The NUP1 gene encodes an essential component of the yeast nuclear pore complex.

Monoclonal antibodies generated against a family of related nuclear pore complex proteins (nucleoporins) from rat liver nuclei cross-react with several proteins in the yeast S. cerevisiae and show punctate nuclear envelope staining similar to the pattern seen in mammalian cells. We have cloned a gene encoding one of these proteins (NUP1) and have confirmed the localization of the NUP1 protein to the pore complex by immunofluorescence, using an epitope-tagged construct to differentiate it from other members of this family. The NUP1 protein is essential for cell viability, and overexpression from the yeast GAL10 promoter prevents further cell growth. The central domain of NUP1 consists of a series of degenerate repeats similar to those found in the nucleoskeletal protein NSP1, a protein that cross-reacts with monoclonal antibodies against NUP1. We propose that the repetitive domain is a feature common to the nucleoporins.

Association of gold-labelled nucleoplasmin with the centres of ring components of Xenopus oocyte nuclear pore complexes.

We have used heavy-metal shadowing to study the interaction of morphological components of Xenopus oocyte nuclear pore complexes with nucleoplasmin conjugated to colloidal gold. When microinjected into Xenopus oocytes, gold-labelled nucleoplasmin accumulated on the axis of the pores. Envelopes partially disrupted by treatment with low ionic strength buffer produced isolated islands of pores together with substantial quantities of rings deriving from the cytoplasmic and nucleoplasmic faces of the pores. In preparations from oocytes in which nucleoplasmin-gold had been microinjected, most (238/288) of the rings examined had also been labelled and, in the majority of these (60%), the label was located centrally within isolated rings. The central positioning of the nucleoplasmin-gold in isolated rings indicated that these morphological components of the pores were probably involved in the transport of nucleoplasmin into the nucleus, either by way of the initial binding of the molecule or by way of its translocation across the nuclear envelope. Although more work is required to resolve the precise stage at which the rings are involved, a number of lines of evidence suggested that they were more likely to be involved in the translocation step rather than in initial binding of nucleoplasmin.

NSP1: a yeast nuclear envelope protein localized at the nuclear pores exerts its essential function by its carboxy-terminal domain.

NSP1 is located at the nuclear periphery in yeast and is essential for cell growth. Employing immunoelectron microscopy on yeast cells, we show that NSP1 is located at the nuclear pores. The molecular analysis of the NSP1 protein points to a two domain model: a nonessential domain (the first 603 amino acids) composed of repetitive sequences common to other nuclear proteins and an essential, carboxy-terminal domain (residues 604-823) mediating the vital function of NSP1. The NSP1 carboxy-terminal domain, which shows a heptad repeat organization, affected the correct location of two nuclear proteins: site-specific amino acid substitutions within a predicted alpha-helical structure of this domain caused a temperature-sensitive growth arrest at 37 degrees C and the appearance of NSP1 and NOP1, a nucleolar protein, in the cytosol.

Isolation and characterization of the Drosophila nuclear envelope otefin cDNA.

We have recently identified and characterized a 53-kDa inner nuclear membrane-associated protein in Drosophila and termed it otefin. Here we report the isolation and characterization of cDNA and genomic clones of the otefin gene. Based on sequence analysis, we deduced that the primary translation product has a calculated mass of 45 kDa, contains many serine and threonine residues, and is mostly hydrophilic. However, in the carboxyl terminus, there is a hydrophobic region which may serve as a membrane anchoring domain. RNA blot analysis indicated that the otefin gene codes for a single poly(A+) transcript of 1.6 kilobases and that relatively large amounts of this transcript are present during developmental stages in which many nuclear divisions occur. Polyclonal antibodies raised against the cDNA translation product react with a 58-kDa mammalian nuclear envelope protein, demonstrating evolutionary conservation.

A 41-42 bp tandemly repeated sequence isolated from nuclear envelopes of chicken erythrocytes is located predominantly on microchromosomes.

