Strains and antibodies
Yeast strains used in this study are listed in Supplemental Table S1. Monoclonal α-HA antibodies (HA.11) were purchased from Covance Research Products (Richmond, CA). Monoclonal α-Pgk1p antibodies were purchased from Molecular Probes (Eugene, OR). Polyclonal α-Kar2p and α-Sec61p antibodies were generously provided by Peter Walter (University of California, San Francisco, San Francisco, CA). Polyclonal α-Gas1p antibodies were raised against a glutathione S
-transferase fusion protein containing the N-terminal amino acids 40–289 of Gas1p (Spear and Ng, 2003
). α-DPAP B antibodies were a kind gift of Tom Stevens (University of Oregon, Eugene, OR). Goat horseradish peroxidase (HRP)–conjugated α-mouse immunoglobulin G (IgG) antibodies were purchased from Pierce Biotechnology (Rockford, IL). Donkey HRP-conjugated α-rabbit IgG antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Goat α-rabbit IRDye 800 and goat α-mouse IRDye 680 were purchased from LI-COR Biosciences (Lincoln, NB). Alexa Fluor 488 goat α-mouse IgG and Alexa Fluor 594 goat α-rabbit IgG were purchased from Molecular Probes.
Plasmids used in this study
. pES28 carries the GAL1
promoter–regulated, HA-tagged CPY* gene in YCp50 (Spear and Ng, 2003
pCB11 and pCH49
. An Eco
I fragment containing the intact, GAL1
promoter–regulated, HA-tagged CPY* gene from pES67 was inserted into pRS313 and pRS314 to create pCB11 and pCH49, respectively (Sikorski and Hieter, 1989
; Spear and Ng, 2003
promoter–regulated, HA-tagged abcD-CPY* was constructed by digestion of pES147 (Spear and Ng, 2005
) with Acc
I and treatment with T4 DNA polymerase, followed by digestion with Sph
I. The fragment was ligated into Bam
HI (T4 DNA polymerase [DNAP]-treated)/Sph
I–digested pTS210, which carries the GAL1/10
promoter cassette (Marschall et al., 1996
Construction of upred-KAR2 and UPRE-KAR2 strains
gene is toxic to Escherichia coli
if inserted into bacterial high-copy-number vectors (Rose et al., 1989
). This required the exclusive use of low copy plasmids. To generate the upred-KAR2
strain, we used pMR397 as a base, which contains the intact KAR2
gene in pMR366 shuttle vector (Vogel et al., 1990
). PCR-based mutagenesis was used to replace sequences containing unfolded protein response element (5′-GGAACTGGACAGCGTGTCGA-3′; Mori et al., 1992
) with the nonspecific sequence 5′-GTTCTCATGTTTGACAGCTT-3′ derived from a pBR322 intergenic region. The resulting plasmid, pCB10, is a functioning bacterial–yeast shuttle vector that carries the upred-KAR2
allele. To generate a genome-integrating version of pCB10, pMR397 was digested with Bgl
II and Kpn
I to release sequences containing the autonomous replicating sequence and centromere from the plasmid. The vector was ligated following treatment with T4 DNAP, resulting in the pCB20 intermediate, which also functions as an integrating vector for the wild-type UPRE-KAR2
allele. A Pvu
I fragment containing KAR2
and flanking sequences from pCB20 were replaced with the corresponding sequences from pCB10 to generate pCB18, the upred-KAR2
To create the upred-KAR2
strain, MS785 (MATα, kar2L148::LEU2
, pMR397; Rose et al., 1989
) was crossed to YJL183 (MATa, ura3Δ99, leu2Δ1, trpΔ99, ade2-101ochre
