driven by unfolded protein response elements-upre s). E. coli BL21(DE3) lysogens

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1 Supporting Online Material Materials and Methods Strains and Plasmids: The ire1 yeast strain PWY260 ( ire1::trp1; his3-11,-15::his + UPRE-lacZ; leu2-3,- 112::LEU2 + UPRE-lacZ; ura3-1) used in this study is in a W303 background (R. Rothstein, Columbia Univ.) and has two integrated β-galactosidase-encoding genes driven by unfolded protein response elements-upre s). E. coli BL21(DE3) lysogens were used for expression of Ire1*, using the His 6 -tag-encoding bacterial expression vector pet-28a(+) (Novagen). All CEN-ARS low copy yeast-shuttle vectors, used to carry IRE1 variant genes, were derivatives of YCplac33 (S1), and were used to transform PWY260 using lithium acetate. The IRE1 gene used in all shuttle vectors was contained on a 5 kb XhoI/HindIII genomic fragment. A 2-step PCR was used to insert a DNA cassette encoding one HA epitope at the 3 end of all IRE1 gene ORF s. All yeast methodologies were based on standard protocols (S2). Cloning was through standard protocols (S3), and site-directed mutagenesis employed the Quikchange kit (Stratagene). Mutant genes were sequenced in their entirety on both strands. IRE1 C derived through random PCR mutagenesis of the ER lumenal domain contains nine missense mutations (R2G, I21V, E69V, S103P, E167D, Q272R, F377S, L466S, K510R); these mutations are currently being deconvoluted for their contribution to the constitutive-on phenotype. Enzyme Assays: β-galactosidase assays were carried out through modification of a published protocol (S4). Yeast cells growing logarithmically in SD minus uracil + 100µg/ml inositol media

2 were treated with 20µM 1NM-PP1 (1NM-PP1 co-factor stimulation of 1NM-PP1- sensitized Ire1-containing cells was found to increase linearly from 500nM concentrations till it peaked at 20µM) diluted from a 10mM stock in DMSO or with an equivalent volume of DMSO for 5 minutes, before DTT was added (or not) to a final concentration of 2 mm from a freshly made 1M stock solution. Cells were harvested 40 minutes later by centrifugation, lysed in 400µL of a solution of 40% v/v Y-PER (Pierce) in 1x Z buffer containing 0.8 mg/ml ONPG. After incubation at 32 o C, reactions were quenched with an equal volume of 1M Na 2 CO 3. LacZ arbitrary units (a.u.) are defined as (OD 420 x 1000)/(OD 600 x t x v), where v is the volume of the sample used for the assay, and t is the time of incubation of the reaction at 32 o C. Values for LacZ a.u. s were expressed as the mean + SEM of three independent transformants for each condition tested. Recombinant Ire1* proteins were purified from BL21(DE3) lysogenic E. coli cells harboring pet28-a(+) plasmids (see above) which encoded wild-type and mutant Ire cytosolic domains (kinase+rnase), residues L of logarithmically-growing cells in LB-kanamycin were induced with 1mM IPTG for 4 hrs and the culture incubated overnight at 4 o C before harvest (we found increased enzymatic activity with this manipulation). Cells were lysed with lysozyme and sonication using buffers described in the Qiexpressionist protocol (Qiagen). PMSF at 100µM and Complete Protease Inhibitors (Roche) were used in all buffers. Purification was through Ni-NTA agarose (Qiagen). Elution was with 250mM Imidizole containing buffer. Proteins were exchanged into kinase buffer (9) though two consecutive centrifugations using 30kD-cutoff Centricon

