The GLFG Regions of Nup116p and Nup100p Serve as Binding Sites for Both Kap95p and Mex67p at the Nuclear Pore Complex

Abstract

Our previous studies have focused on a family of Saccharomyces cerevisiae nuclear pore complex (NPC) proteins that contain domains composed of repetitive tetrapeptide glycine-leucine-phenylalanine-glycine (GLFG) motifs. We have previously shown that the GLFG regions of Nup116p and Nup100p directly bind the karyopherin transport factor Kap95p during nuclear protein import. In this report, we have further investigated potential roles for the GLFG region in mRNA export. The subcellular localizations of green fluorescent protein (GFP)-tagged mRNA transport factors were individually examined in yeast cells overexpressing the Nup116-GLFG region. The essential mRNA export factors Mex67-GFP, Mtr2-GFP, and Dbp5-GFP accumulated in the nucleus. In contrast, the localizations of Gle1-GFP and Gle2-GFP remained predominantly associated with the NPC, as in wild type cells. The localization of Kap95p was also not perturbed with GLFG overexpression. Coimmunoprecipitation experiments from yeast cell lysates resulted in the isolation of a Mex67p-Nup116p complex. Soluble binding assays with bacterially expressed recombinant proteins confirmed a direct interaction between Mex67p and the Nup116-GLFG or Nup100-GLFG regions. Mtr2p was not required for in vitro binding of Mex67p to the GLFG region. To map the Nup116-GLFG subregion(s) required for Kap95p and/or Mex67p association, yeast two-hybrid analysis was used. Of the 33 Nup116-GLFG repeats that compose the domain, a central subregion of nine GLFG repeats was sufficient for binding either Kap95p or Mex67p. Interestingly, the first 12 repeats from the fulllength region only had a positive interaction with Mex67p, whereas the last 12 were only positive with Kap95p. Thus, the GLFG domain may have the capacity to bind both karyopherins and an mRNA export factor simultaneously. Taken together, our in vivo and in vitro results define an essential role for a direct Mex67p-GLFG interaction during mRNA export.To move between the nuclear and cytoplasmic compartments of a eukaryotic cell, all molecules must pass through nuclear pore complexes (NPCs) 1 embedded in the nuclear envelope.Ions, metabolites, and small proteins may diffuse through an ϳ9-nm aqueous channel in the NPC. In contrast, the movement of large macromolecules, including proteins and RNA, is energy-dependent and facilitated (reviewed in Refs. 1-3). The central channel of the proteinaceous NPC is formed by a symmetrical, 8-fold assembly of spoke-like structures sandwiched between nuclear and cytoplasmic rings. Distinct filamentous structures extend from these rings on both the nuclear and cytoplasmic faces, with the filaments on the nuclear side culminating in a basket-like structure. Overall, a vertebrate NPC measures ϳ200 nm from the tips of the cytoplasmic filaments to the base of the nucleoplasmic basket (4, 5). Unraveling the mechanism for active transport between the cytoplasmic and nucleoplasmic NPC faces will require an in-depth understanding of both the NPC itself and soluble transport factors. The Saccharomyces cerev...

Nuclear Export of the Yeast mRNA-binding Protein Nab2 Is Linked to a Direct Interaction with Gfd1 and to Gle1 Function

“…2, lane 3). Our previous studies have shown that the anti-GFP antibodies are specific only for the GFP-tagged proteins (29). Taken together, these results strongly suggest that Gfd1 and Nab2 associate in a complex in vivo.…”

“…The bound protein was eluted with column buffer containing 10 mM maltose. MBP-Gle2 (kindly provided by L. Strawn) was purified as described previously (29).…”

“…Mex67 forms a heterodimer with another export factor, Mtr2, binds the hnRNP-like factor Yra1, and interacts with FG (phenylalanine, glycine) repeats in several Nups (27)(28)(29). Func-tional associations have also been established between Mex67 and the TREX complex through Yra1 and the splicing factor Sub2 (30 -32).…”

Mutations in Tap Uncouple RNA Export Activity from Translocation through the Nuclear Pore Complex

“…Additional evidence that transport receptors rely on distinct FG repeats and/or nucleoporins comes from yeast where mutations or over expression of individual nucleoporins can affect the translocation of particular transport receptors without affecting the movement of others (Bastos et al, 1996;Belgareh et al, 1998;Balasundaram et al, 1999). Mex67p, the Tap homologue in Saccharomyces cerevisiae, and Kap95p bind distinct repeats within Nup116 (Strawn et al, 2001). Moreover, the interaction between Tap and Nup98 is confined to a subset of the GLFG repeats in Nup98 (Blevins et al, 2003).…”

“…It is estimated that more than 3500 FG repeats are distributed throughout each NPC (Strawn et al, 2004). Recent evidence suggests that transport receptors interact with distinct subsets of FG repeats (Clarkson et al, 1997;Damelin and Silver, 2000;Strawn et al, 2001;Blevins et al, 2003;Strawn et al, 2004). This could provide part of the basis for coordinating bidirectional flow of traffic through the NPC, which is estimated to approach 1000 molecules per second (Ribbeck and Gorlich, 2001).…”