A novel pre-mRNP retention factor at NPC Abstract Using a genetic screen, we have identified a previously uncharacterized S. cerevisiae ORF (renamed PML39) that displays a specific interaction with nucleoporins of the Nup84 complex. Localization of a Pml39-GFP fusion and two-hybrid studies revealed that Pml39 is mainly docked to a subset of nuclear pore complexes opposite to the nucleolus through interactions with Mlp1 and Mlp2. The absence of Pml39 leads to a specific leakage of unspliced mRNAs that is not enhanced upon MLP1 deletion. In addition, overexpression of PML39-GFP induces a specific trapping of mRNAs transcribed from an intron-containing reporter and of the hnRNP Nab2 within discrete nuclear domains. In a nup60∆ mutant, Pml39 is mislocalized together with Mlp1 and Mlp2 in intranuclear foci that also recruit Nab2. Moreover, pml39∆ partially rescues the thermosensitive phenotypes of mRNP assembly mutants, indicating that PML39 deletion also bypasses the requirement for normally assembled mRNPs. Altogether, these data indicate that Pml39 is an upstream effector of the Mlps, involved in the retention of improper mRNPs in the nucleus prior to their export.

Using a genetic screen, we have identified a previously uncharacterized Saccharomyces cerevisiae open reading frame (renamed PML39) that displays a specific interaction with nucleoporins of the Nup84 complex. Localization of a Pml39-green fluorescent protein (GFP) fusion and two-hybrid studies revealed that Pml39 is mainly docked to a subset of nuclear pore complexes opposite to the nucleolus through interactions with Mlp1 and Mlp2. The absence of Pml39 leads to a specific leakage of unspliced mRNAs that is not enhanced upon MLP1 deletion. In addition, overexpression of PML39-GFP induces a specific trapping of mRNAs transcribed from an intron-containing reporter and of the heterogenous nuclear ribonucleoprotein Nab2 within discrete nuclear domains. In a nup60delta mutant, Pml39 is mislocalized together with Mlp1 and Mlp2 in intranuclear foci that also recruit Nab2. Moreover, pml39delta partially rescues the thermosensitive phenotypes of messenger ribonucleoparticles (mRNPs) assembly mutants, indicating that PML39 deletion also bypasses the requirement for normally assembled mRNPs. Together, these data indicate that Pml39 is an upstream effector of the Mlps, involved in the retention of improper mRNPs in the nucleus before their export.

The yeast RAD27 gene encodes a functional homolog of the mammalian FEN1 protein, a structure-specific endo/exonuclease which plays an important role in DNA replication and repair. Previous genetic interaction studies, including a synthetic genetic array (SGA) analysis, showed that the survival of rad27Δ cells requires several DNA metabolic processes, in particular those mediated by all members of the Rad52-dependent recombinational repair pathway. Here, we report the results of our SGA analysis of the collection of non-essential yeast genes against the rad27Δ mutation, which resulted in the identification of a novel synthetic lethal interaction conferred by mutations affecting the Nup84 nuclear pore subcomplex (nup133Δ, nup120Δ and nup84Δ). Additional screens showed that all Rad52 group genes are required for the survival of the nup133Δ and nup120Δ mutants, which are defective in nuclear pore distribution and mRNA export, but not of the nup133ΔN mutant, which is solely defective in pore distribution. This requirement for the DNA double-strand break (DSB) repair pathway is consistent with the observation that, like rad27Δ, the nup133Δ, nup120Δ and nup84Δ mutants are sensitive to methyl methanesulfonate (MMS). Furthermore, nup133Δ cells exhibit an increased number of spontaneous DNA repair foci containing Rad52. Altogether, these data suggest that the pathological interactions between the rad27Δ and specific nupΔ mutations result from the accumulation of unrepaired DNA damages.