; Ng et al., 1996
) to generate a KAR2/kar2L148
heterozygous diploid. The pMR397 plasmid was dropped from the diploid strain by counterselection using 5-fluoroorotic acid. Next pCB18 was cleaved at its unique Nco
I site in the URA3
marker gene and transformed into the diploid strain. Uracil prototrophs containing the integrated allele URA3::upred-KAR2
were sporulated for tetrad dissection. A haploid strain containing the kar2L148::LEU2
alleles (scored as leucine and uracil prototrophs) was isolated and backcrossed to the W303 strain (MATa
, ura3-1, leu2-3, his3-11, trp1-1, can1-100, ade2-1
) six times. The identical procedure was performed in parallel using pCB20 to generate the UPRE-KAR2
control strain. Because the kar2L148::LEU2
allele is not a complete knockout, we discovered that an amino-terminal fragment of Kar2p containing the full-length signal sequence was expressed in these cells and causing ER stress. To eliminate the expression of this mutant fragment, the Δkar2::KanMX
allele (complete deletion of KAR2
-coding sequences) was amplified by PCR from a KAR2/Δkar2::KanMX
heterozygous strain (Winzeler et al., 1999
) using CH20 (5′-AGGAACTGGACAGCGTGTCGAA-3′) and CH21 (5′-CAACCTTGAAGCTTCCAGCAGC-3′) primers. The purified PCR product was used directly to knock out the kar2L148::LEU2
alleles in both upred-KAR2
strains to create CHY220 and CHY438, respectively.
Analysis of HAC1 mRNA splicing
Cells were grown at 30°C in YPD and harvested at early log phase (≤0.4 OD600/ml). Cells were treated with 2.5 μg/ml tunicamycin or DMSO for 1 h. Two OD600 units of cells were harvested, and total RNA was isolated using an RNeasy Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. cDNA was prepared from 1 μg of total RNA using the SuperScript III First Strand Synthesis system (Invitrogen, Carlsbad, CA) and oligo(dT) primers according to the manufacturer's protocol. Unspliced and spliced cDNAs were amplified by PCR using primers flanking the intron: HAC1F (GT97), 5′-TCGCACTCGTCGTCTGATA, and HAC1R (GT102), 5′-TCATGAAGTGATGAAGAAATCATTCACT1, cDNA was amplified as a loading control by using primers, Act1F (GT103), 5′-GGTTGCTGCTTTGGTTATTGA, and Act1R (GT104), 5′-TTTTGACCCATACCGACCAT. Products were separated by 1% agarose gel electrophoresis and images acquired with a Gel Doc system (Bio-Rad, Richmond, CA). Quantification was performed using ImageQuant TL software (GE Healthcare Life Sciences, Uppsala, Sweden). All data reflect three independent experiments, with the SD indicated as error bars. A representative gel image from a single experiment is shown.
Activation of ER stress using galactose-inducible CPY*
Test strains were transformed with pCB11 and pCH49 or a single copy of pES28 or pCH66 (each of these plasmids carry a single copy of GAL1-CPY*). Cells were grown at 30°C overnight in SC medium containing the appropriate amino acids and 3% raffinose to mid log phase. Cells were harvested, washed, and transferred into SC medium containing the appropriate amino acids and 2% galactose for 6 h to induce CPY* synthesis before further processing. Δpep4 and upred-KAR2/Δpep4 cells were grown in galactose-containing media for 8 h before processing for indirect immunofluorescence microscopy.
