Strains and antibodies
strains used in this study are listed in Tables S3
. Anti-HA (HA.11) antibody, HA probe, and anti-Myc antibody were purchased from Covance. Anti-hexokinase antibody was purchased from United States Biological Inc. Anti-Kar2p rabbit polyclonal antiserum was provided by P. Walter (University of California, San Francisco, San Francisco, CA).
Plasmids used in this study
Plasmids and primers used in this study are listed in Table S2. Plasmids were constructed using standard cloning protocols. All coding sequences of constructs used in this study were sequenced in their entirety. Unless indicated, all substrate proteins described in this study contain a single C-terminal HA epitope tag used for detection (Spear and Ng, 2005
PrA expression vectors and variants
Plasmids pKK129, pKK159, pKK163, and pKK261 carry PrA*-Ab, PrA*-AbΔ147–183, PrA*-AbΔ295–331, and ngPrAΔ295–331 driven by their endogenous promoter, respectively, were described previously (Xie et al., 2009
). Plasmid pKK249, carrying ngPrA*Δ295–331, was created as follows: a 1.6-kb fragment encoding PrA*-Ab and its promoter was released from plasmid pKK150 by digesting with ClaI. This fragment was ligated into pKK163 digested with ClaI to create pKK249.
Yos9 and Yos9R200A expression vectors
Plasmid pKK278, carrying Yos9 driven by its endogenous promoter, was created as follows: HA epitope–tagged Yos9p driven by the endogenous YOS9
promoter was described previously (Kim et al., 2005
). HA epitope tag was deleted from it by site-directed mutagenesis using KKN303 primer to create the plasmid pKK278. Plasmid pKK284, carrying Yos9R200A driven by the endogenous YOS9
promoter, was created by site-directed mutagenesis to make a point mutation R200A using KKN306 primer. Plasmid pKK278 was used as template for site-directed mutagenesis.
CPY* expression vector
Plasmid pKK286, carrying CPY* driven by a galactose-inducible promoter, was created by site-directed mutagenesis to delete HA epitope tag sequence from plasmid pES67 using KKN307 primer (Spear and Ng, 2003
Hmg2p expression vector
Plasmid pRH244 was provided by R. Hampton (University of California, San Diego, La Jolla, CA). The following plasmids were generated using site-directed mutagenesis of ERAD substrate clones (Sawano and Miyawaki, 2000
). Substrate variant names are indicated in parentheses. Primer sequences are listed in Table S4. All substrate genes are controlled by the endogenous promoter and encode the HA epitope tag at their C termini.
Using the KKN136 primer, a segment encoding Thr295 through Arg331 was deleted from pKK159.
Using the KKN228 primer, the N-linked glycosylation site was mutagenized on pKK214.
Using the WXN41 primer, a segment encoding Lys23 through Leu71 was deleted from pKK232.
Using the WXN29 primer, a segment encoding Asp72 through Ala142 was deleted from pKK232.
Using the WXN30 primer, a segment encoding Glu143 through Glu213 was deleted from pKK232.
Using the WXN31 primer, a segment encoding Ser214 through Ile284 was deleted from pKK232.
Using the WXN31 primer, a segment encoding Ser214 through Ile284 was deleted from pKK214.
Using the KKN279 primer, a segment encoding Lys26 through Ile284 was deleted from pKK150.
Strains were grown to early log phase in synthetic complete media lacking the appropriate component for plasmid selection. Formaldehyde (analytical grade; Merck) was added directly to 3–9 ml medium to 3.7% at 30°C for 90 min. After fixation, cells were collected by centrifugation and washed with 5 ml ice-cold 0.1 M potassium phosphate buffer, pH 7.5, for 5 min. Cells were resuspended and incubated in 90 µl 0.1 M potassium phosphate buffer, pH 7.5, containing 1.2 M sorbitol and 1 mg/ml zymolyase 20T (United States Biological Inc.) for 10–20 min at room temperature to digest the cell wall.
