Figure 3. Analysis of minimal Gle1-binding domain in Nup42.

(A) nup42^408–424 is required for interaction with Gle1. Indicated plasmids were transformed into the Y2H reporter strain, and struck to –Trp –Leu –His –Ade synthetic media for growth at 23°C. Growth on quadruple drop-out media is indicated as “+”. (B) nup42^408–424 lies in a conserved region of the protein. Clustal Omega³⁵ alignment between nup42-CTD and hnup42-CTD. Identical (black) and similar (grey) residues are indicated. The black line above the sequence indicates residues 408–424 of Nup42. (C) Affinity measurements between gle1-CTD and nup42^408–424. BioLayer Interferometry was performed with a biotin-nup42^408–424 peptide and recombinant gle1-CTD protein. Calculated K_D and R² correlation is indicated. (D) GFP-nup42^408–424 localization at the nuclear envelope is disrupted in gle1^QKEE>AAAA mutants. Strains containing the indicated plasmids were grown to mid-log phase at 23°C and live cells were imaged by wide-field microscopy. Scale bar = 5μm. (E) nup42^408–424 does not rescue the growth defect of nup42Δ ipk1Δ mutants. nup42Δ ipk1Δ was transformed with the indicated GFP-tagged constructs, grown to mid-log phase, and plated on –Leu synthetic media for growth at the indicated temperatures. (F) nup42^408–424 is not sufficient for heat shock mRNA export. nup42Δ mutants were transformed with the indicated GFP-tagged constructs, grown at 25°C to early-log phase in synthetic media, kept at 25°C or shifted to 42°C for 15 min, labeled with [³⁵S]methionine for an additional 15 min, and lysed. Lysates were separated by SDS-PAGE, and proteins were visualized by autoradiography. Hsp proteins are indicated by asterisks.