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J A Hanover

Publications and source records attributed to J A Hanover.

At least 19 recordsLinked to original sources
PubMed1992

Regulation of macromolecular traffic mediated by the nuclear pore complex.

A sophisticated selective mechanism that regulates nuclear-cytoplasmic traffic has evolved in eukaryotes which circumvents the formidable barrier presented by the nuclear envelope. The sites of RNA and protein exchanges are the nuclear pore complexes (NPCs), 125 MDa supramolecular assemblies inserted into the envelope (see recent reviews by Dingwall, 1991; Goldfarb and Michaud, 1991; Miller et al., 1991; Nigg et al., 1991). In this article, the role NPCs play in regulating intracellular macromolecular traffic will be discussed.

Animals↗
PubMed1992

The nuclear pore: at the crossroads.

The nuclear pore complex is at the crossroads of macromolecular traffic across the nuclear envelope. Our knowledge of the mechanism whereby nuclear transport is mediated by the nuclear pore complex is also at a crossroads; a molecular understanding of this process has major implications for applied medical sciences. This becomes obvious with the realization that nuclear proteins are synthesized in the cytoplasm and yet function in the nucleus, and that RNA is transcribed in the nucleus but translated in the cytoplasm. Thus, control of macromolecular traffic across the nuclear membrane is an important means for altering the levels and activities of such molecules as steroid hormone receptors, transcription factors, and enzymes involved in DNA replication. Nuclear proteins have been found to contain nuclear localization sequences (NLS) rich in basic amino acids, which target them for transport through the nuclear pore to the nucleus. It is also clear that a group of novel glycoproteins having a unique carbohydrate modification are required for transport across the nuclear pore complex. However, the mechanism by which the NLS is recognized to mediate transport across the nuclear envelope is poorly understood. It is the aim of this brief review to attempt a synthesis of what is known of this mechanism and what may be newly inferred on the basis of current experimental data.

Amino Acid Sequence↗
PubMed1991

The gene encoding rat nuclear pore glycoprotein p62 is intronless.

Glycoproteins of the nuclear pore complex are thought to play an important role in the transport of regulatory proteins and ribonucleoproteins across the nuclear envelope. However, the genetic elements and signals that control the expression of nuclear pore glycoproteins are poorly understood. To study the transcriptional regulation of mammalian nuclear pore glycoprotein biosynthesis, we have isolated the gene coding for the major rat nuclear pore glycoprotein p62. The p62 gene consists of a 2941-base pair region that is linear with the full length p62 cDNA with no intervening sequences. Quantitative Southern analysis revealed that the gene is present in single copy. The p62 gene encodes a 525-amino acid open reading frame that directs the synthesis of the 62-kDa pore glycoprotein in vitro and in transfected cultured cells. The 5'-flanking region contains two potential transcription start sites; primer extension analysis revealed that the furthest upstream site is preferentially used in vivo. When linked to a reporter gene, the 5'-flanking region of the p62 gene serves as an active promoter.

Animals↗
PubMed1991

Nuclear targeting of prothymosin alpha.

Prothymosin alpha is a highly acidic protein which lacks an amino-terminal signal peptide, yet was once thought to be a precursor for thymosin alpha 1, a putative peptide hormone secreted by the thymus. Here, two lines of evidence are presented that strongly implicate prothymosin alpha as a nuclear protein: 1) in COS cells transfected with the human prothymosin alpha gene copious amounts of prothymosin alpha were present in sealed nuclei obtained by treating these cells with cytochalasin B and enucleating them centrifugally. 2) Constructs in which human prothymosin alpha nucleic acid sequences were fused in-frame either near the amino terminus of the beta-galactosidase gene in pCH110 or at the carboxyl terminus, when expressed in COS cells, resulted in nuclear localization of the fusion protein; indirect immunofluorescence in situ was used as the assay. The basic cluster of amino acids at the carboxyl terminus of prothymosin alpha, TKKQKT, has been identified as part of the nuclear targeting signal, whereas the basic cluster of amino acids situated within the thymosin alpha 1 sequence at the amino terminus failed to effect nuclear transport.

Amino Acid Sequence↗
PubMed1991

Antibodies against the SV40 large T antigen nuclear localization sequence.

