Figure 7. Ssa proteins interact with a certain nucleoporin.
(A) Interaction between Ssa proteins and nucleoporins was analyzed by the pull-down assay. 200 pmol of either GST, GST-Nup100(1–640)p, GST-Nup116(165–715)p or GST-Nsp1(1–601)p were mixed with 40 pmol of either Ssa1p or Ssa2p, and were pull-downed with Glutathione Sepharose. The same portions of total (T), unbound (U), and bound (B) samples were subjected to SDS-PAGE. Ssa proteins in each fraction were detected by Western blotting with anti-Ssa protein antibodies (upper, WB), and total proteins were visualized by CBB staining (lower, CBB). Positions of GST fusions were indicated by arrowheads. (B) tRNA interaction with Nups were assayed by a variant of the low affinity binding assay. Alexa 488-labeled tRNA-ProUGG (0.50 µg) was incubated with GST- or GST-Nup116(165–715)p-coated Glutathione Sepharose in the absence (none) or presence of Ssa1p (Ssa1p) or Ssa2p (Ssa2p). Binding of the fluorescent tRNA to the beads was monitored with a fluorescence microscope.