The conserved Nup107-160 complex is critical for nuclear pore complex assembly

Nuclear pore complexes (NPCs) are large multiprotein assemblies that allow traffic between the cytoplasm and the nucleus. During mitosis in higher eukaryotes, the Nuclear Envelope (NE) breaks down and NPCs disassemble. How NPCs reassemble and incorporate into the NE upon mitotic exit is poorly understood. We demonstrate a function for the conserved Nup107-160 complex in this process. Partial in vivo depletion of Nup133 or Nup107 via RNAi in HeLa cells resulted in reduced levels of multiple nucleoporins and decreased NPC density in the NE. Immunodepletion of the entire Nup107-160 complex from in vitro nuclear assembly reactions produced nuclei with a continuous NE but no NPCs. This phenotype was reversible only if Nup107-160 complex was readded before closed NE formation. Depletion also prevented association of FG-repeat nucleoporins with chromatin. We propose a stepwise model in which postmitotic NPC assembly initiates on chromatin via early recruitment of the Nup107-160 complex.     

The integral membrane nucleoporin pom121 functionally links nuclear pore complex assembly and nuclear envelope formation

The metazoan nuclear envelope (NE) breaks down and reforms at each mitosis. Nuclear pore complexes (NPCs), which allow nucleocytoplasmic transport during interphase, assemble into the reforming NE at the end of mitosis. Using in vitro NE assembly assays, we show that one of the two transmembrane nucleoporins, pom121, is essential for NE formation, whereas the second, gp210, is dispensable. Depletion of either pom121-containing membrane vesicles or the protein alone does not affect vesicle binding to chromatin but prevents their fusion to form a closed NE. When the Nup107-160 complex, which is essential for integration of NPCs into the NE, is also depleted, pom121 becomes dispensable for NE formation, suggesting a close functional link between NPC and NE formation and the existence of a checkpoint that monitors NPC assembly state.     

ELYS/MEL-28 chromatin association coordinates nuclear pore complex assembly and replication licensing

Xenopus egg extract supports all the major cell-cycle transitions in vitro. We have used a proteomics approach to identify proteins whose abundance on chromatin changes during the course of an in vitro cell cycle. One of the proteins we identified was ELYS/MEL-28, which has recently been described as the earliest-acting factor known to be required for nuclear pore complex (NPC) assembly [1-4]. ELYS interacts with the Nup107-160 complex and is required for its association with chromatin. ELYS contains an AT-hook domain, which we show binds to chromatin with a high affinity. This domain can compete with full-length ELYS for chromatin association, thereby blocking NPC assembly. This provides evidence that ELYS interacts directly with chromatin and that this interaction is essential for NPC assembly and compartmentalization of chromosomal DNA within the cell. Furthermore, we detected a physical association on chromatin between ELYS and the Mcm2-7 replication-licensing proteins. ELYS chromatin loading, NPC assembly, and nuclear growth were delayed when Mcm2-7 was prevented from loading onto chromatin. Because nuclear assembly is required to shut down licensing prior to entry into S phase, our results suggest a mechanism by which these two early cell-cycle events are coordinated with one another.     

Capture of AT-rich chromatin by ELYS recruits POM121 and NDC1 to initiate nuclear pore assembly

Assembly of the nuclear pore, gateway to the genome, from its component subunits is a complex process. In higher eukaryotes, nuclear pore assembly begins with the binding of ELYS/MEL-28 to chromatin and recruitment of the large critical Nup107-160 pore subunit. The choreography of steps that follow is largely speculative. Here, we set out to molecularly define early steps in nuclear pore assembly, beginning with chromatin binding. Point mutation analysis indicates that pore assembly is exquisitely sensitive to the change of only two amino acids in the AT-hook motif of ELYS. The dependence on AT-rich chromatin for ELYS binding is borne out by the use of two DNA-binding antibiotics. AT-binding Distamycin A largely blocks nuclear pore assembly, whereas GC-binding Chromomycin A(3) does not. Next, we find that recruitment of vesicles containing the key integral membrane pore proteins POM121 and NDC1 to the forming nucleus is dependent on chromatin-bound ELYS/Nup107-160 complex, whereas recruitment of gp210 vesicles is not. Indeed, we reveal an interaction between the cytoplasmic domain of POM121 and the Nup107-160 complex. Our data thus suggest an order for nuclear pore assembly of 1) AT-rich chromatin sites, 2) ELYS, 3) the Nup107-160 complex, and 4) POM121- and NDC1-containing membrane vesicles and/or sheets, followed by (5) assembly of the bulk of the remaining soluble pore subunits.     