TheDrosophila(fruit fly) model system has been instrumental in our current understanding of human biology, development, and diseases. Here, we used a high-throughput yeast two-hybrid (Y2H)-based technology to screen 102 bait proteins fromDrosophila melanogaster, most of them orthologous to human cancer-related and/or signaling proteins, against high-complexity fly cDNA libraries. More than 2300 protein-protein interactions (PPI) were identified, of which 710 are of high confidence. The computation of a reliability score for each protein-protein interaction and the systematic identification of the interacting domain combined with a prediction of structural/functional motifs allow the elaboration of known complexes and the identification of new ones. The full data set can be visualized using a graphical Web interface, the PIMRider (http://pim.hybrigenics.com), and is also accessible in the PSI standard Molecular Interaction data format. Our fly Protein Interaction Map (PIM) is surprisingly different from the one recently proposed by Giot et al. with little overlap between the two data sets. Analysis of the differences in data sets and methods suggests alternative strategies to enhance the accuracy and comprehensiveness of the post-genomic generation of broad-scale protein interaction maps.

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 (a and /) have been previously identified whose increasingly phosphorylated forms migrate as a set of isoelectric variant spots (molecular mass 19 kDa; pl 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.

In eukaryotes, bidirectional transport of macromolecules between the cytoplasm and the nucleus occurs through elaborate supramolecular structures embedded in the nuclear envelope, the nuclear pore complexes (NPCs). NPCs are composed of multiple copies of approximately 30 different proteins termed nucleoporins, of which several can be biochemically isolated as subcomplexes. One such building block of the NPC, termed the Nup107-160 complex in vertebrates, was so far demonstrated to be composed of six different nucleoporins. Here, we identify three WD (Trp-Asp)-repeat nucleoporins as new members of this complex, two of which, Nup37 and Nup43, are specific to higher eukaryotes. The third new member Seh1 is more loosely associated with the Nup107-160 complex biochemically, but its depletion by RNA interference leads to phenotypes similar to knock down of other constituents of this complex. By combining green fluorescent protein-tagged nucleoporins and specific antibodies, we show that all the constituents of this complex, including Nup37, Nup43, Seh1, and Sec13, are targeted to kinetochores from prophase to anaphase of mitosis. Together, our results indicate that the entire Nup107-160 complex, which comprises nearly one-third of the so-far identified nucleoporins, specifically localizes to kinetochores in mitosis.

Until now, regulation of nucleocytoplasmic transport of macromolecules has been thought to occur mainly through modifications of the cargo molecules. However, in the December 26 issue of Cell, Makhnevych et al. describe a novel mechanism controlling cell cycle progression in yeast that involves subtle molecular rearrangements within the nuclear pore complexes.

We have characterized Schizosaccharomyces pombe open reading frames encoding potential orthologues of constituents of the evolutionarily conserved Saccharomyces cerevisiae Nup84 vertebrate Nup107-160 nuclear pore subcomplex, namely Nup133a, Nup133b, Nup120, Nup107, Nup85, and Seh1. In spite of rather weak sequence conservation, in vivo analyses demonstrated that these S. pombe proteins are localized at the nuclear envelope. Biochemical data confirmed the organization of these nucleoporins within conserved complexes. Although examination of the S. cerevisiae and S. pombe deletion mutants revealed different viability phenotypes, functional studies indicated that the involvement of this complex in nuclear pore distribution and mRNA export has been conserved between these highly divergent yeasts. Unexpectedly, microscopic analyses of some of the S. pombe mutants revealed cell division defects at the restrictive temperature (abnormal septa and mitotic spindles and chromosome missegregation) that were reminiscent of defects occurring in several S. pombe GTPase Ran (Ran(Sp))/Spi1 cycle mutants. Furthermore, deletion of nup120 moderately altered the nuclear location of Ran(Sp)/Spi1, whereas overexpression of a nonfunctional Ran(Sp)/Spi1-GFP allele was specifically toxic in the Deltanup120 and Deltanup133b mutant strains, indicating a functional and genetic link between constituents of the S. pombe Nup107-120 complex and of the Ran(Sp)/Spi1 pathway.

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.

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.