Quantitative immunoblot analysis
For each strain, 3.0 A600 OD equivalents of cells were disrupted in 1 ml of 10% trichloroacetic acid (TCA) and 0.4 ml of 0.5-mm zirconium beads with 2× 30-s pulses in a Mini-Beadbeater-8 cell homogenizer (BioSpec Products, Bartlesville, OK). The homogenate was transferred to a new tube and pooled with a 150 μl of bead wash. After centrifugation, the pellet was resuspended in 150 μl TCA resuspension buffer (100 mM Tris base, 3% SDS, 1 mM phenylmethylsulfonyl fluoride [PMSF]) and incubated at 100°C for 10 min. Insoluble particles are removed by centrifugation. A lysate equivalent of 0.02 A600 OD units of cells was used for each sample in Western analysis. The samples were resolved by SDS–PAGE and then transferred to nitrocellulose membrane (GE Healthcare Life Sciences). The membrane was blotted in blotting buffer (5% nonfat dry milk in PBST [137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4·2H2O, 2 mM KH2PO4, 0.1% Tween-20]). α-Kar2p and α-Pgk1p were used at 1:10,000 and 1:1000 dilutions in blotting buffer, respectively. Secondary antibodies were α-rabbit IRDye 800 and α-mouse IRDye 680 used at 1:10,000 in 0.5% nonfat dry milk in PBST. Immunoblots were dried and scanned using the LI-COR Odyssey Infrared Imaging system, which allows for simultaneous dual-wavelength detection (169-μm resolution, 0.0-nm focus offset, 1.5 intensity for 700 nm, 2.5 intensity for 800 nm).
CPY* expression shut-off assay
The GAL1-CPY* gene was induced as described. After 6 h, glucose was added to the culture media to repress synthesis and incubated for the times indicated. CPY* levels were analyzed by immunoblotting as described, except that detection was performed using enhanced chemiluminescence (ECL) according to manufacturer's protocols (Pierce Biotechnology). α-HA (to detect CPY*) and α-Kar2p antibodies were used at 1:10,000 dilutions. HRP-conjugated goat α-mouse IgG and HRP-conjugated donkey and HRP-conjugated α-rabbit IgG were used at 1:10,000 dilutions.
Metabolic pulse-chase analysis
The 3.0 A600 OD cell equivalents grown to log phase were collected and resuspended in 0.9 ml of synthetic media lacking methionine and cysteine. After 30 min of incubation at the appropriate temperature, 150 μCi of [35S]Met/Cys (Pro-Mix; Amersham Pharmacia Biotech, Piscataway, NJ) was added to cells for 5 or 10 min, as indicated. A cold chase was initiated by adding unlabeled methionine/cysteine to 2 mM. A 100 μl amount of ice-cold 100% trichloroacetic acid was added to terminate the chase. Cells were homogenized by adding 0.4 cc of 0.5-mm zirconium beads and agitation in a Mini-Beadbeater-8 cell disrupter for 2× 30-s cycles (BioSpec Products). The homogenate was transferred to a fresh tube and pooled with a subsequent 10% TCA bead wash. Following centrifugation, the pellet was resuspended in 120 μl of TCA resuspension buffer (100 mM Tris base, 3% SDS, 1 mM PMSF) and heated to 100°C for 5 min. Insoluble debris was pelleted, and 40 μl of the detergent lysate was transferred to 560 μl of IPS II (1% Triton X-100, 50 mM Tris pH 7.5, 1 mM PMSF, 1 μl of yeast protease inhibitor cocktail [Sigma-Aldrich, St. Louis, MO]) and the appropriate antibody. Following a 120-min incubation at 4°C, the sample was centrifuged and the supernatant transferred to a fresh tube containing protein A–Sepharose beads. The tube was rotated for 30 min and washed 5× with IPS I (0.2% SDS, 1% Triton X-100, 50 mM Tris pH 7.5) and 1× with phosphate-buffered saline. Immunoprecipitated proteins were eluted with gel sample buffer, separated by SDS-gel electrophoresis, and visualized/quantified using a Typhoon phosphorimager (GE Healthcare Life Sciences).
Analysis of protein folding using Mal-PEG
Cell labeling, homogenization, and TCA precipitation were carried out as described. The TCA precipitate pellet was washed once with cold acetone and resuspended in Mal-PEG reaction solution (5 mM Mal-PEG [Fluka, Sigma-Aldrich], 100 mM Tris, pH 7.4, 2% SDS) at 50 μl per A600 OD cell equivalent. The mixture was incubated at 100°C for 10 min with occasional agitation on a vortex mixer. The tube was then left on ice for 50 min and again heated to 100°C for 10 min. After centrifugation, the cell lysate was used for immunoprecipitation as described. For DTT-treated cells, DTT was added directly to the culture media to 10 mM for 20 min prior to metabolic labeling.
