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The nucleus is a defining hallmark in cells of all the higher organisms: yeast, animals, and plants. As the repository of the genome, it both encloses the chromatin and regulates its accessibility. It is also the site of nucleic acid synthesis, including replica-tion of DNA, transcription and editing of messenger RNA, synthesis of ribosomal RNAs, and assembly of ribosomal subunits. By contrast, the cytoplasm is the site of protein synthesis, where functional ribosomes translate mRNA into polypeptides. The nuclear envelope defines the border between these two distina biochemical worlds. The nuclear pores (or nuclear pore complexes, NPCs) serve as guardians of this border, acting as the gateway for molecular ex-change between the two major cellular compartments. They are deeply integrated to the physi-ological function of every cellular pathway involving communication between enzymatic, sig-naling, or regtdatory activities on one hand, and gene expression on the other. The nuclear pore complex is also a fascinating molecular machine, facilitating the passage of specific macromol-ecules in one direction while ferrying others in the opposite sense.

The nuclear envelope contains three distinct membrane domains. The outer nuclear membrane faces the cytoplasm and is continuous with the rough endoplasmic reticulum (ER). Like the rough ER, the nuclear outer membrane is covered with ribosomes engaged in translating secreted and integral membrane proteins. The inner nuclear membrane faces the nucleoplasm, has its own unique protein composition and interacts with the fibrous meshwork of the nuclear lamina (reviewed in ref 6). The inner and outer nuclear membranes fuse to form the third membrane domain, termed the pore membrane domain. Nuclear pore complexes (NPCs) are anchored at the pore membrane domain and mediate both passive diffusion and active nucleocytoplasmic transport. Aaive transport requires signals on the imported or ex-ported macromolecules, termed nuclear localization signals (NLS) and nuclear export signals (NES), respectively. Transport is mediated by soluble NLS and NES receptors (termed importins/exportins/karyopherins/transportins), whose direction of movement is determined by Ran, a small GTP-binding protein (reviewed in refs. 22, 48 and 50). NPC structure includes soluble proteins, termed nucleoporins (nups) and integral membrane proteins, termed POMs. The NPC is anchored to the pore membrane by binding to POMs. POMs are also proposed to have roles in nuclear pore assembly, nucleocytoplasmic transport and NPC organization (see below).

The case has been made for the presence of sequences different from nuclear localization signals that determine the fate of a protein once inside the nucleus. The number of examples where such signals have been studied in detail is still small, and it is too early to draw conclusions about their similarity or multiplicity. If the described signals act like other targeting domains, they will most likely function by providing a surface for protein-protein interactions. If transcription factors come with signals for specific subnuclear “addresses”, and if their disruption can compromise transcription factor function, then one ought to think about what those addresses are, and how they relate to the position and/or compartmentalization of the promoters regulated by these factors. The prevailing evidence for the association of specific chromatin regions (MARs) with the nuclear matrix, and the positive effect MARs have on the transcription of flanking genes has led to the model that the association of genes with the nuclear matrix increases their ability to be expressed, possibly by providing a more “open” chromatin environment. It might be equally attractive to think about another mechanism, by which association with the nuclear matrix of both promoters and transcription factors might increase the probability of productive assembly of transcription initiation complexes. The fact that “transcriptosomes” appear to have specific locations in the nucleus, and that they can function on the isolated nuclear matrix encourages to think about how such complexes might assemble in specific places. It will be highly informative to investigate whether three-way interactions between genes, sequence-specific transcription factors, and nuclear matrix components play a role in their assembly.

Eukaryotic cells are separated into two large compartments, namely the nucleus and the cytoplasm, by the nuclear envelope. As a result, macromolecules including RNAs, which are transcribed in the nucleus and nuclear proteins, which are translated in the cytoplasm must cross the double Upid bilayer to reach the intracellular sites where they function. In addition, cumulative evidence suggests that trafficking between the nucleus and the cytoplasm is rather dynamic and some proteins and RNAs cross the nuclear envelope again after being transported to one compartment.

The study of protein import in plants is beginning to yield insight into not only the similarities with other kingdoms, but also the interesting differences that we have described throughout this Chapter. Plants are essential to all life on our planet and are the foundation for our food chain. Protein import processes and their role in development and environmental responses are essential to our understanding of plant biology, an important goal in itself. As our knowledge increases about nuclear protein import in plants, contributions to our general understanding of these processes in all organisms will increase.

Some of the important areas to be addressed in the future include:

There is much to be learned and the future will surely present opportunities for new discovery.

-mediated genetic transformation is a process by which genetic material is transported from the bacterium into the host nucleus, where it stably integrates. The transferred DNA (T-DNA) is escorted, by two bacterial proteins, as a single-stranded DNA-protein complex (a T-complex), which mediate its transport to the host nucleus. The large size and mass of this DNA-protein complex raise questions as to the molecular machinery and mechanism by which the T-complex passes the nuclear pore barrier. Recent studies have revealed the important role of specific host proteins in interacting with and guiding the T-complex through the nuclear pore, and to its point of integration. In this chapter, we summarize our knowledge of the function of T-DNA bacterial and host protein chaperones, and draw a model for their action during the nuclear import and intranuclear transport of T-DNA.

