Structure Of Nuclear Pore Complex Research Articles

Wrestling with ubiquitin-like modifiers and the structure and function of the mammalian nuclear pore complex

Wrestling with ubiquitin-like modifiers and the structure and function of the mammalian nuclear pore complex

RAE1 is a shuttling mRNA export factor that binds to a GLEBS-like NUP98 motif at the nuclear pore complex through multiple domains.

Gle2p is implicated in nuclear export of poly(A)+ RNA and nuclear pore complex (NPC) structure and distribution in Saccharomyces cerevisiae. Gle2p is anchored at the nuclear envelope (NE) via a short Gle2p-binding motif within Nup116p called GLEBS. The molecular mechanism by which Gle2p and the Gle2p-Nup116p interaction function in mRNA export is unknown. Here we show that RAE1, the mammalian homologue of Gle2p, binds to a GLEBS-like NUP98 motif at the NPC through multiple domains that include WD-repeats and a COOH-terminal non-WD-repeat extension. This interaction is direct, as evidenced by in vitro binding studies and chemical cross-linking. Microinjection experiments performed in Xenopus laevis oocytes demonstrate that RAE1 shuttles between the nucleus and the cytoplasm and is exported from the nucleus in a temperature-dependent and RanGTP-independent manner. Docking of RAE1 to the NE is highly dependent on new mRNA synthesis. Overexpression of the GLEBS-like motif also inhibits NE binding of RAE1 and induces nuclear accumulation of poly(A)+ RNA. Both effects are abrogated either by the introduction of point mutations in the GLEBS-like motif or by overexpression of RAE1, indicating a direct role for RAE1 and the NUP98-RAE1 interaction in mRNA export. Together, our data suggest that RAE1 is a shuttling transport factor that directly contributes to nuclear export of mRNAs through its ability to anchor to a specific NUP98 motif at the NPC.

Molecular architecture of the yeast nuclear pore complex: localization of Nsp1p subcomplexes.

The nuclear pore complex (NPC), a supramolecular assembly of approximately 100 different proteins (nucleoporins), mediates bidirectional transport of molecules between the cytoplasm and the cell nucleus. Extensive structural studies have revealed the three- dimensional (3D) architecture of Xenopus NPCs, and eight of the approximately 12 cloned and characterized vertebrate nucleoporins have been localized within the NPC. Thanks to the power of yeast genetics, 30 yeast nucleoporins have recently been cloned and characterized at the molecular level. However, the localization of these nucleoporins within the 3D structure of the NPC has remain elusive, mainly due to limitations of preparing yeast cells for electron microscopy (EM). We have developed a new protocol for preparing yeast cells for EM that yielded structurally well-preserved yeast NPCs. A direct comparison of yeast and Xenopus NPCs revealed that the NPC structure is evolutionarily conserved, although yeast NPCs are 15% smaller in their linear dimensions. With this preparation protocol and yeast strains expressing nucleoporins tagged with protein A, we have localized Nsp1p and its interacting partners Nup49p, Nup57p, Nup82p, and Nic96p by immuno-EM. Accordingly, Nsp1p resides in three distinct subcomplexes which are located at the entry and exit of the central gated channel and at the terminal ring of the nuclear basket.

A novel fluorescence-based genetic strategy identifies mutants of Saccharomyces cerevisiae defective for nuclear pore complex assembly.

