42Noda Institute for Scientiﬁc Research GRANT
2011 Research Grant

Activation mechanism of ER stress in yeast and mammaliancells
Kenji KOHNO
Nara Institute of Science and Technology, Graduate School of Biological Sciences, Laboratory of Molecular and Cell Genetics
Research aims
Eukaryotic cells activate the unfolded protein response(UPR) upon endoplasmic reticulum (ER) stress, where thestress is assumed to be accumulation of unfolded proteinsin the ER1). Of the ER-membrane proteins that mediate theintracellular signal for the UPR, only Ire1 is known to be
evolutionarily conserved throughout eukaryotes1,2). Ire1 is avery unique type-I transmembrane protein carrying both kinase and endoribonuclease activities in its cytosolic region(Fig. 1)2). The luminal portion of yeast Ire1 has been precisely analyzed and core stress-sensing region (CSSR) in
Ire1 luminal domain plays a central role in the recognitionof the accumulation of misfolded proteins in the ER (Fig.
1)3). Recent our in vitro study showed that unfolded model
proteins directly bound the Ire1 CSSR domain4). However
there has been no evidence that unfolded protein could directly bind Ire1 luminal region in vivo. To address thisFig. 1. Structure of the luminal domain of S. cerevisiae Ire1. The luminal domain of yeast Ire1 can be divided into ﬁve subregions. Subregions I(aa 32–111), III (aa 243–272), and V (aa 455–524) are loosely folded,
while subregions II (aa 112–242) and IV (aa 273–454) form the tightly
folded CSSR3,6). The CSSR dimer associated via interface I forms agroove, by which unfolded proteins may be captured6). Mutation of Interface 1 (X: T226A/F247A) and another mutation of Interface II (Y:W426A) disrupt the groove structure and the cluster formation, respectively, resulting in the great reduction of full activation of UPR under ERstress3,6).

question, we tried to detect in vivo physical interaction between Ire1 and a misfolded model protein CPY*, which is a
R255G missense mutant of carboxypeptidase Y.
Methods5)
Plasmids: S. cerevisiae IRE1 plasmid pRS313-IRE1 andC-terminally HA-tagged IRE1 (Ire1-HA) plasmids
pRS315-IRE1-HA and pRS423-IRE1-HA were respectively produced from centromeric vectors pRS313, pRS315
and 2 m vector pRS423. Plasmid pRS426-IRE1-HA is theURA3-marker variant of pRS423-IRE1-HA. To introduce
point or partial-deletion mutations into the IRE1 gene onthese plasmids, overlap PCR and in vivo homologous
recombination (gap repair) techniques were employed3,4).
Results
In order to support the idea of a physical interaction between unfolded proteins and Ire1, we investigated whether
such complexes are formed in yeast cells. The R225G mutant of Prc1 carboxypeptidase Y (CPY) known as CPY*
fails to be correctly folded and transported to the vacuole.
In Figure 2, GFP-tagged wild-type CPY (CPY-GFP) or
CPY* (CPY*-GFP) was constitutively produced from thestrong TEF1 promoter. Activation of the UPR by these proteins was checked by induction of a lacZ reporter controlled
by the UPR promoter element (UPRE), which the Hac1
protein directly activates. As expected, the reporter was induced by expression of CPY*-GFP and less strongly by
CPY-GFP (Fig. 2A). This observation was reproduced byan assay for Ire1-dependent HAC1i mRNA production, in
which cellular RNA samples were used for reverse transcriptase (RT)-PCR ampliﬁcation of the HAC1 mRNAs. We
thus think that CPY-GFP may be somewhat unfolded, while
CPY*-GFP acts as a more obviously unfolded protein
model. In Figure 2B, cells were treated with the chemical
crosslinker dithiobis(succinimydylpropionate) (DSP) before
cell lysis and anti-GFP immunoprecipitation (IP). In agreement with the ER retention of CPY*-GFP, this protein appeared as a single protein band in an anti-GFP Western blotof the lysate and the anti-GFP IP samples. Also consistent
with the above result, CPY-GFP partially converted to thefast-mobility vacuolar form. Importantly, co-expressed HAepitope-tagged Ire1 (Ire1-HA) was co-immunoprecipitatedwith CPY*-GFP but less abundantly with CPY-GFP (Fig.
2B). Next we performed a reverse immunoprecipitation ex-43Fig. 2. In vivo association of CPY*-GFP with Ire1. (A) An ire1D strain KMY1015 carrying both the wild-type IRE1 (WT Ire1) plasmid pRS315-IRE1-HA
and the UPRE-lacZ reporter plasmid pCZY1 was further transformed with the CPY-GFP or the CPY*-GFP expression plasmid (pRS313-TEF1pr-CPY-GFP
or pRS313-TEF1pr-CPY*-GFP) or empty vector pRS313 (Vector). The transformant strains were then assayed for cellular b -galactosidase activity, the values of which are normalized against that of vector control cells (set at 1.00). In the “no ire1” sample, cells carried vector plasmids pRS315 and pRS313.
Error bars represent the SDs from three independent transformants. According to Student’s t test, all values are statistically different from each other(p⬍0.05). (B) The ire1D strain KMY1015 carrying both HA-tagged Ire1 plasmid pRS426-IRE1-HA and pRS313-TEF1pr-CPY-GFP or pRS313-TEF1prCPY*-GFP (or empty vector pRS313; Vector) was incubated with protein cross-linker DSP before cell lysis and anti-GFP IP. Subsequently, the lysate and theanti-GFP IP samples were analyzed by anti-HA or anti-GFP Western blotting. (C) The ire1D strain transformed with both pRS426-IRE1-HA (or empty vector pRS426 for lanes 1 and 7) and a GAL1 promoter-inducible CPY-GFP or CPY*-GFP plasmid, pRS313-GAL1pr-CPY-GFP or pRS313-GAL1pr-CPY*GFP, was cultured in galactose-containing medium. After incubation with DSP, cells were lysed and analyzed by anti-HA IP, followed by anti-HA or antiGFP Western blotting. In lanes 2, 3, and 4 and 8, 9, and 10, samples from three independent clones were analyzed. Cells for lane 6 carried an empty vector
pRS313 instead of the CPY-GFP or CPY*-GFP plasmid. A molecular mass marker (M) was loaded in lane 5.