POM121 and Sun1 play a role in early steps of interphase NPC assembly

Nuclear pore complexes (NPCs) assemble at the end of mitosis during nuclear envelope (NE) reformation and into an intact NE as cells progress through interphase. Although recent studies have shown that NPC formation occurs by two different molecular mechanisms at two distinct cell cycle stages, little is known about the molecular players that mediate the fusion of the outer and inner nuclear membranes to form pores. In this paper, we provide evidence that the transmembrane nucleoporin (Nup), POM121, but not the Nup107-160 complex, is present at new pore assembly sites at a time that coincides with inner nuclear membrane (INM) and outer nuclear membrane (ONM) fusion. Overexpression of POM121 resulted in juxtaposition of the INM and ONM. Additionally, Sun1, an INM protein that is known to interact with the cytoskeleton, was specifically required for interphase assembly and localized with POM121 at forming pores. We propose a model in which POM121 and Sun1 interact transiently to promote early steps of interphase NPC assembly.     

An InCytes from MBC Selection: Importin β Regulates the Seeding of Chromatin with Initiation Sites for Nuclear Pore Assembly

The nuclear envelope of higher eukaryotic cells reforms at the exit from mitosis, in concert with the assembly of nuclear pore complexes (NPCs). The first step in postmitotic NPC assembly involves the “seeding” of chromatin with ELYS and the Nup107-160 complex. Subsequent steps in the assembly process are poorly understood and different mechanistic models have been proposed to explain the formation of the full supramolecular structure. Here, we show that the initial step of chromatin seeding is negatively regulated by importin β. Direct imaging of the chromatin attachment sites reveals single sites situated predominantly on the highest substructures of chromatin surface and lacking any sign of annular structures or oligomerized pre-NPCs. Surprisingly, the inhibition by importin β is only partially reversed by RanGTP. Importin β forms a high-molecular-weight complex with both ELYS and the Nup107-160 complex in cytosol. We suggest that initiation sites for NPC assembly contain single copies of chromatin-bound ELYS/Nup107-160 and that the lateral oligomerization of these subunits depends on the recruitment of membrane components. We predict that additional regulators, besides importin β and Ran, may be involved in coordinating the initial seeding of chromatin with subsequent steps in the NPC assembly pathway.     

The discovery of a Werner helicase interacting protein (WHIP) association with the nuclear pore complex

The gateway for molecular trafficking between the cytoplasm and the nucleus is the Nuclear Pore Complex (NPC). Through mass spectral analysis of the isolated Nuclear Pore Nup107-160 subcomplex, we discovered an in vivo interaction with Werner's Helicase Interacting Protein 1, (WRNIP1 or WHIP). WHIP was originally identified as a binding partner of Werner protein (WRN), which functions to maintain genome stability and is responsible for the progeria disease, Werner syndrome. We established the reciprocal isolation of Nup107 by α-WHIP. WHIP was found in purified Nuclear Envelope (NE) fractions treated with DNase/RNase/Heparin. We demonstrated by immunofluorescence microscopy that WHIP is located at the nuclear rim as well as punctate regions in the nuclear matrix. Ultimately, synchronized cells show a dynamic association between WHIP and the Nup107-160 subcomplex through the cell cycle without an interaction with WRN. We thus identify WHIP as a partner/component of the NE/NPC and set forth to investigate a role for the protein positioned at the NPC.     