This assay was modified from that previously described by Carvalho et al. (2006
). Briefly, 40 A600
OD units of log-phase cells were harvested, washed once with ice-cold water, and resuspended in 500 μl of TBS IP buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM PMSF, 0.3 μl of yeast protease inhibitor cocktail [Sigma-Aldrich] per A600
OD unit). Cell disruption was performed by agitation with 0.5 mm zirconium beads in the Mini-Beadbeater-8 (3× 15 s with 5-min intervals on ice). The lysate was transferred to a new tube and pooled with a subsequent 500 μl of TBS IP buffer bead wash. After centrifugation at 30,000 × g
for 30 min, the pellet was collected, resuspended in 1 ml of TBS IP buffer containing 1% Triton X-100, and incubated for 30 min on ice. The detergent lysate was clarified by centrifugation at 30,000 × g
for 10 min. Five μl of HA-probe mouse monoclonal IgG (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the supernatant and incubated for 30 min. Twenty-five microliters of protein A–Sepharose beads (Sigma-Aldrich) was added into the tube and incubated for 2 h with rocking. Beads were washed three times with 1 ml of TBS IP buffer containing 1% Triton X-100 and once with 1 ml of TBS IP buffer (1×). Immunoprecipitated proteins were eluted by boiling in SDS-loading buffer for 10 min, resolved on SDS–PAGE, and analyzed by immunoblotting.
Analysis of CPY* protein aggregates
Wild-type, Δire1, and upred-KAR2 cells carrying the GAL1-CPY* gene were grown and galactose induced as described, except that Δire1 cell media was supplemented with 50 μg/ml myoinositol. Five OD600 units of cells were harvested, washed once in water, and resuspended in 500 μl of TNE buffer (50 mM Tris, pH7.4, 150 mM NaCl, 5 mM EDTA) containing 1 mM PMSF and 1.5 μl of yeast protease inhibitor cocktail. Cells were disrupted by beating with 0.5-mm zirconium beads (10× 1 min, with 5 min between each interval on ice) using a vortex mixer at full speed at 4°C. Low-speed centrifugation (750 × g, 5 min) was performed twice to remove cell debris. Membranes in the supernatant fraction were solubilized by adding Triton X-100 1% followed by incubation at room temperature for 5 min. Fifty microliters of this material (T, total) was saved. The remaining lysate was centrifuged at 100,000 × g for 15 min at 4°C. The supernatant fraction (S) was removed and saved. The pellet fraction (P) was resuspended in 450 μl of 3% SDS and 50 mM Tris, pH 7.5, and boiled at 100°C for 5 min. Fifty microliters of each fraction (T, S, and P) was resolved by SDS–PAGE and proteins detected using ECL-based Western analysis as described.
