FIGURE 7.

Conserved residues within the N‐terminus of Nup2 are required for proper Nup2 function in vivo and for binding to importin‐α in vitro. A, Nup2 variant protein function in vivo was assessed by a plasmid shuffle assay ⁴² as described in Section 4. ΔNUP2/ΔNUP133 cells (ACY1552) maintained by a plasmid encoding wild‐type Nup2 and expressing wild‐type Nup2, Δ50 Nup2, or mutant Nup2 were spotted onto 5‐FOA plates and grown at 25°C. Cells from the 5‐FOA plates were grown to saturation at 25°C, serially diluted, spotted onto selective plates, and grown at 30°C. B, In vitro binding between variants of Nup2‐GFP and importin‐α‐myc was assessed by co‐immunoprecipitation as described in Section 4. The unbound (U) and bound (B) fractions were probed with α‐GFP antibodies to detect the Nup2‐GFP and with α‐myc antibodies to detect the importin‐α‐myc. C, ΔNUP2 cells transformed with wild‐type Nup2, Δ50 Nup2, or one of the Nup2 variants and expressing integrated importin‐α‐GFP were examined by direct fluorescence microscopy (imp‐α‐GFP). Corresponding DIC images are shown. Scale bar is 5 μm