To study whether specific DNA sequences are associated with nuclear membranes, residual DNA was extracted from DNase-treated nuclear envelopes prepared from erythrocytes of adult chickens (Gallus domesticus). This DNA was then blunt-end ligated into a bacterial plasmid vector. DNA blot analysis and nucleotide sequence determination revealed that approximately 30% of the cloned fragments consisted of different multiples of a 41-42 bp tandemly repeated, partially symmetrical sequence. In situ hybridization to chicken chromosomes demonstrated that the sequence was located primarily on microchromosomes, although some hybridization was also observed to macrochromosomes 7 and 8. Digestion of chicken DNA with any of a number of restriction enzymes did not completely reduce the intensity of a high molecular weight band to which the repeated sequence hybridized. These results, along with those obtained from in situ hybridization, suggested that many copies of this sequence are organized into large tandem arrays, and are not dispersed in many shorter repetitive blocks throughout the chicken genome. Although the repetitive sequence constituted approximately 10% of the chicken genome, it did not hybridize to quail or turkey DNA.

The mitosis-specific monoclonal antibody MPM-2 recognizes phosphoproteins associated with the nuclear envelope in Chlamydomonas reinhardtii cells.

The monoclonal antibody MPM-2 recognizes a family of phosphorylated proteins present in mitotic cells. In a number of organisms it stains nuclei and also cytoskeletal structures which contain or organize tubulin. In mitotic Chlamydomonas reinhardtii cells MPM-2 reacts with phosphoproteins associated with the nuclear envelope (NE). Staining of the NE region appears in preprophase, reaches a maximum intensity in metaphase/anaphase and disappears rapidly in telophase. Localized hyperphosphorylation of the anterior NE region is apparent in many cells throughout mitosis. The distribution and timing of MPM-2 labeling suggests that in Chlamydomonas MPM-2 may be interacting with lamin-like phosphoproteins.

Tissue-specific differences in the compartmentalization of rat nuclear triiodothyronine receptors.

In addition to the recognized rat liver nuclear T3 receptor extractable with hypertonic salt, recent studies have described nucleoplasmic receptors extractable with isotonic KCl and salt-resistant receptors localized to the nuclear matrix. A method was developed for the determination of intra-nuclear receptor distribution in small samples of nuclei dispersed within glass wool matrices. After in vitro labelling with 6 nmol/l [125I]T3, dispersed nuclei were sequentially extracted with 0.15 mol/l KCl (yielding nucleoplasmic receptors), 0.4 mol/l KCl. and 2 mol/l KCl (the latter two concentrations yielding hypertonic salt-extractable receptors). The salt-resistant receptors were retained within the glass wool columns. The intra-nuclear distribution of in vivo labelled receptors was very similar to that obtained by in vitro labelling. The equilibrium association constants for L-T3 binding among the receptor pools ranged from 0.6 X 10(9) to 1.0 X 10(9) l/mol. The distribution of total nuclear receptors within each nuclear compartment was (percentage of nucleoplasmic, hypertonic salt-extractable, and salt-resistant receptors): Cerebrum: 23.6, 52.2, 24.2; Liver: 25.2, 57.2, 17.5; Kidney: 45.9, 33.5, 20.6; Testis: 65.5, 14.7, 19.7; and Spleen: 66.7, 18.7, 14.6. The rank order of percentage of hypertonic salt-extractable receptors approximates the rank order of thyroid hormone-responsiveness by traditional criteria. The inverse is true for the percentage of nucleoplasmic receptors. The percentage of salt-resistant receptors was very similar in all of the tissues.

Localization of an acetylcholine receptor intron to the nuclear membrane.

The first intron of the RNA for the acetylcholine receptor (AChR) alpha subunit shows a ringlike distribution around nuclei in multinucleated myotubes by in situ hybridization. This pattern is not observed for an actin intron or U1 RNA. Quantitation of the intron sequences reveals large variations in the amount of both the AChR and actin introns between nuclei within the same myotube, although all nuclei express equivalent amounts of U1 RNA. This differential RNA expression indicates that nuclei can individually control expression of messenger RNAs. The restricted distribution of the AChR intron RNA suggests a previously unknown step in RNA processing.

