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PubMed1992

The single transmembrane segment of gp210 is sufficient for sorting to the pore membrane domain of the nuclear envelope.

The glycoprotein gp210 is located in the "pore membrane," a specialized domain of the nuclear envelope to which the nuclear pore complex (NPC) is anchored. gp210 contains a large cisternal domain, a single transmembrane segment (TM), and a COOH-terminal, 58-amino acid residue cytoplasmic tail (CT) (Wozniak, R. W., E. Bartnik, and G. Blobel. 1989. J. Cell Biol. 108:2083-2092; Greber, U. F., A. Senior, and L. Gerace. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:1495-1502). To locate determinants for sorting of gp210 to the pore membrane, we constructed various cDNAs coding for wild-type, mutant, and chimeric gp210, and monitored localization of the expressed protein in 3T3 cells by immunofluorescence microscopy using appropriate antibodies. The large cisternal domain of gp210 (95% of its mass) did not reveal any sorting determinants. Surprisingly, the TM of gp210 is sufficient for sorting to the pore membrane. The CT also contains a sorting determinant, but it is weaker than that of the TM. We propose specific lateral association of the transmembrane helices of two proteins to yield either a gp210 homodimer or a heterodimer of gp210 and another protein. The cytoplasmically oriented tails of these dimers may bind cooperatively to the adjacent NPCs. In addition, we demonstrate that gp210 co-localizes with cytoplasmically dispersed nucleoporins, suggesting a cytoplasmic association of these components.

Amino Acid Sequence↗
PubMed2006

Nuclear gene delivery: the Trojan horse approach.

The nuclear envelope represents a formidable barrier to the transfer of plasmids to the cell nucleus, particularly in nondividing cells. The probability of intact plasmids arriving in the nucleus by a passive process is extremely low. There is substantial evidence in the literature that describes the transport of macromolecules, including plasmids, to the nucleus as a very inefficient process, and so far attempts to affect the active transport through the nuclear pores have achieved limited success. Several approaches have been attempted to improve nuclear transport of plasmids, including the condensation of plasmids to unimolecular complexes of minimal hydrodynamic diameter to favour passive transport through the nuclear pore complex, and the incorporation of nuclear localisation signals in the plasmid or in the delivery system to enhance the active transport of plasmids through the nuclear pores.

Active Transport, Cell Nucleus↗
PubMed1996

Identification of different roles for RanGDP and RanGTP in nuclear protein import.

The importin-alpha/beta heterodimer and the GTPase Ran play key roles in nuclear protein import. Importin binds the nuclear localization signal (NLS). Translocation of the resulting import ligand complex through the nuclear pore complex (NPC) requires Ran and is terminated at the nucleoplasmic side by its disassembly. The principal GTP exchange factor for Ran is the nuclear protein RCC1, whereas the major RanGAP is cytoplasmic, predicting that nuclear Ran is mainly in the GTP form and cytoplasmic Ran is in the GDP-bound form. Here, we show that nuclear import depends on cytoplasmic RanGDP and free GTP, and that RanGDP binds to the NPC. Therefore, import might involve nucleotide exchange and GTP hydrolysis on NPC-bound Ran. RanGDP binding to the NPC is not mediated by the Ran binding sites of importin-beta, suggesting that translocation is not driven from these sites. Consistently, a mutant importin-beta deficient in Ran binding can deliver its cargo up to the nucleoplasmic side of the NPC. However, the mutant is unable to release the import substrate into the nucleoplasm. Thus, binding of nucleoplasmic RanGTP to importin-beta probably triggers termination, i.e. the dissociation of importin-alpha from importin-beta and the subsequent release of the import substrate into the nucleoplasm.

Animals↗
PubMed1998

Characterization of HIV-1 vpr nuclear import: analysis of signals and pathways.

While the Vpr protein of HIV-1 has been implicated in import of the viral preintegration complex across the nuclear pore complex (NPC) of nondividing cellular hosts, the mechanism by which Vpr enters the nucleus remains unknown. We now demonstrate that Vpr contains two discrete nuclear targeting signals that use two different import pathways, both of which are distinct from the classical nuclear localization signal (NLS)- and the M9-dependent pathways. Vpr import does not appear to require Ran-mediated GTP hydrolysis and persists under conditions of low energy. Competition experiments further suggest that Vpr directly engages the NPC at two discrete sites. These sites appear to form distal components of a common import pathway used by NLS- and M9-containing proteins. Together, our data suggest that Vpr bypasses many of the soluble receptors involved in import of cellular cargoes. Rather, this viral protein appears to directly access the NPC, a property that may help to ensure the capacity of HIV to replicate in nondividing cellular hosts.

