Assembly of the nuclear pore: biochemically distinct steps revealed with NEM, GTP gamma S, and BAPTA

A key event in nuclear formation is the assembly of functional nuclear pores. We have used a nuclear reconstitution system derived from Xenopus eggs to examine the process of nuclear pore assembly in vitro. With this system, we have identified three reagents which interfere with nuclear pore assembly, NEM, GTP gamma S, and the Ca++ chelator, BAPTA. These reagents have allowed us to determine that the assembly of a nuclear pore requires the prior assembly of a double nuclear membrane. Inhibition of nuclear vesicle fusion by pretreatment of the membrane vesicle fraction with NEM blocks pore complex assembly. In contrast, NEM treatment of already fused double nuclear membranes does not block pore assembly. This indicates that NEM inhibits a single step in pore assembly--the initial fusion of vesicles required to form a double nuclear membrane. The presence of GTP gamma S blocks pore assembly at two distinct steps, first by preventing fusion between nuclear vesicles, and second by blocking a step in pore assembly that occurs on already fused double nuclear membranes. Interestingly, when the Ca2+ chelator BAPTA is added to a nuclear assembly reaction, it only transiently blocks nuclear vesicle fusion, but completely blocks nuclear pore assembly. This results in the formation of a nucleus surrounded by a double nuclear membrane, but devoid of nuclear pores. To order the positions at which GTP gamma S and BAPTA interfere with pore assembly, a novel anchored nuclear assembly assay was developed. This assay revealed that the BAPTA-sensitive step in pore assembly occurs after the second GTP gamma S-sensitive step. Thus, through use of an in vitro nuclear reconstitution system, it has been possible to biochemically define and order multiple steps in nuclear pore assembly.     

Nuclear envelope transport capacity and the cell cycle in yeast (Saccharomyces cerevisiae).

Changes in the nuclear envelope transport capacity, as measured by the number of nuclear pore complexes/unit nuclear volume/cell, were followed during the Saccharomyces cerevisiae cell cycle using data obtained by freeze-fracture electron microscopy. Pore number per unit nuclear volume decreased sharply in early G0, remained steady from mid-GO through S to G2, and showed a further slight decrease at M and G1. These periods of decline apparently resulted from nuclear enlargement without sufficient formation of new nuclear pore complexes to maintain the pore number to nuclear volume ratio. However, marked nuclear pore formation did accompany both increases in nuclear volume. The significance of these changes in relation to other events in the cell cycle is discussed. The validity of using nuclear pore number/unit nuclear volume and other pore number data as indices of nuclear envelope transport capacity and cell activity is critically examined.     

The nuclear pore and plant development

All macromolecules that traffic between the nucleus and the cytoplasm traverse the nuclear pore. While yeast and mammalian nuclear pore structure and function have recently been substantially refined, our understanding of the plant nuclear pore is still far from comprehensive. Nevertheless, a number of nuclear pore and nucleocytoplasmic trafficking components have recently been identified as required for diverse developmental and signaling pathways. In addition, some aspects of the nuclear pore composition itself now appear under developmental control and nuclear pore components have recently surfaced as novel players in plant cytokinesis. Here, we review these new findings in context and attempt to correlate molecular functions with developmental processes.     

Reconstitution of biochemically altered nuclear pores: transport can be eliminated and restored

Biochemically altered nuclear pores specifically lacking the N-acetylglucosamine-bearing pore proteins were constructed in a nuclear assembly extract in order to assign function to these proteins. The depleted pores do not bind nuclear signal sequences or actively import nuclear proteins, but they are functional for diffusion. These defects can be fully repaired by assembly with readded Xenopus pore glycoproteins. Strikingly, isolated rat pore glycoproteins also restore transport. Electron microscopy reveals that depleted pores have largely normal morphology. Thus, the pore glycoproteins are not required for assembly of the nuclear envelope, the major structures of the pore, or a pore diffusional channel. Instead, they are essential for active protein import and, unexpectedly, for construction of the part of the pore necessary for signal sequence recognition.     