Nuclear pore complexes (NPCs) are large proteinaceous portals for exchanging macromolecules between the nucleus and the cytoplasm. Revealing how this transport apparatus is assembled will be critical for understanding the nuclear transport mechanism. To address this issue and to identify factors that regulate NPC formation and dynamics, a novel fluorescence-based strategy was used. This approach is based on the functional tagging of NPC proteins with the green fluorescent protein (GFP), and the hypothesis that NPC assembly mutants will have distinct GFP-NPC signals as compared with wild-type (wt) cells. By fluorescence-activated cell sorting for cells with low GFP signal from a population of mutagenized cells expressing GFP-Nup49p, three complementation groups were identified: two correspond to mutant nup120 and gle2 alleles that result in clusters of NPCs. Interestingly, a third group was a novel temperature-sensitive allele of nup57. The lowered GFP-Nup49p incorporation in the nup57-E17 cells resulted in a decreased fluorescence level, which was due in part to a sharply diminished interaction between the carboxy-terminal truncated nup57pE17 and wt Nup49p. Interestingly, the nup57-E17 mutant also affected the incorporation of a specific subset of other nucleoporins into the NPC. Decreased levels of NPC-associated Nsp1p and Nup116p were observed. In contrast, the localizations of Nic96p, Nup82p, Nup159p, Nup145p, and Pom152p were not markedly diminished. Coincidentally, nuclear import capacity was inhibited. Taken together, the identification of such mutants with specific perturbations of NPC structure validates this fluorescence-based strategy as a powerful approach for providing insight into the mechanism of NPC biogenesis.

FEISEM, Form and Function in the Nuclear Pore Complex

Recent initiatives have resulted in a considerable increase in our understanding of the structure of the nuclear pore complex (NPC). The biochemical factors involved in both import and export have been rapidly characterised, with steady progress in the molecular dissection of the structural elements of the NPC, which is a unit of considerable molecular architecture (MW 125 kD), comprising an estimated 50- 100 different proteins. Despite this progress, the crucial molecular interactions involved in the mechanics of transport through the central transporter of the NPC remain unclear. NPC structure in Diptera, fish, (Fig 1) amphibians, birds and mammals shows a high degree of evolutionary conservation. 3D reconstructions of isolated yeast NPCs, show that the core structure is very similar to ‘higher’ organisms, but peripheral structures may be considerably reduced in structural complexity (1).Individual NPC components have been accessed in FEISEM by a variety of methods, including proteolysis,

Molecular dissection of nuclear pore complex structure and nucleocytoplasmic transport

Molecular dissection of nuclear pore complex structure and nucleocytoplasmic transport

The integral membrane protein snl1p is genetically linked to yeast nuclear pore complex function.

Integral membrane proteins are predicted to play key roles in the biogenesis and function of nuclear pore complexes (NPCs). Revealing how the transport apparatus is assembled will be critical for understanding the mechanism of nucleocytoplasmic transport. We observed that expression of the carboxyl-terminal 200 amino acids of the nucleoporin Nup116p had no effect on wild-type yeast cells, but it rendered the nup116 null strain inviable at all temperatures and coincidentally resulted in the formation of nuclear membrane herniations at 23 degrees C. To identify factors related to NPC function, a genetic screen for high-copy suppressors of this lethal nup116-C phenotype was conducted. One gene (designated SNL1 for suppressor of nup116-C lethal) was identified whose expression was necessary and sufficient for rescuing growth. Snl1p has a predicted molecular mass of 18.3 kDa, a putative transmembrane domain, and limited sequence similarity to Pom152p, the only previously identified yeast NPC-associated integral membrane protein. By both indirect immunofluorescence microscopy and subcellular fractionation studies, Snl1p was localized to both the nuclear envelope and the endoplasmic reticulum. Membrane extraction and topology assays suggested that Snl1p was an integral membrane protein, with its carboxyl-terminal region exposed to the cytosol. With regard to genetic specificity, the nup116-C lethality was also suppressed by high-copy GLE2 and NIC96. Moreover, high-copy SNL1 suppressed the temperature sensitivity of gle2-1 and nic96-G3 mutant cells. The nic96-G3 allele was identified in a synthetic lethal genetic screen with a null allele of the closely related nucleoporin nup100. Gle2p physically associated with Nup116p in vitro, and the interaction required the N-terminal region of Nup116p. Therefore, genetic links between the role of Snl1p and at least three NPC-associated proteins were established. We suggest that Snl1p plays a stabilizing role in NPC structure and function.