MEL-28, a novel nuclear-envelope and kinetochore protein essential for zygotic nuclear-envelope assembly in C. elegans

The nuclear envelope (NE) of eukaryotic cells separates nucleoplasm from cytoplasm, mediates nucleo-cytoplasmic transport, and contributes to the control of gene expression. The NE consists of three major components: the nuclear membranes, the nuclear pore complexes (NPCs), and the nuclear lamina. The list of identified NE proteins has increased considerably during recent years but is most likely not complete. In most eukaryotes, the NE breaks down and is then reassembled during mitosis. The assembly of NPCs and the association and fusion of nuclear membranes around decondensing chromosomes are tightly coordinated processes. Here, we report the identification and characterization of MEL-28, a large protein essential for the assembly of a functional NE in C. elegans embryos. RNAi depletion or genetic mutation of mel-28 severely impairs nuclear morphology and leads to abnormal distribution of both integral NE proteins and NPCs. The structural defects of the NE were associated with functional defects and lack of nuclear exclusion of soluble proteins. MEL-28 localizes to NPCs during interphase, to kinetochores in early to middle mitosis then is widely distributed on chromatin late in mitosis. We show that MEL-28 is an early-assembling, stable NE component required for all aspects of NE assembly.     

Nup53 is required for nuclear envelope and nuclear pore complex assembly

Transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). These structures are composed of various subcomplexes of proteins that are each present in multiple copies and together establish the eightfold symmetry of the NPC. One evolutionarily conserved subcomplex of the NPC contains the nucleoporins Nup53 and Nup155. Using truncation analysis, we have defined regions of Nup53 that bind to neighboring nucleoporins as well as those domains that target Nup53 to the NPC in vivo. Using this information, we investigated the role of Nup53 in NE and NPC assembly using Xenopus egg extracts. We show that both events require Nup53. Importantly, the analysis of Nup53 fragments revealed that the assembly activity of Nup53 depleted extracts could be reconstituted using a region of Nup53 that binds specifically to its interacting partner Nup155. On the basis of these results, we propose that the formation of a Nup53-Nup155 complex plays a critical role in the processes of NPC and NE assembly.     

The nucleoporin Nup188 controls passage of membrane proteins across the nuclear pore complex

All transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). Despite their enormous size, ∼60 MD in vertebrates, they are comprised of only ∼30 distinct proteins (nucleoporins or Nups), many of which form subcomplexes that act as building blocks for NPC assembly. One of these evolutionarily conserved subcomplexes, the Nup93 complex, is a major structural component linking the NPC to the membranes of the NE. Using in vitro nuclear assembly assays, we show that two components of the Nup93 complex, Nup188 and Nup205, are dispensable for NPC formation. However, nuclei lacking Nup188 increase in size by several fold compared with wild type. We demonstrate that this phenotype is caused by an accelerated translocation of integral membrane proteins through NPCs, suggesting that Nup188 confines the passage of membrane proteins and is thus crucial for the homeostasis of the different nuclear membranes.     

Structural and functional studies of Nup107/Nup133 interaction and its implications for the architecture of the nuclear pore complex

Nuclear pore complexes (NPCs) are 40-60 MDa protein assemblies embedded in the nuclear envelope of eukaryotic cells. NPCs exclusively mediate all transport between cytoplasm and nucleus. The nucleoporins that build the NPC are arranged in a stable core of module-like subcomplexes with eight-fold rotational symmetry. To gain insight into the intricate assembly of the NPC, we have solved the crystal structure of a protein complex between two nucleoporins, human Nup107 and Nup133. Both proteins form elongated structures that interact tightly via a compact interface in tail-to-tail fashion. Additional experiments using structure-guided mutants show that Nup107 is the critical anchor for Nup133 to the NPC, positioning Nup133 at the periphery of the NPC. The significant topological differences between Nup107 and Nup133 suggest that *-helical nucleoporin domains of the NPC scaffold fall in different classes and fulfill largely nonredundant functions.     

