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.     

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.     

A major glycoprotein of the nuclear pore complex is a membrane-spanning polypeptide with a large lumenal domain and a small cytoplasmic tail.

One of a small number of polypeptides of the nuclear pore complex that have been identified is a major glycoprotein called gp210. Since it is very resistant to chemical extractions from membranes, gp210 was suggested to be integrated into nuclear membranes. In this study we have determined the membrane topology of this protein by biochemical and immunological approaches. We found that limited proteolysis of isolated nuclear envelopes with papain released a 200 kd water-soluble fragment of gp210 containing concanavalin A-reactive carbohydrate. Immunogold electron microscopy with a monoclonal antibody showed that this domain is localized on the lumenal side of nuclear membranes at pore complexes. Anti-peptide antibodies against two sequences near the C-terminus of gp210 were used to map possible membrane spanning and cytoplasmically disposed regions of this protein. From analysis of the protease sensitivity of these epitopes in sealed membrane vesicles, we determined that gp210 contains a small cytoplasmic tail and only a single membrane-spanning region. Thus, gp210 is a transmembrane protein with most of its mass, including the carbohydrate, located in the perinuclear space. This topology suggests that gp210 is involved primarily in structural organization of the pore complex, for which it may provide a membrane attachment site.     

Nuclear pore protein gp210 is essential for viability in HeLa cells and Caenorhabditis elegans

Gp210 is an evolutionarily conserved membrane protein of the nuclear pore complex (NPC). We studied the phenotypes produced by RNAi-induced downregulation of gp210 in both human (HeLa) cells and Caenorhabditis elegans embryos. HeLa cell viability requires Gp210 activity. The dying cells accumulated clustered NPCs and aberrant membrane structures at the nuclear envelope, suggesting that gp210 is required directly or indirectly for nuclear pore formation and dilation as well as the anchoring or structural integrity of mature NPCs. Essential roles for gp210 were confirmed in C. elegans, where RNAi-induced reduction of gp210 caused embryonic lethality. The nuclear envelopes of embryos with reduced gp210 also had aberrant nuclear membrane structures and clustered NPCs, confirming that gp210 plays critical roles at the nuclear membrane through mechanisms that are conserved from nematodes to humans.     

Dynamic properties of nuclear pore complex proteins in gp210 deficient cells

Gp210, an integral membrane protein of the nuclear pore complex (NPC), is believed to be involved in NPC biogenesis. To test this hypothesis, we have investigated dynamic properties of the NPC and distribution of NPC proteins in NIH/3T3 cells lacking gp210. POM121 (the other integral NPC protein) and NUP107 (of the NUP107/160 complex) were correctly distributed at the nuclear pores in the absence of gp210. Furthermore, fluorescence recovery after photobleaching experiments showed that POM121 and NUP107 remained stably associated at the NPCs. We conclude that gp210 cannot be required for incorporation of POM121 or NUP107 or be required for maintaining NPC stability.     

Limited expression of nuclear pore membrane glycoprotein 210 in cell lines and tissues suggests cell-type specific nuclear pores in metazoans

The nuclear pore complex (NPC) is the only known gateway for nucleocytoplasmic traffic. The nuclear pore membrane glycoprotein 210 (POM210/gp210) is considered to be important for the assembly and structure of pore complexes in metazoan cells. However, here we demonstrate cell-type specific expression of the gp210 protein during mouse organogenesis. As shown previously for its mRNA, distinct expression of the gp210 was seen in developing epithelia and some other cell types, whereas it was undetectable in nuclei of several other embryonic tissue compartments. In sharp contrast, monoclonal antibody 414 recognizing four non-membrane nucleoporins, stained the nuclear envelope of all cell types. In four cultured mouse cell lines, gp210 mRNA and protein were below detection levels, in contrast to some other nucleoporins tested. Distinct expression of gp210 mRNA and protein was seen in cultured mouse embryonic stem (ES) cells. These findings support the view of cell-type specific NPCs in metazoans and that the gp210 gene is regulated by cell-type specific control elements not shared by other nucleoporins. Although it cannot be excluded that very low expression levels of gp210 are sufficient to allow attachment of NPCs, a more likely alternative is that it has cell-type specific functions.     

