Nuclear Pore Complex Assembly Research Articles

Integral membrane proteins Brr6 and Apq12 link assembly of the nuclear pore complex to lipid homeostasis in the endoplasmic reticulum

Cells of Saccharomyces cerevisiae lacking Apq12, a nuclear envelope (NE)-endoplasmic reticulum (ER) integral membrane protein, are defective in assembly of nuclear pore complexes (NPCs), possibly because of defects in regulating membrane fluidity. We identified BRR6, which encodes an essential integral membrane protein of the NE-ER, as a dosage suppressor of apq12 Delta. Cells carrying the temperature-sensitive brr6-1 allele have been shown to have defects in nucleoporin localization, mRNA metabolism and nuclear transport. Electron microscopy revealed that brr6-1 cells have gross NE abnormalities and proliferation of the ER. brr6-1 cells were hypersensitive to compounds that affect membrane biophysical properties and to inhibitors of lipid biosynthetic pathways, and displayed strong genetic interactions with genes encoding non-essential lipid biosynthetic enzymes. Strikingly, brr6-1 cells accumulated, in or near the NE, elevated levels of the two classes of neutral lipids, steryl esters and triacylglycerols, and over-accumulated sterols when they were provided exogenously. Although neutral lipid synthesis is dispensable in wild-type cells, viability of brr6-1 cells was fully dependent on neutral lipid production. These data indicate that Brr6 has an essential function in regulating lipid homeostasis in the NE-ER, thereby impacting NPC formation and nucleocytoplasmic transport.

Formation of the Nuclear Envelope Permeability Barrier Studied by Sequential Photoswitching and Flux Analysis

In higher eukaryotes, the nuclear envelope breaks down during mitosis. It reforms during telophase, and nuclear import is reestablished within <10 min after anaphase onset. It is widely assumed that import functionality simultaneously leads to the exclusion of bulk cytoplasmic proteins. However, nuclear pore complex assembly is not fully completed when import capacity is regained, which raises the question of whether the transport and permeability barrier functions of the nuclear envelope are indeed coupled. In this study, we therefore analyzed the reestablishment of the permeability barrier of the nuclear envelope after mitosis in living cells by monitoring the flux of the reversibly photoswitchable fluorescent protein Dronpa from the cytoplasm into the nucleus after photoactivation. We performed many consecutive flux measurements in the same cell to directly monitor changes in nuclear envelope permeability. Our measurements at different time points after mitosis in individual cells show that contrary to the general view and despite the rapid reestablishment of facilitated nuclear import, the nuclear envelope remains relatively permeable for passive diffusion for the first 2 h after mitosis. Our data demonstrate that reformation of the permeability barrier of nuclear pore complexes occurs only gradually and is uncoupled from regaining active import functionality.

Transmembrane Protein-free Membranes Fuse into Xenopus Nuclear Envelope and Promote Assembly of Functional Pores

Post-mitotic reassembly of nuclear envelope (NE) and the endoplasmic reticulum (ER) has been reconstituted in a cell-free system based on interphase Xenopus egg extract. To evaluate the relative contributions of cytosolic and transmembrane proteins in NE and ER assembly, we replaced a part of native membrane vesicles with ones either functionally impaired by trypsin or N-ethylmaleimide treatments or with protein-free liposomes. Although neither impaired membrane vesicles nor liposomes formed ER and nuclear membrane, they both supported assembly reactions by fusing with native membrane vesicles. At membrane concentrations insufficient to generate full-sized functional nuclei, addition of liposomes and their fusion with membrane vesicles resulted in an extensive expansion of NE, further chromatin decondensation, restoration of the functionality, and spatial distribution of the nuclear pore complexes (NPCs), and, absent newly delivered transmembrane proteins, an increase in NPC numbers. This rescue of the nuclear assembly by liposomes was inhibited by wheat germ agglutinin and thus required active nuclear transport, similarly to the assembly of full-sized functional NE with membrane vesicles. Mechanism of fusion between liposomes and between liposomes and membrane vesicles was investigated using lipid mixing assay. This fusion required interphase cytosol and, like fusion between native membrane vesicles, was inhibited by guanosine 5'-3-O-(thio)triphosphate, soluble N-ethylmaleimide-sensitive factor attachment protein, and N-ethylmaleimide. Our findings suggest that interphase cytosol contains proteins that mediate the fusion stage of ER and NE reassembly, emphasize an unexpected tolerance of nucleus assembly to changes in concentrations of transmembrane proteins, and reveal the existence of a feedback mechanism that couples NE expansion with NPC assembly.

