Fig. 2
Swi6 contains a leucine-rich domain that is necessary for efficient nuclear export. (A) Cartoon diagram depicting truncation mutants lacking amino acids 274–803 (Swi61–273-GFP) and 182–803 (Swi61–181-GFP) from the carboxy-terminus of Swi6 fused with GFP. Sites of NLSs interacting with Kap95 [43, 44, 50] and with Kap120 [40] are shown, along with the location of the Crm1-NES investigated in this study. The NES sequence in Swi6 recognized by Msn5 has not been determined and thus is not depicted here. (B) Swi6 nuclear export decreases upon deletion of amino acids 182–274 or mutagenesis of leucines to alanines within the NES-like sequence at amino acids 250–258. Cells expressing Swi61–273-GFP (left), Swi61–181-GFP (middle), or Swi61–273ΔNES-GFP (right) were arrested with α-factor, then released from arrest to observe Swi6 localization in synchronous cell populations. Cells expressing the Swi6-GFP fusions were observed by direct fluorescence (GFP) and phase-contrast (Phase) microscopy at the timepoints indicated after release from arrest. Bar, 5 μm. (C) Nuclear export of Swi61–273ΔNES-GFP is intermediate between Swi61–273-GFP and Swi61–181-GFP. A plasmid encoding Swi6 amino acids 1–273 fused with GFP was subjected to site directed mutagenesis so that codons 250, 254, 257, and 258 encode alanines rather than leucines, generating Swi61–273ΔNES-GFP. Plasmids expressing Swi61–273ΔNES-GFP, Swi61–273-GFP, and Swi61–181-GFP were expressed in synchronized cells and the percent of cells with nuclear GFP fluorescence was determined for each Swi6 fusion at 20-min intervals after release from α-factor. Cells were synchronized as in (B) and observed by direct fluorescence (GFP) and phase-contrast (phase) microscopy at the time intervals indicated after release from α-factor arrest. Error bars depict s.e.m. from at least three experiments. (D) Alignment of Swi6 amino acids 250–258 with leucine-rich nuclear export signals (NESs) from other shuttling proteins. Leucine residues are underlined. (E) Alignment of Saccharomyces cerevisiae Swi6 leucine-rich sequence and flanking residues with homologous regions from fungi Saccharomyces paradoxus, Saccharomyces pastorianus, Zygosaccharomyces bailii, Candida glabrata, and Kluyveromyces lactis. Leucine-rich Crm1-NES-like sequences aligning with amino acids 250–258 from S. cerevisiae are highlighted in yellow. Amino acids that differ from S. cerevisiae Swi6 are red