Binding, Competitive↗
PubMed1996

[Vimentin and Nup 180 in vitro binding assay].

In order to investigate relationship between vimentin and nuclear pore complex, we examined binding ability of vimentin, expressed in E. coli, with nucleoporin, isolated from rat liver nuclei, in vitro. Negative staining electron microscopy showed that the vimentin expressed in bacteria assembled 10 nm filament in vitro. SDS-PAGE and western blotting showed that Nup 180 bind to vimentin in binding assay in vitro. Combining immunogold labeling and negative staining electron microscopy techniques, we showed that Nup 180 bind on the 10 nm vimentin filaments. The experiment results indicated that vimentin filament may be anchored on nuclear pore complex in vivo by binding with Nup 180.

Animals↗
PubMed1999

The direction of transport through the nuclear pore can be inverted.

Transport of macromolecules across the nuclear envelope is an active process that depends on soluble factors including the GTPase Ran. Ran-GTP is predominantly located in the nucleus and has been shown to regulate cargo binding and release of import and export receptors in their respective target compartments. Recently, it was shown that transport of receptor-cargo complexes across the nuclear pore complex (NPC) does not depend on GTP-hydrolysis by Ran; however, the mechanism of translocation is still poorly understood. Here, we show that the direction of transport through the NPC can be inverted in the presence of high concentrations of cytoplasmic Ran-GTP. Under these conditions, two different classes of export cargoes are transported into the nucleus in the absence of GTP hydrolysis. The inverted transport is very rapid and can be blocked by known inhibitors of nuclear protein export. These results suggest that the NPC functions as a facilitated transport channel, allowing the selective translocation of receptor-cargo complexes. We conclude that the directionality of nucleocytoplasmic transport is determined mainly by the compartmentalized distribution of Ran-GTP.

Biological Transport↗
PubMed2000

The nucleoporin Nup98 is a site for GDP/GTP exchange on ran and termination of karyopherin beta 2-mediated nuclear import.

Karyopherin beta2 (Kapbeta2, transportin) binds the M9 sequence of human ribonucleoprotein A1 and mediates its nuclear import. Here we show a role for the nucleoporin Nup98 in the disassembly of Kapbeta2 import complexes at the nuclear side of the nuclear pore complex (NPC). Kapbeta2 bound to a region at the N terminus of Nup98 that contains an M9-like sequence. The human ribonucleoprotein A1 M9 sequence competed with Nup98 for binding to Kapbeta2, indicating that Nup98 can dissociate Kapbeta2 from its substrate. Binding of Kapbeta2 to Nup98 was inhibited by Ran loaded with guanylyl imidophosphate, suggesting that RanGTP dissociates Kapbeta2 from Nup98. RanGTP is produced from RanGDP through nucleotide exchange mediated by RanGEF (RCC1). Immunoelectron microscopy and nucleotide exchange assays revealed functional RanGEF on both sides of the NPC. On the nuclear side, the localization of RanGEF coincided with that of Nup98. RanGEF bound to Nup98 at a region adjacent to the Kapbeta2-binding site. These findings suggest a model where 1) import substrate is released from Kapbeta2 at the nucleoplasmic side of the NPC by competition with the Nup98 M9-like site, 2) Nup98-bound RanGEF catalyzes the formation of RanGTP, and 3) RanGTP dissociates Kapbeta2 from Nup98 allowing repeated cycles of import.

Amino Acid Sequence↗
PubMed1999

Association with the cellular export receptor CRM 1 mediates function and intracellular localization of Epstein-Barr virus SM protein, a regulator of gene expression.