Early embryonic requirement for nucleoporin Nup35/NPP-19 in nuclear assembly

Nuclear pore complexes (NPCs) are gateways for transport between the nucleus and cytoplasm of eukaryotic cells and play crucial roles in regulation of gene expression. NPCs are composed of multiple copies of ∼ 30 different nucleoporins (nups) that display both ubiquitous and cell type specific functions during development. Vertebrate Nup35 (also known as Nup53) was previously described to interact with Nup93, Nup155 and Nup205 and to be required for nuclear envelope (NE) assembly in vitro. Here, we report the first in vivo characterization of a Nup35 mutation, npp-19(tm2886), and its temperature-dependent effects on Caenorhabditis elegans embryogenesis. At restrictive temperature, npp-19(tm2886) embryos exhibit chromosome missegregation, nuclear morphology defects and die around mid-gastrulation. Depletion of Nup35/NPP-19 inhibits NE localization of Nup155/NPP-8, NPC assembly and nuclear lamina formation. Consequently, nuclear envelope function, including nucleo-cytoplasmic transport, is impaired. In contrast, recruitment of Nup107/NPP-5, LEM-2 and nuclear membranes to the chromatin surface is Nup35/NPP-19-independent, suggesting an uncoupling of nuclear membrane targeting and NPC assembly in the absence of Nup35/NPP-19. We propose that Nup35/NPP-19 has an evolutionary conserved role in NE formation and function, and that this role is particularly critical during the rapid cell divisions of early embryogenesis.     

Pom121 links two essential subcomplexes of the nuclear pore complex core to the membrane

Nuclear pore complexes (NPCs) control the movement of molecules across the nuclear envelope (NE). We investigated the molecular interactions that exist at the interface between the NPC scaffold and the pore membrane. We show that key players mediating these interactions in mammalian cells are the nucleoporins Nup155 and Nup160. Nup155 depletion massively alters NE structure, causing a dramatic decrease in NPC numbers and the improper targeting of membrane proteins to the inner nuclear membrane. The role of Nup155 in assembly is likely closely linked to events at the membrane as we show that Nup155 interacts with pore membrane proteins Pom121 and NDC1. Furthermore, we demonstrate that the N terminus of Pom121 directly binds the β-propeller regions of Nup155 and Nup160. We propose a model in which the interactions of Pom121 with Nup155 and Nup160 are predicted to assist in the formation of the nuclear pore and the anchoring of the NPC to the pore membrane.     

The nucleoporin Nup153 is required for nuclear pore basket formation, nuclear pore complex anchoring and import of a subset of nuclear proteins

The nuclear pore complex (NPC) is a large proteinaceous structure through which bidirectional transport of macromolecules across the nuclear envelope (NE) takes place. Nup153 is a peripheral NPC component that has been implicated in protein and RNP transport and in the interaction of NPCs with the nuclear lamina. Here, Nup153 is localized by immunogold electron microscopy to a position on the nuclear ring of the NPC. Nuclear reconstitution is used to investigate the role of Nup153 in nucleo- cytoplasmic transport and NPC architecture. NPCs assembled in the absence of Nup153 lacked several nuclear basket components, were unevenly distributed in the NE and, unlike wild-type NPCs, were mobile within the NE. Importin alpha/beta-mediated protein import into the nucleus was strongly reduced in the absence of Nup153, while transportin-mediated import was unaffected. This was due to a reduction in import complex translocation rather than to defective receptor recycling. Our results therefore reveal functions for Nup153 in NPC assembly, in anchoring NPCs within the NE and in mediating specific nuclear import events.     

Inner/Outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis

Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.     