The nucleoporin gp210/Nup210 controls muscle differentiation by regulating nuclear envelope/ER homeostasis

Previously, we identified the nucleoporin gp210/Nup210 as a critical regulator of muscle and neuronal differentiation, but how this nucleoporin exerts its function and whether it modulates nuclear pore complex (NPC) activity remain unknown. Here, we show that gp210/Nup210 mediates muscle cell differentiation in vitro via its conserved N-terminal domain that extends into the perinuclear space. Removal of the C-terminal domain, which partially mislocalizes gp210/Nup210 away from NPCs, efficiently rescues the differentiation defect caused by the knockdown of endogenous gp210/Nup210. Unexpectedly, a gp210/Nup210 mutant lacking the NPC-targeting transmembrane and C-terminal domains is sufficient for C2C12 myoblast differentiation. We demonstrate that the endoplasmic reticulum (ER) stress-specific caspase cascade is exacerbated during Nup210 depletion and that blocking ER stress-mediated apoptosis rescues differentiation of Nup210-deficient cells. Our results suggest that the role of gp210/Nup210 in cell differentiation is mediated by its large luminal domain, which can act independently of NPC association and appears to play a pivotal role in the maintenance of nuclear envelope/ER homeostasis.     

Energy- and temperature-dependent transport of integral proteins to the inner nuclear membrane via the nuclear pore

Resident integral proteins of the inner nuclear membrane (INM) are synthesized as membrane-integrated proteins on the peripheral endoplasmic reticulum (ER) and are transported to the INM throughout interphase using an unknown trafficking mechanism. To study this transport, we developed a live cell assay that measures the movement of transmembrane reporters from the ER to the INM by rapamycin-mediated trapping at the nuclear lamina. Reporter constructs with small (<30 kD) cytosolic and lumenal domains rapidly accumulated at the INM. However, increasing the size of either domain by 47 kD strongly inhibited movement. Reduced temperature and ATP depletion also inhibited movement, which is characteristic of membrane fusion mechanisms, but pharmacological inhibition of vesicular trafficking had no effect. Because reporter accumulation at the INM was inhibited by antibodies to the nuclear pore membrane protein gp210, our results support a model wherein transport of integral proteins to the INM involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of 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.     

Stepwise reassembly of the nuclear envelope at the end of mitosis

The nuclear envelope consists of three distinct membrane domains: the outer membrane with the bound ribosomes, the inner membrane with the bound lamina, and the pore membrane with the bound pore complexes. Using biochemical and morphological methods, we observed that the nuclear membranes of HeLa cells undergoing mitosis are disassembled in a domain-specific manner, i.e., integral membrane proteins representing the inner nuclear membrane (the lamin B receptor) and the nuclear pore membrane (gp210) are segregated into different populations of mitotic vesicles. At the completion of mitosis, the inner nuclear membrane- derived vesicles associate with chromatin first, beginning in anaphase, whereas the pore membranes and the lamina assemble later, during telophase and cytokinesis. Our data suggest that the ordered reassembly of the nuclear envelope is triggered by the early attachment of inner nuclear membrane-derived vesicles to the chromatin.     

Removal of a single pore subcomplex results in vertebrate nuclei devoid of nuclear pores

The vertebrate nuclear pore complex, 30 times the size of a ribosome, assembles from a library of soluble subunits and two membrane proteins. Using immunodepletion of Xenopus nuclear reconstitution extracts, it has previously been possible to assemble nuclei lacking pore subunits tied to protein import, export, or mRNA export. However, these altered pores all still possessed the bulk of pore structure. Here, we immunodeplete a single subunit, the Nup107-160 complex, using antibodies to Nup85 and Nup133, two of its components. The resulting reconstituted nuclei are severely defective for NLS import and DNA replication. Strikingly, they show a profound defect for every tested nucleoporin. Even the integral membrane proteins POM121 and gp210 are absent or unorganized. Scanning electron microscopy reveals pore-free nuclei, while addback of the Nup107-160 complex restores functional pores. We conclude that the Nup107-160 complex is a pivotal determinant for vertebrate nuclear pore complex assembly.     

