An evolutionarily conserved NPC subcomplex, which redistributes in part to kinetochores in mammalian cells.

The nuclear pore complexes (NPCs) are evolutionarily conserved assemblies that allow traffic between the cytoplasm and the nucleus. In this study, we have identified and characterized a novel human nuclear pore protein, hNup133, through its homology with the Saccharomyces cerevisiae nucleoporin scNup133. Two-hybrid screens and immunoprecipitation experiments revealed a direct and evolutionarily conserved interaction between Nup133 and Nup84/Nup107 and indicated that hNup133 and hNup107 are part of a NPC subcomplex that contains two other nucleoporins (the previously characterized hNup96 and a novel nucleoporin designated as hNup120) homologous to constituents of the scNup84 subcomplex. We further demonstrate that hNup133 and hNup107 are localized on both sides of the NPC to which they are stably associated at interphase, remain associated as part of a NPC subcomplex during mitosis, and are targeted at early stages to the reforming nuclear envelope. Throughout mitosis, a fraction of hNup133 and hNup107 localizes to the kinetochores, thus revealing an unexpected connection between structural NPCs constituents and kinetochores. Photobleaching experiments further showed that the mitotic cytoplasm contains kinetochore-binding competent hNup133 molecules and that in contrast to its stable association with the NPCs the interaction of this nucleoporin with kinetochores is dynamic.

A mechanism for nuclear positioning in fission yeast based on microtubule pushing.

The correct positioning of the nucleus is often important in defining the spatial organization of the cell, for example, in determining the cell division plane. In interphase Schizosaccharomyces pombe cells, the nucleus is positioned in the middle of the cylindrical cell in an active microtubule (MT)-dependent process. Here, we used green fluorescent protein markers to examine the dynamics of MTs, spindle pole body, and the nuclear envelope in living cells. We find that interphase MTs are organized in three to four antiparallel MT bundles arranged along the long axis of the cell, with MT plus ends facing both the cell tips and minus ends near the middle of the cell. The MT bundles are organized from medial MT-organizing centers that may function as nuclear attachment sites. When MTs grow to the cell tips, they exert transient forces produced by plus end MT polymerization that push the nucleus. After an average of 1.5 min of growth at the cell tip, MT plus ends exhibit catastrophe and shrink back to the nuclear region before growing back to the cell tip. Computer modeling suggests that a balance of these pushing MT forces can provide a mechanism to position the nucleus at the middle of the cell.

Nuclear pore complexes in the organization of silent telomeric chromatin.

The functional regulation of chromatin is closely related to its spatial organization within the nucleus. In yeast, perinuclear chromatin domains constitute areas of transcriptional repression. These 'silent' domains are defined by the presence of perinuclear telomere clusters. The only protein found to be involved in the peripheral localization of telomeres is Yku70/Yku80. This conserved heterodimer can bind telomeres and functions in both repair of DNA double-strand breaks and telomere maintenance. These findings, however, do not address the underlying structural basis of perinuclear silent domains. Here we show that nuclear-pore-complex extensions formed by the conserved TPR homologues Mlp1 and Mlp2 are responsible for the structural and functional organization of perinuclear chromatin. Loss of MLP2 results in a severe deficiency in the repair of double-strand breaks. Furthermore, double deletion of MLP1 and MLP2 disrupts the clustering of perinuclear telomeres and releases telomeric gene repression. These effects are probably mediated through the interaction with Yku70. Mlp2 physically tethers Yku70 to the nuclear periphery, thus forming a link between chromatin and the nuclear envelope. We show, moreover, that this structural link is docked to nuclear-pore complexes through a cleavable nucleoporin, Nup145. We propose that, through these interactions, nuclear-pore complexes organize a nuclear subdomain that is intimately involved in the regulation of chromatin metabolism.

Overexpression of yeast karyopherin Pse1p/Kap121p stimulates the mitochondrial import of hydrophobic proteins in vivo.