The nuclear envelope separates the theatres of two major cellular processes in eukaryotes: transcription takes place in the nucleus whereas proteins are synthesized in the cytoplasm. The localization of these processes in two different compartments of the cell implies that macromolecules must be exchanged very rapidly and efficiendy between the nucleus and the cytoplasm in order to ensure proper regulation of signaling and metabolism of a living cell.

One of the distinguishing features of eukaryotic cells is the compartmentalization of genetic information within a membrane-enclosed nucleus. The double membrane of the nuclear envelope separates the nucleus and the cytoplasm, and all macromolecu-lar exchange across the nuclear envelope takes place through large protein channels termed the nuclear pore complexes (NPCs). The molecules that are exchanged between these two com-partments range in size from ions and other small molecules to large complexes such as the 5OS ribosome and other large ribonucleoprotein complexes. In contrast to ions and small proteins that diffuse across the NPC, macromolecular movement is an active process. Active nucleocy-toplasmic transport allows for the proper compartmentalization of nuclear proteins involved in transcription, replication of DNA, and remodeling of chromatin. Transport also is necessary for mRNAs, tRNAs, and rRNAs that are transcribed in the nucleus but ultimately function in the cytoplasm. This growing awareness of the role of nuclear transport in regulating gene ex-pression has paralleled a remarkable increase in our knowledge of the nuclear transport process itself. Far from acting as static “localization signals” the sequences specifying nuclear location act in combination with other signals to alter the steady-state distribution of nuclear proteins. Thus, the concept of nuclear proteins shuttling between nucleus and cytoplasm has emerged as a dominant principle in understanding nuclear import and export. It is impossible to look at nuclear export in isolation without considering nuclear import rates. This has proven to be a barrier in understanding nuclear export as a process with distinct features and requirements. The number of import and export carriers identified has grown to include factors specific for classes of nuclear components and more general factors. In addition, it is clear that movement through the nuclear pore complex is dictated by properties of both the pore and the carrier molecules themselves. These properties are reflected in binding interactions that may facilitate movement across the nuclear pore and perhaps provide for directionality of transport. In this brief chapter, we will give a brief overview of the nuclear pore complex, methods for examining nuclear export and shutding, the nature of the transport machinery and nuclear export carri-ers. We will then attempt to combine these into a coherent model for understanding nuclear import and export. We do not claim that this is a comprehensive overview. A number of excel-lent reviews of this type have recendy appeared (see for example refs. 1,2). What we hope to do is point out some novel aspects of nuclear export and emphasize some recent findings which suggest that factors other than traditional transport carriers may be involved and regulate the process of nuclear shuttling.

Entry into the eukaryotic cell nucleus occurs through multiple pathways involving specific targeting signals, and intracellular receptor molecules of the importin/karyopherin superfamily which recognise and dock the nuclear import substrates carrying these signals at the nuclear pore. Subsequent to translocation through the pore via a series of importin-mediated docking steps at multiple sites within it, release into the nucleus is effected by the monomeric guanine nucleotide binding protein Ran. Different importins possess distinct target sequence-binding specificities, meaning that different importins mediate the nuclear import of different classes of proteins. This extends to different classes of transcription factors which are recognised by distinct importins, and whose transport to the nucleus is modulated by specific regulatory mechanisms. The first step of nuclear import is of central importance, with the affinity of the importin: targeting signal interaction being a critical parameter in determining transport efficiency. In the whole cell context, target signal recognition can be modulated through differential expression of the importins themselves, as well as through competition between different importins for the same nuclear import substrate, and between different nuclear import substrates for the same importin. In addition, there are specific mechanisms to modulate targeting sequence-importin interaction directly through phosphorylation. The fact that there are distinct nuclear import pathways for different types of nuclear import substrates enables the cell to regulate these pathways specifically, ensuring efficient nuclear import of particular proteins as and when required.

Ageneral paradigm for nuclear transport was established primarily through studies of protein import and export. Until recendy, this paradigm was generally presumed also to apply to the process of RNA export from the nucleus. In particular, it was assumed that general mRNA export was mediated by one or more transport receptors of the importin-β family and that the RanGTP/GDP gradient was required to impart directionality to the process. The highly abundant class of nuclear RNA-binding proteins—the hnRNP proteins—were regarded as primary candidates for mRNA export adapter proteins that could link mRNAs to importin-ß family export factors. Within the past few years, however, an explosion of data has largely disproven prior assumptions about the mechanisms of mRNA export, permanently changing the face of the field. The dust is still settling, but what we now see, albeit incompletely, is the outline of a probable major route of mRNA export that is independent of the importin-ß family and the Ran GTPase system.