Gate-crashing the nuclear pore complex

As a third in a series of MD simulations investigating the binding dynamics between nuclear transport receptors and FG-repeats, Isgro and Schulten (2007b) unveil that close, physical intimacy between partners is likely to ensure a hassle-free passage through the nuclear pore complex.     

The nuclear pore complex

Nuclear pore complexes, the conduits for information exchange between the nucleus and cytoplasm, appear broadly similar in eukaryotes from yeast to human. Precisely how nuclear pore complexes regulate macromolecular and ionic traffic remains unknown, but recent advances in the identification and characterization of components of the complex by proteomics and genomics have provided new insights.     

The nuclear pore complex

The nuclear pore complex is the largest supramolecular complex that assembles in the eukaryotic cell. This structure is highly dynamic and must disassemble prior to mitosis and reassemble after the event. The directed movement of macromolecules into and out of the nucleus occurs through the nuclear pore complex, a potentially regulatory point for translocation. Using biochemical and genetic approaches, several nuclear pore complex proteins from yeast and vertebrates have been well characterized. Although very little is known about plant nuclear pore proteins, research is providing new information that indicates that plant nuclear pore complexes may have some unique features.     

Translocation through the nuclear pore complex

The nuclear transport field has completed a decade of fast-paced research dominated by the discovery of transport signals, receptors, and regulators. What might be considered the Holy Grail of nuclear transport – the physical basis of translocation through the nuclear pore – is now under close scrutiny. Recent publications describe structural and biochemical approaches that help address key aspects of the translocation mechanism. These studies have led to the affinity gradient, Brownian affinity gate and selective phase models of translocation.     

Lighting up the nuclear pore complex

It is generally accepted that transport through the nuclear pore complex (NPC) involves an abundance of phenylalanine-glycine rich protein domains (FG-domains) that serve as docking sites for soluble nuclear transport receptors (NTRs) and their cargo complexes. But the precise mechanism of translocation through the NPC allowing for high speed and selectivity is still vividly debated. To ultimately decipher the underlying gating mechanism it is indispensable to shed more light on the molecular arrangement of FG-domains and the distribution of NTR-binding sites within the central channel of the NPC. In this review we revisit current transport models, summarize recent results regarding translocation through the NPC obtained by super-resolution microscopy and finally discuss the status and potential of optical methods in the analysis of the NPC.     

An agent-based model for mRNA export through the nuclear pore complex

mRNA export from the nucleus is an essential step in the expression of every protein- coding gene in eukaryotes, but many aspects of this process remain poorly understood. The density of export receptors that must bind an mRNA to ensure export, as well as how receptor distribution affects transport dynamics, is not known. It is also unclear whether the rate-limiting step for transport occurs at the nuclear basket, in the central channel, or on the cytoplasmic face of the nuclear pore complex. Using previously published biophysical and biochemical parameters of mRNA export, we implemented a three-dimensional, coarse-grained, agent-based model of mRNA export in the nanosecond regime to gain insight into these issues. On running the model, we observed that mRNA export is sensitive to the number and distribution of transport receptors coating the mRNA and that there is a rate-limiting step in the nuclear basket that is potentially associated with the mRNA reconfiguring itself to thread into the central channel. Of note, our results also suggest that using a single location-monitoring mRNA label may be insufficient to correctly capture the time regime of mRNA threading through the pore and subsequent transport. This has implications for future experimental design to study mRNA transport dynamics.     

Isolation of the yeast nuclear pore complex

Nuclear pore complexes (NPCs) have been isolated from the yeast Saccharomyces. Negative stain electron microscopy of the isolated NPCs and subsequent image reconstruction revealed the octagonal symmetry and many of the ultrastructural features characteristic of vertebrate NPCs. The overall dimensions of the yeast NPC, both in its isolated form as well as in situ, are smaller than its vertebrate counterpart. However, the diameter of the central structures are similar. The isolated yeast NPC has a sedimentation coefficient of approximately 310 S and an M(r) of approximately 66 MD. It retains all but one of the eight known NPC proteins. In addition it contains as many as 80 uncharacterized proteins that are candidate NPC proteins.     

HIV-1 remodels the nuclear pore complex

Human immunodeficiency virus type 1 (HIV-1) commandeers host cell proteins and machineries for its replication. Our earlier work showed that HIV-1 induced the cytoplasmic retention of nucleocytoplasmic shuttling and ribonucleic acid (RNA)-binding proteins. This retention is dependent on nuclear export of the viral genomic RNA and on changes in the localization and expression level of the nucleoporin (Nup) p62 (Nup62). To further characterize the extent of perturbation induced by HIV-1, we performed proteomics analyses of nuclear envelopes (NEs) isolated from infected T cells. Infection induced extensive changes in the composition of the NE and its associated proteins, including a remarkable decrease in the abundance of Nups. Immunogold electron microscopy revealed the translocation of Nups into the cytoplasm. Nup62 was identified as a component of purified virus, and small interfering RNA depletion studies revealed an important role for this Nup in virus gene expression and infectivity. This detailed analysis highlights the profound effects on NE composition induced by HIV-1 infection, providing further evidence of the magnitude of viral control over the cell biology of its host.     