Splicing and posttranscriptional processing of eukaryotic gene transcripts are linked to their nuclear export and cytoplasmic expression. Unspliced pre-mRNAs and intronless transcripts are thus inherently poorly expressed. Nevertheless, human and animal viruses encode essential genes as single open reading frames or in the intervening sequences of other genes. Many retroviruses have evolved mechanisms to facilitate nuclear export of their unspliced mRNAs. For example, the human immunodeficiency virus RNA-binding protein Rev associates with the soluble cellular export receptor CRM 1 (exportin 1), which mediates nucleocytoplasmic translocation of Rev-HIV RNA complexes through the nuclear pore. The transforming human herpesvirus Epstein-Barr virus (EBV) expresses a nuclear protein, SM, early in its lytic cycle; SM binds RNA and posttranscriptionally activates expression of certain intronless lytic EBV genes. Here we show that both the trans-activation function and cytoplasmic translocation of SM are dependent on association with CRM 1 in vivo. SM is also shown to be associated in vivo with other components of the CRM 1 export pathway, including the small GTPase Ran and the nucleoporin CAN/Nup214. SM is shown to be present in the cytoplasm, nucleoplasm, and nuclear envelope of transfected cells. Mutation of a leucine-rich region (LRR) of SM inhibited CRM 1-mediated cytoplasmic translocation and SM activity, as did leptomycin B, an inhibitor of CRM 1 complex formation. Surprisingly, however, leptomycin B treatment and mutation of the LRR both led to SM becoming more tightly attached to intranuclear structures. These findings suggest a model in which SM is not merely a soluble carrier protein for RNA but rather is bound directly to intranuclear proteins, possibly including the nuclear pore complex.

Animals↗
PubMed2006

SAGA interacting factors confine sub-diffusion of transcribed genes to the nuclear envelope.

Changes in the transcriptional state of genes have been correlated with their repositioning within the nuclear space. Tethering reporter genes to the nuclear envelope alone can impose repression and recent reports have shown that, after activation, certain genes can also be found closer to the nuclear periphery. The molecular mechanisms underlying these phenomena have remained elusive. Here, with the use of dynamic three-dimensional tracking of a single locus in live yeast (Saccharomyces cerevisiae) cells, we show that the activation of GAL genes (GAL7, GAL10 and GAL1) leads to a confinement in dynamic motility. We demonstrate that the GAL locus is subject to sub-diffusive movement, which after activation can become constrained to a two-dimensional sliding motion along the nuclear envelope. RNA-fluorescence in situ hybridization analysis after activation reveals a higher transcriptional activity for the peripherally constrained GAL genes than for loci remaining intranuclear. This confinement was mediated by Sus1 and Ada2, members of the SAGA histone acetyltransferase complex, and Sac3, a messenger RNA export factor, physically linking the activated GAL genes to the nuclear-pore-complex component Nup1. Deleting ADA2 or NUP1 abrogates perinuclear GAL confinement without affecting GAL1 transcription. Accordingly, transcriptional activation is necessary but not sufficient for the confinement of GAL genes at the nuclear periphery. The observed real-time dynamic mooring of active GAL genes to the inner side of the nuclear pore complex is in accordance with the 'gene gating' hypothesis.

Diffusion↗
PubMed2000

Epstein-Barr virus EB2 protein exports unspliced RNA via a Crm-1-independent pathway.

Human herpesviruses encode posttranscriptional activators that are believed to up-regulate viral replication by facilitating early and late gene expression. We have reported previously that the Epstein-Barr virus protein EB2 (also called M or SM) promotes nuclear export of RNAs that are poor substrates for spliceosome assembly, an effect that closely resembles the human immunodeficiency virus type 1 Rev-dependent nuclear export of unspliced viral RNA. Here we present experimental data showing that EB2 efficiently promotes the nuclear export of unspliced RNA expressed from a Rev reporter construct. Site-directed mutagenesis as well as domain swapping experiments indicate that a leucine-rich region found in the EB2 protein, which matches the consensus sequence for the leucine-rich nuclear export signal, is not a nuclear export signal per se. Accordingly, leptomycin B (LMB), a specific Crm-1 inhibitor, impairs Rev- but not EB2-dependent nuclear export of unspliced RNA. Moreover, EB2 nucleocytoplasmic shuttling visualized by a heterokaryon assay is, unlike Rev shuttling, not affected by LMB. We also show that overexpression of an N-terminal deletion mutant of Nup214/can, a major nucleoporin of the nuclear pore complex involved in several aspects of nuclear transport, blocks both Rev- and EB2-dependent nuclear export of RNA. These results strongly suggest that EB2 nuclear export of unspliced RNA is mediated by a Crm-1-independent pathway.

3T3 Cells↗
PubMed1989

Surface views of nuclear pores in isolated rat liver nuclei as revealed by fracture-flip/Triton-X.

