FIGURE 7.

GST pulldown assays of the interaction of Kap60 and Kap95 with Hxk2 in the absence of functional Kap95 and Kap60 proteins, respectively. The GST-Kap95 and GST-Kap60 fusion proteins were purified on glutathione-Sepharose columns. a, equal amounts of GST-Kap95 were incubated with cell extracts from wild-type, JCY1410 (kap60^ts), and FMY306 (kap60^tsHxk2nls1(Ala)) strains. b, equal amounts of GST-Kap60 were incubated with cell extracts from wild-type and JCY1407 (kap95^ts) strains. The yeast strains were grown in YEPD media until an A_{600 nm} of 0.8 was reached and then shifted to low (L-Glc) glucose conditions for 1 h. The wild-type strain was grown at 28 °C and the mutant cells were grown at 25 °C (permissive temperature) and then shifted to 37 °C (nonpermissive temperature) for 5 h. After exhaustive washing the proteins were separated by 12% SDS-PAGE, and the retained Hxk2 variants were visualized on a Western blot with polyclonal anti-Hxk2 antibody. For the control samples, GST protein was also incubated with the high-glucose (H-Glc) cell extracts, but no signals were detected. The level of Hxk2 present in the different extracts used in a and b was determined by Western blot using anti-Hxk2 antibody. The Western blots shown are representative of results obtained from four independent experiments.