Nuclear import of histone H2A and H2B is mediated by a network of karyopherins.

The first step in the assembly of new chromatin is the cell cycle-regulated synthesis and nuclear import of core histones. The core histones include H2A and H2B, which are assembled into nucleosomes as heterodimers. We show here that the import of histone H2A and H2B is mediated by several members of the karyopherin (Kap; importin) family. An abundant complex of H2A, H2B, and Kap114p was detected in cytosol. In addition, two other Kaps, Kap121p and Kap123p, and the histone chaperone Nap1p were isolated with H2A and H2B. Nap1p is not necessary for the formation of the Kap114p-H2A/H2B complex or for import of H2A and H2B. We demonstrate that both histones contain a nuclear localization sequence (NLS) in the amino-terminal tail. Fusions of the NLSs to green fluorescent protein were specifically mislocalized to the cytoplasm in kap mutant strains. In addition, we detected a specific mislocalization in a kap95 temperature-sensitive strain, suggesting that this Kap is also involved in the import of H2A and H2B in vivo. Importantly, we show that Kap114p, Kap121p, and Kap95 interact directly with both histone NLSs and that RanGTP inhibits this association. These data suggest that the import of H2A and H2B is mediated by a network of Kaps, in which Kap114p may play the major role.

Nuclear import of the TATA-binding protein: mediation by the karyopherin Kap114p and a possible mechanism for intranuclear targeting.

Binding of the TATA-binding protein (TBP) to the promoter is the first and rate limiting step in the formation of transcriptional complexes. We show here that nuclear import of TBP is mediated by a new karyopherin (Kap) (importin) family member, Kap114p. Kap114p is localized to the cytoplasm and nucleus. A complex of Kap114p and TBP was detected in the cytosol and could be reconstituted using recombinant proteins, suggesting that the interaction was direct. Deletion of the KAP114 gene led to specific mislocalization of TBP to the cytoplasm. We also describe two other potential minor import pathways for TBP. Consistent with other Kaps, the dissociation of TBP from Kap114p is dependent on RanGTP. However, we could show that double stranded, TATA-containing DNA stimulates this RanGTP-mediated dissociation of TBP, and is necessary at lower RanGTP concentrations. This suggests a mechanism where, once in the nucleus, TBP is preferentially released from Kap114p at the promoter of genes to be transcribed. In this fashion Kap114p may play a role in the intranuclear targeting of TBP.

A novel nuclear import pathway for the transcription factor TFIIS.

We have identified a novel pathway for protein import into the nucleus. We have shown that the previously identified but uncharacterized yeast protein Nmd5p functions as a karyopherin. It was therefore designated Kap119p (karyopherin with Mr of 119 kD). We localized Kap119p to both the nucleus and the cytoplasm. We identified the transcription elongation factor TFIIS as its major cognate import substrate. The cytoplasmic Kap119p exists as an approximately stoichiometric complex with TFIIS. RanGTP, not RanGDP, dissociated the isolated Kap119p/TFIIS complex and bound to Kap119p. Kap119p also bound directly to a number of peptide repeat containing nucleoporins in overlay assays. In wild-type cells, TFIIS was primarily localized to the nucleus. In a strain where KAP119 has been deleted, TFIIS was mislocalized to the cytoplasm indicating that TFIIS is imported into the nucleus by Kap119p. The transport of various substrates that use other karyopherin-mediated import or export pathways was not affected in a kap119Delta strain. Hence Kap119p is a novel karyopherin that is responsible for the import of the transcription elongation factor TFIIS.

Nuclear import and the evolution of a multifunctional RNA-binding protein.

La (SS-B) is a highly expressed protein that is able to bind 3'-oligouridylate and other common RNA sequence/structural motifs. By virtue of these interactions, La is present in a myriad of nuclear and cytoplasmic ribonucleoprotein complexes in vivo where it may function as an RNA-folding protein or RNA chaperone. We have recently characterized the nuclear import pathway of the S. cerevisiae La, Lhp1p. The soluble transport factor, or karyopherin, that mediates the import of Lhp1p is Kap108p/Sxm1p. We have now determined a 113-amino acid domain of Lhp1p that is brought to the nucleus by Kap108p. Unexpectedly, this domain does not coincide with the previously identified nuclear localization signal of human La. Furthermore, when expressed in Saccharomyces cerevisiae, the nuclear localization of Schizosaccharomyces pombe, Drosophila, and human La proteins are independent of Kap108p. We have been able to reconstitute the nuclear import of human La into permeabilized HeLa cells using the recombinant human factors karyopherin alpha2, karyopherin beta1, Ran, and p10. As such, the yeast and human La proteins are imported using different sequence motifs and dissimilar karyopherins. Our results are consistent with an intermingling of the nuclear import and evolution of La.