Does amiodarone inhibit T3 binding to solubilized nuclear receptors in vitro?

One of the proposed mechanisms of action of amiodarone is by interfering with T3-receptor interactions. However, reports in the literature on this matter are contradictory and, in an attempt to resolve these contradictions, the effects of amiodarone on T3 binding to solubilized nuclear receptors from rat liver were investigated. A drug concentration-related competitive inhibition of T3 binding occurred in the presence of considerable amiodarone precipitation. Measurement of the proportion of soluble amiodarone over the range where significant inhibition of T3 binding was observed, revealed that the soluble amiodarone decreased over this range. Since the amiodarone preparation was free of any contaminant, these findings suggested that a product generated from amiodarone during incubation was responsible for the inhibition. HPLC analysis of the soluble amiodarone fraction indicated that, during incubation, two substances were generated from the parent compound. However, only the concentration of one of the substances continually increased whereas the other decreased with increasing concentrations of amiodarone added to the samples. These findings suggest that the inhibition of T3 binding to solubilized nuclear receptors in the presence of amiodarone results from the generation of a specific product from the amiodarone under certain conditions during incubation.

Identification of specific polypeptides of the nuclear envelope by iodination of mouse liver nuclei.

A sensitive technique is described for the rapid identification of nuclear-envelope proteins. Mouse liver nuclei (purified on sucrose gradients) were iodinated with Na125I by the immobilized water-insoluble reagent Iodogen. Iodinated nuclei were digested with RNAase A and DNAase I and then salt-extracted to obtain labelled nuclear envelopes. Nuclear envelopes were characterized by morphological and biochemical criteria and by SDS/polyacrylamide-gel electrophoresis. In all, 13 polypeptides of molecular masses 145, 115, 98, 85, 75, 70, 65, 54, 50, 45, 40, 38 and 36 kDa were identified in the labelled nuclear envelopes. The labelled polypeptides were localized to the nuclear envelope by extraction of the envelope with Triton X-100 and different concentrations of salt. Iodination of intact nuclei was shown to be specific for the nuclear envelope by the absence of labelling of histones and cytoplasmic contaminants.

Additional members of the rat liver lamin polypeptide family. Structural and immunological characterization.

In addition to lamins A, B, and C (Gerace, L., Blum, A., and Blobel, G. (1978) J. Cell Biol. 79, 546-566), the rat liver nuclear lamina has recently been shown to contain two higher molecular weight (70,000-75,000, pI 5.7-5.8) quantitatively less prominent nuclear polypeptides (Lehner, C.F., Kurer, V., Eppenberger, H. M., and Nigg, E. A. (1986) J. Biol. Chem. 261, 13293-13301). In the present study two-dimensional tryptic peptide mapping and Western blotting with affinity-purified chicken and human sera have been utilized to examine the structural relationships and the tissue distribution of these quantitatively less prominent mammalian lamins (termed lamins D and E in this study). Lamins D and E have indistinguishable one- and two-dimensional proteolytic maps. Whereas the one-dimensional proteolytic maps of lamins D and E show several degradation products which are of similar molecular mass to polypeptides seen in one-dimensional proteolytic maps of lamins A, B, and C, the two-dimensional tryptic maps of D and E are distinct from those of lamins A, B, and C, suggesting that lamins D and E are produced by transcription of one or more unique genes. Nonetheless, affinity-purified anti-D/E antibodies (raised against lamin E) cross-react with lamin B, suggesting the presence of a shared epitope. Moreover, a human autoantibody cross-reacts with all five lamins after affinity elution from any of the five, suggesting the presence of another epitope which is shared by all five polypeptides. All five lamins were undetectable in rodent epididymal sperm. In contrast, lamins D and E were readily detected in a variety of rat somatic tissues (liver, kidney, prostate, brain, heart) including lymphoid cells, a cell type depleted of lamins A and C. During mitogenic stimulation of lymphocytes, the signal for lamins A and C increased 5-fold, the signal for lamins D and E increased 2-fold, but the signal for lamin B remained unchanged, suggesting that levels of lamins D and E are regulated independently from those of the major lamins.

