title: The unfolded protein response transducer Ire1p contains a nuclear localization sequence recognized by multiple beta importins.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Melbourne, Victoria 3010, Australia.
authors.forename: Laurence
authors.initials: L
authors.lastname: Goffin
authors.forename: Sadanand
authors.initials: S
authors.lastname: Vodala
authors.forename: Christine
authors.initials: C
authors.lastname: Fraser
authors.forename: Joanne
authors.initials: J
authors.lastname: Ryan
authors.forename: Mark
authors.initials: M
authors.lastname: Timms
authors.forename: Sarina
authors.initials: S
authors.lastname: Meusburger
authors.forename: Bruno
authors.initials: B
authors.lastname: Catimel
authors.forename: Edouard C
authors.initials: EC
authors.lastname: Nice
authors.forename: Pamela A
authors.initials: PA
authors.lastname: Silver
authors.forename: Chong-Yun
authors.initials: CY
authors.lastname: Xiao
authors.forename: David A
authors.initials: DA
authors.lastname: Jans
authors.forename: Mary-Jane H
authors.initials: MJ
authors.lastname: Gething
mesh_terms: D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism; D016384:Consensus Sequence; D015966:Gene Expression Regulation, Fungal; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding; D017510:Protein Folding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012326:RNA Splicing / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D028961:beta Karyopherins / Q000378:metabolism*; D020931:ran GTP-Binding Protein / Q000378:metabolism
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; C091341:HAC1 protein, S cerevisiae; D008562:Membrane Glycoproteins; D019913:Nuclear Localization Signals; D012333:RNA, Messenger; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D028961:beta Karyopherins; C082863:IRE1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D020931:ran GTP-Binding Protein