To terminate digestion, cells were washed twice in 0.1 M potassium phosphate buffer, pH 7.5, containing 1.2 M sorbitol. Cells were resuspended in 30 µl 0.1 M potassium phosphate buffer, pH 7.5, containing 1.2 M sorbitol and applied to a clean glass slide (precoated with 0.1% poly L-lysine) for 10 min at room temperature. Slides were washed once with ice-cold TBS buffer (50 mM Tris–HCl and 150 mM NaCl, pH 7.4) for 3 min, soaked in methanol (−20°C) for 6 min followed by acetone for 30 s, rinsed in ice-cold TBS buffer for 3 min, and air dried. Subsequent steps were performed at room temperature. 30 µl of blocking buffer (TBS buffer with 5% nonfat milk and 0.05% Tween 20) was added to each well and incubated for 30 min followed by a TBS buffer wash for 10 min. Primary antibodies HA.11 mAb (Covance) and polyclonal rabbit α-Kar2p were applied at 1:500 and 1:1,000 dilutions, respectively, in 30 µl of blocking buffer for 90 min. Wells were washed twice for 10 min with TBS buffer. 30 µl of blocking buffer containing Alexa Fluor 488 goat anti–mouse and Alexa Fluor 594 goat anti–rabbit (Invitrogen) as secondary antibodies was added to each well and incubated for 90 min in the dark. Slides were washed for 10 min twice with TBS buffer. Each well was applied with 15 µl of mounting medium (PBS buffer, pH 9.0, 90% glycerol, and 0.05 µg/ml DAPI) and a glass coverslip. Slides were sealed with clear nail polish and stored at −20°C in a dry and dark container.
Cells were visualized using an inverted microscope (LSM 510 META; Carl Zeiss, Inc.) with a Plan Apochromat 100× 1.4 NA Ph3 objective (Carl Zeiss, Inc.) in immersion oil (Immersol 518F; Carl Zeiss, Inc.) at room temperature. Image acquisition was performed using standard photomultiplier tube with LSM 510. Images were archived using LSM 5 Image Examiner (Carl Zeiss, Inc.) and Photoshop (version 7.0; Adobe), and no additional software adjustments were performed on images after acquisition other than cropping.
Cell labeling and immunoprecipitation (IP)
3.0 A600 OD units of cells grown to log phase were harvested by centrifugation and resuspended in 0.9 ml of synthetic complete media lacking methionine and cysteine. After a 30-min incubation at the appropriate temperature, 150 µCi [35S]methionine/cysteine (Pro-mix; GE Healthcare) was added to cells for 10 min. A chase was initiated by adding cold methionine/cysteine to a final concentration of 2 mM. Ice-cold TCA was added to a final concentration of 10% to terminate each time point. Cells were homogenized by the addition of 0.4 ml of 0.5-mm zirconium beads followed with agitation in a mini-bead beater cell disrupter two times for 30 s (Biospec Products). The homogenate was transferred to a fresh tube, pooled with a subsequent 10% TCA bead wash, and centrifuged at 18,000 g for 15 min. The pellet was resuspended in 120 µl of TCA suspension buffer (100 mM Tri-HCl, 3% SDS, and 3 mM DTT) and heated to 100°C for 5 min. Insoluble debris was pelleted, and 40 µl detergent lysate was added to 560 µl IPS II (13.3 mM Tris–HCl, 150 mM NaCl, 1% Triton X-100, 0.02% NaN3, 1 mM PMSF, and 1 µl protease inhibitor cocktail; Sigma-Aldrich) and the appropriate antiserum. After a 2-h incubation at 4°C, the sample was centrifuged for 15 min at 18,000 g, and the supernatant transferred to a fresh tube containing protein A–Sepharose beads. The tube was rotated for 1 h and washed three times with IPS I (0.2% SDS, 1% Triton X-100, 20 mM Tris–HCl, pH 7.4, 150 mM NaCl, and 0.02% NaN3) and once with TBS (20 mM Tris–HCl, pH 7.4, and 150 mM NaCl). Immunoprecipitated proteins were eluted with SDS gel sample buffer and resolved by gel electrophoresis. SDS-PAGE gels were exposed to phosphor screens for 24–48 h. Exposed screens were scanned using a phosphorimager (Typhoon; GE Healthcare) and quantified using ImageQuant TL software (GE Healthcare). All data plots reflect three independent experiments with SEM indicated with error bars.