Formation of the postmitotic nuclear envelope from extended ER cisternae precedes nuclear pore assembly

During mitosis, the nuclear envelope merges with the endoplasmic reticulum (ER), and nuclear pore complexes are disassembled. In a current model for reassembly after mitosis, the nuclear envelope forms by a reshaping of ER tubules. For the assembly of pores, two major models have been proposed. In the insertion model, nuclear pore complexes are embedded in the nuclear envelope after their formation. In the prepore model, nucleoporins assemble on the chromatin as an intermediate nuclear pore complex before nuclear envelope formation. Using live-cell imaging and electron microscope tomography, we find that the mitotic assembly of the nuclear envelope primarily originates from ER cisternae. Moreover, the nuclear pore complexes assemble only on the already formed nuclear envelope. Indeed, all the chromatin-associated Nup107-160 complexes are in single units instead of assembled prepores. We therefore propose that the postmitotic nuclear envelope assembles directly from ER cisternae followed by membrane-dependent insertion of nuclear pore complexes.     

EFFECT OF METABOLIC INHIBITORS ON NUCLEAR PORE FORMATION DURING THE HELA S3 CELL CYCLE

The effect of various antimetabolites on nuclear pore formation was studied in synchronized HeLa S₃ cells. The nuclear size was determined by light microscopy and the pore number per unit area of nuclear surface by the freeze-etching technique and electron microscopy. It was found that the inhibition of DNA replication or ribosomal RNA synthesis has no effect on nuclear size increase or pore formation. However, the inhibition of ATP synthesis effectively stops nuclear pore formation. Cycloheximide blocks nuclear pore formation at the same time during G₁ phase of the cell cycle when nuclear size increase is blocked by high concentrations of actinomycin D. This suggests that certain proteins or other factors leading to pore formation and nuclear size increase are transcribed and synthesized at about 3–4 h after mitosis, i.e., about 1–2 h before S phase begins.     

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.     

The Mechanism of a Nuclear Pore Assembly: A Molecular Biophysics View

The basic problem of nuclear pore assembly is the big perinuclear space that must be overcome for nuclear membrane fusion and pore creation. Our investigations of ternary complexes: DNA–PC liposomes–Mg²⁺, and modern conceptions of nuclear pore structure allowed us to introduce a new mechanism of nuclear pore assembly. DNA-induced fusion of liposomes (membrane vesicles) with a single-lipid bilayer or two closely located nuclear membranes is considered. After such fusion on the lipid bilayer surface, traces of a complex of ssDNA with lipids were revealed. At fusion of two identical small liposomes (membrane vesicles) <100 nm in diameter, a “big” liposome (vesicle) with ssDNA on the vesicle equator is formed. ssDNA occurrence on liposome surface gives a biphasic character to the fusion kinetics. The “big” membrane vesicle surrounded by ssDNA is the base of nuclear pore assembly. Its contact with the nuclear envelope leads to fast fusion of half of the vesicles with one nuclear membrane; then ensues a fusion delay when ssDNA reaches the membrane. The next step is to turn inside out the second vesicle half and its fusion to other nuclear membrane. A hole is formed between the two membranes, and nucleoporins begin pore complex assembly around the ssDNA. The surface tension of vesicles and nuclear membranes along with the kinetic energy of a liquid inside a vesicle play the main roles in this process. Special cases of nuclear pore formation are considered: pore formation on both nuclear envelope sides, the difference of pores formed in various cell-cycle phases and linear nuclear pore clusters.     

Caspases target only two architectural components within the core structure of the nuclear pore complex

Caspases were recently implicated in the functional impairment of the nuclear pore complex during apoptosis, affecting its dual activity as nucleocytoplasmic transport channel and permeability barrier. Concurrently, electron microscopic data indicated that nuclear pore morphology is not overtly altered in apoptotic cells, raising the question of how caspases may deactivate nuclear pore function while leaving its overall structure largely intact. To clarify this issue we have analyzed the fate of all known nuclear pore proteins during apoptotic cell death. Our results show that only two of more than 20 nuclear pore core structure components, namely Nup93 and Nup96, are caspase targets. Both proteins are cleaved near their N terminus, disrupting the domains required for interaction with other nucleoporins actively involved in transport and providing the permeability barrier but dispensable for maintaining the nuclear pore scaffold. Caspase-mediated proteolysis of only few nuclear pore complex components may exemplify a general strategy of apoptotic cells to efficiently disable huge macromolecular machines.     