Primary structure analysis of an integral membrane glycoprotein of the nuclear pore.

The complete primary structure of an integral membrane glycoprotein of the nuclear pore was deduced from the cDNA sequence. The cDNA encodes a polypeptide of 204,205 D containing a 25-residue-long signal sequence, two hydrophobic segments that could function as transmembrane segments, and 13 potential N-linked oligosaccharide addition sites. Endoglycosidase H reduces the molecular mass by approximately 9 kD suggesting that not all of these 13 sites are used. We discuss possible models for the topology of this protein in the pore membrane as well as a possible role in the formation of pores and pore complexes.

Localization of Epstein-Barr virus envelope glycoproteins on the inner nuclear membrane of virus-producing cells.

Epstein-Barr virus-producing cells were used as a model to analyze, with a fracture-immunolabel technique, the distribution, behavior on fracture, and extent of glycosylation of viral transmembrane glycoproteins at the inner nuclear membrane. Surface and fracture immunolabeling with two monoclonal antibodies directed against the carbohydrate or polypeptide portions of the major viral envelope glycoproteins gp350/220 showed the following. (i) The glycoproteins present on the inner and outer nuclear membranes were labeled only with the monoclonal antibody directed against the polypeptide chain, whereas over the surface of virus-producing cells and on mature virions the labeling was dense and uniformly distributed with both monoclonal antibodies. (ii) The glycoproteins were nonuniformly distributed only over the inner nuclear membranes; at the sites of viral budding, the glycoproteins showed a preferential partition with the protoplasmic face. Since fully glycosylated glycoproteins were not present on the nuclear membranes, our observations support the proposed model of herpesvirus maturation. The peculiar distribution and partition on fracture of the envelope glycoproteins on the inner nuclear membrane are similar to those of Sindbis virus envelope glycoproteins on the plasma membrane of infected cells. Therefore, our results suggest that inner nuclear membranes may behave like plasma membranes during viral assembly.

Partial cDNA sequence encoding a nuclear pore protein modified by O-linked N-acetylglucosamine.

The nuclear pore complex contains a family of proteins ranging in molecular mass from 35 to 220 kDa that are glycosylated with O-linked N-acetylglucosamine (GlcNAc) residues. We sought to determine the primary sequence of a nuclear pore protein modified by O-linked GlcNAc. The major (62 kDa) nuclear pore glycoprotein (np62) was purified from rat liver nuclear envelopes by immunoaffinity chromatography and preparative gel electrophoresis. After CNBr fragmentation, a glycopeptide was isolated and microsequenced. An oligonucleotide probe based on this sequence information was used to screen a lambda gt11 cDNA library constructed from poly(A) mRNA of the rat thyroid cell line FRTL-5. A clone (B5) was isolated and shown to hybridize to a single 2.5-kilobase species in poly(A) mRNA from rat liver and FRTL-5. This insert was sequenced and found to contain a 691-base-pair cDNA encoding a 155-amino acid open reading frame. This open reading frame contained a CNBr fragment identical to the original glycopeptide sequence and a second CNBr fragment corresponding to a nonglycosylated peptide that was also isolated from the purified pore glycoprotein. The B5 cDNA produced a beta-galactosidase fusion protein of the size predicted by the open reading frame. Analysis of the residues making up a presumptive glycosylation site suggests that the sequence is unlike any known sites for enzymatic N- or O-linked glycosylation. The partial sequence of the 62-kDa nuclear pore glycoprotein shows little similarity to other characterized proteins and elucidates structural features of a member of the family of nuclear pore glycoproteins.

Integral membrane proteins specific to the inner nuclear membrane and associated with the nuclear lamina.

