Two regulatory steps of ER-stress sensor Ire1 involving its cluster formation and interaction with unfolded proteins

Kimata, Yukio; Ishiwata-Kimata, Yuki; Ito, Tatsuhiko; Hirata, Aiko; Suzuki, Tamaki; Oikawa, Daisuke; Takeuchi, Masato; Kohno, Kenji

doi:10.1083/jcb.200704166

Cited by 282 publications

(365 citation statements)

References 36 publications

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“…Given that cellular clustering can be a consequence of protein self‐association (Kimata et al , 2007; Li et al , 2010), we next asked whether SPOP self‐association influences its ability to localize to nuclear speckles. Mutations that disrupt SPOP oligomerization through the BTB (Zhuang et al , 2009) and BACK domain (van Geersdaele et al , 2013) have been previously described.…”

Section: Resultsmentioning

confidence: 99%

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

et al. 2016

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Membrane‐less organelles in cells are large, dynamic protein/protein or protein/RNA assemblies that have been reported in some cases to have liquid droplet properties. However, the molecular interactions underlying the recruitment of components are not well understood. Herein, we study how the ability to form higher‐order assemblies influences the recruitment of the speckle‐type POZ protein (SPOP) to nuclear speckles. SPOP, a cullin‐3‐RING ubiquitin ligase (CRL3) substrate adaptor, self‐associates into higher‐order oligomers; that is, the number of monomers in an oligomer is broadly distributed and can be large. While wild‐type SPOP localizes to liquid nuclear speckles, self‐association‐deficient SPOP mutants have a diffuse distribution in the nucleus. SPOP oligomerizes through its BTB and BACK domains. We show that BTB‐mediated SPOP dimers form linear oligomers via BACK domain dimerization, and we determine the concentration‐dependent populations of the resulting oligomeric species. Higher‐order oligomerization of SPOP stimulates CRL3SPOP ubiquitination efficiency for its physiological substrate Gli3, suggesting that nuclear speckles are hotspots of ubiquitination. Dynamic, higher‐order protein self‐association may be a general mechanism to concentrate functional components in membrane‐less cellular bodies.

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Section: Resultsmentioning

confidence: 99%

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

et al. 2016

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“…The triggers for this release are unclear. Structural studies of IRE1, which is also controlled by BiP, have posited direct recognition of misfolded proteins that induces a conformation change to the active state (9,10). Whether ATF6 is triggered by a similar mechanism of misfolded protein binding is unclear.…”

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confidence: 99%

In vitro reconstitution of ER-stress induced ATF6 transport in COPII vesicles

Schindler

Schekman

2009

Proc. Natl. Acad. Sci. U.S.A.