During evolution, cellular processes leading to the transfer of genetic information failed to send all the mitochondrial genes into the nuclear genome. Two mitochondrial genes are still exclusively located in the mitochondrial genome of all living organisms. They code for two highly hydrophobic proteins: the apocytochrome b and the subunit I of cytochrome oxidase. Assuming that the translocation machinery could not efficiently transport long hydrophobic fragments, we searched for multicopy suppressors of this physical blockage. We demonstrated that overexpression of Pse1p/Kap121p or Kap123p, which belong to the superfamily of karyopherin beta proteins, facilitates the translocation of chimeric proteins containing several stretches of apocytochrome b fused to a reporter mitochondrial gene. The effect of PSE1/KAP121 overexpression (in which PSE1 is protein secretion enhancer 1) on mitochondrial import of the chimera is correlated with an enrichment of the corresponding transcript in cytoplasmic ribosomes associated with mitochondria. PSE1/KAP121 overexpression also improves the import of the hydrophobic protein Atm1p, an ABC transporter of the mitochondrial inner membrane. These results suggest that in vivo PSE1/KAP121 overexpression facilitates, either directly or indirectly, the co-translational import of hydrophobic proteins into mitochondria.

Functional characterization of a Nup159p-containing nuclear pore subcomplex.

Nup159p/Rat7p is an essential FG repeat-containing nucleoporin localized at the cytoplasmic face of the nuclear pore complex (NPC) and involved in poly(A)+ RNA export and NPC distribution. A detailed structural-functional analysis of this nucleoporin previously demonstrated that Nup159p is anchored within the NPC through its essential carboxyl-terminal domain. In this study, we demonstrate that Nup159p specifically interacts through this domain with both Nsp1p and Nup82p. Further analysis of the interactions within the Nup159p/Nsp1p/Nup82p subcomplex using the nup82Delta108 mutant strain revealed that a deletion within the carboxyl-terminal domain of Nup82p prevents its interaction with Nsp1p but does not affect the interaction between Nup159p and Nsp1p. Moreover, immunofluorescence analysis demonstrated that Nup159p is delocalized from the NPC in nup82Delta108 cells grown at 37 degrees C, a temperature at which the Nup82Delta108p mutant protein becomes degraded. This suggests that Nup82p may act as a docking site for a core complex composed of the repeat-containing nucleoporins Nup159p and Nsp1p. In vivo transport assays further revealed that nup82Delta108 and nup159-1/rat7-1 mutant strains have little if any defect in nuclear protein import and protein export. Together our data suggest that the poly(A)+ RNA export defect previously observed in nup82 mutant cells might be due to the loss from the NPCs of the repeat-containing nucleoporin Nup159p.

From nucleoporins to nuclear pore complexes.

One of the largest supramolecular assemblies in the eukaryotic cell, the nuclear pore complex, is now being dissected into its numerous molecular constituents. The combined use of biochemistry and genetics in yeast has made this rapid development possible. Although less is known about vertebrate nucleoporins, the first clues are now emerging about their in vivo function also. Much remains to be learned about nuclear pore complex assembly and function, however.

Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA and nuclear pores.

An essential cellular factor for nuclear mRNA export called Mex67p which has homologous proteins in human and Caenorhabditis elegans was identified through its genetic interaction with nucleoporin Nup85p. In the thermosensitive mex67-5 mutant, poly(A)+ RNA accumulates in intranuclear foci shortly after shift to the restrictive temperature, but NLS-mediated nuclear protein import is not inhibited. In vivo, Mex67p tagged with green fluorescent protein (GFP) is found at the nuclear pores, but mutant mex67-5-GFP accumulates in the cytoplasm. Upon purification of poly(A)+ RNA derived from of UV-irradiated yeast cells, Mex67p, but not nucleoporins Nup85p and Nup57p, was crosslinked to mRNA. In a two-hybrid screen, a putative RNA-binding protein with RNP consensus motifs was found to interact with the Mex67p carboxy-terminal domain. Thus, Mex67p is likely to participate directly in the export of mRNA from the nucleus to the cytoplasm.

Dynamics of nuclear pore distribution in nucleoporin mutant yeast cells.

