Deciphering Networks of Protein Interactions at the Nuclear Pore Complex

Abstract

The nuclear pore complex (NPC) gates the only known conduit for molecular exchange between the nucleus and cytoplasm of eukaryotic cells. Macromolecular transport across the NPC is mediated by nucleocytoplasmic shuttling receptors termed karyopherins (Kaps). Kaps interact with NPC proteins (nucleoporins) that contain FG peptide repeats (FG Nups) and altogether carry hundreds of different cargoes across the NPC. Previously we described a biochemical strategy to identify proteins that interact with individual components of the nucleocytoplasmic transport machinery. We used bacterially expressed fusions of glutathione S-transferase with nucleoporins or karyopherins as bait to capture interacting proteins from yeast extracts. Forty-five distinct proteins were identified as binding to one or several FG Nups and Kaps. Most of the detected interactions were expected, such as Kap-Nup interactions, but others were unexpected, such as the interactions of the multisubunit Nup84p complex with several of the FG Nups. Also unexpected were the interactions of various FG Nups with the nucleoporins Nup2p and Nup133p, the Gsp1p-GTPase-activating protein Rna1p, and the mRNA-binding protein Pab1p. Here we resolve how these interactions occur. We show that Pab1p associates nonspecifically with immobilized baits via RNA.More interestingly, we demonstrate that the Nup84p complex contains Nup133p as a subunit and binds to the FG repeat regions of Nups directly via the Nup85p subunit. Binding of Nup85p to the GLFG region of Nup116p was quantified in vitro (K D ‫؍‬ 1.5 M) and was confirmed in vivo using the yeast two-hybrid assay. We also demonstrate that Nup2p and Rna1p can be tethered directly to FG Nups via the importin Kap95p-Kap60p and the exportin Crm1p, respectively. We discuss possible roles of these novel interactions in the mechanisms of nucleocytoplasmic transport.

Molecular Basis for the Rapid Dissociation of Nuclear Localization Signals from Karyopherin α in the Nucleoplasm

“…This implies that the KaRF domain of Nup2p and the Nup60p binding domain are physically separable, consistent with the notion that the Nup2p KaRF domain is exposed while Nup2p is bound to Nup60p. However, under conditions of low Gsp1p⅐GTP, Nup2p forms complexes with Kap95p monomers and Kap95p⅐Kap60p heterodimers, which cause its dissociation from Nup60p (9,26) and probably facilitate its nucleocytoplasmic shuttling (36). As discussed below, the KaRF activity of Nup2p is inhibited by Kap95p wherever the concentration of free Gsp1p-GTP is low.…”

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

“…The fact that human Seh1 may be lost during our purification processes without affecting the assembly of the other constituents of this complex, suggests that this WD nucleoporin is likely to be a peripheral constituent of the vertebrate Nup107-160 complex. Noteworthy, S. cerevisiae Nup133 was not initially identified in the ScNup84 complex, and its interaction with components of this complex could only be demonstrated in vitro or by immunoprecipitations performed after enrichment of the complex on Nup100-coated beads (Allen et al, 2002). In contrast, vertebrate Nup133 seems to be stably associated with the Nup107-160 complex.…”

“…In S. cerevisiae, this complex is formed by seven constituents: Nup84; Nup85; Nup120; the in vivo-cleaved carboxyterminal domain of Nup145 (Nup145-C); Seh1; a fraction of Sec13; and Nup133 (Siniossoglou et al, 1996;Teixeira et al, 1997;Allen et al, 2002;Lutzmann et al, 2002). 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).…”

Arx1 Is a Nuclear Export Receptor for the 60S Ribosomal Subunit in Yeast

“…The cleaved protein was then passed over a glutathione Sepharose column equilibrated in 50 mM Tris⅐HCl, 50 mM NaCl, and 10% glycerol to remove glutathione S-transferase (GST) and uncleaved GST-Arx1. Binding of Arx1 to GST-Nups was essentially as described in Allen et al (2002).…”