Transport routes through the nuclear pore complex.

The nuclear pore complex can be considered to be the stationary phase of bidirectional traffic between the nucleus and the cytoplasm. The mobile phase consists of karyopherins, transport substrates, and the small GTPase Ran and its modulators. Recently, the family of karyopherins was expanded with the recognition of numerous open reading frames with limited homology to karyopherin beta 1. In several cases, the specific substrates transported by the new karyopherins have been identified, allowing the characterization of new pathways into and out of the nucleus. However, the mechanisms of transport, particularly the role of Ran, remain poorly understood.

Characterization of the Wtm proteins, a novel family of Saccharomyces cerevisiae transcriptional modulators with roles in meiotic regulation and silencing.

Transcription is regulated by the complex interplay of repressors and activators. Much of this regulation is carried out by, in addition to gene-specific factors, complexes of more general transcriptional modulators. Here we present the characterization of a novel family of transcriptional regulators in yeast. Wtm1p (WD repeat-containing transcriptional modulator) was identified as a protein present in a large nuclear complex. This protein has two homologs, Wtm2p and Wtm3p, which probably arose by gene duplications. Deletion of these genes affects transcriptional repression at several loci, including derepression of IME2, a meiotic gene normally repressed in haploid cells. Targeting of these proteins to DNA resulted in a dramatic repression of activated transcription. In common with a mutation in the histone deacetylase RPD3, wtm mutants showed increased repression at the silent mating-type locus, HMR, and at telomeres. Although all three Wtm proteins could act as transcriptional repressors, Wtm3p, which is the least homologous, appeared to have functions separate from those of the other two. Wtm3p did not appear to be complexed with the other two proteins, was essential for IME2 repression, and could not efficiently repress transcription in the absence of the other Wtm proteins. These data suggested that Wtm1p and Wtm2p are repressors and that Wtm3p has different effects on transcription at different loci.

A distinct and parallel pathway for the nuclear import of an mRNA-binding protein.

Three independent pathways of nuclear import have so far been identified in yeast, each mediated by cognate nuclear transport factors, or karyopherins. Here we have characterized a new pathway to the nucleus, mediated by Mtr10p, a protein first identified in a screen for strains defective in polyadenylated RNA export. Mtr10p is shown to be responsible for the nuclear import of the shuttling mRNA-binding protein Npl3p. A complex of Mtr10p and Npl3p was detected in cytosol, and deletion of Mtr10p was shown to lead to the mislocalization of nuclear Npl3p to the cytoplasm, correlating with a block in import. Mtr10p bound peptide repeat-containing nucleoporins and Ran, suggesting that this import pathway involves a docking step at the nuclear pore complex and is Ran dependent. This pathway of Npl3p import is distinct and does not appear to overlap with another known import pathway for an mRNA-binding protein. Thus, at least two parallel pathways function in the import of mRNA-binding proteins, suggesting the need for the coordination of these pathways.

A nuclear import pathway for a protein involved in tRNA maturation.

A limited number of transport factors, or karyopherins, ferry particular substrates between the cytoplasm and nucleoplasm. We identified the Saccharomyces cerevisiae gene YDR395w/SXM1 as a potential karyopherin on the basis of limited sequence similarity to known karyopherins. From yeast cytosol, we isolated Sxm1p in complex with several potential import substrates. These substrates included Lhp1p, the yeast homologue of the human autoantigen La that has recently been shown to facilitate maturation of pre-tRNA, and three distinct ribosomal proteins, Rpl16p, Rpl25p, and Rpl34p. Further, we demonstrate that Lhp1p is specifically imported by Sxm1p. In the absence of Sxm1p, Lhp1p was mislocalized to the cytoplasm. Sxm1p and Lhp1p represent the karyopherin and a cognate substrate of a unique nuclear import pathway, one that operates upstream of a major pathway of pre-tRNA maturation, which itself is upstream of tRNA export in wild-type cells. In addition, through its association with ribosomal proteins, Sxm1p may have a role in coordinating ribosome biogenesis with tRNA processing.

The epithelial mucin MUC1 contains at least two discrete signals specifying membrane localization in cells.

The MUC1 gene product (PEM, polymorphic epithelial mucin) is a cell-associated glycoprotein expressed on the apical surface of most simple secretory epithelia. The transmembrane and cytoplasmic domains of MUC1 have been shown to be highly conserved between mammalian species, and it has been shown that this molecule interacts with the actin cytoskeleton. Apical targeting signals in polarized cells have yet to be defined. The mechanism by which MUC1 is targeted and maintained on the apical surface is not known; correct localization, however, would be predicted to be crucial for function. In order to determine which domains of MUC1 were important for this localization, mutational analysis of the protein was undertaken. Using cytoplasmic tail deletion mutants, fusion proteins of MUC1 and CD2, and site-directed mutagenesis, it could be shown that MUC1 appeared to contain at least two motifs involved in apical localization. The first was located in the extracellular domain and was sufficient to confer apical localization on the fusion protein. The second was the Cys-GlnCys (CQC) motif at the junction of the cytoplasmic and transmembrane domains. This sequence was necessary for surface expression. These results suggest that MUC1 contains two discrete motifs important in its apical localization.