To follow the dynamics of nuclear pore distribution in living yeast cells, we have generated fusion proteins between the green fluorescent protein (GFP) and the yeast nucleoporins Nup49p and Nup133p. In nup133- dividing cells that display a constitutive nuclear pore clustering, in vivo analysis of GFP-Nup49p localization revealed changes in the distribution of nuclear pore complex (NPC) clusters. Furthermore, upon induction of Nup133p expression in a GAL-nup133 strain, a progressive fragmentation of the NPC aggregates was observed that in turn led to a wild-type nuclear pore distribution. To try to uncouple Nup133p-induced NPC redistribution from successive nuclear divisions and nuclear pore biogenesis, we devised an assay based on the formation of heterokaryons between nup133- mutants and cells either expressing or overexpressing Nup133p. Under these conditions, the use of GFP-Nup133p and GFP-Nup49p fusion proteins revealed that Nup133p can be rapidly targeted to the clustered nuclear pores, where its amino-terminal domain is required to promote the redistribution of preexisting NPCs.

Nic96p is required for nuclear pore formation and functionally interacts with a novel nucleoporin, Nup188p.

The amino-terminal domain of Nic96p physically interacts with the Nsp1p complex which is involved in nucleocytoplasmic transport. Here we show that thermosensitive mutations mapping in the central domain of Nic96p inhibit nuclear pore formation at the nonpermissive temperature. Furthermore, the carboxyterminal domain of Nic96p functionally interacts with a novel nucleoporin Nup188p in an allele-specific fashion, and when ProtA-Nup188p was affinity purified, a fraction of Nic96p was found in physical interaction. Although NUP188 is not essential for viability, a null mutant exhibits striking abnormalities in nuclear envelope and nuclear pore morphology. We propose that Nic96p is a multivalent protein of the nuclear pore complex linked to several nuclear pore proteins via its different domains.

A novel complex of nucleoporins, which includes Sec13p and a Sec13p homolog, is essential for normal nuclear pores.

In a genetic screen for nucleoporin-interacting components, a novel nuclear pore protein Nup84p, which exhibits homology to mammalian Nup107p, was isolated. Nup84p forms a complex with five proteins, of which Nup120p, Nup85p, Sec13p, and a Sec13p homolog were identified. Upon isolation of Sec13p-ProtA, nucleoporins were still associated, but the major copurifying band was a 150 kDa protein, showing that Sec13p occurs in two complexes. Disruption of any of the genes encoding Nup84p, Nup85p, or Nup120p caused defects in nuclear membrane and nuclear pore complex organization, as well as in poly(A)+ RNA transport. Thus, the Nup84p complex in conjunction with Sec13-type proteins is required for correct nuclear pore biogenesis.

Genetic approaches to nuclear pore structure and function.

The major features of nucleocytoplasmic transport through nuclear pore complexes are conserved from yeast to humans. This transport machinery, which includes the 125 MDa nuclear pore complex structure, has been molecularly dissected in the yeast Saccharomyces cerevisiae by genetic approaches. Here, we summarize the genetic analyses used to elucidate structure-function relationships within this large supramolecular assembly.

A novel nuclear pore protein Nup133p with distinct roles in poly(A)+ RNA transport and nuclear pore distribution.

Temperature-sensitive nucleoporin nup49-316 mutant cells accumulate poly(A)+ RNA inside the nucleus when shifted to restrictive temperature. We performed a synthetic lethal screen with this mutant allele to identify further components of the mRNA export machinery. A synthetic lethal mutant slv21 was isolated, which exhibited a ts phenotype and showed nuclear accumulation of poly(A)+ RNA at 37 degrees C. The wild-type gene complementing slv21 was cloned and sequenced. It encodes a novel protein Nup133p which is located at the nuclear pore complex. NUP133 is not an essential gene, but cells in which NUP133 is disrupted grow slowly at permissive temperatures and stop growing at 37 degrees C. Concomitant with the growth inhibition, nup133- cells accumulate poly(A)+ RNA inside the nucleus whereas nuclear import of a karyophilic reporter protein is not altered. Strikingly, nup133- cells display extensive clustering of nuclear pore complexes at a few sites on the nuclear envelope. However, the nuclear pore clustering phenotype and intranuclear accumulation of poly(A)+ RNA are not obligatorily linked, since an amino-terminally truncated Nup133p allows normal poly(A)+ RNA export, but does not complement the clustering phenotype of nup133- cells.

Reconstitution of nuclear protein transport with semi-intact yeast cells.