Integral Membrane Proteins of the Nuclear Envelope Are Dispersed throughout the Endoplasmic Reticulum during Mitosis

We have analyzed the fate of several integral membrane proteins of the nuclear envelope during mitosis in cultured mammalian cells to determine whether nuclear membrane proteins are present in a vesicle population distinct from bulk ER membranes after mitotic nuclear envelope disassembly or are dispersed throughout the ER. Using immunofluorescence staining and confocal microscopy, we compared the localization of two inner nuclear membrane proteins (laminaassociated polypeptides 1 and 2 [LAP1 and LAP2]) and a nuclear pore membrane protein (gp210) to the distribution of bulk ER membranes, which was determined with lipid dyes (DiOC₆ and R6) and polyclonal antibodies. We found that at the resolution of this technique, the three nuclear envelope markers become completely dispersed throughout ER membranes during mitosis. In agreement with these results, we detected LAP1 in most membranes containing ER markers by immunogold electron microscopy of metaphase cells. Together, these findings indicate that nuclear membranes lose their identity as a subcompartment of the ER during mitosis. We found that nuclear lamins begin to reassemble around chromosomes at the end of mitosis at the same time as LAP1 and LAP2 and propose that reassembly of the nuclear envelope at the end of mitosis involves sorting of integral membrane proteins to chromosome surfaces by binding interactions with lamins and chromatin.     

Function and assembly of nuclear pore complex proteins.

Nuclear pore complexes (NPCs) are extremely elaborate structures that mediate the bidirectional movement of macromolecules between the nucleus and cytoplasm. The current view of NPC organization features a massive symmetrical framework that is embedded in the double membranes of the nuclear envelope. It embraces a central channel of as yet ill-defined structure but which may accommodate particles with diameters up to 26 nm provided that they bear specific import/export signals. Attached to both faces of the central framework are peripheral structures, short cytoplasmic filaments, and a nuclear basket assembly, which interact with molecules transiting the NPC. The mechanisms of assembly and the nature of NPC structural intermediates are still poorly understood. However, mutagenesis and expression studies have revealed discrete sequences within certain NPC proteins that are necessary and sufficient for their appropriate targeting. In addition, some details are emerging from observations on cells undergoing mitosis where the nuclear envelope is disassembled and its components, including NPC subunits, are dispersed throughout the mitotic cytoplasm. At the end of mitosis, all of these components are reutilized to form nuclear envelopes in the two daughter cells. To date, it has been possible to define a time course of postmitotic assembly for a group of NPC components (CAN/Nup214, Nup153, POM121, p62 and Tpr) relative to the integral inner nuclear membrane protein LAP2 and the NPC membrane glycoprotein gp210. Nup153, a dynamic component of the nuclear basket, associates with chromatin towards the end of anaphase coincident with, although independent of, the inner nuclear membrane protein, LAP2. Assembly of the remaining proteins follows that of the nuclear membranes and occurs in the sequence POM121, p62, CAN/Nup214 and gp210/Tpr. Since p62 remains as a complex with three other NPC proteins (p58, p54, p45) during mitosis, and CAN/Nup214 maintains a similar interaction with its partner, Nup84, the relative timing of assembly of these additional four proteins may also be inferred. These observations suggest that there is a sequential association of NPC proteins with chromosomes during nuclear envelope reformation and the recruitment of at least eight of these precedes that of gp210. These findings support a model in which it is POM121 rather than gp210 that defines initial membrane-associated NPC assembly intermediates and which may therefore represent an essential component of the central framework of the NPC.     

