Conditional and specific trapping of newly synthesized nups. (A) Schematic of the “anchor-away” strategy where nup genes are genomically tagged with ORFs encoding FRB or FRB-GFP and expressed in cells containing a plasma membrane–localized anchor (Pma1-FKBP12; hook). In the presence of rapamycin, only newly synthesized/cytoplasmic nups (Nup-FRB-GFPCYT, green ovals) are sequestered, but not those bound to NPCs (Nup-FRB-GFPNPC, red ovals). (B) Yeast strains containing the indicated Nup-FRB fusions (PCCPL260, PCCPL251) with wild-type control (HHY110), and indicated plasmids were plated in 10-fold serial dilutions on YPD plates containing rapamycin or carrier (DMSO) and imaged after 48 h at 30°C. (C) Deconvolved fluorescence micrographs (with bright field) of a top and middle z section of cells expressing the indicated Nup-FRB-GFP fusions (PCCPL259, PCCPL302, CPL1238, and CPL1239) incubated for 4 h with rapamycin or with rapamycin and cycloheximide. Bars, 2 µm.