We report here the use of fracture-flip (Anderson Forsman, C., P. Pinto da Silva, J. Cell Sci. 90, 531-541 (1988] and fracture-flip/Triton X-100 to examine the cytoplasmic surface of the outer nuclear membrane, and the perinuclear (exoplasmic) surface of the inner nuclear membrane of rat liver nuclei. After freeze-fracture, carbon stabilization and thawing, unfixed nuclei were treated with Triton X-100 to dissolve unfractured portions of the outer nuclear membrane. Flipping of the casts with their carbon-stabilized membrane halves (cytoplasmic half of the outer nuclear membrane; exoplasmic half of the inner nuclear membrane) exposed the cytoplasmic surface of the outer nuclear membrane and the surface of the inner nuclear membrane facing the perinuclear space. After drying, these membrane surfaces were visualized by Pt/C shadowing. We show that the cytoplasmic surface of outer nuclear membrane is covered by numerous large, oblong "kernel-like" globular particles (30-35 nm length, 25-30 nm width), often aligned into curved chains or grouped into clusters. Because of their size and characteristic arrays, we interpret these particles as ribosomes. Over the cytoplasmic surfaces of the outer nuclear membrane the nuclear pore complexes (outer diameter: 120-130 nm) appear as relatively inconspicuous annular structures, only slightly raised against the surrounding areas of membrane surface. Observation of the surface of the inner nuclear membrane facing the perinuclear space reveals prominent, sharply raised, "doughnut-like" nuclear pore complexes (outer diameter: 120-130 nm; inner diameter: 40 nm). Our results indicate the absence of large, protruding, ribosome-like structures lining the nuclear pore at its cytoplasmic surface, as proposed before.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

In vivo analysis of the stability and transport of nuclear poly(A)+ RNA.

We have studied the distribution of poly(A)+ RNA in the mammalian cell nucleus and its transport through nuclear pores by fluorescence and electron microscopic in situ hybridization. Poly(A)+ RNA was detected in the nucleus as a speckled pattern which includes interchromatin granule clusters and perichromatin fibrils. When cells are fractionated by detergent and salt extraction as well as DNase I digestion, the majority of the nuclear poly(A)+ RNA was found to remain associated with the nonchromatin RNP-enriched fraction of the nucleus. After inhibition of RNA polymerase II transcription for 5-10 h, a stable population of poly(A)+ RNA remained in the nucleus and was reorganized into fewer and larger interchromatin granule clusters along with pre-mRNA splicing factors. This stable population of nuclear RNA may play an important role in nuclear function. Furthermore, we have observed that, in actively transcribing cells, the regions of poly(A)+ RNA which reached the nuclear pore complexes appeared as narrow concentrations of RNA suggesting a limited or directed pathway of movement. All of the observed nuclear pores contained poly(A)+ RNA staining suggesting that they are all capable of exporting RNA. In addition, we have directly visualized, for the first time in mammalian cells, the transport of poly(A)+ RNA through the nuclear pore complexes.

Animals↗
PubMed1996

In vivo nuclear transport kinetics in Saccharomyces cerevisiae: a role for heat shock protein 70 during targeting and translocation.

The transport of proteins into the nucleus is a receptor-mediated process that is likely to involve between 50-100 gene products, including many that comprise the nuclear pore complex. We have developed an assay in Saccharomyces cerevisiae for the nuclear transport of green fluorescent protein fused to the SV-40 large T antigen nuclear localization signal (NLS-GFP). This assay allows the measurement of relative NLS-GFP nuclear import rates in wild-type and mutant cells under various physiological conditions. Probably the best understood component of the nuclear transport apparatus is Srp1p, the NLS receptor, which binds NLS-cargo in the cytoplasm and accompanies it into the nucleus. When compared to SRP1+ cells, NLS-GFP import rates in temperature-sensitive srp1-31 cells were slower and showed a lower temperature optimum. The in vivo transport defect of the srp1-31 cells was correlated with the purified protein's thermal sensitivity, as assayed by in vitro NLS peptide binding. We show that the kinetics of NLS-directed nuclear transport in wild-type cells is stimulated by the elevated expression of SSA1, which encodes a cytoplasmic heat shock protein 70 (Hsp70). Elevated Hsp70 levels are sufficient to suppress the NLS-GFP import defects in srp1-31 and nup82-3 cells. NUP82 encodes a protein that functions within the nuclear pore complex subsequent to docking. These results provide genetic evidence that Hsp70 acts during both targeting and translocation phases of nuclear transport, possibly as a molecular chaperone to promote the formation and stability of the Srp1p-NLS-cargo complex.

