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

Abstract

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.

The Entire Nup107-160 Complex, Including Three New Members, Is Targeted as One Entity to Kinetochores in Mitosis

“…Serums were depleted of GST antibodies and affinity purified using GST-Nup85, GST-Nup37 or a mixture of GST-Nup160-N and GSTNup160-C immobilized on a NHS-activated columns. The others antibodies used were affinity-purified rabbit polyclonal antibodies directed against human Nup133 and Nup107 (Belgareh et al, 2001), Nup96/p87 serum affinitypurified by using recombinant Nup96/p87 (residues 1291-1482 of Nup196) immobilized on nitrocellulose (Fontoura et al, 1999), affinity-purified antiNup96 (directed against a synthetic peptide corresponding to aa 1743-1763 of Nup196) (Hase and Cordes, 2003), affinity-purified anti-Sec13 (Tang et al, 1997) and Sec31 (Tang et al, 2000), anti-Tpr (Kuznetsov et al, 2002), or anti-myc epitope (Euromedex, Souffelweyersheim, France); monoclonal antibodies anti-myc (clone 9E10; Sigma-Aldrich, St. Louis, MO), anti-GFP (clone 7.1 and 13.1; Roche Diagnostics, Indianapolis, IN), anti-SC-35 (Sigma-Aldrich), anti-cyclin B1 (Santa Cruz Biotechnology), anti-mitosin (BD Biosciences, San Jose, CA), and mAb414 (Babco, Richmond, CA); the autoimmune CREST serum was obtained from J.C. Courvalin (Institut J. Monod, Paris, France). To detect Giantin, the TA10 scFv (Nizak et al, 2003), obtained from F. Perez (Institut Curie, Paris, France), was used at 1/100 and mixed with a goat anti-myc antibody (Santa Cruz Biotechnology).…”

“…Immunofluorescence on paraformaldehyde (PFA) fixed cells was performed essentially as described previously (Belgareh et al, 2001). In the course of this study, we noticed that the use of a slightly basic PFA was critical for a clear detection of Nup96 at both the NPCs and kinetochores.…”

“…RNA interference approaches in Caenorhabditis elegans early embryo and HeLa cells further revealed the involvement of RanBP2/ Nup358 in chromosome congression and segregation, stable kinetochore-microtubule association, and kinetochore assembly (Askjaer et al, 2002;Salina et al, 2003;Joseph et al, 2004). Finally, a fraction of human Nup133, Nup107, and Nup85/75 was shown to associate with kinetochores in mitosis (Belgareh et al, 2001;Harel et al, 2003).…”

“…Constituents of this complex are localized on both sides of the NPCs in yeast (Rout et al, 2000) and vertebrates (Belgareh et al, 2001;Enninga et al, 2003). This complex was shown to be involved in NPC distribution in S. cerevisiae, and in mRNA export both in yeast (reviewed in Doye and Hurt, 1997) and vertebrates (Vasu et al, 2001;Boehmer et al, 2003;Walther et al, 2003a).…”

“…Isolation under native conditions and in vitro reconstitution have further revealed the Y-shaped structure of this complex (Siniossoglou et al, 2000;Lutzmann et al, 2002). The vertebrate Nup107-160 complex was so far demonstrated to consist of Nup107 (homologous to ScNup84), Nup133 (homologous to ScNup133), Nup160 (homologous to ScNup120 and thus also referred in human cells as Nup120), Nup96 (homologous to ScNup145-C), Sec13 and Nup85/75 (Fontoura et al, 1999;Belgareh et al, 2001;Vasu et al, 2001;Harel et al, 2003). In addition, previous studies reported the cofractionation on sucrose gradients of Nup107, Nup96, and Sec13 together with a 37-kDa WD-repeat containing protein (Fontoura et al, 1999), subsequently identified as Nup37 (Cronshaw et al, 2002).…”