Figure 7.

The nup57-E17 allele results in a C-terminal truncation of nup57p^E17 and temperature-dependent degradation of nup57p^E17 and GFP-Nup49p. (A) Immunoblot analysis reveals the truncated nup57p^E17 protein is unstable at 37°C. Total cell lysates prepared from wt (SWY809) and nup57-E17 (SWY1586) cells were separated on 8% SDS-polyacrylamide gels, transferred to nitrocellulose, and analyzed by immunoblotting with the indicated antibody (upper, affinity-purified polyclonal anti-GLFG; lower, polyclonal anti-GFP). The samples were prepared from cells grown at 23°C (lanes 1 and 8) or shifted to growth at 37°C for the indicated times (lanes 2–7 and 9–14). For each sample, identical cell number equivalents were loaded in each well making the protein levels directly comparable. Lysates from wt cells have similar levels of Nup116p, Nup57p, and GFP-Nup49p at all time points. In nup57-E17 cells, the levels of nup57p^E17 and GFP-Nup49p decrease with increasing time at 37°C. (B) Diagram of wt Nup57p and mutated nup57p^E17. Sequencing of the nup57-E17 allele revealed a single point mutation resulting in a stop codon after the codon for residue 515 and truncation of 26 residues from the C terminus. Numbers reflect amino acid positions within Nup57p. Vertical boxes designate individual GLFG repeats.