We have developed an in vitro nuclear protein import reaction from semi-intact yeast cells. The reaction uses cells that have been permeabilized by freeze-thaw after spheroplast formation. Electron microscopic analysis and antibody-binding experiments show that the nuclear envelope remains intact but the plasma membrane is perforated. In the presence of ATP and cytosol derived from yeast or mammalian cells, a protein containing the nuclear localization sequence (NLS) of SV40 large T-antigen is transported into the nucleus. Proteins with mutant NLSs are not imported. In the absence of cytosol, binding of NLS-containing proteins occurs at the nuclear envelope. N-ethylmaleimide treatment of the cytosol as well as antibodies to the nuclear pore protein Nsp1 inhibit import but not binding to the nuclear envelope. Yeast mutants defective in nuclear protein transport were tested in the in vitro import reaction. Semi-intact cells from temperature-sensitive nsp1 mutants failed to import but some binding to the nuclear envelope was observed. On the other hand, no binding and thus no import into nuclei was observed in semi-intact nsp49 cells which are mutated in another nuclear pore protein. Np13 mutants, which are defective for nuclear protein import in vivo, were also deficient in the binding step under the in vitro conditions. Thus, the transport defect in these mutants is at the level of the nucleus and the point at which nuclear transport is blocked can be defined.

Stathmin: cellular localization of a major phosphoprotein in the adult rat and human CNS.

Stathmin is a ubiquitous, 19 kDa cytoplasmic protein the phosphorylation of which is associated with many cellular signaling pathways. It is particularly abundant in neurons and reaches a peak of expression in the neonatal period, although it remains highly expressed in the adult brain. In order to determine whether this abundant expression is associated with discrete cellular populations that are still at an immature stage during adulthood, as suggested by others, the cellular localization of stathmin was investigated in the adult rat and human central nervous system. Western blotting with a specific antiserum indicated that stathmin was ubiquitous in the brain and spinal cord but that its relative concentration varied up to 2.6 times between regions. To characterize the distribution of stathmin within the brain, its cellular localization was analyzed by immunocytochemistry. Highly immunoreactive neurons and oligodendrocytes were observed, and stathmin immunoreactivity was localized to the perikaryon and all processes, but not the nucleus. Most brain and spinal cord cell groups showed stathmin immunoreactivity, although the extent and intensity of labeling differed largely from one place to another. Particularly numerous stathmin-immunoreactive neuronal cell bodies were found in the pyriform, cingulate, and neocortex, as well as in many cholinergic nuclei of the basal forebrain and brainstem, in the medial thalamus, in various brainstem nuclei, in the dorsalmost layers of the spinal cord, and in brain areas lacking a blood-brain barrier to macromolecules. In addition to neuronal populations, stathmin-antibodies intensely labeled choroid plexuses. Many other brain regions exhibited moderate neuronal immunostaining. The distribution of stathmin-immunoreactive processes was in some areas relatively heterogeneous. Intense immunoreactivity was observed in some fiber tracts (corpus callosum, anterior commissure, inferior cerebellar peduncle, etc.) but was missing in others (internal capsule, posterior commissure, etc.). Some brain areas rich in immunoreactive neurons also displayed an intense immunoreactivity of the neuropile, whereas others contained either immunoreactive cells or fibers. In the human brain, stathmin immunostaining occurred in many areas, corresponding to those identified in the rat, with the exception of the cerebral cortex, the hippocampal fascia dentata, and the substantia nigra. The present results support our suggestion that, in addition to its involvement in cell proliferation and differentiation, stathmin may also be related to regulation of differentiated cell functions, as it appears to be a major signaling protein in widespread areas of the adult brain in both rat and human.

Purification of NSP1 reveals complex formation with 'GLFG' nucleoporins and a novel nuclear pore protein NIC96.

The essential C-terminal domain of NSP1 mediates assembly into the nuclear pore complex (NPC). To identify components which interact physically with this yeast nucleoporin, the tagged C-terminal domain of NSP1 (ProtA-NSP1) was isolated by affinity chromatography under non-denaturing conditions. The purified complex contains ProtA-NSP1, two previously identified 'GLFG' nucleoporins, NUP49 (NSP49) and p54 and a novel protein designated NIC96 (for Nucleoporin-Interacting Component of 96 kDa). Conversely, affinity purification of tagged NSP49 enriches for NSP1, the p54 and the NIC96 component. The NIC96 gene was cloned; it encodes a novel 839 amino acid protein essential for cell growth. By immunofluorescence, protein A-tagged NIC96 exhibits a punctate nuclear membrane staining indicative of nuclear pore location. Therefore, affinity purification of tagged nucleoporins has allowed the definition of a subcomplex of the NPC and analysis of physical interactions between nuclear pore proteins.

