Exportin Crm1 is important for Swi6 nucleocytoplasmic shuttling and MBF transcription activation inSaccharomyces cerevisiae

Abstract

AbstractThe Swi6 protein acts as a transcription factor in budding yeast, functioning in two different heterodimeric complexes, SBF and MBF, that activate the expression of distinct but overlapping sets of genes. Swi6 undergoes regulated changes in nucleocytoplasmic localization throughout the cell cycle that correlate with changes in gene expression. While the process of Swi6 nuclear import is well understood, mechanisms underlying its nuclear export remain unclear. Here we investigate Swi6 nuclear export and its impact on Swi6 function. We show that the exportin Crm1, in addition to three other karyopherins previously shown to affect Swi6 localization, is important for Swi6 nuclear export and activity. A truncation of Swi6 that removes a putative Crm1 nuclear export signal results in the loss of changes in nucleocytoplasmic Swi6 localization that normally occur during progression through the cell cycle. Mutagenesis of the NES-like sequence or removal of Crm1 activity using leptomycin B results in a similar decrease in nuclear export as cells enter S-phase. Using two-hybrid analysis, we also show that Swi6 associates with Crm1in vivo. Alteration of the Crm1 NES in Swi6 results in a decrease in MBF-mediated gene expression, but does not affect expression of an SBF reporter, suggesting that export of Swi6 by Crm1 regulates a subset of Swi6 transcription activation activity. Finally, alteration of the Crm1 NES in Swi6 results in cells that are larger than wild type, but not to the extent of those with a complete Swi6 deletion. Expressing a Swi6 NES mutant in combination with a deletion of Msn5, an exportin involved in Swi6 nuclear export and specifically affecting SBF activation, further increases the large cell phenotype, but still not to the extent observed in a Swi6 deletion mutant. These data suggest that Swi6 has at least two different exportins, Crm1 and Msn5, each of which interacts with a distinct nuclear export signal and influences expression of a different subset of Swi6-controlled genes.Summary StatementPrecise intracellular localization is important for the proper activity of proteins. Here we provide evidence that the Swi6 transcription factor important for cell cycle progression shuttles between the cell nucleus and cytoplasm, its nuclear export is important for its activity, and that it contains a nuclear export signal (NES) recognized by the Crm1 nuclear transport factor.