How proteins enter the nucleus

Nuclear protein import is a selective process. Proteins destined for the nucleus contain NLSs. These short stretches of amino acids interact with proteins located in the cytoplasm, on the nuclear envelope, and/or at the nuclear pore complex. Following binding at the pore complex, proteins are translocated through the pore into the nucleus in a manner requiring ATP. The biochemical dissection of the nuclear pore complex has begun. Alteration of protein import into the nucleus is emerging as a new and complex form of regulation. However, we are left with the following problems: How do proteins move through the cytoplasm to reach the nuclear pore? How does the nuclear pore complex open and close in a selective manner? How is ATP utilized during import? And finally, how is bi-directional traffic of both proteins and RNA through the pore regulated?     

Nuclear envelope proteins from Spisula solidissima germinal vesicles

Biochemical characterization of the nuclear pore complex requires quantities of highly enriched nuclear pore complex material which could not be obtained with available procedures. We have developed a technique for the mass isolation of nuclear envelopes from germinal vesicles of Spisula solidissima oocytes. The nuclear pore complex is intact after isolation as judged ultrastructurally. The nuclear envelope and the pore complex fibrous lamina fraction are highly purified with respect to nuclear and cytoplasmic protein contaminants. The fibrous lamina pore complex (FLPC) as presently isolated consists of about eight major proteins, three of which are phosphorylated. Comparison of the FLPC of clams with that of rat reveals three proteins of similar molecular weights, which may be pore complex-specific proteins. The clam nuclear envelope has only one protein (67000) in the molecular weight range which is comparable to the three lamina of rat nuclei. The solubility, intermolecular cross-linking and in vitro phosphorylation of this protein resemble that of one of the lamina of rat nuclei. The other lamina of the rat nuclear envelope are not essential proteins of the pore complex because they are not present in the clam FLPC preparation. They also seem non-essential for the maintenance of the fibrous lamina.     

The maximal size of protein to diffuse through the nuclear pore is larger than 60kDa

It has generally been believed that the diffusion limit set by the nuclear pore for protein is 60kDa. We here studied the cellular localization of several artificial proteins and found that the diffusion limit set by the nuclear pore is not as small as previously thought. The results indicate that the maximal size of protein to diffuse through the nuclear pore complex could be quite larger than 60kDa, thus greatly extending the diffusion limit that the nuclear pore can accommodate.     

Interference with the cytoplasmic tail of gp210 disrupts "close apposition" of nuclear membranes and blocks nuclear pore dilation

We tested the hypothesis that gp210, an integral membrane protein of nuclear pore complexes (NPCs), mediates nuclear pore formation. Gp210 has a large lumenal domain and small COOH-terminal tail exposed to the cytoplasm. We studied the exposed tail. We added recombinant tail polypeptides to Xenopus nuclear assembly extracts, or inhibited endogenous gp210 tails using anti-tail antibodies. Both strategies had no effect on the formation of fused flattened nuclear membranes, but blocked NPC assembly and nuclear growth. Inhibited nuclei accumulated gp210 and some nucleoporin p62, but failed to incorporate nup214/CAN, nup153, or nup98 and were defective for nuclear import of lamin B3. Scanning and transmission EM revealed a lack of "closely apposed" inner and outer membranes, and the accumulation of novel arrested structures including "mini-pores." We conclude that gp210 has early roles in nuclear pore formation, and that pore dilation is mediated by gp210 and its tail-binding partner(s). We propose that membrane fusion and pore dilation are coupled, acting as a mechanism to control nuclear pore size.     

Purification of the vertebrate nuclear pore complex by biochemical criteria

The nuclear pore is a large and complex biological machine, mediating all signal-directed transport between the nucleus and the cytoplasm. The vertebrate pore has a mass of ∼120 million daltons or 30 times the size of a ribosome. The large size of the pore, coupled to its tight integration in the nuclear lamina, has hampered the isolation of pore complexes from vertebrate sources. We have now developed a strategy for the purification of nuclear pores from in vitro assembled annulate lamellae (AL), a cytoplasmic mimic of the nuclear envelope that lacks a lamina, nuclear matrix, and chromatin-associated proteins. We find that purified pore complexes from annulate lamellae contain every nuclear pore protein tested. In addition, immunoblotting reveals the presence of soluble transport receptors and factors known to play important roles in the transport of macromolecules through the pore. While transport factors such as Ran and NTF2 show only transient interaction with the pores, a number of soluble transport receptors, including importin β, show a tight association with the purified pores. In summary, we report that we have purified the vertebrate pore by biochemical criteria; silver staining reveals ∼40–50 distinct protein bands.     