We obtained a monoclonal antibody (RL13) that identifies three integral membrane proteins specific to the nuclear envelope of rat liver, a major 75-kD polypeptide and two more minor components of 68 and 55 kD. Immunogold labeling of isolated nuclear envelopes demonstrates that these antigens are localized specifically to the inner nuclear membrane, and that the RL13 epitope occurs on the inner membrane's nucleoplasmic surface where the nuclear lamina is found. When nuclear envelopes are extracted with solutions containing nonionic detergent and high salt to solubilize nuclear membranes and pore complexes, most of these integral proteins remain associated with the insoluble lamina. Since the polypeptides recognized by RL13 are relatively abundant, they may function as lamina attachment sites in the inner nuclear membrane. Major cross-reacting antigens are found by immunoblotting and immunofluorescence microscopy in all rat cells examined. Therefore, these integral proteins are biochemical markers for the inner nuclear membrane and will be useful models for studying nuclear membrane biogenesis.

A histone 1-like antigen is a component of the nuclear envelope.

Rabbit antibodies have been raised against rat liver nuclear envelopes. An enzyme-linked immunosorbent assay (ELISA) demonstrated high titer antiserum specific for the nuclear envelope preparation. Immunocytochemical studies showed that the antiserum stained the nuclear envelopes, but not intra-nuclear components of HEp-2 (human malignant epithelial) cells. When electrophoretically separated peptides were tested by immunoblotting techniques, the rabbit antiserum specifically stained proteins with molecular masses of 26 and 28 kD. These peptides had similar mobilities to purified histone 1 (H1). Indeed purified calf thymus H1 recognized the antiserum. The antigens are not loosely bound to the nuclear envelope, as they could not be extracted with low salt. Therefore, we have established that the 26 and 28 kD nuclear envelope peptides are not contaminants of the nuclear envelope preparation and that they express determinants that are immunologically cross-reactive with purified H1, but not with intra-nuclear H1.

The fates of chicken nuclear lamin proteins during mitosis: evidence for a reversible redistribution of lamin B2 between inner nuclear membrane and elements of the endoplasmic reticulum.

In chicken, three structurally distinct nuclear lamin proteins have been described. According to their migration on two-dimensional gels, these proteins have been designated as lamins A, B1, and B2. To investigate the functional relationship between chicken lamins and their mammalian counterparts, we have examined here the state of individual chicken lamin proteins during mitosis. Current models proposing functional specializations of mammalian lamin subtypes are in fact largely based on the observation that during mitosis mammalian lamin B remains associated with membrane vesicles, whereas lamins A and C become freely soluble. Cell fractionation experiments combined with immunoblotting show that during mitosis both chicken lamins B1 and B2 remain associated with membranes, whereas lamin A exists in a soluble form. In situ immunoelectron microscopy carried out on mitotic cells also reveals membrane association of lamin B2, whereas the distribution of lamin A is random. From these results we conclude that both chicken lamins B1 and B2 may functionally resemble mammalian lamin B. Interestingly, immunolabeling of mitotic cells revealed an association of lamin B2 with extended membrane cisternae that resembled elements of the endoplasmic reticulum. Quantitatively, we found that all large endoplasmic reticulum-like membranes present in metaphase cells were decorated with lamin B2-specific antibodies. Given that labeling of these mitotic membranes was lower than labeling of interphase nuclear envelopes, it appears likely that during mitotic disassembly and reassembly of the nuclear envelope lamin B2 may reversibly distribute between the inner nuclear membrane and the endoplasmic reticulum.

Nucleolar and nuclear envelope proteins of the yeast Saccharomyces cerevisiae.

We have developed a fast and reliable purification protocol to obtain yeast nuclei in intact and pure form and in a reasonable yield. The purified nuclei appear homogeneous at the light and electron microscopic level, are highly enriched in the nuclear marker histone H2B and devoid of mitochondrial, vacuolar and cytosolic marker proteins. On sodium dodecyl sulfate (SDS)-polyacrylamide gels, the nuclear fraction contains unique proteins which distinguishes them from the major yeast subcellular fractions. Yeast nuclei were separated by detergent/salt extraction into soluble, insoluble and membrane fractions. Antibodies raised against subnuclear fractions lead to the identification of an integral nuclear membrane protein and a high-abundance 38-kDa protein which is located in the yeast nucleolus.