Transport of large proteins into the nucleus requires both a nuclear localization signal (NLS) and exposure of that signal to components of the transport machinery. In this report, polyclonal and monoclonal antibodies were generated against the NLS of SV40 large T antigen. Several of these antibodies immunoprecipitated large T antigen produced by in vitro transcription-translation and recognized T antigen expressed in cultured cells. Binding of the antibodies to T antigen was quantified using an indirect radioimmunoassay and found to be specifically inhibited by peptides corresponding to the T antigen NLS. The ability of NLS-specific antibodies to recognize large T antigen suggests that the NLS is exposed on the surface of T antigen. When one of the NLS-specific monoclonal antibodies was introduced into the cytoplasm of cells expressing T antigen, the antibody remained cytoplasmic. These results suggested either that cytoplasmic components compete for binding to the NLS or that the antibody dissociates from T antigen during transport into the nucleus. When an antibody directed against an epitope distinct from the NLS was microinjected into the cytoplasm of cells expressing large T antigen, both the antibody and antigen were transported into the nucleus. The observed stability of the antigen-antibody complex strongly suggest protein unfolding is not required for nuclear protein transport.

Amino Acid Sequence↗
PubMed1990

Glycosylation of nuclear pore protein p62. Reticulocyte lysate catalyzes O-linked N-acetylglucosamine addition in vitro.

The addition of O-linked N-acetylglucosamine (GlcNAc) to the major nuclear pore complex glycoprotein p62 was examined. Expression of the rat p62 cDNA in transfected monkey cells was detected using a rat p62-specific antipeptide antiserum and two previously described nuclear pore-specific monoclonal antibodies which require O-linked GlcNAc for binding. Although the p62 cDNA was predicted to encode a 54-kDa polypeptide, the product expressed in monkey cells migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two species of 62 and 59-kDa. Cell-free translation of the p62 in vitro transcript yielded a 59-kDa polypeptide using wheat germ extract and a 62-kDa product using a commercially available rabbit reticulocyte lysate. Several lines of evidence indicated that the 62-kDa rabbit reticulocyte lysate translation product was modified by O-linked N-acetylglucosamine; the protein bound specifically to a wheat germ agglutinin affinity column and was converted to 59 kDa when treated with jack bean beta-acetylglucosaminidase. The 59-kDa unglycosylated wheat germ translation product was converted to the 62-kDa glycosylated form upon incubation with reticulocyte lysate demonstrating that O-linked GlcNAc can be added to p62 post-translationally.

Acetylglucosamine↗
PubMed1990

Structure and function of the nuclear pore complex: new perspectives.

The double membrane of the nuclear envelope is a formidable barrier separating the nucleus and cytoplasm of eukaryotic cells. However, movement of specific macromolecules across the nuclear envelope is critical for embryonic development, cell growth and differentiation. Transfer of molecules between the nucleus and cytoplasm occurs through the aqueous channel formed by the nuclear pore complex (NPC). Although small molecules may simply diffuse across the NPC, transport of large proteins and RNA requires specific transport signals and is energy dependent. A family of pore glycoproteins modified by O-linked N-acetylglucosamine moieties are essential for transport through the NPC. Recent evidence suggests that the regulation of nuclear transport may also involve the interaction of RNA and nuclear proteins with specific binding proteins that recognize these transport signals. Are these nuclear pore glycoproteins and signal binding proteins the 'gatekeepers' that control access to the genetic material? Recent evidence obtained from a combination of biochemical and genetic approaches suggests--perhaps.

Animals↗
PubMed1990

Primary sequence and heterologous expression of nuclear pore glycoprotein p62.

The major nuclear pore protein p62 is modified by O-linked N-acetylglucosamine and functions in nuclear transport. We have cloned, sequenced, and expressed the full-length rat p62 cDNA. The rat p62 mRNA is 2,941 nucleotides long and encodes a protein of 525 amino acids containing 30% serine and threonine residues. The amino acid sequence near the amino-terminus contains unique tetrapeptide repeats while the carboxy-terminus consists of a series of predicted alpha-helical regions with hydrophobic heptad repeats. Heterologous expression of rat p62 in African Green Monkey Kidney COS-1 cells and CV-1 cells was detected using a species-specific antipeptide serum. When transiently expressed in COS-1 cells, rat p62 binds wheat germ agglutinin and concentrates at the spindle poles during mitosis. In CV-1 cells cotransfected with rat p62 cDNA and SV40 viral DNA, rat p62 associates with the nuclear membrane without interfering with the nuclear transport of SV40 large T antigen. The ability to express p62 in tissue culture cells will facilitate analysis of the role of this pore protein in nuclear transport.