Cycloheximide chase analysis
Cells were grown logarithmically at 30°C in synthetic media. Cessation of protein synthesis was initiated with the addition of cycloheximide to 200 µg/ml. 1.08 A600 OD U cells were collected at each time point by low speed centrifugation and resuspended in 1 ml 10% TCA. Cells were homogenized by agitation in a mini-bead beater cell disrupter with 0.4 ml 0.5-mm zirconium beads (Biospec Products). The homogenate was transferred to a fresh tube and pooled with a subsequent 10% TCA bead wash. After precipitation in 10% TCA, proteins were resuspended in urea SDS sample buffer (8 M urea, 5% SDS, 200 mM Tris–HCl, pH 6.8, 0.1 mM EDTA, and 100 mM DTT) and incubated for 30 min at room temperature. Detergent lysates were clarified by centrifugation (18,000 g for 10 min). Before loading on gels, proteins were incubated for 10 min at 37°C in sample buffer. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated in blocking buffer provided by the manufacturer (LI-COR Biosciences). After incubation with anti-Myc and anti-hexokinase antibodies, membranes were washed in PBS containing 0.1% Tween 20. Secondary antibodies conjugated to IRDye 680 (infrared dye) or 800 were diluted 15,000-fold in blocking buffer containing 0.1% Tween 20. After washing, membranes were scanned for direct infrared fluorescence using an infrared imaging system (Odyssey) and quantified using the manufacturer’s software (LI-COR Biosciences).
Induction of CPY* expression by galactose
Wild-type strains expressing CPY* using the GAL1 promoter (or empty vector as control) were preincubated overnight in synthetic complete media with 3% raffinose as the sole carbon source. Cells were harvested by low speed centrifugation (3,000 g for 5 min), inoculated in synthetic complete media with 2% galactose as carbon source, and grown to log phase. 9.0 OD600 equivalents of cells were harvested by low speed centrifugation (3,000 g for 5 min) and resuspended in 3.0 ml synthetic complete media with 2% galactose followed by metabolic pulse-chase analysis.
Purification of proteins for mass spectrometry
Strains were grown to late log phase in synthetic complete media lacking uracil. 4,000 OD600 equivalents of cell culture were harvested by low speed centrifugation (3,000 g for 10 min) and washed once in ice-cold water. The cells were collected by centrifugation (3,000 g for 10 min), and the pellet was resuspended in 40 ml ice-cold 50 mM Tris–HCl, pH 7.4. The cells were collected by centrifugation (3,000 g for 10 min) and transferred in a mortar filled with liquid nitrogen. The cells were ground by a pestle to a fine powder in liquid nitrogen. The disrupted cells were resuspended in 20 ml Tris-–IP buffer (50 mM Tris–HCl, pH 7.4, and 150 mM NaCl) containing 1.0 mM PMSF. After a low speed spin (800 g for 5 min) to pellet debris, the lysate was subjected to ultracentrifugation (30,000 g for 30 min). The pellet was resuspended in 20 ml Tris–IP buffer containing 0.5% Triton X-100 and 1 mM PMSF on ice for 1 h. The solubilized microsome fraction was collected by ultracentrifugation (30,000 g for 10 min). 50 µl agarose-immobilized anti-HA antibody (Santa Cruz Biotechnology, Inc.) was added to the supernatant and incubated at 4°C with rocking for 2 h. The beads were washed gently three times in ice-cold Tris–IP buffer with 0.5% Triton X-100 and once in ice-cold Tris–IP buffer. Proteins were eluted from the beads by boiling in SDS loading buffer. Proteins were boiled for 5 min, resolved by SDS-PAGE, and visualized by Coomassie brilliant blue staining. Protein bands were excised for in-gel trypsinization and identified by matrix-assisted laser desorption/ionization mass spectrometry/mass spectrometry (National University of Singapore Mass Spectrometry Laboratory). Analysis of ngPrAΔ295–331-binding proteins is shown in Table S1.
Online supplemental material
Fig. S1 shows PrA variants with reduced dependence on the glycan-dependent ERAD factor Htm1p for degradation. Fig. S2 shows the degradation profile of the ERAD-M substrate Hmg2p (single Myc tagged). Table S1 lists the peptide mass data of the ngPrAΔ295–331-binding protein. Tables S2–S4 list the strains, plasmids, and oligonucleotide primers used in this study. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200907055/DC1