Disruption of the nucleoporin gene NUP133 results in clustering of nuclear pore complexes.

We have characterized a protein with an estimated molecular mass of 130 kDa that is contained in a highly enriched yeast nuclear pore complex (NPC) fraction. Partial amino acid sequence from this protein has led us to a previously identified open reading frame on chromosome XI of Saccharomyces cerevisiae encoding a protein of 133 kDa. Due to its coenrichment with NPCs during cell fractionation and the phenotype observed in the disrupted strain, we propose to term the gene encoding this protein NUP133. Cells carrying a disrupted copy of NUP133 were temperature sensitive for growth. In addition, abnormal clustering of NPCs was observed. This phenotype is similar to that previously observed in the disruption of another nucleoporin gene, NUP145. We speculate that the gene product of NUP133, Nup133p, may functionally overlap with the NUP145 gene product, Nup145p, and that these proteins may be involved in maintaining the position of the NPC within the nuclear envelope.

Spatial and temporal expression of an epithelial mucin, Muc-1, during mouse development.

The Muc-1 mucin is found as a transmembrane protein in the apical surface of glandular epithelia. To provide insight into possible functions, we have assessed the timing of expression and the distribution of the Muc-1 protein during mouse embryogenesis using three different techniques: RT-PCR, northern blots and immunohistochemistry. Our results indicate that Muc-1 expression correlates with epithelial differentiation in stomach, pancreas, lung, trachea, kidney and salivary glands. Once started, Muc-1 synthesis continually increases with time, mainly due to epithelial area growth. Our data suggest that expression of the Muc-1 gene is under spatial and temporal control during organogenesis. Although Muc-1 is present in different organs, its expression is not induced systemically, but according to the particular onset of epithelial polarization and branching morphogenesis of each individual organ. It is of particular interest that Muc-1 protein can be detected lining the apical surfaces of the developing lumens when the epithelium of these organs is still undergoing folding and branching, and glandular activity has not yet started. We speculate that Muc-1 may participate in epithelial sheet differentiation/lumen formation during early development of the organs known to express it. This speculation is based on: (1) the detection of Muc-1 expression early during organogenesis, (2) the defined apical localization in different epithelia, (3) the decrease in cell-cell interactions when Muc-1 protein is highly expressed and (4) the possible interaction of its cytoplasmic tail with the actin cytoskeleton. However, it remains to be established using in vitro systems, whether the temporal and local expression of the Muc-1 gene coincident with the morphogenetic events described here is relevant for the process.

Monoclonal antibody 3F8 recognises the neural cell adhesion molecule (NCAM) in addition to the ganglioside GD2.

The monoclonal antibody 3F8 has been described as binding to the ganglioside GD2. This antibody, of the IgG3 isotype, has been used in immunotherapy, radioimmunolocalisation and targeted radiation therapy. 3F8 was originally observed to have a binding profile similar to two monoclonal antibodies, UJ13A and 5.1.H11, characterised as binding to the neural cell adhesion molecule (NCAM). This observation has also been confirmed using a hetero-antiserum prepared against purified NCAM. The cross-reactivity of 3F8 with NCAM has been confirmed by cross-blocking studies with an anti-NCAM antiserum, and by direct immunoprecipitation and gel electrophoresis. In addition, we show that 3F8 binds to human NCAM from 3T3 fibroblasts transfected with NCAM cDNA constructs. It is possible that the common epitope shared by GD2 ganglioside and NCAM involves sialic acid residues common to both the ganglioside and the glycoprotein.

N-myc gene expression and oncoprotein characterisation in medulloblastoma.

Although medulloblastoma and neuroblastoma share many common biological, histological and immunological features, the frequency of N-myc amplification differs markedly between the two tumours. In this study, Southern blot analysis revealed that the N-myc gene was not amplified in any of the nine medulloblastoma samples analysed. In contrast, over-expression of the gene was found in six of 11 samples as determined by immunocytochemistry and/or Western blot analysis, using an antiserum raised against a synthetic peptide representing a sequence unique to the N-myc gene product. The specificity of this reagent was demonstrated by studies on a variety of cell lines expressing N-myc and/or c-myc oncoproteins. Of the 12 medulloblastoma samples collected over a two-year period and analysed in the course of this project, a trend towards longer disease-free survival was noted in the patients having low levels of the N-myc protein in their tumour.