Investigation of Ternary Complexes: DNA–Phosphatidylcholine Liposomes–Mg2+ by Freeze-Fracture Method and Their Role in the Formation of Some Cell Structures

Long-term investigations of ternary complexes: DNA-zwitterionic liposomes-divalent metal cations have revealed many details of their structure; but some questions need additional study. The conditions under which fusion or aggregation of liposomes occurs during such complex formation remain obscure. The DNA structure in the ternary complex is still unclear. In this work, using a freeze-fracture method, author demonstrate the thin structure of a complex (early attempts to observe this structure employing other electron microscopic methods, in particular cryo-TEM, have not met with success). After treatment of ternary complexes with nuclease S1, which is able to digest single-stranded DNA, local DNA unwinding in such complexes was confirmed. Author describe how the curvature of liposomes as the main factor may determine the interaction between liposomes and DNA, especially aggregation or fusion of liposomes during ternary complex formation. Therefore, interaction between lipids of membrane vesicles in cell and chromatin DNA can be the first stage of a nuclear envelope and pore complex assembly.

The transmembrane nucleoporin NDC1 is required for targeting of ALADIN to nuclear pore complexes

NDC1 is a transmembrane nucleoporin that is required for NPC assembly and nucleocytoplasmic transport. We show here that NDC1 directly interacts with the nucleoporin ALADIN, mutations of which are responsible for triple-A syndrome, and that this interaction is required for targeting of ALADIN to nuclear pore complexes (NPCs). Furthermore, we show that NDC1 is required for selective nuclear import. Our findings suggest that NDC1-mediated localization of ALADIN to NPCs is essential for selective nuclear protein import, and that abrogation of the interaction between ALADIN and NDC1 may be important for the development of triple-A syndrome.

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 beta. 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 beta is only partially reversed by RanGTP. Importin beta 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 beta and Ran, may be involved in coordinating the initial seeding of chromatin with subsequent steps in the NPC assembly pathway.

Lipid–nucleic acids interactions as base for organization and expression of cellular genome

AbstractAlthough lipid–nucleic acid interactions have been studied, with certain or little progress, for more than 30 years, it is only in recent years that the problem has received particular attention. It should, however, be noted that most studies deal with DNA‐cationic surfactants interactions, whereas DNA‐zwitterionic interactions, which are more complex and close to nature, are poorly investigated. The long‐standing studies of the triple complexes: DNA–phosphatidylcholine liposomes–divalent metal cations allow us to confirm that these complexes are responsible for the formation of not only the structures existing in DNA–cationic liposome complexes but also some other cellular structures. The author proposed hypothesis about the involvement of direct DNA–lipid interactions in the nuclear pore assembly. Only taking into account interactions between DNA and lipids of cellular membrane, one can explain the origin of such structures as nucleoid, nuclear pore, and nuclear matrix. The formation of triple complexes was accompanied by the aggregation and partial fusion of liposomes as was shown by cryo‐TEM technique. The author has presented new data on the structure of triple complexes, which were obtained by phase contrast cryo‐TEM. Biophysical data on the liposomes fusion during triple complex formation and perspective of their computer simulation are also presented. DNA acts as a fusogen in this process and it unwinds in the region of liposomes fusion. The nuclear envelope and pore complexes assembly is provided by membrane vesicles fusion. Author has proposed that the DNA‐induced fusion of zwitterionic liposomes in vitro may suggest the involvement of direct lipids–DNA interaction in nuclear envelope assembly. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2010

Piecing together nuclear pore complex assembly during interphase

All nucleocytoplasmic traffic of macromolecules occurs through nuclear pore complexes (NPCs), which function as stents in the nuclear envelope to keep nuclear pores open but gated. Three studies in this issue (Flemming, D., P. Sarges, P. Stelter, A. Hellwig, B. Böttcher, and E. Hurt. 2009. J. Cell Biol. 185:387–395; Makio, T., L.H. Stanton, C.-C. Lin, D.S. Goldfarb, K. Weis, and R.W. Wozniak. 2009. J. Cell Biol. 185:459–491; Onishchenko, E., L.H. Stanton, A.S. Madrid, T. Kieselbach, and K. Weis. 2009. J. Cell Biol. 185:475–491) further our understanding of the NPC assembly process by reporting what happens when the supply lines of key proteins that provide a foundation for building these marvelous supramolecular structures are disrupted.

Role of the Ndc1 interaction network in yeast nuclear pore complex assembly and maintenance

The nuclear pore complex (NPC) mediates all nucleocytoplasmic transport, yet its structure and biogenesis remain poorly understood. In this study, we have functionally characterized interaction partners of the yeast transmembrane nucleoporin Ndc1. Ndc1 forms a distinct complex with the transmembrane proteins Pom152 and Pom34 and two alternative complexes with the soluble nucleoporins Nup53 and Nup59, which in turn bind to Nup170 and Nup157. The transmembrane and soluble Ndc1-binding partners have redundant functions at the NPC, and disruption of both groups of interactions causes defects in Ndc1 targeting and in NPC structure accompanied by significant pore dilation. Using photoconvertible fluorescent protein fusions, we further show that the depletion of Pom34 in cells that lack NUP53 and NUP59 blocks new NPC assembly and leads to the reversible accumulation of newly made nucleoporins in cytoplasmic foci. Therefore, Ndc1 together with its interaction partners are collectively essential for the biosynthesis and structural integrity of yeast NPCs.

