of review articles, we focus on our rapid growing knowledge of nucleocytoplasmic transport, especially the translocation of proteins through the NPC and mRNA export, and review this exciting field from various points of view including cell biology, structural biology and yeast genetics.     

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 large number of viral proteins. Usually they contain short stretches of lysine or arginine residues. These signals are recognized by the importin super-family (importin α and β) proteins that mediate the transport across the nuclear envelope through Ran-GTP. In contrast, only one class of the leucine-rich nuclear export signal (NES) on viral proteins is known at present. Chromosome region maintenance 1 (CRM1) protein mediates nuclear export of hundreds of viral proteins through the recognition of the leucine-rich NES.     

Calcium regulation of nucleocytoplasmic transport

Bidirectional trafficking of macromolecules between the cytoplasm and the nucleus is mediated by the nuclear pore complexes (NPCs) embedded in the nuclear envelope (NE) of eukaryotic cell. The NPC functions as the sole pathway to allow for the passive diffusion of small molecules and the facilitated translocation of larger molecules. Evidence shows that these two transport modes and the conformation of NPC can be regulated by calcium stored in the lumen of nuclear envelope and endoplasmic reticulum. However, the mechanism of calcium regulation remains poorly understood. In this review, we integrate data on the observations of calcium-regulated structure and function of the NPC over the past years. Furthermore, we highlight challenges in the measurements of dynamic conformational changes and transient transport kinetics in the NPC. Finally, an innovative imaging approach, single-molecule super-resolution fluorescence microscopy, is introduced and expected to provide more insights into the mechanism of calcium-regulated nucleocytoplasmic transport.     

Enriching the Pore: Splendid Complexity from Humble Origins

The nucleus is the defining intracellular organelle of eukaryotic cells and represents a major structural innovation that differentiates the eukaryotic and prokaryotic cellular form. The presence of a nuclear envelope (NE) encapsulating the nucleus necessitates a mechanism for interchange between the contents of the nuclear interior and the cytoplasm, which is mediated via the nuclear pore complex (NPC), a large protein assembly residing in nuclear pores in the NE. Recent advances have begun to map the structure and functions of the NPC in multiple organisms, and to allow reconstruction of some of the evolutionary events that underpin the modern NPC form, highlighting common and differential NPC features across the eukaryotes. Here we discuss some of these advances and the questions being pursued, consider how the evolution of the NPC has been constrained, and finally propose a model for how the NPC evolved.      

The nuclear pore complex: a protein machine bridging the nucleus and cytoplasm

Compositional analysis of nuclear pore complexes (NPCs) is nearing completion, and efforts are now focused on understanding how these protein machines work. Recent analysis of soluble transport factor interactions with NPC proteins reveals distinct and overlapping pathways for movement between the nucleus and cytoplasm. New fluorescence- and microscopy-based strategies have been used to monitor the pathway of NPC assembly and to reveal the dynamics of the NPC during transport.     

Molecular trafficking across the nuclear pore complex.

The nuclear pore complex is the gateway for protein and RNA transport between the cytoplasm and nucleus. Recent work has characterized signals and components involved in nuclear import of macromolecules and has described mechanisms for transport regulation. Advances in understanding the structure of the pore complex are starting to provide a framework for interpreting the biochemistry of nuclear import. Information on the export of RNA from the nucleus is only beginning to emerge.     

Isolation and fractionation of rat liver nuclear envelopes and nuclear pore complexes

The nuclear envelope is a double lipid bilayer that physically separates the functions of the nucleus and the cytoplasm of eukaryotic cells. Regulated transport of molecules between the nucleus and the cytoplasm is essential for normal cell metabolism and is mediated by large protein complexes, termed nuclear pore complexes (NPCs), which span the inner and outer membranes of the nuclear envelope. Significant progress has been made in the past 10 years in identifying the protein composition of NPCs and the basic molecular mechanisms by which these complexes facilitate the selective exchange of molecules between the nucleus and the cytoplasm. However, many fundamentally important questions about the functions of NPCs, the specific functions of individual NPC-associated proteins, and the assembly and disassembly of NPCs, remain unanswered. This review describes approaches for isolating and characterizing nuclear envelopes and NPC-associated proteins from mammalian cells. It is anticipated that these procedures can be used as a starting point for further molecular and biochemical analysis of the mammalian nuclear envelope, NPCs, and NPC-associated proteins.