Active nuclear pore complexes in Chironomus: visualization of transporter configurations related to mRNP export.

The Nuclear Pore Complex (NPC) regulates nucleocytoplasmic transport by providing small channels for passive diffusion and multiple docking surfaces that lead to a central translocation channel for active transport. In this study we have investigated by high resolution scanning and transmission electron microscopy the dynamics of NPC structure in salivary gland nuclei from Chironomus during Balbiani ring (BR) mRNP translocation, and present evidence of rearrangement of the transporter related to mRNP export. Analysis of the individual NPC components verified a strong evolutionary conservation of NPC structure between vertebrates and invertebrates. The transporter is an integral part of the NPC and is composed of a central short double cylinder that is retained within the inner spoke ring, and two peripheral globular assemblies which are tethered to the cytoplasmic and nucleoplasmic coaxial rings by eight conserved internal ring filaments. Distinct stages of BR mRNP nuclear export through the individual NPC components were directly visualized and placed in a linear transport sequence. The BR mRNP first binds to the NPC basket, which forms an expanded distal basket ring. In this communication we present stages of BR mRNP transport through the nucleoplasmic, central and cytoplasmic transporter subunits, which change their conformation during mRNP translocation, and the emergence of mRNP into the cytoplasm. We propose that the reorganization of the basket may be driven, in part, by an active translocation process at the transporter. Furthermore, the images provide dramatic evidence that the transporter functions as a central translocation channel with transiently open discrete gates in its globular assemblies. A model of NPC transporter reorganization accompanied with mRNP translocation is discussed.

High-resolution field-emission scanning electron microscopy of nuclear pore complex.

The nuclear pore complex (NPC) is a large macromolecular assembly inserted into the nuclear envelope (NE). It controls the traffic of proteins, RNA, and RNA proteins between nucleus and cytoplasm. It chemical composition and function are now intensively investigated in many organisms. To understand this unique membrane transport system, we must know the supramolecular organization of the NPC. In recent years, high-resolution field-emission scanning electron microscopy has made important contributions to our knowledge of NPC structure. It provided the first images of the complex and beautiful fish trap-like structure of its intranuclear surface, documented in this review. It also has provided the first images of a new intranuclear structure, a system of branching hollow cables connecting the nuclear interior with the NPCs at the nuclear surface. Most likely this is an intranuclear transport system, assuring efficient exchange between the nuclear interior and the NE, especially in large nuclei.

A role for the divergent actin gene, ACT2, in nuclear pore structure and function

We have identified a temperature-sensitive allele of the yeast divergent actin gene ACT2, act2-1, which displays defects in nuclear pore complex (NPC) structure and nuclear import at the restrictive temperature. Although defective in nuclear import, act2-1 cells still selectively retain reporter proteins in the nucleus, and by indirect immunofluorescence the actin cytoskeleton appears normal. Previous studies in Acanthamoeba and Saccharomyces cerevisiae reported that the cellular location of Act2p partially overlaps that of conventional actin, indicating that it has a cytoskeletal function. In this study, both immunofluorescence localization and cellular fractionation of different epitope-tagged versions of Act2p also reveal an association with the nucleus, suggesting an independent nuclear function for Act2p. Analysis of act2-1 by electron microscopy, 30 min after a shift to the restrictive temperature (37 degrees C), reveals a striking aberration in NPC morphology; NPCs appear as abnormal densities on either side of, rather than spanning, the nuclear envelope. Immunoelectron microscopy confirms that these densities contain XFXFG nucleoporins. act2-1 is synthetically lethal in combination with a deletion in the XFXFG nucleoporin gene, NUP1, or a mutation in the nuclear localization sequence receptor gene, SRP1. Act2p and Srp1p co-immunoprecipitate, suggesting that the proteins exist in a complex. Together our data argue that Act2p plays an important role in NPC structure and function.

GLE2, a Saccharomyces cerevisiae homologue of the Schizosaccharomyces pombe export factor RAE1, is required for nuclear pore complex structure and function.

