The unfolded protein response transducer Ire1p contains a nuclear localization sequence recognized by multiple beta importins

Department of Biochemistry and Molecular Biology, University of Melbourne, Victoria 3010, Australia.
Molecular Biology of the Cell (Impact Factor: 4.47). 01/2007; 17(12):5309-23. DOI: 10.1091/mbc.E06-04-0292
Source: PubMed


The Ire1p transmembrane receptor kinase/endonuclease transduces the unfolded protein response (UPR) from the endoplasmic reticulum (ER) to the nucleus in Saccharomyces 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 18-residue sequence is capable of targeting green fluorescent protein 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 classical NLS (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. The NLS-dependent nuclear localization of Ire1p would thus seem to be central to its role in UPR signaling.

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    • "Walter and colleagues clearly demonstrated that unspliced HAC1 mRNA is located in the cytoplasm and associated with stalled polysomes, and that splicing of HAC1 can occur even if de novo transcription is inhibited, strongly suggesting that unconventional splicing occurs in the cytoplasm in yeast cells (Ruegsegger et al., 2001). However, Gething and colleagues recently reported that yeast Ire1p contains a nuclear localization sequence, and that HAC1 splicing requires both nuclear localization of Ire1p and components of the nuclear import machinery such as RanGAP and RanGEF (Goffin et al., 2006). From these observations, they argued that newly synthesized unspliced HAC1 mRNA is spliced in the nucleus, whereas the preexisting pool of unspliced HAC1 mRNA is spliced in the cytoplasm. "
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