Fig. 3

Swi6 associates with Crm1 and requires Crm1 activity for efficient nuclear export. Plasmids containing Swi6 fused to a transcription activation domain (Swi6^AD) and Crm1 fused to the LexA DNA-binding domain (Crm1^BD) were co-transformed into two-hybrid reporter yeast strains together or in combination with two-hybrid vectors lacking the fusion (pAD or pOBD-2). (A) Transformed cells containing only empty vectors (pAD + pOBD-2), Swi6^AD and empty vector (pSwi6^AD + pOBD-2), Swi6^AD and Crm1^BD (pSwi6^AD + pCrm1^BD), or empty vector plus Crm1^BD (pAD + pCrm1^BD) were plated on media to select for transformation with both plasmids (−Leu –Trp) and media to select for a two-hybrid interaction by complementation of an ADE2 mutation (−Leu –Trp –Ade). Serial dilutions were plated on selective media and photographed after 72 h at 30 °C. (B) Two-hybrid reporter yeast cells were transformed as above, grown in liquid media to select for both plasmids, lysed, and assayed for β-galactosidase activity as a marker for two-hybrid interaction. Data depict averages of three or more assays. Error bars represent s.e.m. (C) Yeast cells containing a mutation in Crm1 (Crm1^LMB, [57]) that makes the protein susceptible to inhibition by leptomycin-B (LMB) were transformed with a plasmid expressing Swi6^1–273-GFP. Overnight cultures in log-phase growth were arrested in G1 using α-factor and treated with LMB for 1 h (+LMB) or left in media lacking drug (−LMB). Cells were then rinsed with +LMB or –LMB media lacking α-factor and observed by fluorescence microscopy. Cells in the absence (−LMB) or presence (+LMB) of leptomycin-B were photographed in α-factor (0 min) and 20 min, 70 min, and 120 min after release from cell cycle arrest. Bar, 5 μm. (D) Cells were treated as in (C) and the percent of cells with Swi6^1–273-GFP nuclear fluorescence was determined at 20 min intervals after α-factor release. Values shown are averages of two separate experiments