Purification and Activity Assays of the Catalytic Domains of the Kinase/Endoribonuclease Ire1p from Saccharomyces cerevisiae

Silke Nock; Tania N. Gonzalez; Carmela Sidrauski; Maho Niwa; Peter Walter

Introduction

Ire1p is a single-spanning transmembrane protein of the endoplasmic reticulum (ER) of all eukaryotic cells. It is a bifunctional enzyme that exhibits both kinase-and site-specific endoribonuclease activities. Work with the yeast Saccharomyces cerevisiae showed that Ire1p provides a key regulatory switch during an intracellular signaling pathway that originates in the lumen of the ER.1,2 In brief, when unfolded proteins accumulate in the ER, a signal is sent to induce a transcriptional program, termed the unfolded protein response or UPR, that causes an increase in the protein-folding capacity of the ER. An accumulation of unfolded proteins is initially sensed by the ER—lumenal domain of Ire1p by an unknown mechanism.3–5 Ire1p molecules are then thought to laterally oligomerize in the plane of the membrane, which leads to trans-autophosphorylation of their kinase domains and concomitant activation of an endoribonuclease activity.6 Activated Ire1p cleaves the mRNA encoding Hac1p, a UPR-specific transcription factor, at two positions, thereby excising an intron from the RNA.7 A second enzyme, tRNA ligase, joins the two exons liberated by Ire1p cleavage to produce spliced HAC1 mRNA that is now efficiently translated to produce Hac1p, which in turn drives the transcriptional programs that comprise the UPR.8 The presence of the intron in the unspliced HAC1 mRNA blocks its translation.9 Removal of the intron by the spliceosome-independent Ire1p/tRNA ligase-mediated splicing reaction is necessary to induce the UPR.

Ire1 in S. cerevisiae is a 1115-amino acid protein. It is initially synthesized with an N-terminal signal sequence followed by an ER–lumenal "unfolded protein-sensing" domain [amino acids (aa) 1–526], a single-transmembrane α helix (aa 527–556), a linker domain (aa 557–678), a kinase domain (aa 679–973), and a C-terminal domain (aa 974–1115) that is presumed to harbor the endonuclease activity. Homologs of Ire1p have been identified in Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, and mammalian cells. By sequence analysis, the enzymes are most conserved in the kinase and presumed nuclease domains; and site-specific endoribonuclease activity has been demonstrated for two mammalian isoforms10 and shown to be indistinguishable from that of S. cerevisiae Ire1p. In HAC1 mRNA, Ire1p recognizes conserved RNA stem–loop structures, which it cleaves after invariant G residues found in the third position of a seven-nucleotide loop. RNA oligonucleotides that form such stem–loop structures are cleaved by Ire1p and hence provide convenient "minisubstrates" to characterize the reaction. In this way, it was determined that Ire1p cleaves RNA to leave a 2′,3′-phosphate on the 5′ fragment and a free 5′-hydroxyl group on the 3′ fragment.11

We here describe the overexpression in two different systems, purification, and activity assays for fusion proteins containing the cytosolic domains of Ire1p from S. cerevisiae. All expressed proteins truncate Ire1p just after the transmembrane region and hence contain the linker, kinase, and presumed nuclease domains. Because C-terminal tags reduce Ire1p activity in vivo (C. Shamu and P. Walter, unpublished data, 1995), all fusion proteins were constructed containing N-terminal glutathione S-transferase (GST) or hexahistidine (His6) tags to facilitate purification. Detailed methods for analysis of Ire1p endonuclease cleavage products can be found elsewhere.11

Solutions

Buffer A 20 mM HEPES (pH 7.5), 150 mM KCl, 1 mM dithiothreitol (DTT), 5 mM MgCl2, 10% (v/v) glycerol

Buffer B 20 mM HEPES (pH 7.5), 1 mM DTT

Buffer C 20 mM HEPES (pH 7.5), 1 mM DTT, 500 mM KCl

Buffer D 50 mM HEPES (pH 7.5), 150 mM KCl, 1 mM EDTA, 1 mM DTT

Buffer E 20 mM HEPES (pH 7.5), 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM DTT

Buffer F 0.3 M sodium acetate (pH 5.2), 10 mM magnesium acetate

Urea buffer (2×) Combine 28 ml of 5 M NaCl, 4 ml of 1 M Tris (pH 7.5), 8 ml of 0.5 M EDTA, 40 ml of 10% (w/v) sodium dodecyl sulfate (SDS); add water to 200 ml

Buffer G Combine 21 g of urea and 25 ml of 2× urea buffer; add water up to 50 ml; heat (~50°) to dissolve

Buffer H 50 mM Tris (pH 8.5), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM 2-mercaptoethanol, 1% (v/v) Triton X-100

Buffer I 50 mM sodium phosphate (pH 8), 300 mM NaCl, 10% (v/v) glycerol

Cell Growth and Protein Expression in Escherichia coli

For expression of GST-Ire1(l + k + t) (linker plus kinase plus tail, amino acids 556–1115) in Escherichia coli, the vector pCF2107 is constructed by subcloning the Ire1p fragment into pGEX-6P-2 (Amersham Pharmacia, Piscataway, NJ). This construct contains a PreScission protease cleavage site that allows removal of the GST tag linked to the amino-terminal end of Ire1(l + k + t).

We use BL21(DE3)pLysS cells (Stratagene, La Jolla, CA), which consistently give better expression yields than DH5α or BL21(DE3) cells. Irep1 expression vectors can be unstable in E. coli cells; we therefore always transform cells freshly with the expression vector. Five colonies are used to start a 50-ml preculture in Luria—Bertani (LB) medium, containing carbenicillin (100 µg/ml) (GIBCO-BRL Gaithersburg, MD). The preculture is grown for 14 hr to late log phase and used to inoculate larger batches at a dilution of 1:200 (5 to 1000 ml) in LB medium containing carbenicillin at 100 µg/ml. Typically, cells are grown in six 1-liter batches of LB medium in shaking flasks (225 rpm) at 37° to an OD600 of 0.6 to 0.8. Cells are then induced with 0.7 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) (Denville Scientific, Metuchen, NJ) and shifted to 30°. About 4 hr after induction, cells are harvested by centrifugation with a GSA rotor (Sorvall, Newtown, CT) at 16,000g at 4° for 15 min, yielding about 20g of E. coli cell paste (wet weight). The cell pellet is resuspended in buffer A (150 ml), supplemented with protease inhibitors (one tablet/50 ml, Protease Inhibitor Complete; Boehringer Mannheim, Indianapolis, IN) and 1% (v/v) Triton X-100 (Calbiochem, La Jolla, CA,). The suspension is quick-frozen in liquid nitrogen and stored at −80°.

Purification of GST-Ire1(l+k+t)

The cell suspension is thawed and kept on ice. Fresh DTT (to a final concentration of 1 mM) and additional protease inhibitor mix (three inhibitor cocktail tablets) are added, and the cells are lysed in an ice-cold Microfluidizer (Microfluidics, Newton, MA) for three cycles. The BL21(DE3)pLysS cells used do not require the addition of lysozyme. The crude extract is centrifuged in an SS-34 rotor at 31,000g at 4° for 30 min to remove the cell debris. To the resulting supernatant is added a 2-ml aliquot of a slurry of 50% glutathione—Sepharose 4B (Amersham Pharmacia; equilibrated in 20 mM HEPES, pH 7.0) and the mixture is incubated with gentle...