Function of the CysD domain of the gel-forming MUC2 mucin

The colonic human MUC2 mucin forms a polymeric gel by covalent disulfide bonds in its N- and C-termini. The middle part of MUC2 is largely composed of two highly O-glycosylated mucin domains that are interrupted by a CysD domain of unknown function. We studied its function as recombinant proteins fused to a removable immunoglobulin Fc domain. Analysis of affinity-purified fusion proteins by native gel electrophoresis and gel filtration showed that they formed oligomeric complexes. Analysis of the individual isolated CysD parts showed that they formed dimers both when flanked by two MUC2 tandem repeats and without these. Cleavages of the two non-reduced CysD fusion proteins and analysis by MS revealed the localization of all five CysD disulfide bonds and that the predicted C-mannosylated site was not glycosylated. All disulfide bonds were within individual peptides showing that the domain was stabilized by intramolecular disulfide bonds and that CysD dimers were of non-covalent nature. These observations suggest that CysD domains act as non-covalent cross-links in the MUC2 gel, thereby determining the pore sizes of the mucus.     

Nuclear protein import is inhibited by an antibody to a lumenal epitope of a nuclear pore complex glycoprotein

Gp210 is a major transmembrane glycoprotein associated with the nuclear pore complex that is suggested to be important for organizing pore complex architecture and assembly. A mouse monoclonal IgG directed against an epitope in the lumenal domain of rat gp210 was expressed in cultured rat cells by microinjection of mRNA prepared from a hybridoma cell line. The expressed IgG, which becomes assembled into a functional antibody in the lumen of the endoplasmic reticulum, bound to the nuclear envelope in vivo. Expression of anti-gp210 antibody in interphase cells specifically reduced approximately fourfold the mediated nuclear import of a microinjected nuclear protein (nucleoplasmin) coupled to gold particles. The antibody also significantly decreased nuclear influx of a 10-kD dextran by passive diffusion. This transport inhibition did not result from removal of pore complexes from nuclear membranes or from gross alterations in pore complex structure, as shown by EM and immunocytochemistry. A physiological consequence of this transport inhibition was inhibition of cell progression from G2 into M phase. Hence, binding of this antibody to the lumenal side of gp210 must have a transmembrane effect on the structure and functions of the pore complex. These data argue that gp210 is directly or indirectly connected to pore complex constituents involved in mediated import and passive diffusion.     

NDC1: a crucial membrane-integral nucleoporin of metazoan nuclear pore complexes

POM121 and gp210 were, until this point, the only known membrane-integral nucleoporins (Nups) of vertebrates and, thus, the only candidate anchors for nuclear pore complexes (NPCs) within the nuclear membrane. In an accompanying study (Stavru et al.), we provided evidence that NPCs can exist independently of POM121 and gp210, and we predicted that vertebrate NPCs contain additional membrane-integral constituents. We identify such an additional membrane protein in the NPCs of mammals, frogs, insects, and nematodes as the orthologue to yeast Ndc1p/Cut11p. Human NDC1 (hNDC1) likely possesses six transmembrane segments, and it is located at the nuclear pore wall. Depletion of hNDC1 from human HeLa cells interferes with the assembly of phenylalanine-glycine repeat Nups into NPCs. The loss of NDC1 function in Caenorhabditis elegans also causes severe NPC defects and very high larval and embryonic mortality. However, it is not ultimately lethal. Instead, homozygous NDC1-deficient worms can be propagated. This indicates that none of the membrane-integral Nups is universally essential for NPC assembly, and suggests that NPC biogenesis is an extremely fault-tolerant process.     

