((SWY1313) strains were transformed with the plasmid expressing NLSC-galactosidase under (pNLS-E1)

((SWY1313) strains were transformed with the plasmid expressing NLSC-galactosidase under (pNLS-E1). mutant, and immunofluorescence microscopy experiments showed that the mutated Kap95p was not recycled but instead localized in the nucleus and at the nuclear envelope. Srp1p, the yeast nuclear localization signal-receptor, also accumulated in the nuclei of the arrested mutant cells. Wild-type and NES-mutated Kap95p both bound Gsp1p (the yeast Ran/TC4 homologue), Srp1p, and the FXFG repeat region of the nucleoporin Nup1p. In contrast, the NES mutation abolished Kap95p interaction with the GLFG repeat regions from the nucleoporins Nup116p and Nup100p. In vivo FMK 9a interaction was demonstrated by isolation of Kap95p from yeast nuclear lysates in either protein ACtagged Nup116p or protein ACtagged Nup100p complexes. The protein ACtagged Nup116p complex also specifically contained Gle2p. These results support a model in which a step in the recycling of Kap95p is mediated by interaction of an NES with GLFG regions. Analysis of genetic interactions suggests Nup116p has a primary role in Kap95p recycling, with Nup100p compensating in the absence of Nup116p. FMK 9a This finding highlights an important role for a subfamily of GLFG nucleoporins in nuclear export processes. Communication between the nucleus and cytoplasm is mediated by large proteinaceous structures called nuclear pore complexes (NPCs).1 Cytosolic factors, as well as NPC proteins (nucleoporins), are required to mediate signal-dependent transport into the nucleus (for reviews see Moore and Blobel, 1994; Powers and Forbes, 1994; Melchior and Gerace, 1995; Simos and Hurt, 1995; Gorlich and Mattaj, 1996). After translation, a nuclear localization signal (NLS)Cbearing protein binds in the cytoplasm to a heterodimeric complex comprised of a 60-kD NLS-receptor (importin /karyopherin /hSRP1/ Srp1p/Kap60p) (Adam and Gerace, 1991; Gorlich et al., 1994; Imamoto et al., 1995to was completed as follows: pSW271 (Iovine et al., 1995) was used as the template for sequential PCRs. The first reaction used primers 259 and 279, with the 3 279 primer incorporating base pair changes for leucine to alanine (cta to gca) at nucleotides 2,830 and 2,831 of The second reaction used primers 278 and 262, with the 5 278 primer incorporating the same two nucleotide changes. After mixing the products of these two reactions as a template, PCR was conducted with primers 259 (annealing at nucleotide 1,702) and 262 (at 4,357). The final product was digested with BamHI and ligated into pRS315 (pSW507). The AatII/NcoI fragment from pSW507 was ligated into the AatII/NcoI backbone of vector pSW503 to generate pSW509 harboring The site of the mutation was confirmed by DNA sequence analysis. A plasmid bearing the allele (pSW696) with an in-frame deletion of the sequence encoding residues 55C65 was constructed in a similar manner with the following modifications: the first reaction used primers 259 and 281 (with deletion of 30 nucleotides of sequence), the second reaction used primers 280 (with deletion of 30 nucleotides of sequence) and 262. The AatII/NcoI fragment from the NES product was ligated into the AatII/NcoI backbone of vector pSW503 to generate pSW510. Nuclear Lysates and Coimmunoprecipitation Nuclei were purified from yeast strain SWY960, SWY1381, or W303a/ cells as described by Rout and Kilmartin (1990). Lysates were prepared from 4 109 nuclei as described in Rout and Blobel (1993), and 400 l of lysate was incubated with 50 l of IgG-Sepharose (spin for 20 min, and then incubated with 2 ml of packed Ni-NTA resin (Qiagen, Chatsworth, CA) for 1 h at 4C. The resin was washed with 15 vol of sonication buffer and then with 20 vol of wash buffer (50 mM sodium phosphate, pH 6.0, 300 mM NaCl, 10% glycerol, 10 mM imidazole). The fusion protein was eluted with 20 ml MYO7A of 0.15 FMK 9a M imidazole in wash buffer. The purified 6x-His-Kap95p was transferred into binding buffer (20 mM Hepes, pH 6.8, 150 mM potassium acetate, 2 mM magnesium acetate, 2 mM DTT, 0.1% Tween-20, 0.1% casaminoacids) using a Centricon 30 (Amicon Inc., Beverly, MA) or by dialysis. The soluble binding assay was conducted as described in Rexach and Blobel (1995). -Galactosidase Assays for Two-Hybrid Interactions Yeast strain L40 (MATa, Manneheim Biochemicals, Indianapolis, IN). After washing with 40 mM K2HPO4, 10 mM KH2PO4, 150 mM NaCl, 0.1% NaN3, 0.1% Tween 20, 2% nonfat dry milk (M buffer) alone, detection of bound antibody was accomplished by incubation with either FMK 9a affinity-purified Texas redCconjugated goat FMK 9a antiCrabbit IgG (Cappel Laboratories, Organon Teknika Corp., Durham, NC) at a 1: 200 dilution, or affinity purified FITC-conjugated goat antiCmouse IgG (1: 200) for 1 h at room temperature. The final washes in M buffer and then 1% BSA-PBS were followed by mounting with 90% glycerol, 1 mg/ml (protein A) and expressed from the promoter in an null strain. The protein ACNup116p completely complemented the null phenotype, and it localized exclusively at NPCs as determined by indirect immunofluorescence microscopy (data not shown). Lysates.