The nucleoporins Nup170p and Nup157p are essential for nuclear pore complex assembly

We have established that two homologous nucleoporins, Nup170p and Nup157p, play an essential role in the formation of nuclear pore complexes (NPCs) in Saccharomyces cerevisiae. By regulating their synthesis, we showed that the loss of these nucleoporins triggers a decrease in NPCs caused by a halt in new NPC assembly. Preexisting NPCs are ultimately lost by dilution as cells grow, causing the inhibition of nuclear transport and the loss of viability. Significantly, the loss of Nup170p/Nup157p had distinct effects on the assembly of different architectural components of the NPC. Nucleoporins (nups) positioned on the cytoplasmic face of the NPC rapidly accumulated in cytoplasmic foci. These nup complexes could be recruited into new NPCs after reinitiation of Nup170p synthesis, and may represent a physiological intermediate. Loss of Nup170p/Nup157p also caused core and nucleoplasmically positioned nups to accumulate in NPC-like structures adjacent to the inner nuclear membrane, which suggests that these nucleoporins are required for formation of the pore membrane and the incorporation of cytoplasmic nups into forming NPCs.

Nuclear pore complex structure and assembly involve the ER membrane protein Rtn1

Nucleocytoplasmic transport occurs exclusively through nuclear pore complexes (NPCs) which have been implicated in proper embryonic development. Although structural studies have resolved the complex NPC architecture, the mechanism that directs pore membrane fusion and NPC biogenesis remains unclear. Reticulons (RTNs) and Yop1/DP1 are conserved membrane protein families required to form and maintain the tubular endoplasmic reticulum (ER). Remarkably, whereas NPC membrane proteins such as Pom34 and Pom152 are not required for viability, an rtn1δ yop1δ pom34δ pom152δ quadruple null (δ) mutant is lethal. This suggests that Rtn1 and/or Yop1 play a role in NPC structure, assembly and/or maintenance. This lethality is rescued by expression of wild type RTN1, as well as by the more nuclear localized point mutant rtn1‐K48I. The exogenous expression of RTN1 and rtn1‐K48I also rescues a lethal pom34δ nup59δ double mutant. Nup59 is a structural NPC protein linked to NPC assembly. These results indicate that Rtn1 has at least a partial redundancy in function with NPC structural and transmembrane proteins. In addition, this suggests a function for the reticulons at the NE that is independent of RTNs role at the tubular ER. We hypothesize that RTNs and/or Yop1/DP1 stabilize the membrane curvature of the nuclear pore during NPC assembly.Grant Funding SourceIRACDA and NIH

ER membrane–bending proteins are necessary for de novo nuclear pore formation

Nucleocytoplasmic transport occurs exclusively through nuclear pore complexes (NPCs) embedded in pores formed by inner and outer nuclear membrane fusion. The mechanism for de novo pore and NPC biogenesis remains unclear. Reticulons (RTNs) and Yop1/DP1 are conserved membrane protein families required to form and maintain the tubular endoplasmic reticulum (ER) and the postmitotic nuclear envelope. In this study, we report that members of the RTN and Yop1/DP1 families are required for nuclear pore formation. Analysis of Saccharomyces cerevisiae prp20-G282S and nup133Δ NPC assembly mutants revealed perturbations in Rtn1–green fluorescent protein (GFP) and Yop1-GFP ER distribution and colocalization to NPC clusters. Combined deletion of RTN1 and YOP1 resulted in NPC clustering, nuclear import defects, and synthetic lethality with the additional absence of Pom34, Pom152, and Nup84 subcomplex members. We tested for a direct role in NPC biogenesis using Xenopus laevis in vitro assays and found that anti-Rtn4a antibodies specifically inhibited de novo nuclear pore formation. We hypothesize that these ER membrane–bending proteins mediate early NPC assembly steps.

Assembly of nuclear pore complexes mediated by major vault protein

During interphase growth of eukaryotic cells, nuclear pore complexes (NPCs) are continuously incorporated into the intact nuclear envelope (NE) by mechanisms that are largely unknown. De novo formation of NPCs involves local fusion events between the inner and outer nuclear membrane, formation of a transcisternal membranous channel of defined diameter and the coordinated assembly of hundreds of nucleoporins into the characteristic NPC structure. Here we have used a cell-free system based on Xenopus egg extract, which allows the experimental separation of nuclear-membrane assembly and NPC formation. Nuclei surrounded by a closed double nuclear membrane, but devoid of NPCs, were first reconstituted from chromatin and a specific membrane fraction. Insertion of NPCs into the preformed pore-free nuclei required cytosol containing soluble nucleoporins or nucleoporin subcomplexes and, quite unexpectedly, major vault protein (MVP). MVP is the main component of vaults, which are ubiquitous barrel-shaped particles of enigmatic function. Our results implicate MVP, and thus also vaults, in NPC biogenesis and provide a functional explanation for the association of a fraction of vaults with the NE and specifically with NPCs in intact cells.