To identify and characterize novel factors required for nuclear transport, a genetic screen was conducted in the yeast Saccharomyces cerevisiae. Mutations that were lethal in combination with a null allele of the gene encoding the nucleoporin Nup100p were isolated using a colony-sectoring assay. Three complementation groups of gle (for GLFG lethal) mutants were identified. In this report, the characterization of GLE2 is detailed. GLE2 encodes a 40.5-kDa polypeptide with striking similarity to that of Schizosaccharomyces pombe RAE1. In indirect immunofluorescence and nuclear pore complex fractionation experiments, Gle2p was associated with nuclear pore complexes. Mutated alleles of GLE2 displayed blockage of polyadenylated RNA export; however, nuclear protein import was not apparently diminished. Immunofluorescence and thin-section electron microscopic analysis revealed that the nuclear pore complex and nuclear envelope structure was grossly perturbed in gle2 mutants. Because the clusters of herniated pore complexes appeared subsequent to the export block, the structural perturbations were likely indirect consequences of the export phenotype. Interestingly, a two-hybrid interaction was detected between Gle2p and Srp1p, the nuclear localization signal receptor, as well as Rip1p, a nuclear export signal-interacting protein. We propose that Gle2p has a novel role in mediating nuclear transport.

Immunolocalization of lamins and nuclear pore complex proteins by atomic force microscopy.

The nuclear envelope functions as a selective barrier separating the nuclear from the cytosolic compartment. Nuclear pore complexes (NPCs) mediate nuclear import and export of macromolecules and, therefore, are potential regulators of gene expression. In this study we applied atomic force microscopy (AFM) to visualize the three dimensional (3D) structure of individual NPCs in the absence and presence of two different antibodies, one directed against a pore protein (gp62) and another directed against Xenopus lamin LIII, a component of the nuclear lamina, a filament meshwork localized on the nucleoplasmic side of the nuclear envelope (NE) adjacent to and interacting with NPCs. Using 12-nm gold-labelled secondary antibodies and transmission electron microscopy we could clearly localize the primary single anti-gp62 antibody on NPCs and the primary single anti-LIII antibody between NPCs. Using AFM, the secondary antibodies against anti-gp62 could be detected as particles 7 nm in height on the nucleoplasmic face of NPCs. The secondary antibodies against anti-LIII could be clearly identified between NPCs. The secondary antibodies, attached to a 12-nm colloidal gold particle and visualized on glass, revealed similar shapes and heights as found on NEs. According to the 3D images, the volume of a single gold particle conjugated with secondary antibodies was 10203 nm3. This volume is equivalent to the volume of 38 IgG molecules associated with one individual gold particle. A similar volume of 11987 nm3 was calculated from a model assuming that the 150-kDa IgG molecules perfectly cover the spherical gold particle. We conclude that AFM can be used for identifying antibodies or other macromolecules associated with biomembranes.

The nuclear envelope and nuclear pore complex as seen by high-resolution field emission SEM and high voltage TEM

The nuclear envelope (NE) consists of an inner membrane and an outer membrane continuous with the endoplasmic reticulum. The membranes are fused at the nuclear pores, which connect cytoplasm with nuclear space. Located in these pores is the nuclear pore complex (NPC) which controls the transport of macromolecules across the NE. Attached to the inner surface of the NE is a network of 10 nm lamin filaments, the nuclear lamina. Recent studies using high voltage TEM and field emission “in lens” SEM have greatly advanced our knowledge of NPC structure, particularly the components facing the cytoplasm and nuclear space. The cytoplasmic face consists of a 120 nm ring carrying 8 twisted filaments condensed into 20×40 nm cylinders. On the nuclear side is another 120 nm ring, from which 8 filaments, 5 nm thick and 50 nm long, extend into the nuclear space. Each filament ends in a 10 nm bead. The 8 beads are joined into a 50 nm ring, forming the top of a fishtrap-like structure. Usually this ring is obscured by distorted pore connecting fibers.