Determination of the intracellular state of soluble macromolecules by gel filtration in vivo in the cytoplasm of amphibian oocytes

A method to examine the diffusible state and the sizes of major cytoplasmic proteins in a living cell is described. Small (40-300 microns) commercially available gel filtration beads of a broad range of Mr exclusion limits were microsurgically implanted into the cytoplasm of oocytes of the frog, Xenopus laevis, usually after metabolic labeling of oocyte proteins with [35S]methionine. After equilibration in vivo for several hours, the appearance of the implanted cells, notably the bead-cytoplasm boundary, was examined by light and electron microscopy of sections and found to be unaffected. After incubation the beads were isolated, briefly rinsed, and their protein contents examined by one- or two-dimensional gel electrophoresis. We show that diffusible proteins can be identified by their inclusion in the pores of the gel filtration beads used and that their approximate sizes can be estimated from the size exclusion values of the specific materials used. The application of this method to important cell biological questions is demonstrated by showing that several "karyophobic proteins," i.e., proteins of the cytosolic fraction which accumulate in the cytoplasm in vivo, are indeed diffusible in the living oocyte and appear with sizes similar to those determined in vitro. This indicates that the nucleo-cytoplasmic distribution of certain diffusible proteins is governed, in addition to size exclusion at nuclear pore complexes and karyophilic "signals," by other, as yet unknown forces. Some possible applications of this method of gel filtration in vivo are discussed.     

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.     

A glycoprotein modified with terminal N-acetylglucosamine and localized at the nuclear rim shows sequence similarity to aldose-1-epimerases.

Several glycoproteins that are present at the nuclear rim and at the nuclear pore complex of tobacco suspension-cultured cells are modified by O-linked oligosaccharides with terminal N-acetylglucosamine (GlcNAc). Here, we report on the purification of several of these glycoproteins, which are referred to as terminal GlcNAc (tGlcNAc) proteins. In vitro galactosylation of the tGlcNAc proteins generated glycoproteins with terminal galactosyl-beta-1, 4-GlcNAc and thus permitted their isolation by Erythrina crystagalli agglutinin affinity chromatography. Peptide sequence information derived from one tGlcNAc protein with an apparent molecular mass of 40 to 43 kD, designated gp40, made it possible to clone its gene. Interestingly, gp40 has 28 to 34% amino acid identity to aldose-1-epimerases from bacteria, and no gene encoding an aldose-1-epimerase has been isolated previously from higher organisms. Polyclonal antibodies were generated against recombinant gp40. Consistent with its purification as a putative nuclear pore complex protein, gp40 was localized to the nuclear rim, as shown by biochemical fractionation and immunofluorescence microscopy.     

Disassembly and characterization of the nuclear pore complex-lamina fraction from bovine liver nuclei

The nuclear pore complex-lamina (PCL), composed of nuclear pore structures attached to fibrous lamina, was isolated from bovine liver nuclei. We found that the highly aggregated PCL was disrupted and 75% of the constituent polypeptides could be solubilized by extraction for 1 h with 2% deoxycholate (DOC) and 3% 2-mercaptoethanol. While some differential solubilization was observed at lower detergent concentrations, all PCL proteins were solubilized equally at 2% DOC. The reducing agent was necessary to achieve maximum dispersal of the PCL and to prevent aggregation of the solubilized proteins. No tightly bound phospholipid or Triton X-100 could be detected in these preparations. Rapid removal of DOC, by dialysis or gel filtration, resulted in aggregation and precipitation of the PCL proteins, but the detergent could be removed by centrifugation through sucrose gradients. The sedimentation profiles indicated that the three major polypeptides, lamins A, B, and C, each sedimented as a single peak with a shoulder of more rapidly sedimenting material, possibly higher oligomeric forms. The sedimentation coefficient of lamins B and C, in the presence and absence of detergent, was 4.5 S. In the presence of DOC, lamin A had a sedimentation coefficient of 5.6 S, but this value was decreased to 4.1 S, when DOC was omitted from the gradient. These studies suggested that lamins B and C do not interact with or bind DOC, while lamin A may bind appreciable amounts of the detergent. The Stokes radii of lamins A, B, and C were found by gel filtration to be 75, 75, and 70 A, respectively. The molecular weights and frictional ratios estimated from the sedimentation and gel filtration data indicated that the lamins are dimeric, rod-shaped molecules.