A new subclass of nucleoporins that functionally interact with nuclear pore protein NSP1.

NSP1 is a nuclear pore protein (nucleoporin) essential for cell growth. To identify the components that functionally interact with NSP1 in the living cell, we developed a genetic screen for mutants that are lethal in a genetic background of mutated, but not wild type NSP1. Fourteen synthetic lethal mutants were obtained, belonging to at least four different complementation groups. The genes of two complementation groups, NSP116 and NSP49, were cloned. Like the previously described nucleoporins, these genes encode proteins with many repeat sequences. NSP116 and NSP49, however, contain a new repetitive sequence motif 'GLFG', which classifies them as a subclass of nucleoporins. NSP116 and NSP49, tagged with the IgG binding domain of protein A and expressed in yeast, are located at the nuclear envelope. These data provide in vivo evidence that distinct subclasses of nucleoporins physically interact or share overlapping function in nuclear pore complexes.

Expression of transfected stathmin cDNA reveals novel phosphorylated forms associated with developmental and functional cell regulation.

Stathmin is a ubiquitous, highly conserved phosphoprotein, which most likely acts as an intracellular relay integrating various transduction pathways triggered by extracellular signals. Two post-translational isoforms (alpha and beta) have been previously identified whose increasingly phosphorylated forms migrate as a set of isoelectric variant spots (molecular mass 19 kDa; pI 6.2-5.6) on two-dimensional electrophoretic gels. In parallel with the phosphorylation of these forms of stathmin, two sets of three proteins migrating with slightly higher apparent molecular masses (21 and 23 kDa respectively) also incorporated radioactive phosphate in response to cell regulation through various transduction pathways. These phosphoproteins, previously referred to as proteins '16' and '17', share several biochemical properties with stathmin and are recognized by antibodies directed to stathmin or to stathmin peptides. Furthermore, when rat stathmin cDNA was transfected into mouse myogenic C2 cells, it directed the expression of protein sets 16 and 17 together with the 19 kDa forms of stathmin, as detected with a species-specific anti-stathmin antiserum. Proteins 16 and 17 are thus novel phosphorylated derivatives of stathmin, encoded by the same cDNA as its previously identified 19 kDa forms. These results increase the known complexity and diversity of stathmin patterns, which may yield the molecular support for its proposed role as a relay integrating various signals which regulate the proliferation, differentiation and functions of cells during development and adult life.

High expression of stathmin in multipotential teratocarcinoma and normal embryonic cells versus their early differentiated derivatives.

Stathmin is a ubiquitous cytoplasmic protein, phosphorylated in response to agents regulating the proliferation, the differentiation and the specialized functions of cells, in a way possibly integrating the actions of diverse concomitant regulatory signals. Its expression is also regulated in relation with cell proliferation and differentiation and reaches a peak at the neonatal stage. To assess the possible role of stathmin at earlier stages of development, we examined its expression and regulation in embryonal carcinoma (EC) and derived cell lines as well as in the early mouse embryo. Interestingly, stathmin is highly abundant in the undifferentiated, multipotential cells of the F9, 1003 and 1009 EC cell lines. Its high expression markedly decreased, both at the protein and mRNA levels, when F9 cells were induced to differentiate into endodermal-like cells with retinoic acid and dibutyryl-cAMP. Stathmin was also much less abundant in differentiated cell lines such as the trophectodermal line TDM-1, as well as in several F9- and 1003-derived cell lines committed to differentiate towards the mesodermal and neuroectodermal lineages but still proliferating. Therefore, the observed decrease of stathmin expression is not related to the reduced proliferation rate but rather to the differentiation of the multipotential EC cells. The immunocytochemical pattern of stathmin expression during early mouse development indicated that stathmin is also highly abundant in the multipotential cells of the inner cell mass of the blastula, whereas it is much lower in the differentiated trophectodermal cells. These results confirm the physiological relevance of the observations with EC cells, and suggest that stathmin, in addition to its high expression at later stages of development and in the adult nervous system, may be considered as a new marker of the multipotential cells of the early mouse embryo.