The Nup107-160 nucleoporin complex is required for correct bipolar spindle assembly

The Nup107-160 complex is a critical subunit of the nuclear pore. This complex localizes to kinetochores in mitotic mammalian cells, where its function is unknown. To examine Nup107-160 complex recruitment to kinetochores, we stained human cells with antisera to four complex components. Each antibody stained not only kinetochores but also prometaphase spindle poles and proximal spindle fibers, mirroring the dual prometaphase localization of the spindle checkpoint proteins Mad1, Mad2, Bub3, and Cdc20. Indeed, expanded crescents of the Nup107-160 complex encircled unattached kinetochores, similar to the hyperaccumulation observed of dynamic outer kinetochore checkpoint proteins and motors at unattached kinetochores. In mitotic Xenopus egg extracts, the Nup107-160 complex localized throughout reconstituted spindles. When the Nup107-160 complex was depleted from extracts, the spindle checkpoint remained intact, but spindle assembly was rendered strikingly defective. Microtubule nucleation around sperm centrosomes seemed normal, but the microtubules quickly disassembled, leaving largely unattached sperm chromatin. Notably, Ran-GTP caused normal assembly of microtubule asters in depleted extracts, indicating that this defect was upstream of Ran or independent of it. We conclude that the Nup107-160 complex is dynamic in mitosis and that it promotes spindle assembly in a manner that is distinct from its functions at interphase nuclear pores.     

The conserved transmembrane nucleoporin NDC1 is required for nuclear pore complex assembly in vertebrate cells

Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in the nuclear envelope (NE), through which exchange of molecules between the nucleus and cytosol occurs. Biogenesis of NPCs is complex and poorly understood. In particular, almost nothing is known about how NPCs are anchored in the NE. Here, we characterize vertebrate NDC1--a transmembrane nucleoporin conserved between yeast and metazoans. We show by RNA interference (RNAi) and biochemical depletion that NDC1 plays an important role in NPC and NE assembly in vivo and in vitro. RNAi experiments suggest a functional link between NDC1 and the soluble nucleoporins Nup93, Nup53, and Nup205. Importantly, NDC1 interacts with Nup53 in vitro. This suggests that NDC1 function involves forming a link between the NE membrane and soluble nucleoporins, thereby anchoring the NPC in the membrane.     

Dimerization and direct membrane interaction of Nup53 contribute to nuclear pore complex assembly

Nuclear pore complexes (NPCs) fuse the two membranes of the nuclear envelope (NE) to a pore, connecting cytoplasm and nucleoplasm and allowing exchange of macromolecules between these compartments. Most NPC proteins do not contain integral membrane domains and thus it is largely unclear how NPCs are embedded and anchored in the NE. Here, we show that the evolutionary conserved nuclear pore protein Nup53 binds independently of other proteins to membranes, a property that is crucial for NPC assembly and conserved between yeast and vertebrates. The vertebrate protein comprises two membrane binding sites, of which the C‐terminal domain has membrane deforming capabilities, and is specifically required for de novo NPC assembly and insertion into the intact NE during interphase. Dimerization of Nup53 contributes to its membrane interaction and is crucial for its function in NPC assembly.     

The entire Nup107-160 complex, including three new members, is targeted as one entity to kinetochores in mitosis

In eukaryotes, bidirectional transport of macromolecules between the cytoplasm and the nucleus occurs through elaborate supramolecular structures embedded in the nuclear envelope, the nuclear pore complexes (NPCs). NPCs are composed of multiple copies of approximately 30 different proteins termed nucleoporins, of which several can be biochemically isolated as subcomplexes. One such building block of the NPC, termed the Nup107-160 complex in vertebrates, was so far demonstrated to be composed of six different nucleoporins. Here, we identify three WD (Trp-Asp)-repeat nucleoporins as new members of this complex, two of which, Nup37 and Nup43, are specific to higher eukaryotes. The third new member Seh1 is more loosely associated with the Nup107-160 complex biochemically, but its depletion by RNA interference leads to phenotypes similar to knock down of other constituents of this complex. By combining green fluorescent protein-tagged nucleoporins and specific antibodies, we show that all the constituents of this complex, including Nup37, Nup43, Seh1, and Sec13, are targeted to kinetochores from prophase to anaphase of mitosis. Together, our results indicate that the entire Nup107-160 complex, which comprises nearly one-third of the so-far identified nucleoporins, specifically localizes to kinetochores in mitosis.