Amino Acid Sequence↗
PubMed1989

Overexpression and biosynthesis of CD4 in Chinese hamster ovary cells: coamplification using the multiple drug resistance gene.

CD4 is a T-cell surface glycoprotein and serves as the receptor for the human immunodeficiency virus. Glycosylation of CD4 has been shown to be necessary for proper surface expression. To study the biosynthesis and assembly of CD4, wild-type and glycosylation-deficient mutant Chinese hamster ovary (CHO) cells were cotransfected with a cDNA encoding CD4 and a cDNA for the human multiple drug resistance gene, which allowed the amplification of the transfected CD4 cDNA sequences. Clones were isolated that exhibited high-level expression of CD4 resulting from the integration of several copies of CD4 cDNA. CD4 synthesized by these cells acquired resistance to endoglycosidase H after 20-30 min of chase, suggesting a rapid translocation of the glycoprotein from the rough endoplasmic reticulum to the medial Golgi apparatus. The sensitivity of CD4 to glycosidases suggested the presence of biantennary unsialylated complex-type oligosaccharides. Consistent with this, CD4 synthesized by the Lec2 mutant, which does not add sialic acid to oligosaccharides, was identical to the glycoprotein produced by wild-type CHO cells. The amplification strategy used to express CD4 at high levels in wild-type and mutant CHO cells will have general utility.

Animals↗
PubMed1988

Glycosylation of CD4. Tunicamycin inhibits surface expression.

The T-cell surface glycoprotein CD4 plays an important role in mediating cellular immunity and serves as the receptor for human immunodeficiency virus. We have examined the glycosylation of CD4 and asked whether carbohydrate addition is essential for proper expression of the glycoprotein on the cell membrane. Under conditions where treatment of CD4+ human acute lymphoblastic leukemia cells (CEM-CM3 cells) with the glycosylation inhibitor tunicamycin decreased surface expression of CD4 in a time- and concentration-dependent manner, the surface expression of several other glycoproteins was unaffected. Incubation with tunicamycin for 48 h inhibited mannose incorporation by 98%, caused a 76% decrease in CD4 surface expression as judged by flow cytometry, and had little effect on methionine incorporation. Scatchard analysis showed a decrease in the total number of CD4 molecules on the cell surface from 17,000 to 8,900 after 24 h of tunicamycin treatment. Immunoprecipitation of metabolically labeled CD4 revealed the presence of an unglycosylated precursor in tunicamycin-treated cells. The observed difference between the Mr of the glycoprotein and its precursor is consistent with glycosylation at two potential N-linked sites. However, this precursor could not be detected by measuring steady state levels by immunoblotting. Also, no intracellular accumulation of CD4 in tunicamycin-treated cells was detectable using immunofluorescence microscopy. We conclude that surface expression of CD4 depends on glycosylation of the protein and that the unglycosylated precursor is preferentially degraded.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1988

Intracellular transport of VSV G protein occurs in cells lacking a nuclear envelope.

The intracellular transport of the G protein of a temperature sensitive vesicular stomatitis virus (VSV) mutant (ts 045) was examined in nucleated chinese hamster ovary (CHO) cells and in CHO cells subjected to enucleation with cytochalasin B. Although protein synthesis was virtually unaffected, enucleated cells synthesized only 30-45% of the amount of G protein assembled in control cells. As measured by acquisition of endoglycosidase H resistance, the rate of transport of the G protein from the RER to the medial golgi was found to be similar for nucleated and enucleated cells (t1/2 = 15 min). These data suggest that elements of the RER may directly transfer their contents to the Golgi, without the obligatory involvement of an intact NE.

Animals↗
PubMed1988

Nuclear protein import: specificity for transport across the nuclear pore.