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.

Mutation of the Zebrafish Nucleoporin elys Sensitizes Tissue Progenitors to Replication Stress

The recessive lethal mutation flotte lotte (flo) disrupts development of the zebrafish digestive system and other tissues. We show that flo encodes the ortholog of Mel-28/Elys, a highly conserved gene that has been shown to be required for nuclear integrity in worms and nuclear pore complex (NPC) assembly in amphibian and mammalian cells. Maternal elys expression sustains zebrafish flo mutants to larval stages when cells in proliferative tissues that lack nuclear pores undergo cell cycle arrest and apoptosis. p53 mutation rescues apoptosis in the flo retina and optic tectum, but not in the intestine, where the checkpoint kinase Chk2 is activated. Chk2 inhibition and replication stress induced by DNA synthesis inhibitors were lethal to flo larvae. By contrast, flo mutants were not sensitized to agents that cause DNA double strand breaks, thus showing that loss of Elys disrupts responses to selected replication inhibitors. Elys binds Mcm2-7 complexes derived from Xenopus egg extracts. Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine. These in vivo data indicate a role for Elys in Mcm2-chromatin interactions. Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

Nuclear pore complex assembly through the cell cycle: Regulation and membrane organization

In eukaryotes, all macromolecules traffic between the nucleus and the cytoplasm through nuclear pore complexes (NPCs), which are among the largest supramolecular assemblies in cells. Although their composition in yeast and metazoa is well characterized, understanding how NPCs are assembled and form the pore through the double membrane of the nuclear envelope and how both processes are controlled still remains a challenge. Here, we summarize what is known about the biogenesis of NPCs throughout the cell cycle with special focus on the membrane reorganization and the regulation that go along with NPC assembly.

Systematic kinetic analysis of mitotic dis- and reassembly of the nuclear pore in living cells

During mitosis in higher eukaryotes, nuclear pore complexes (NPCs) disassemble in prophase and are rebuilt in anaphase and telophase. NPC formation is hypothesized to occur by the interaction of mitotically stable subcomplexes that form defined structural intermediates. To determine the sequence of events that lead to breakdown and reformation of functional NPCs during mitosis, we present here our quantitative assay based on confocal time-lapse microscopy of single dividing cells. We use this assay to systematically investigate the kinetics of dis- and reassembly for eight nucleoporin subcomplexes relative to nuclear transport in NRK cells, linking the assembly state of the NPC with its function. Our data establish that NPC assembly is an ordered stepwise process that leads to import function already in a partially assembled state. We furthermore find that nucleoporin dissociation does not occur in the reverse order from binding during assembly, which may indicate a distinct mechanism.

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.

NEP-A and NEP-B both contribute to nuclear pore formation in Xenopus eggs and oocytes

In vertebrates, the nuclear envelope (NE) assembles and disassembles during mitosis. As the NE is a complex structure consisting of inner and outer membranes, nuclear pore complexes (NPCs) and the nuclear lamina, NE assembly must be a controlled and systematic process. In Xenopus egg extracts, NE assembly is mediated by two distinct membrane vesicle populations, termed NEP-A and NEP-B. Here, we re-investigate how these two membrane populations contribute to NPC assembly. In growing stage III Xenopus oocytes, NPC assembly intermediates are frequently observed. High concentrations of NPC assembly intermediates always correlate with fusion of vesicles into preformed membranes. In Xenopus egg extracts, two integral membrane proteins essential for NPC assembly, POM121 and NDC1, are exclusively associated with NEP-B membranes. By contrast, a third integral membrane protein associated with the NPCs, gp210, associates only with NEP-A membranes. During NE assembly, fusion between NEP-A and NEP-B led to the formation of fusion junctions at which >65% of assembling NPCs were located. To investigate how each membrane type contributes to NPC assembly, we preferentially limited NEP-A in NE assembly assays. We found that, by limiting the NEP-A contribution to the NE, partially formed NPCs were assembled in which protein components of the nucleoplasmic face were depleted or absent. Our data suggest that fusion between NEP-A and NEP-B membranes is essential for NPC assembly and that, in contrast to previous reports, both membranes contribute to NPC assembly.

ELYS/MEL-28 Chromatin Association Coordinates Nuclear Pore Complex Assembly and Replication Licensing

SummaryXenopus 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.