Patch clamp and atomic force microscopy demonstrate TATA-binding protein (TBP) interactions with the nuclear pore complex.

The universal TATA-binding protein, TBP, is an essential component of the multiprotein complex known as transcription factor IID (TFIID). This complex, which consists of TBP and TBP-associated factors (TAFs), is essential for RNA polymerase II-mediated transcription. The molecular size of human TBP (37.7 kD) is close to the passive diffusion limit along the transport channel of the nuclear pore complex (NPC). Therefore, the possibility exists that NPCs restrict TBP translocation to the nuclear interior. Here we show for the first time, with patch-clamp and atomic force microscopy (AFM), that NPCs regulate TBP movement into the nucleus and that TBP (10(-15)-10(-10)M) is capable of modifying NPC structure and function. The translocation of TBP was ATP-dependent and could be detected as a transient plugging of the NPC channels, with a concomitant transient reduction in single NPC channel conductance, gamma, to a negligible value. NPC unplugging was accompanied by permanent channel opening at concentrations greater than 250 pM. AFM images demonstrated that the TBP molecules attached to and accumulated on the NPC cytosolic side. NPC channel activity could be recorded for more than 48 hr. These observations suggest that three novel functions of TBP are: to stabilize NPC, to force the NPC channels into an open state, and to increase the number of functional channels. Since TBP is a major component of transcription, our observations are relevant to the understanding of the gene expression mechanisms underlying normal and pathological cell structure and function.

The peptide repeat domain of nucleoporin Nup98 functions as a docking site in transport across the nuclear pore complex

We report the cDNA deduced primary structure of a wheat germ agglutinin-reactive nuclear pore complex (NPC) protein of rat. The protein, termed Nup98 (for nucleoporin of 98 kDa), contains numerous GLFG and FG repeats and some FXFG repeats and is thus a vertebrate member of a family of GLFG nucleoporins that were previously discovered in yeast. Immunoelectron microscopy showed Nup98 to be asymmetrically located at the nucleoplasmic side of the NPC. Nup98 functions as one of several docking site nucleoporins in a cytosolic docking activity-mediated binding of a model transport substrate. The docking site of Nup98 was mapped to its N-terminal half, which contains all of the peptide repeats. A recombinant segment of this region depleted the docking activity of cytosol. We suggest that the peptide repeat domain of Nup98, together with peptide repeat domains of other nucleoporins, forms an array of sites for mediated docking of transport substrate, and that bidirectional transport across the NPC proceeds by repeated docking and undocking reactions.

Exploring nuclear pore complex structure and function in molecular detail

Bidirectional molecular trafficking between the nucleus and the cytoplasm of eukaryotic cells occurs through the nuclear pore complexes (NPCs), approximately 120 megadalton supramolecular assemblies embedded in the double-membraned nuclear envelope. Significant progress has been made in elucidating the three-dimensional (3-D) architecture of the NPC, and in identifying, characterizing, and cloning and sequencing NPC proteins. Several of these have now been localized within the 3-D structure of the NPC. Nevertheless, there still remain major questions relating to the conformation, molecular composition and functional roles of distinct NPC components. Here we review recent structural studies from our group and others which have contributed toward dissecting the molecular architecture of the NPC. We also present our results on the molecular characterization of some NPC components, and on the elucidation of their functional roles in mediated nucleocytoplasmic transport.