Previous articleNext article No AccessJournal ArticleMicrotubule-dependent nuclear positioning and nuclear-dependent septum positioning in the fission yeast Saccharomyces pombePT Tran, V Doye, F Chang, and S InouePT Tran, V Doye, F Chang, and S InoueCorrections to this articleErratumPDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by The Biological Bulletin Volume 199, Number 2October 2000 Published in association with the Marine Biological Laboratory Article DOIhttps://doi.org/10.2307/1542900 Views: 25Total views on this site Copyright © 2000 by Marine Biological LaboratoryPDF download Crossref reports no articles citing this article.Related articlesErratum28 Mar 2017The Biological Bulletin

Summary The nuclear-pore complex (NPC), which consists of ca. 50 proteins called nucleoporins, is a huge macromolecular structure that spans the nuclear envelope and is an obligatory passage for molecules in transit between the cytoplasm and the nucleus. In the last years, major progress has allowed the characterization of the so-called “soluble phase’ of nucleocytoplasmic transport, that involves transport substrates, import and export receptors of which some belong to the karyopherin-β family, and the small GTPase Ran and its modulators. In addition, the knowledge of the NPC architecture, the identification of its constituents, and the determination of the hierarchy of interactions within the pore should help to understand how nucleoporins are assembled, and how they give rise to a functional NPC through interactions with specific transport factors. In this review, we will focus on recent insights into the stationary phase of nucleocytoplasmic transport (i.e., the NPCs) that have been gained from exploiting the benefits of several organisms, such as Xenopus laevis oocytes, mammalian cell lines, and the yeast Saccharomyces cerevisiae .

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 β 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.

Biology of the CellVolume 90, Issue 1 p. 112-112 The yeast importin/karyopherin-β PSE1P facilitates the mitochondrial import of hydrophobic proteins M. Corral-Debrinski, M. Corral-Debrinski Coole PJormale Supbrieure, Laboratoire de Génétique Moleéculaire URA CNRS 1302 46, Rue d'ULm 75230 Paris Cedex 05 France.Search for more papers by this authorN. Belgareh, N. Belgareh Institut Curie, CNRS UMR 144 26 Rue d'Ulm 75248 Paris Cedex 05 FranceSearch for more papers by this authorC. Blugeon, C. Blugeon Coole PJormale Supbrieure, Laboratoire de Génétique Moleéculaire URA CNRS 1302 46, Rue d'ULm 75230 Paris Cedex 05 France.Search for more papers by this authorM.G. Claros, M.G. Claros Department de Bioquimica y Biolagla Molecular facltad de Ciencias e lnstituto Andaluz de Biotecnologia, Universidad de Malaga E-29071 Malaga-Spain.Search for more papers by this authorV. Doye, V. Doye Institut Curie, CNRS UMR 144 26 Rue d'Ulm 75248 Paris Cedex 05 FranceSearch for more papers by this authorC. Jacq, C. Jacq Coole PJormale Supbrieure, Laboratoire de Génétique Moleéculaire URA CNRS 1302 46, Rue d'ULm 75230 Paris Cedex 05 France.Search for more papers by this author M. Corral-Debrinski, M. Corral-Debrinski Coole PJormale Supbrieure, Laboratoire de Génétique Moleéculaire URA CNRS 1302 46, Rue d'ULm 75230 Paris Cedex 05 France.Search for more papers by this authorN. Belgareh, N. Belgareh Institut Curie, CNRS UMR 144 26 Rue d'Ulm 75248 Paris Cedex 05 FranceSearch for more papers by this authorC. Blugeon, C. Blugeon Coole PJormale Supbrieure, Laboratoire de Génétique Moleéculaire URA CNRS 1302 46, Rue d'ULm 75230 Paris Cedex 05 France.Search for more papers by this authorM.G. Claros, M.G. Claros Department de Bioquimica y Biolagla Molecular facltad de Ciencias e lnstituto Andaluz de Biotecnologia, Universidad de Malaga E-29071 Malaga-Spain.Search for more papers by this authorV. Doye, V. Doye Institut Curie, CNRS UMR 144 26 Rue d'Ulm 75248 Paris Cedex 05 FranceSearch for more papers by this authorC. Jacq, C. Jacq Coole PJormale Supbrieure, Laboratoire de Génétique Moleéculaire URA CNRS 1302 46, Rue d'ULm 75230 Paris Cedex 05 France.Search for more papers by this author First published: 01 February 2012 https://doi.org/10.1016/S0248-4900(98)80261-XAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume90, Issue1January 1998Pages 112-112 RelatedInformation