Nup2p Dynamically Associates with the Distal Regions of the Yeast Nuclear Pore Complex

Abstract

Nucleocytoplasmic transport is mediated by the interplay between soluble transport factors and nucleoporins resident within the nuclear pore complex (NPC). Understanding this process demands knowledge of components of both the soluble and stationary phases and the interface between them. Here, we provide evidence that Nup2p, previously considered to be a typical yeast nucleoporin that binds import- and export-bound karyopherins, dynamically associates with the NPC in a Ran-facilitated manner. When bound to the NPC, Nup2p associates with regions corresponding to the nuclear basket and cytoplasmic fibrils. On the nucleoplasmic face, where the Ran–GTP levels are predicted to be high, Nup2p binds to Nup60p. Deletion of NUP60 renders Nup2p nucleoplasmic and compromises Nup2p-mediated recycling of Kap60p/Srp1p. Depletion of Ran–GTP by metabolic poisoning, disruption of the Ran cycle, or in vitro by cell lysis, results in a shift of Nup2p from the nucleoplasm to the cytoplasmic face of the NPC. This mobility of Nup2p was also detected using heterokaryons where, unlike nucleoporins, Nup2p was observed to move from one nucleus to the other. Together, our data support a model in which Nup2p movement facilitates the transition between the import and export phases of nucleocytoplasmic transport.

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

“…While bound to the nuclear basket of the NPC, however, it is likely that Nup2p is active as a KaRF. For instance, a Nup2p mutant (Nup2p ⌬aa1-50) that cannot bind Kap60p (and therefore lacks KaRF activity) binds normally to Nup60p and their auxiliary "bridging factor" Gsp1p⅐GTP (9,26). 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.…”

“…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.…”

“…Nup2p is normally tethered to the nuclear basket of the yeast NPC via Nup60p, but it becomes mobile (i.e. shuttles between the nucleoplasm and cytoplasm) when the cellular concentration of Gsp1p⅐GTP is low (9,26). While bound to the nuclear basket of the NPC, however, it is likely that Nup2p is active as a KaRF.…”

Yeast nuclear pore complexes have a cytoplasmic ring and internal filaments

“…Therefore, the methods could be biased against structural components that are variable between NPCs or which are flexible and hence do not occupy a clearly defined position. Such structural components would be predicted from the protein composition of the NPC, which includes several proteins which appear to be "mobile" in vertebrates (Nakielny et al, 1999;Griffis et al, 2002) and in yeast (Dilworth et al, 2001;Denning et al, 2001). It is also possible that isolation of NPCs, necessary for these structural studies could disrupt particularly peripheral components.…”

Feedback control of stochastic noise in the yeast galactose utilization pathway

“…To do this, the gene encoding GFP+ followed by the Schizosaccharomyces pombe gene HIS5, was amplified from pGFP+/HIS5, generated by replacing EGFP in pGFP/HIS5 [42] with GFP+ [43]. The GAL1 promoter consisting of nucleotides -12 to -690 was amplified from genomic DNA and the two PCR products were fused by PCR using flanking oligomers containing ~60 bp sequences homologous to HIS3.…”