Exploring native nuclear pore complex structure and conformation by scanning force microscopy in physiological buffers

We have used the scanning force microscope to image the cytoplasmic and nuclear faces of the nuclear pore complex (NPC) in physiological buffer environment. In agreement with previous electron micrographs recorded from dehydrated specimens, we have been able to reproducibly distinguish a high degree of asymmetry between the nuclear and cytoplasmic surfaces of the nuclear pore complex. Very much like seen in the electron microscope, the cytoplasmic face of the nuclear pore complex appears ‘‘donutlike’’ with a massive, ∼18 nm high annulus surrounding the membrane pore. In contrast, the nuclear face of the pore complex looks like a ∼36 nm high ‘‘dome’’ in the scanning force microscope, which in the electron microscope is resolved into a tenuous ring from which eight thin filaments emanate that are joined distally by a terminal ring, thus forming a ‘‘basket’’ or ‘‘fishtrap.’’ In addition, we were able to visualize distinct structural changes occurring upon mechanical manipulation of the nuclear pore complex periphery with the scanning tip. These preliminary data provide us with the intriguing future possibility to directly correlate NPC structure with function.

Evolutionary conservation of ‘Basket’ structure in nuclear pore complexes, and their possible role in mRNA export

The nuclear pore complex (NPC) is a macromolecular complex which provides transport through the nuclear envelope. Each NPC has a diameter of around 120 nms, and extends outwards into the cytoplasm by some 20 nms via eight cytoplasmic filaments of granular substructure. On the nucleoplasmic face, NPC ‘cages’ or ‘baskets’ project some 60 nms into the nuclear interior where they terminate in an inner basket ring that supports a further structure, the nuclear envelope lattice (NEL), a network of filaments. Both the NEL and NPC baskets have been visualised to date only in amphibian germinal vesicle nuclear envelopes. All current models of NPC structure are based on data from amphibian oocytes, mainly due to the convenience of isolating clean and well preserved nuclear envelopes. It is important therefore to determine how widely these structures are conserved in other groups throughout the animal kingdom, as a basis for the overall evolutionary conservation of NPC structure.

The nuclear pore complex: three-dimensional surface structure revealed by field emission, in-lens scanning electron microscopy, with underlying structure uncovered by proteolysis

The structure of the nuclear pore complex (NPC) has been previously studied by many different electron microscopic techniques. Recently, scanning electron microscopes have been developed that can visualise biologically relevant structural detail at the same level of resolution as transmission electron microscopes and have been used to study NPC structure. We have used such an instrument to visualise directly the structure of both cytoplasmic and nucleoplasmic surfaces of the NPC of manually isolated amphibian oocyte nuclear envelopes that have been spread, fixed, critical point dried and coated with a thin fine-grained film of chromium or tantalum. We present images that directly show features of the NPC that are visible at each surface, including coaxial rings, cytoplasmic particles, plug/spoke complexes and the nucleoplasmic basket or fishtrap. Some cytoplasmic particles are rod-shaped or possibly "T"-shaped, can be quite long structures extending into the cytoplasm and may be joined to the coaxial ring at a position between each subunit. Both coaxial rings, which are proud of the membranes, can be exposed by light proteolytic digestion, revealing eight equal subunits each of which may be bipartite. We have determined that the nucleoplasmic filaments that make up the baskets are attached to the outer periphery of the coaxial ring at a position between each of its subunits. These filaments extend into the nucleoplasm and insert at the distal end to the smaller basket ring. The space left between adjacent basket filaments would exclude particles bigger than about 25 nm, which is consistent with the exclusion limit previously found for NPC-transported molecules.

Nuclear transport and nuclear pores in yeast.

The central features of nuclear import have been conserved during evolution. In yeast the nuclear accumulation of proteins follows the same selective and active transport mechanisms known from higher eukaryotes. Yeast nuclear proteins contain nuclear localization sequences (NLS) which are presumably recognized by receptors in the cytoplasm and the nuclear envelope. Subsequent to this recognition step, nuclear proteins are translocated into the nucleus via the nuclear pore complexes. The structure of the yeast nuclear pore complex resembles that of higher eukaryotes. Recently, the first putative components of the yeast nuclear import machinery have been cloned and sequenced. The genetically amenable yeast system allows for an efficient structural and functional analysis of these components. Due to the evolutionary conservation potential insights into the nuclear import mechanisms in yeast can be transferred to higher eukaryotes. Thus, yeast can be considered as a eukaryotic model system to study nuclear transport.