Antigens, Viral, Tumor↗
PubMed2006

Ultrastructural nuclear import assay.

Electron microscopy (EM) has been used for several decades to study the mechanisms of nuclear transport. In early studies of nuclear import, gold-conjugated nuclear proteins were microinjected into cells and followed by EM. As the components of the nuclear pore complex (NPC) and soluble mediators of nuclear import were cloned and characterized, gold-conjugated antibodies were utilized to sublocalize the components of the nuclear transport machinery by immuno-EM. Further, gold-conjugated recombinant proteins were used to probe permeabilized cells or isolated nuclear envelopes and characterize binding sites for these proteins at the NPC. More recently, recombinant gold-conjugated nuclear proteins were used in in vitro nuclear import assays to help dissect the mechanisms of nuclear import. We have used this ultrastructural nuclear import assay to study the nuclear import of the transcription factor PU.1. The results showed that this import requires energy but is carrier-independent. In the presence of energy, gold-conjugated PU.1 shifted to the nuclear side of the NPC and the inside of the nucleus. In conjunction with biochemical assays, these results indicated that this shift involved Ran-dependent binding of PU.1 to NUP153, a nucleoporin situated at the nuclear side of the NPC. Here we describe in detail the methods used in the ultrastructural nuclear import assay including preparation of recombinant protein, gold conjugation, in vitro nuclear import assay, electron microscopy, and data analysis.

Active Transport, Cell Nucleus↗
PubMed1996

The product of the Saccharomyces cerevisiae RSS1 gene, identified as a high-copy suppressor of the rat7-1 temperature-sensitive allele of the RAT7/NUP159 nucleoporin, is required for efficient mRNA export.

RAT7/NUP159 was identified previously in a screen for genes whose products are important for nucleocytoplasmic export of poly(A)+ RNA and encodes an essential nucleoporin. We report here the identification of RSS1 (Rat Seven Suppressor) as a high-copy extragenic suppressor of the rat7-1 temperature-sensitive allele. Rss1p encodes a novel essential protein of 538 amino acids, which contains an extended predicted coiled-coil domain and is located both at nuclear pore complexes (NPCs) and in the cytoplasm. RSS1 is the first reported high-copy extragenic suppressor of a mutant nucleoporin. Overexpression of Rss1p partially suppresses the defects in nucleocytoplasmic export of poly(A)+ RNA, rRNA synthesis and processing, and nucleolar morphology seen in rat7-1 cells shifted to the nonpermissive temperature of 37 degrees C and, thus, restores these processes to levels adequate for growth at a rate approximately one-half that of wild-type cells. After a shift to 37 degrees C, the mutant Rat7-1p/Nup159-1p is lost from the nuclear rim of rat7-1 cells and NPCs, which are clustered together in these cells grown under permissive conditions become substantially less clustered. Overexpression of Rss1p did not result in retention of the mutant Rat7-1p/Nup159-1p in NPCs, but it did result in partial maintenance of the NPC-clustering phenotype seen in mutant cells. Depletion of Rss1p by placing the RSS1 open reading frame (ORF) under control of the GAL1 promoter led to cessation of growth and nuclear accumulation of poly(A)+ RNA without affecting nuclear protein import or nuclear pore complex distribution, suggesting that RSS1 is directly involved in mRNA export. Because both rat7-1 cells and cells depleted for Rss1p are defective in mRNA export, our data are consistent with both gene products playing essential roles in the process of mRNA export and suggest that Rss1p overexpression suppresses the growth defect of rat7-1 cells at 37 degrees C by acting to maintain mRNA export.

Alleles↗
PubMed2000

Nup2p, a yeast nucleoporin, functions in bidirectional transport of importin alpha.