Integral membrane proteins and dynamic organization of the nuclear envelope

The nuclear envelope is a complex structure consisting of nuclear membranes, nuclear pore complexes and lamina. Several integral membrane proteins specific to the nuclear pore membrane and the inner nuclear membrane are known. Pore membrane proteins are probably important for organization and assembly of the nuclear pore complex, while proteins of the inner nuclear membrane are likely to play major roles in the structure and dynamics of the nuclear lamina and chromatin. Biochemical studies are now identifying potential binding partners for some of these integral membrane proteins, and analysis of nuclear envelope assembly at the end of mitosis is providing important insights into their functions.     

FORMATION AND DISTRIBUTION OF NUCLEAR PORE COMPLEXES IN INTERPHASE

The possibility of nuclear pore formation in the interphase nucleus was investigated in control and phytohemagglutinin (PHA) stimulated lymphocytes by the freeze-etching technique. 48 hr after the addition of PHA, the newly formed blasts which had not as yet divided had at least twice the number of pores per nucleus as controls. This clearly demonstrates that in lymphocytes nuclear pore formation can take place during interphase. It has generally been assumed that the distribution of nuclear pore complexes in somatic animal cells is random. However, we have utilized freeze etched rat kidney cells and a computer program to evaluate pore distribution. We find a minimum pore center-to-center spacing of approximately 1300 A and multiples thereof with high frequency. This is strong evidence for a nonrandom distribution of nuclear pores. The nonrandomness may be related to an underlying chromosomal organization in interphase. Using three criteria for identifying prospective pore sites (membrane specialization, nonrandomness, and alteration of heterochromatin distribution), we have found forming pores in sectioned material from cultured human melanoma cells. While nuclear pore formation may take place in conjunction with reformation of the nuclear membrane, a mechanism also exists for their formation during interphase.     

Nuclear pore complexes exceeding eightfold rotational symmetry

Nuclear pore complexes are rotationally symmetric structures that span the nuclear envelope and provide channels for nucleocytoplasmic traffic. These large complexes normally consist of eight spokes arranged around a central channel, although, occasionally, 9- and 10-fold nuclear pore complexes are found in preparations of Xenopus oocyte macronuclei. Here we examine these unusual nuclear pore complexes by negative stain electron microscopy and image analysis and compare the results with data previously obtained from 8-fold structures. The details in two-dimensional and three-dimensional maps indicate that the substructure of the spoke is the same in 8-, 9- and 10-fold nuclear pore complexes: therefore, the spoke is likely an immutable structural component. In all three variant forms, the spacing between adjacent annular subunits, which surround the central channel, is identical. Distances between spokes at higher radius decrease in the 9- and 10-fold nuclear pore complexes. These data imply that the most important connections holding the nuclear pore complex together are those between adjacent annular subunits and that these interactions may play a predominant role in nuclear pore complex assembly. Circumferential connections mediated by ring subunits and radial arms presumably further stabilize the structure and are flexible enough to accommodate additional spokes.     

Inhibition of Nuclear Protein Import by a Monoclonal Antibody against a Novel Class of Nuclear Pore Proteins

Nuclear import of proteins is mediated by the nuclear pore complexes in the nuclear envelope and requires the presence of a nuclear localization signal (NLS) on the karyophilic protein. In this paper, we describe studies with a monoclonal antibody, Mab E2, which recognizes a class of nuclear pore proteins of 60-76 kDa with a common phosphorylated epitope on rat nuclear envelopes. The Mab E2-reactive proteins fractionated with the relatively insoluble pore complex-containing component of the envelope and gave a finely punctate pattern of nuclear staining in immunofluorescence assays. The antibody did not bind to any cytosolic proteins. Mab E2 inhibited the interaction of a simian virus 40 large T antigen NLS peptide with a specific 60-kDa NLS-binding protein from rat nuclear envelopes in photoaffinity labeling experiments. The antibody blocked the nuclear import of NLS--albumin conjugates in an in vitro nuclear transport assay with digitonin-permeabilized cells, but did not affect passive diffusion of a small non-nuclear protein, lysozyme, across the pore. Mab E2 may inhibit protein transport by directly interacting with the 60-kDa NLS-binding protein, thereby blocking signal-mediated nuclear import across the nuclear pore complex.     

