Figure 3.

dbp5^RR mutant does not bind Nup159 and displays no in vivo growth defects. (A) Amino acid residue alignment for a portion of S. cerevisiae Dbp5 and Homo sapiens hDBP5. Black stars designate conserved arginine residues that were changed to aspartic acid residues (R256D and R259D) in Dbp5 to yield dbp5^RR. The open white star represents a conserved glutamate residue that was changed to glutamine (E240Q) to yield dbp5^EQ. Conserved DEAD-box family sequence motifs are boxed and identified below alignment. (B) Cytoplasmic mislocalization of GFP-dbp5^RR by live-cell microscopy: S. cerevisiae strains with DBP5 chromosomally deleted, expressing either GFP-DBP5 or GFP-dbp5^RR on plasmids, were used in live-cell fluorescent microscopy experiments to visualize subcellular localization of each protein. (Left) DIC image. (Right) GFP. Western blot analysis of yeast whole-cell extracts was conducted to test for relatively equal levels of expression of GFP-Dbp5 and GFP-dbp5^RR. Nup116 protein level was used as a loading control. (*) Degradation products of Nup116. (C) Nup159-NTD interaction in vitro with dbp5^RR is not detected: Soluble binding assays were conducted with 200 pmol of bacterially expressed recombinant His₆-Nup159-NTD and immobilized on Ni-NTA agarose resin and either recombinant Dbp5, dbp5^RR, or dbp5^EQ. Input and bound fractions were analyzed by SDS-PAGE and Coomassie staining. (*) Nup159-NTD degradation product. (D) Swapping the salt bridge interaction allows nup159^DD and dbp5^RR interaction: Soluble binding assays were conducted as in C with His₆-Nup159-NTD, His₆-nup159^VI-NTD, His₆-nup159^DD-NTD, Dbp5, or dbp5^RR. (E) dbp5^RR strain does not have growth defects: S. cerevisiae strains with DBP5 chromosomally deleted, expressing either wild-type DBP5 or dbp5^RR, were used to perform serial dilution growth assays at 23°C, 30°C, and 37°C.