3 devices (Amicon). Glycerol was added to protein solutions at 10% final concentration before flash-freezing in liquid nitrogen. We found that Ire1* aliquots could be stored for several months at -80 o C without loss of activity. Kinase assays were as described (9), except that 20µM 1NM-PP1, or an equivalent volume of DMSO carrier was added for 5 minutes prior to incubation with [γ- 32 P] labeled ATP at 30 o C. The kinase reactions with [γ- 32 P]-labeled N-6 benzyl ATP utilized 100µM cold N-6 benzyl ATP, to which 5 µci of [γ- 32 P]-labeled N-6 benzyl ATP (specific activity 6,000Ci/mmol, 5mCi/mL) was added. Ire1 RNase assays using in vitro transcribed HAC1 600 RNA have been described (9). The only modification of the reported protocol for this study was the addition of either 20µM 1NM-PP1 or 2mM ADP for 5 minutes prior to incubation with the RNA substrate. DMSO concentrations were adjusted to equivalent amounts in all reactions. Velocity sedimentation analysis used 25µg Ire1*, which was centrifuged through 4 ml 25-35% glycerol density gradients in kinase buffer (9) for 20 hrs at 60,000 rpm in a SW60 rotor (Beckman). After fractionation using a Foxy Jr. fraction collector (Isco) into 250µL fractions, proteins were electrophoresed through SDS-PAGE gels, blotted to nitrocellulose, probed with an anti-yeast Ire1 polyclonal antibody, and developed by ECL. 1NM-PP1 synthesis has been described (12).

4 Northern and Western Analysis: Northern blot analysis to detect HAC1 u and HAC1 i mrna species and western analysis to detect Hac1 protein has been described (8). A 5 exon HAC1 32 P body-labelled probe was used for detection of HAC1 mrna splicing. All protein samples from yeast cells were isolated by glass bead lysis in 8M urea, 2.5% SDS, 50mM HEPES (ph 7.6). After clarification of unbroken cells and debris in a microfuge, the extracts were flash-frozen in liquid nitrogen and stored at -80 o C. The anti-hac1 i polyclonal antibody was used at 1:10,000. A donkey anti-rabbit HRP conjugate (Amersham) secondary antibody was used for detection using ECL (Pierce). Anti-HA HRP conjugate used to detect HA-tagged Ire1p s was purchased from Roche and used at 1:1500 dilution. Microarray Analysis: The yeast strain PWY260 harboring either a wild-type or kinase-dead, 1NM-PP1- sensitized IRE1 gene (L745A,D828A) on the low-copy Ycplac33 vector (or a Ycplac33 vector alone control) was grown at 30 C in SD minus uracil+100µg/ml inositol to an OD 600 of µM 1NM-PP1 in DMSO was added to all cultures and growth continued for 5 minutes, before 2mM DTT (diluted from a freshly made 1M solution) was added. Growth of cultures was continued for either 25 or 50 minutes, cells harvested by filtration, flash-frozen in liquid nitrogen, and stored at -80 C overnight. Total and poly(a) RNA were isolated as described, and poly(a) RNA was reverse-transcribed with StrataScript (Stratagene) incorporating amino-allyl dutp (Sigma) at a ratio of 3:2 with dttp (19). The resulting cdnas were labeled by using monofunctional reactive Cy3 and

5 Cy5 dyes (Amersham Pharmacia) in the presence of sodium bicarbonate. All other manipulations were through standard protocols (19). The analysis and presentation of the data were performed using the software tools GENEPIX PRO (Axon Instruments, Union City, CA), and Microsoft EXCEL. Figures S1, S2, S3. Excel spreadsheets, and corresponding x-y scatter plots (log 10 ), showing mrna levels for all yeast genes, analyzed through genomic microarray analysis (see above) for the following genotypes: S1: IRE1(wt) vs. ire1, S2: IRE1(L745A,D828A) vs. ire1, and S2: IRE1(wt) vs. IRE1(L745A,D828A). For all three experiments, pink dots are genes >2 fold expressed in the IRE1(wt) vs. ire1 experiment. These include the previously described canonical UPR targets (20) see individual spreadsheets. Blue dots represent the rest of the transcriptome. Total fluorescence in the Cy3 and Cy5 channels was equalized during the analyses for normalization. References S1. R. D. Gietz, A. Sugino, Gene 74, 527 (1988). S2. F. Sherman, G. R. Fink, J. B. Hicks, Methods in Yeast Genetics (Cold Spring Harbor Laboratory, New York, 1986). S3. F. M. Ausubel, e. al., Current Protocols in Molecular Biology (John Wiley and Sons, New York, 1989). S4. H. O. Park, E. A. Craig, Mol Cell Biol 9, 2025 (1989).

Yeast Nuclei Isolation Kit

Yeast Nuclei Isolation Kit Catalog Number KA3951 50 assays Version: 02 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Intended Use... 3 Background... 3 General Information...