Identification of a major polypeptide of the nuclear pore complex

The nuclear pore complex is a prominent structural component of the nuclear envelope that appears to regulate nucleoplasmic molecular movement. Up to now, none of its polypeptides have been defined. To identify possible pore complex proteins, we fractionated rat liver nuclear envelopes and microsomal membranes with strong protein perturbants into peripheral and intrinsic membrane proteins, and compared these fractions on SDS gels. From this analysis, we identified a prominent 190-kilodalton intrinsic membrane polypeptide that occurs specifically in nuclear envelopes. Lectin binding studies indicate that this polypeptide (gp 190) is the major nuclear envelope glycoprotein. Upon treatment of nuclear envelopes with Triton X-100, gp 190 remains associated with a protein substructure of the nuclear envelope consisting of pore complexes and nuclear lamina. We prepared monospecific antibodies to gp 190 for immunocytochemical localization. Immunofluorescence staining of tissue culture cells suggests that gp 190 occurs exclusively in the nucleus during interphase. This polypeptide becomes dispersed throughout the cell in mitotic prophase when the nuclear envelope is disassembled, and subsequently returns to the nuclear surfaces during telophase when the nuclear envelope is reconstructed. Immunoferritin labeling of Triton-treated rat liver nuclei demonstrates that gp 190 occurs exclusively in the nuclear pore complex, in the regions of the cytoplasmic (and possibly nucleoplasmic) pore complex annuli. A polypeptide that cross-reacts with gp 190 is present in diverse vertebrate species, as shown by antibody labeling of nitrocellulose SDS gel transfers. On the basis of its biochemical characteristics, we suggest that gp 190 may be involved in anchoring the pore complex to nuclear envelope membranes.     

Nuclear envelope breakdown is coordinated by both Nup358/RanBP2 and Nup153, two nucleoporins with zinc finger modules

When higher eukaryotic cells transition into mitosis, the nuclear envelope, nuclear pore complexes, and nuclear lamina are coordinately disassembled. The COPI coatomer complex, which plays a major role in membrane remodeling at the Golgi, has been implicated in the process of nuclear envelope breakdown and requires interactions at the nuclear pore complex for recruitment to this new site of action at mitosis. Nup153, a resident of the nuclear pore basket, was found to be involved in COPI recruitment, but the molecular nature of the interface between COPI and the nuclear pore has not been fully elucidated. To better understand what occurs at the nuclear pore at this juncture, we have probed the role of the nucleoporin Nup358/RanBP2. Nup358 contains a repetitive zinc finger domain with overall organization similar to a region within Nup153 that is critical to COPI association, yet inspection of these two zinc finger domains reveals features that also clearly distinguish them. Here, we found that the Nup358 zinc finger domain, but not a zinc finger domain from an unrelated protein, binds to COPI and dominantly inhibits progression of nuclear envelope breakdown in an assay that robustly recapitulates this process in vitro. Moreover, the Nup358 zinc finger domain interferes with COPI recruitment to the nuclear rim. Consistent with a role for this pore protein in coordinating nuclear envelope breakdown, Nup358-specific antibodies impair nuclear disassembly. Significantly, targeting either Nup153 or Nup358 for inhibition perturbs nuclear envelope breakdown, supporting a model in which these nucleoporins play nonredundant roles, perhaps contributing to COPI recruitment platforms on both the nuclear and cytoplasmic faces of the pore. We found that an individual zinc finger is the minimal interface for COPI association, although tandem zinc fingers are optimal. These results provide new information about the critical components of nuclear membrane remodeling and lay the foundation for a better understanding of how this process is regulated.