Transport of proteins into the cell nucleus is thought to require specific localization sequences and may be mediated by nuclear pores. Following microinjection into fused cultured cells, nuclear protein import was directly monitored by fluorescence microscopy using B-phycoerythrin (PE; Mr 240,000) coupled to synthetic peptides corresponding to the simian virus 40 (SV-40) large T antigen nuclear localization signal. Peptides with a single amino acid replacement found in a cytoplasmic mutant of T antigen (cT) failed to promote uptake. Further studies with deletion peptides revealed the minimum sequence requirements for efficient nuclear import of PE conjugates to be similar to those previously defined genetically for large T antigen itself. No competitive inhibition of uptake was observed in cells expressing nuclear or cytoplasmic T antigen. Nuclear import was time- and temperature-dependent. The lectin wheat germ agglutinin (WGA) binds to glycoproteins bearing O-linked GlcNAc on the cytoplasmic face of the nuclear pore in vitro [J.A. Hanover et al. (1987) J. Biol. Chem. 262, 9887-9894] and in vivo. Microinjection of WGA into the cytoplasm of living cells did not alter the diffusion of dextran (Mr 10,000) into the nucleus, but blocked the uptake of PE conjugates. This inhibition was reversed when a competing saccharide was introduced into the cytoplasm.

Amino Acid Sequence↗
PubMed1988

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.

Acetylglucosamine↗
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↗
PubMed1987

A monoclonal antibody against a family of nuclear pore proteins (nucleoporins): O-linked N-acetylglucosamine is part of the immunodeterminant.

Using nuclear envelopes from Chinese hamster ovary (CHO) cells as an antigen, a mouse monoclonal antibody (IgM; designated mAb CHON211) that specifically binds to components of the nuclear pore complex (nucleoporins) was isolated. Immunofluorescence localization of the antigen recognized by mAb CHON211 revealed a punctate pattern restricted to the nuclear envelope; this pattern changed dramatically during the cell cycle. When examined by electron microscopy, the antigen was largely restricted to the nucleoplasmic and cytoplasmic faces of the nuclear pore complex. Immunoblots showed that mAb CHON211 bound to a series of polypeptides enriched in the nuclear envelope fraction with apparent molecular masses ranging from 110 to 35 kDa. Using galactosyltransferase and the lectin wheat germ agglutinin as probes, we have previously shown that proteins bearing O-linked N-acetylglucosamine (GlcNAc) are restricted to the cytoplasmic and nucleoplasmic faces of the nuclear pore complex. The nuclear membrane proteins recognized by mAb CHON211 had properties very similar to those identified by galactosyltransferase and wheat germ agglutinin labeling. Removal of O-linked GlcNAc residues with beta-hexosaminidase greatly reduced the binding of mAb CHON211, strongly suggesting that O-linked GlcNAc moieties are part of the immunodeterminant. This monoclonal antibody defines a new family of antigens that are restricted to the nuclear pore and bear a common modification: O-linked GlcNAc. mAb CHON211 will be useful for defining the role of the nucleoporins in nucleo-cytoplasmic exchange.

Acetylglucosamine↗
PubMed1986

Monoclonal antibodies against a glycoprotein localized in coated pits and endocytic vesicles inhibit alpha 2-macroglobulin binding and uptake.

Eight monoclonal antibodies, all IgG2a, which recognize a 180/90-kDa glycoprotein similar in properties to the receptor for alpha 2-macroglobulin of mouse embryo 3T3 cell plasma membranes, have been tested for their effect on the binding and uptake of alpha 2-macroglobulin by live cells. One antibody directly inhibited binding of 125I-alpha 2-macroglobulin under conditions in which 125I-transferrin binding to the transferrin receptor was unaffected. Another monoclonal antibody decreased alpha 2-macroglobulin binding when preincubated with cells at 37 degrees C. This antibody was also capable of specifically binding to ligand-receptor complexes formed by preincubating 125I-alpha 2-macroglobulin with detergent extracts of Swiss 3T3 cells. Immunoelectron microscopy showed that the 180/90-kDa glycoprotein was localized in coated pits of the cell surface and in intracellular endocytic vesicles (receptosomes/endosomes). The data suggest that the 180/90-kDa glycoprotein is a component of the receptor for alpha 2-macroglobulin.

Animals↗