periment, in which CPY*-GFP was expressed from the inducible GAL1 promoter, since we failed to transform ire1
null strains with the CPY*-GFP constitutive expression
plasmid. Consistent with the results in Figure 2B, Ire1-HA
co-immunoprecipitates CPY*-GFP, and less abundantly theER-retained form of CPY-GFP. The vacuolar form of CPYGFP, which migrates faster on PAGE, was not co-immunoprecipitated with Ire1-HA (Fig. 2C, lanes 2–4).

References
1)
2)

3)

4)

ConclusionConsistent with previous in vitro studies of the ER-luminal domain of the UPR initiator Ire14), the results ofimmnoprecipitation experiments indeed represent in vivointeraction between Ire1-HA and the ER-located model
protein5).

5)6)Walter, P., and Ron, D. (2011) The unfolded protein response: fromstress pathway to homeostatic regulation. Science 334: 1081–1086.
Kimata, Y., and Kohno, K. (2011) Endoplasmic reticulum stresssensing mechanisms in yeast and mammalian cells. Curr. Opin. CellBiol. 23: 135–142.
Kimata, Y., Oikawa, D., Shimizu, Y., Ishiwata-Kimata, Y., andKohno, K. (2004). A role for BiP as an adjustor for the endoplasmic
reticulum stress-sensing protein Ire1. J. Cell Biol. 167: 445–456.
Kimata, Y., et al. (2007) Two regulatory steps of ER-stress sensor
Ire1 involving its cluster formation and interaction with unfolded
proteins. J. Cell Biol. 179: 75–86.
Promlek, T., Ishiwata-Kimata, Y., Shido, M., Sakuramoto, M.,
Kohno, K. and Kimata, Y. (2011) Membrane aberrancy and unfolded
proteins activate the endoplasmic reticulum stress sensor Ire1 in different ways. Mol. Biol. Cell 22: 3520-3532.
Credle, J. J., Finer-Moore, J. S., Papa, F. R., Stroud, R. M., and Walter, P. (2005). On the mechanism of sensing unfolded protein in theendoplasmic reticulum. Proc. Natl. Acad. Sci. USA 102: 18773–18784.