Abstract: Monitoring Editor: Reid Gilmore The Ire1p transmembrane receptor kinase/endonuclease transduces the unfolded protein response (UPR) from the ER to the nucleus in S. cerevisiae. In this study we analyzed the capacity of a highly basic sequence in the linker region of Ire1p to function as a nuclear localization sequence (NLS) both in vivo and in vitro. This 19-residue sequence is capable of targeting GFP to the nucleus of yeast cells in a process requiring proteins involved in the Ran GTPase cycle that facilitates nuclear import. Mutagenic analysis and importin binding studies demonstrate that the Ire1p linker region contains overlapping potential NLSs: at least one cNLS (within sequences 642KKKRKR647 and/or 653KKGR656) that is recognized by yeast importin alpha (Kap60p), and a novel betaNLS (646KRGSRGGKKGRK657) that is recognized by several yeast importin beta homologues. Kinetic binding data suggest that binding to importin beta proteins would predominate in vivo. The UPR, and in particular ER stress-induced HAC1 mRNA splicing, is inhibited by point mutations in the Ire1p NLS that inhibit nuclear localization, and also requires functional RanGAP and Ran GEF proteins. Ire1p's NLS-dependent nuclear localization would thus appear to be central to its role in UPR signaling.