Figure 8.

Nup100p–GLFG may also mediate Kap95p recycling. (A) Synthetic lethal interactions between nup100ΔGLFG and nup 145Δ GLFG alleles. The heterozygous nup100ΔGLFG/NUP100 nup145ΔGLFG/NUP145 strain SWY585 was sporulated and dissected. The nup100ΔGLFG and nup145Δ GLFG alleles cosegregated and resulted in the viable haploid nup100ΔGLFG/nup145Δ GLFG strain SWY588. To demonstrate viability of cells, the double mutant haploid (SWY588), wild-type (W303), and respective single mutant parental strains (nup100ΔGLFG, SWY583; nup145Δ GLFG, SWY581) were grown on YPD at 30°C for 2 d. (B) Synthetic lethal interactions between nup116ΔGLFG, nup100Δ GLFG, and nup145ΔGLFG mutants. Strains containing the respective double ΔGLFG mutation combinations are shown on YPD and 5-FOA at 23°C. Strains harboring double deletions of the GLFG regions of NUP116, NUP100, and NUP145 were obtained by dissecting the appropriate diploid strains (see Table I). Confirmation of the nup116ΔGLFG chromosome segregation was determined by immunoblotting with an anti116 carboxy-terminal antibody (Iovine et al., 1995). On the YPD plate, the nup116Δ GLFG (SWY1407), nup100ΔGLFG/nup116ΔGLFG (SWY1406), and the nup145ΔGLFG/nup116ΔGLFG (SWY1429) cells also carry pSW131 (NUP116/URA3); the nup100ΔGLFG (SWY1401) cells also carry pSW132 (NUP100/URA3); and the nup145ΔGLFG (SWY656) cells also carry pSW190. (C) Protein A–Nup100p coimmunoprecipitates Kap95p from isolated nuclei. Nuclear lysates were prepared from SWY1381 and processed as described for Fig. 1. Immunoblots of the eluate fraction were incubated with either the anti-Kap95p antibody or an anti-IgG antibody. The latter antibody was used to detect the position of protein A–Nup100p (uppermost band) and any of its proteolytic fragments (asterisk).