Proteins connecting the nuclear pore complex with the nuclear interior

While much has been learned in recent years about the movement of soluble transport factors across the nuclear pore complex (NPC), comparatively little is known about intranuclear trafficking. We isolated the previously identified Saccharomyces protein Mlp1p (myosin-like protein) by an assay designed to find nuclear envelope (NE) associated proteins that are not nucleoporins. We localized both Mlp1p and a closely related protein that we termed Mlp2p to filamentous structures stretching from the nucleoplasmic face of the NE into the nucleoplasm, similar to the homologous vertebrate and Drosophila Tpr proteins. Mlp1p can be imported into the nucleus by virtue of a nuclear localization sequence (NLS) within its COOH-terminal domain. Overexpression experiments indicate that Mlp1p can form large structures within the nucleus which exclude chromatin but appear highly permeable to proteins. Remarkably, cells harboring a double deletion of MLP1 and MLP2 were viable, although they showed a slower net rate of active nuclear import and faster passive efflux of a reporter protein. Our data indicate that the Tpr homologues are not merely NPC-associated proteins but that they can be part of NPC-independent, peripheral intranuclear structures. In addition, we suggest that the Tpr filaments could provide chromatin-free conduits or tracks to guide the efficient translocation of macromolecules between the nucleoplasm and the NPC.     

Active genes at the nuclear pore complex

The nucleus is spatially and functionally organized and its architecture is now seen as a key contributor to genome functions. A central component of this architecture is the nuclear envelope, which is studded with nuclear pore complexes that serve as gateways for communication between the nucleoplasm and cytoplasm. Although the nuclear periphery has traditionally been described as a repressive compartment and repository for gene-poor chromosome regions, several recent studies in yeast have demonstrated that repressive and activating domains can both be positioned at the periphery of the nucleus. Moreover, association with the nuclear envelope favors the expression of particular genes, demonstrating that nuclear organization can play an active role in gene regulation.     

Getting across the nuclear pore complex.

The nuclear pore complex (NPC) connects the cytoplasm and nucleus through the nuclear envelope and serves as the pipeline for moving material between the two compartments. Macromolecules that move through the NPC range in size from the very small (for example, ions and ATP) to the very large (for example, ribonucleoprotein particle complexes). Unlike translocation across other organelle membranes, proteins do not have to be unfolded to be transported through the NPC, and the NPC also routinely transports large, multicomponent substrates in both directions. This review focuses on current understanding of the different mechanisms by which macromolecules move across the NPC.     

On the attachment of the nuclear pore complex

Electron microscope examination of isolated rat liver nuclei after treatment with the detergent Triton X-100 revealed the complete removal of both the inner and outer membranes of the nuclear envelope. The envelope-denuded nuclei did not show any change in either shape or internal ultrastructure. Most strikingly, the nuclear pore complexes, which in untreated nuclei appear to be integral components of the nuclear envelope, were retained in their characteristic location at the distal ends of the channels leading through the peripheral heterochromatin. Determination of the chemical composition of detergent-treated nuclei showed that over 95% of the nuclear phospholipid was solubilized, thus corroborating the morphological absence of nuclear membranes. Furthermore, detergent treatment also solubilized approximately 10% of the nuclear protein. Analysis of the solubilized protein by polyacrylamide gel electrophoresis in the presence of SDS indicated that these proteins belong to a few specific classes which presumably represent the major polypeptides of the nuclear membranes. The total absence of the nuclear envelope on both morphological and biochemical grounds supports the idea that the nuclear pore complex does not require the membranes either for attachment to the nucleus or for maintenance of its own structural integrity.     

On the universality of nuclear pore complex structure

Summary Substructural details of the nuclear pore complex were studied in diverse plant and animal cells with both section technique and negative staining of isolated nuclear envelope pieces. The structures observed after the different techniques, including a variety of fixation procedures, are compared and their significance is discussed. It is shown that, down to the 15–20 Å level, the architecture of the nuclear pore complex is universal among such diverse cell types as from, e. g., onion root tips, bean leaves, mammalian liver parenchyma, HeLa cell cultures, and amphibian germ material. The fundamental substructures of the pore complex such as (1) the annular granules, (2) the fibrils attached to the annuli, (3) the central granules, (4) the fibrils in the pore interior including those which make up the inner ring and/or those which connect the central granule to the pore margin, are recognized in all cell types studied. The dynamic variability of the central granule morphology is emphasized and observations are presented which suggest that the view of such centrally located material as representing ribonucleoproteins in a transitory state of nucleocytoplasmic migration can be extended to generality. General concepts of the nuclear pore complex structure are presented as alternative model views revealing either a more compact, predominantly granular, or a more fibrillar aspect.The author gratefully acknowledges the frequent discussions and cooperation with his team-colleagues Drs. H. Falk (in the work on leaf material) and U. Scheer (in working with amphibian oocytes) as well as the skillful technical assistance of Miss Marianne Winter and Miss Sigrid Krien. The work was supported by the Deutsche Forschungsgemeinschaft.