Mapping regions in Ste5 that support Msn5-dependent and -independent nuclear export.

Careful control of the available pool of the MAPK scaffold Ste5 is important for mating-pathway activation and the prevention of inappropriate mating differentiation in haploid Saccharomyces cerevisiae. Ste5 shuttles constitutively through the nucleus, where it is degraded by a ubiquitin-dependent mechanism triggered by G1 CDK phosphorylation. Here we narrow-down regions of Ste5 that mediate nuclear export. Four regions in Ste5 relocalize SV40-TAgNLS-GFP-GFP from nucleus to cytoplasm. One region is N-terminal, dependent on exportin Msn5/Ste21/Kap142, and interacts with Msn5 in 2 hybrid assays independently of mating pheromone, Fus3, Kss1, Ptc1, the NLS/PM, and RING-H2. A second region overlaps the PH domain and Ste11 binding site and 2 others are on the vWA domain and include residues essential for MAPK activation. We find no evidence for dependence on Crm1/Xpo1, despite numerous potential nuclear export sequences (NESs) detected by LocNES and NetNES1.1 predictors. Thus, Msn5 (homolog of human Exportin-5) and one or more exportins or adaptor molecules besides Crm1/Xpo1 may regulate Ste5 through multiple recognition sites.

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