The Unfolded Protein Response Transducer Ire1p Contains a Nuclear Localization Sequence Recognized by Multiple ␤ 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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Available from: Edouard Nice, Oct 01, 2015
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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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    ABSTRACT: XBP1 is a key transcription factor that regulates the mammalian unfolded protein response. Its expression is regulated by unconventional mRNA splicing that is carried out by endonuclease IRE1 and a specific, as yet unknown, RNA ligase in response to the accumulation of unfolded proteins in the ER. Conventional mRNA splicing occurs only in the nucleus, but it has remained unclear whether unconventional splicing of XBP1 mRNA takes place in the nucleus, cytoplasm or both. Here, we show that the catalytic domain of IRE1 contains a nuclear exclusion signal to prevent IRE1 from mislocalizing to the nucleus. In addition, RNA ligase, which joins XBP1 exons cleaved by IRE1 was detected in the cytoplasm but not in the nucleus. Moreover, the cytoplasm contained large amounts of unspliced XBP1 mRNA compared with the nucleus. Most unspliced XBP1 mRNA was converted to spliced mRNA by unconventional splicing even if de novo transcription was blocked, suggesting that cytoplasmic XBP1 mRNA, not nuclear XBP1 mRNA, is a major substrate for unconventional splicing. From these observations, we concluded that unconventional splicing of XBP1 mRNA occurs predominantly in the cytoplasm.
    Journal of Cell Science 09/2009; 122(Pt 16):2877-86. DOI:10.1242/jcs.040584 · 5.43 Impact Factor
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    • "Their nuclear import is directed by a NLS and is also dependent on the function of nuclear pore complex–associated proteins (Nups). Ire1p, a regulator of the unfolded protein response and previously thought be an ER membrane protein, has been shown to contain a NLS and preferentially localize to the INM (Goffin et al., 2006). In contrast, like Doa10p, Zmp- ste24 and Icmt contain no obvious NLS as predicted by the PSORT II algorithm and do not concentrate in the INM, yet they also reside and are active within the nucleus, as shown here. "
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    ABSTRACT: Proteins establish and maintain a distinct intracellular localization by means of targeting, retention, and retrieval signals, ensuring most proteins reside predominantly in one cellular location. The enzymes involved in the maturation of lamin A present a challenge to this paradigm. Lamin A is first synthesized as a 74-kDa precursor, prelamin A, with a C-terminal CaaX motif and undergoes a series of posttranslational modifications including CaaX processing (farnesylation, aaX cleavage and carboxylmethylation), followed by endoproteolytic cleavage by Zmpste24. Failure to cleave prelamin A results in progeria and related premature aging disorders. Evidence suggests prelamin A is imported directly into the nucleus where it is processed. Paradoxically, the processing enzymes have been shown to reside in the cytosol (farnesyltransferase), or are ER membrane proteins (Zmpste24, Rce1, and Icmt) with their active sites facing the cytosol. Here we have reexamined the cellular site of prelamin A processing, and show that the mammalian and yeast processing enzymes Zmpste24 and Icmt exhibit a dual localization to the inner nuclear membrane, as well as the ER membrane. Our findings reveal the nucleus to be a physiologically relevant location for CaaX processing, and provide insight into the biology of a protein at the center of devastating progeroid diseases.
    Molecular biology of the cell 11/2008; 19(12):5398-408. DOI:10.1091/mbc.E08-07-0704 · 4.47 Impact Factor
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    • "The cytosolic domain of yeast Ire1 carries a highly basic sequence, which, according to Goffin et al. (2006), acts as an NLS when fused with other proteins. However, our immunofluorescent analysis indicated that both unclustered and clustered Ire1 variants are distributed not only at the nuclear rim but also at other parts of the ER. "
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    ABSTRACT: Chaperone protein BiP binds to Ire1 and dissociates in response to endoplasmic reticulum (ER) stress. However, it remains unclear how the signal transducer Ire1 senses ER stress and is subsequently activated. The crystal structure of the core stress-sensing region (CSSR) of yeast Ire1 luminal domain led to the controversial suggestion that the molecule can bind to unfolded proteins. We demonstrate that, upon ER stress, Ire1 clusters and actually interacts with unfolded proteins. Ire1 mutations that affect these phenomena reveal that Ire1 is activated via two steps, both of which are ER stress regulated, albeit in different ways. In the first step, BiP dissociation from Ire1 leads to its cluster formation. In the second step, direct interaction of unfolded proteins with the CSSR orients the cytosolic effector domains of clustered Ire1 molecules.
    The Journal of Cell Biology 11/2007; 179(1):75-86. DOI:10.1083/jcb.200704166 · 9.83 Impact Factor
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