The GLFG repetitive region of the nucleoporin Nup116p interacts with Kap95p, an essential yeast nuclear import factor.

Abstract

Abstract. Nup116p is a member of a family of five yeast nuclear pore complex (NPC) proteins that share an amino terminal region of repetitive tetrapeptide "GLFG" motifs. Previous experiments characterized the unique morphological perturbations that occur in a nup116 null mutant: temperature-sensitive formation of nuclear envelope seals over the cytoplasmic face of the NPC (Wente, S. R., and G. Blobel. 1993. J. Cell Biol. 123:275-284). Three approaches have been taken to dissect the structural basis for Nupll6p's role in NPC function. First, deletion mutagenesis analysis of NUPll6 revealed that the GLFG region was required for NPC function. This was not true for the other four yeast GLFG family members (Nup49p, Nup57p, Nupl00p, and Nup145p). Moreover, deletion of either half of Nup116p's GLFG repeats or replacement of Nupll6p's GLFG region with either Nupl00p's GLFG region or Nsplp's FXFG repetitive region abolishes the function of Nupl16p. At a semipermissive growth temperature, the cells lacking Nupl16p's GLFG region displayed a diminished capacity for nuclear import. Second, overexpression of Nup116p's GLFG region severely inhibited cell growth, rapidly blocked polyadenylated-RNA export, and fragmented the nucleolus. Although it inhibited nuclear export, the overexpressed GLFG region appeared predominantly localized in the cytoplasm and NPC/nuclear envelope structure was not perturbed in thin section electron micrographs. Finally, using biochemical and two-hybrid analysis, an interaction was characterized between Nupl16p's GLFG region and Kap95p, an essential yeast homologue of the vertebrate nuclear import factor p97/Imp90/karopherin [3. These data show that Nup116p's GLFG region has an essential role in mediating nuclear transport.

GLE2, a Saccharomyces cerevisiae homologue of the Schizosaccharomyces pombe export factor RAE1, is required for nuclear pore complex structure and function.

“…At short fixation times, the Gle2p-HA was concentrated in a punctate pattern at the nuclear periphery (Figure 3). This is typical of yeast nuclear pore complexes (Davis and Fink, 1990 (Figure 4) with samples of both total yeast cell lysate and enriched NPCs was divided in half and probed with either the anti-Gle2p antibody or an affinitypurified polyclonal antibody recognizing Nupil6p (Iovine et al, 1995). Gle2p migrated with an apparent molecular mass of 40 kDa and was present in the enriched NPC fraction.…”

“…Protein samples were separated by electrophoresis in a 9% SDS-polyacrylamide gel and transferred electrophoretically to nitrocellulose membranes. Blots were probed with affinity-purified rabbit polyclonal anti-Nupll6p carboxyl-terminal antibody (Iovine et al, 1995) at a 1:2500 dilution (1 h at room temperature) or affinity-purified rabbit polyclonal anti-Gle2p antibody at a 1:25 dilution (2 h at room temperature). All dilutions were made in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween 20 (TBST)/2% milk.…”

“…Strains were tested for ,3-galactosidase expression levels using the filter assay described in the study of Breeden and Nasmyth (1985). Plasmids tested for Gle2p interactions include pSW365 (full-length Srplp in LexABD; gift from K. Mishra), pSW310 (full-length Kap95p in LexABD; Iovine et al, 1995), pSW262 (the GLFG region of Nupll6p in Gal4BD; Iovine et al, 1995), pSW546…”

Importin 7 and Importin α/Importin β Are Nuclear Import Receptors for the Glucocorticoid Receptor

“…The localization of GR-GFP was monitored in 14 yeast strains each deficient in a different nuclear transport receptor: CRMI (Stade et al, 1997), CSEI (Xiao et al, 1993), Kap95 (Iovine et al, 1995), Kap104 (Aitchison et al, 1996), Kap123 (Seedorf and Silver, 1997), LOS1 (Hopper et al, 1980), MSN5 (Kaffman et al, 1998a), MTR10 (Kadowaki et al, 1994), NMD5 (He and Jacobson, 1995), PSEI (Seedorf and Silver, 1997), SRP1 (Yano et al, 1992), SXM1 (Seedorf and Silver, 1997), Kap114 (Morehouse et al, 1999;Pemberton et al, 1999), and PDR6 (Titov and Blobel, 1999). Yeast strains PSY580, pse1-1, PSY1200, and PSY902 have been described (Seedorf and Silver, 1997).…”

Protein import into the nucleus

“…Similarly, incubation of immobilized GLFG repeats of Nup98 with a cytosolic subfraction led to depletion of the binding activity [47]; NRI3 was identified as one protein bound to these repeats [12]. Binding of the NLS-receptor to some but not all FG nucleoporins has also been reported by others [20,32,45,[48][49][50] and required only the repeat domains [32,45,49,50].…”