Indirect immunofluorescence assays for CPY* localization and decay
GAL1-CPY* expression was induced in cells as described. CPY* synthesis was terminated by adding 2% glucose and incubated for various times indicated. Formaldehyde was added directly to the culture media to 3.7%. After incubation at 30°C for 90 min, cells were washed once with 0.1 M potassium phosphate, pH 7.5, and treated with zymolyase (1 mg/ml zymolyase 20T [ICN Biomedicals, Irvine, CA] in 0.1 M potassium phosphate, pH 7.5, and 1.2 M sorbitol) for 30 min at room temperature. The resulting spheroplasts were washed twice with 0.1 M potassium phosphate, pH 7.5, and 1.2 M sorbitol. Cells were applied to a 0.1% poly-l-lysine–coated multi-well slide, incubated at room temperature for 10 min, and washed once with TBS buffer (50 mM Tris, pH 7.5, 150 mM NaCl). The slide was immersed in cold methanol for 6 min at −20°C, transferred into cold acetone for 30 s, and allowed to equilibrate in TBS for 3 min at room temperature. Thirty microliters of TBS-blocking buffer (5% nonfat dry milk in TBS buffer) was added to each well, incubated for 30 min, and then washed once with TBS. Thirty microliters of primary antibodies was applied on wells for 90 min and washed twice with TBS afterward. Thirty microliters of secondary antibodies was then applied on wells for 90 min, followed by two TBS washes. After drying, 5 μl of mounting media (phosphate-buffered saline, 90% glycerol, 1 mg/ml p-phenylenediamine, 0.025 μg/ml 4′,6-diamidino-2-phenylindole [DAPI]) was added to each well before sealing. Samples were viewed on Zeiss LSM 5 Exciter upright microscope with a Zeiss PlanApoChromatic 100× oil immersion lens (numerical aperture, 1.4; Carl Zeiss MicroImaging, Jena, Germany). Images were acquired and processed with LSM Image Browser software (Carl Zeiss MicroImaging). Primary antibodies α-HA (for CPY*) and α-Kar2p were used at 1:500 and 1:1000 dilutions, respectively, in TBS-blocking buffer. Secondary antibodies Alexa Fluor 488 goat α-mouse IgG and Alexa Fluor 594 goat α-rabbit were diluted 1:500 in blocking buffer for working concentrations.
UPR activity assay
Cells transformed with pJC31, a plasmid carrying the UPRE-LacZ
reporter gene (Cox and Walter, 1996
), were grown to log phase and treated with 2.5 μg/ml tunicamycin for 1 h. The 3.0 OD600
units were collected, washed with 1 ml of Z buffer (0.06 M Na2
O, 0.04 M NaH2
O, 0.01 M KCl, 0.001 M MgSO4
O, 50 mM β-mercaptoethanol) and pelleted by low-speed centrifugation. The cells were resuspended in 50 μl of Z buffer, and then 50 μl of CHCl3
and 20 μl of 0.1% SDS were added. The mixture was vortexed hard for 20 s. Seven hundred microliters of 2.0 mg/ml ο
-nitrophenyl-β-galactopyranoside (in Z buffer) was added into the mixture and incubated at 30°C for 1–10 min with time of incubation recorded. The reaction was quenched by adding 500 μl of 1 M Na2
. After a quick spin, the A420
OD of the supernatant was measured. β-Galactosidase activity was expressed in Miller units as 1000(A420
OD units of cells)] (Guarente, 1983
Northern blot analysis
To induce the UPR, cells were treated with 2.5 μg/ml tunicamycin or 10 mM DTT for 1 h. Total RNA was isolated using the hot phenol method. Briefly, resuspended cells in 22 ml of 50 mM NaOAc and 10 mM EDTA were disrupted with the addition of 2 ml of 10% SDS and 25 ml of phenol and shaking at 65°C. Following phase separation by centrifugation, the phenol phase was removed and the extraction was repeated by addition of 25 ml of phenol. The aqueous phase was transferred to another tube for extraction using chloroform. Total RNA was collected by ethanol precipitation from the aqueous phase. Equal amounts of RNA were separated by formaldehyde agarose gel electrophoresis and transferred to nitrocellulose using the buffer wicking method. DNA for probe preparation was amplified by PCR and purified from agarose gels. Probes were labeled with [α-32P]dCTP using Ready-To-Go DNA labeling beads (GE Healthcare Life Sciences). The KAR2-specific probe was prepared using the forward primer 5′-ACAGACTAAGCGCTGGCAAGCT-3′ and the reverse primer 5′-CAGCATGGGTAACCTTAGAGCC-3′ to obtain a 550–base pair fragment corresponding to the 5′ end of the coding sequence. The ACT1-specific probe was generated using the forward primer 5′-ATCGTCGGTAGACCAAGA CACC-3′ and the reverse primer 5′-CGAAGTCCAAGGCGACGTAACA-3′ to obtain a 559–base pair fragment corresponding to the 5′-end of the coding sequence.