185

148

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The transcription factor ATF6 is held as a membrane precursor in the endoplasmic reticulum (ER), and is transported and proteolytically processed in the Golgi apparatus under conditions of unfolded protein response stress. We show that during stress, ATF6 forms an interaction with COPII, the protein complex required for vesicular traffic of cargo proteins from the ER. Using an in vitro budding reaction that recapitulates the ER-stress induced transport of ATF6, we show that no cytoplasmic proteins other than COPII are necessary for transport. ATF6 is retained in the ER by association with the chaperone BiP (GRP78). In the in vitro reaction, the ATF6-BiP complex disassembles when membranes are treated with reducing agent and ATP. A hybrid protein with the ATF6 cytoplasmic domain replaced by a constitutive sorting signal (Sec22b SNARE) retains stress-responsive transport in vivo and in vitro. These results suggest that unfolded proteins or an ER luminal ؊SH reactive bond controls BiP-ATF6 stability and access of ATF6 to the COPII budding machinery.regulated transport ͉ unfolded protein response ͉ BiP ͉ prebudding complex E ndoplasmic reticulum (ER) homeostasis can be perturbed by up-regulation of secretory proteins or by disruption of protein processing, a condition termed ER stress (1). ER stress causes increased protein misfolding and leads to activation of the unfolded protein response (UPR), an evolutionarily conserved pathway to alleviate ER stress (2). The UPR leads to a slowdown in protein synthesis, an upregulation of ER chaperones, and an upregulation of ER-associated protein degradation.The three effector proteins for the UPR are IRE1, PERK, and ATF6. During the UPR, IRE and PERK remain in the ER and act via cytoplasmic effectors, whereas ATF6 is transported to the Golgi complex. In the Golgi, ATF6 is cleaved by Site-1 and Site-2 proteases (3). These cleavages release the N-terminal cytoplasmic domain, which contains a bZIP motif that binds DNA at ER stress response elements that control expression of the ER chaperones BiP (GRP78) and GRP94 (4).Transport of ATF6 and other cargo proteins likely involves the COPII coat, a complex of five cytoplasmic proteins that selects cargo at the ER membrane and pinches off membranes to form vesicles. The five proteins are the GTPase Sar1, which is recruited to the ER membrane and initiates coat formation by GDP to GTP exchange; the heterodimeric complex Sec23/Sec24, which binds to Sar1⅐GTP; and a second dimeric complex, Sec13/Sec31, which binds to Sec23/24 and provides the curvature necessary for vesicle fission (5). Cargo is selected by interactions between domains on the Sec24 subunit and cytoplasmic motifs on cargo proteins (6). Inhibition of COPII by overexpression of dominant negative Sar1 has been shown to block transport of ATF6 (7).A central question in ATF6 function is how luminal stress is converted into recognition by the cytoplasmic COPII complex. The luminal chaperone BiP binds ATF6 stably in unstressed cells and dissociates specifically during stress (8)...

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“…Several lines of evidences have suggested that Rot1 is involved in protein folding: ROT1 genetically interacts with genes encoding ER chaperones such as BiP/Kar2, and the unfolded protein response (Kimata et al, 2007;Kohno 2007) was induced in the temperature-sensitive rot1-2 mutant. Furthermore, folding of disulfide-reduced carboxypeptidaseY (CPY) was likely to be severely perturbed in kar2-1 rot1-2 double mutant cells (Takeuchi et al, 2006a).…”

Section: Introductionmentioning

confidence: 99%

Saccharomyces cerevisiaeRot1 Is an Essential Molecular Chaperone in the Endoplasmic Reticulum

Takeuchi

Kohno

2008

MBoC

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Molecular chaperones prevent aggregation of denatured proteins in vitro and are thought to support folding of diverse proteins in vivo. Chaperones may have some selectivity for their substrate proteins, but knowledge of particular in vivo substrates is still poor. We here show that yeast Rot1, an essential, type-I ER membrane protein functions as a chaperone. Recombinant Rot1 exhibited antiaggregation activity in vitro, which was partly impaired by a temperature-sensitive rot1-2 mutation. In vivo, the rot1-2 mutation caused accelerated degradation of five proteins in the secretory pathway via ER-associated degradation, resulting in a decrease in their cellular levels. Furthermore, we demonstrate a physical and probably transient interaction of Rot1 with four of these proteins. Collectively, these results indicate that Rot1 functions as a chaperone in vivo supporting the folding of those proteins. Their folding also requires BiP, and one of these proteins was simultaneously associated with both Rot1 and BiP, suggesting that they can cooperate to facilitate protein folding. The Rot1-dependent proteins include a soluble, type I and II, and polytopic membrane proteins, and they do not share structural similarities. In addition, their dependency on Rot1 appeared different. We therefore propose that Rot1 is a general chaperone with some substrate specificity.

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Two regulatory steps of ER-stress sensor Ire1 involving its cluster formation and interaction with unfolded proteins

Cited by 282 publications

References 36 publications

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles

In vitro reconstitution of ER-stress induced ATF6 transport in COPII vesicles

Saccharomyces cerevisiaeRot1 Is an Essential Molecular Chaperone in the Endoplasmic Reticulum

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