Import of proteins containing a classical nuclear localization signal (NLS) into the nucleus is mediated by importin alpha and importin beta. Srp1p, the Saccharomyces cerevisiae homologue of importin alpha, returns from the nucleus in a complex with its export factor Cse1p and with Gsp1p (yeast Ran) in its GTP-bound state. We studied the role of the nucleoporin Nup2p in the transport cycle of Srp1p. Cells lacking NUP2 show a specific defect in both NLS import and Srp1p export, indicating that Nup2p is required for efficient bidirectional transport of Srp1p across the nuclear pore complex (NPC). Nup2p is located at the nuclear side of the central gated channel of the NPC and provides a binding site for Srp1p via its amino-terminal domain. We show that Nup2p effectively releases the NLS protein from importin alpha-importin and beta and strongly binds to the importin heterodimer via Srp1p. Kap95p (importin beta) is released from this complex by a direct interaction with Gsp1p-GTP. These data suggest that besides Gsp1p, which disassembles the NLS-importin alpha-importin beta complex upon binding to Kap95p in the nucleus, Nup2p can also dissociate the import complex by binding to Srp1p. We also show data indicating that Nup1p, a relative of Nup2p, plays a similar role in termination of NLS import. Cse1p and Gsp1p-GTP release Srp1p from Nup2p, which suggests that the Srp1p export complex can be formed directly at the NPC. The changed distribution of Cse1p at the NPC in nup2 mutants also supports a role for Nup2p in Srp1p export from the nucleus.

Active Transport, Cell Nucleus↗
PubMed1987

O-linked N-acetylglucosamine is attached to proteins of the nuclear pore. Evidence for cytoplasmic and nucleoplasmic glycoproteins.

Glycoproteins bearing a single N-acetylglucosamine (GlcNAc) residue attached by an O-glycosidic linkage to the polypeptide chain (Holt, G. D., and Hart, G. W. (1986) J. Biol. Chem. 261, 8049-8057) have been found to be enriched in the nuclear and soluble fractions of rat liver. Our goal was to determine the localization and membrane topography of proteins bearing O-linked GlcNAc using galactosyltransferase and wheat germ agglutinin (WGA) as membrane-impermeant probes. Latency of the enzyme mannose-6-phosphatase was used to quantitatively confirm the intactness of the nuclear envelope during incubations with galactosyltransferase or WGA. The O-linked GlcNAc residues of nuclei were fully accessible to modification by galactosyltransferase under conditions where the nuclear envelope mannose-6-phosphatase was 70% latent. Addition of detergent destroyed the permeability barrier but did not increase galactosylation of the O-linked GlcnAc. The major polypeptides bearing O-linked GlcNAc residues on nuclei were peripheral rather than integral membrane proteins with apparent molecular masses ranging from 210 to 54 kDa. The proteins were also detected on sealed nuclei using conjugates of WGA. WGA-rhodamine labeled intact nuclei when examined by immunofluorescence; WGA-peroxidase was used to identify the nuclear glycoproteins after transfer to nitrocellulose. WGA-ferritin selectively labels the cytoplasmic and nucleoplasmic faces of the nuclear pore complex when examined by electron microscopy. Taken together, these data strongly suggest that proteins bearing cytoplasmically oriented O-linked GlcNAc are components of the nuclear pore complex, thereby raising the possibility that cytoplasmic and nucleoplasmic glycoproteins are involved in the assembly or functioning of the nuclear pore.

Acetylglucosamine↗
PubMed1997

Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent 'envelope smoothing' event.

We analyzed the pathway of nuclear envelope assembly in Xenopus egg extracts using field emission in-lens scanning electron microscopy. The binding, fusion, and flattening of vesicles onto the chromatin surface were visualized in detail. The first nuclear pore complexes assembled in flattened patches of nuclear envelope, before the chromatin was fully enclosed by membranes. Confirming previous transmission electron microscope observations, two morphologically distinct types of vesicles contributed to the nuclear membranes: ribosome-carrying ('rough') vesicles, many of which bound directly to chromatin, and 'smooth' vesicles, which appeared to associate primarily with other nuclear vesicles or membrane patches. The presence of ribosomes, an outer nuclear membrane marker, on many chromatin-binding vesicles suggested that chromatin-attachment proteins integral to the inner membrane were present on vesicles that also carried markers of the outer membrane and endoplasmic reticulum. Chromatin-associated vesicles also carried pore membrane proteins, since pore complexes formed when these vesicles were incubated with cytosol. A change in nuclear envelope morphology termed 'envelope smoothing' occurred 5-15 minutes after enclosure. Nuclear envelopes that were assembled in extracts depleted of wheat-germ-agglutinin-binding nucleoporins, and therefore unable to form functional pore complexes, remained wrinkled, suggesting that 'smoothing' required active nuclear transport. Lamins accumulated with time when nuclei were enclosed and had functional pore complexes, whereas lamins were not detected on nuclei that lacked functional pore complexes. Very low levels of lamins were detected on nuclear intermediates whose surfaces were substantially covered with patches of pore-complex-containing envelope, suggesting that pore complexes might be functional before enclosure.

Animals↗