Assays for studying normal versus suppressive ERAD-associated retrotranslocation pathways in yeast

Summary In S. cerevisiae, we identified rhomboid pseudoprotease Dfm1 as the major mediator for removing or retrotranslocating misfolded membrane substrates from the ER (endoplasmic reticulum). Long-standing challenges with rapid suppression of dfm1-null cells have limited the biochemical study of Dfm1’s role in ER protein quality control. Here, we provide a protocol for the generation and handling of dfm1-null cells and procedures for studying normal vs. suppressive alternative retrotranslocation pathways. Our methods can be utilized to study other components involved in retrotranslocation. For complete information on the generation and use of this protocol, please refer to Neal et al. (2017, 2018); Neal et al. (2019); Neal et al. (2020).

. Schematic overview of the knock-out strategy by the one-step PCR method Primers are designed to contain ''homologous arms'' that are upstream and downstream of the Dfm1 ORF. (A) An antibiotic gene cassette (e.g., KanMx) is amplified by PCR and the PCR product is transformed to competent yeast cells. Homologous recombination will replace the targeted Dfm1 ORF with the KanMx cassette. (B) Representative gel image of PCR diagnostics on WT negative control strain, dfm1D positive control strain, and dfm1D candiates 1-8. Diagnostic with Primers C and D will yield $150 bp product whereas Primers E and F will yield $200 bp product.
16. For transformants breeding true, inoculate in 3 mL of YPD, rotate overnight (16-20 h) at 30 C and once cells reach saturation (OD 600 $3.0), freeze all cultures immediately in 15% DMSO in 1.5 mL cryo-storage tubes at À80 C. 17. For confirmation of Dfm1 knockout, isolate genomic DNA from transformant as described in (Harju, Fedosyuk and Peterson, 2004). We typically obtain $200 ng/mL with a 260 nm/280 nm absorption of$1.8 ,which indicates pure DNA. The genomic DNA is diluted to 10 ng/mL in deionized sterile water and 1 mL (10 ng) is used for PCR diagnostic amplification. Knockout was confirmed with forward primer C, reverse primer D, forward primer E and reverse primer F (Figures 1A and 1B & key resources table) that are outside of Dfm1 locus and within the KanMx gene.
Note: For PCR amplification, it is also critical to include a negative control, wild-type genomic DNA. Furthermore, DFM1 knockout efficiency is $60%. Accordingly, by screening at least 10 colonies, we typically confirm knockout from $5-6 colonies.
CRITICAL: Because dfm1D cells have been shown to suppress, it is imperative that once transformants are obtained, that you freeze them (we usually freeze 10 transformants) and once validated, a representative set of 2-3 strains are transferred to the lab's yeast storage collection. For freezing, prepare an overnight culture by inoculating 5 mL of YPD with a single colony of transformant and grow overnight (16-20 hours) with rotation at 30 C. Once cultures reach saturation growth (OD 600 $3.0), add 850 mL of culture to 150 mL of DMSO (15%) in 1 mL cryo-storage tubes and place in À80 C freezer.
For additional advice for generating dfm1D strains, see troubleshooting section below.
Day 1: 18. Thaw out fresh dfm1D-null cells from À80 C freezer by plating on YPD plate. Incubate plates at 30 C for $3 days. 19. While dfm1D cells are growing, linearize a yeast integration plasmid pSN105 (key resources table) containing GALpr-Hmg2-GFP by digesting 3 mg of plasmid with restriction digest enzyme (total volume 150 mL) that cuts once at the marker gene (e.g., StuI enzyme was used in our case for the ADE2 marker). Accordingly, the linearized plasmid should integrate at the ade2-101 locus. 20. Check for linearization via 1% agarose gel and quench digestion reaction by incubating at 60 C for 20 min.
Day 4: Prepare dfm1D competent cells by following steps 3-6 in major step above ''Generating dfm1D-null yeast strains.'' Day 6: 21. Mix 100 mL of yeast competent cells, 50 mg fish sperm DNA, and 50 mL of digestion reaction containing 1 mg of linearized plasmid in a 1.5 mL Eppendorf tube. Incubate for 20 min at room temperature (20 C-25 C).

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22. Add 700 mL of 40% PEG-TEL solution, vortex for couple seconds (setting on high) and incubate for 40 min at room temperature (20 C-25 C). 23. Heat shock samples by placing samples in a water bath set at 42 C for 7 min. Immediately after heat shock, spin sample briefly for 10 s (set at 10,000 3 g). Discard supernatant and resuspend pellet in 50 mL of sterilized deionized water. 24. Add sterilized glass beads to SC-His plates ($10 beads per plate) followed by addition of suspension cells. Spread cells by shaking plates for several minutes. Incubate the plates at 30 C for $2-3 days.
Day 9: 25. Once colonies are visible on plates, streak transformants as wagon wheels on another SC-His plate to ensure transformants breed true.
CRITICAL: Because dfm1D cells have been shown to suppress, it is imperative that once transformants are obtained, that they are frozen immediately (we usually freeze 10 transformants) and once validated, transfer a representative 2-3 strains to the lab yeast storage collection.
Check for integration by growth in media and 0.2% galactose induction of GALpr-Hmg2-GFP by flow cytometry (To use flow cytometry, see major step ''Flow Cytometry to analyze for restored membrane substrate, Hmg2-GFP, degradation'' below. Note: Because multiple integrations can occur in a single transformation, this step is critical for scanning transformants with a single integrant of GALpr-Hmg2-GFP (see Figure 2).
26. For transformants breeding true, inoculate in 3 mL of minimal media-His and freeze all cultures immediately with 15% DMSO in 1.5 mL cryo-storage tubes.
Note: See Troubleshooting section below for common problems and solutions that arise in yeast transformation.

MATERIALS AND EQUIPMENT
The following reagents can be prepared ahead of time.
Note: Cycloheximide solution waste should be disposed by appropriate hazardous waste procedures.
Note: G418 and Tryptophan shouldn't be exposed to light upon storage.
Note: Please refer to the product information for the shelf lives of the individual reagents listed here.

Reagent Final concentration Amount
Yeast nitrogen base n/a 14 g

STEP-BY-STEP METHOD DETAILS
Culturing and passaging dfm1D-null +GAL pr -Hmg2-GFP cells to suppression

Timing: [2-3 weeks]
This section outlines how dfm1D-null yeast cells containing GAL pr -Hmg2-GFP are passaged into fresh minimal media overtime to generate completely suppressed cells with restored ERAD-M retrotranslocation ( Figure 3).  8. Dilute cells to OD 600 $0.1 in total volume of 3 mL of minimal media -His supplemented with 1.8% raffinose/0.2% galactose and incubate at 30 C with rotation for 24 h or until cells are grown to saturation (OD 600 >1). Designate this new culture as (P1).

Day 6-21
Continue passaging until dfm1D-null cells are completely suppressed (see major step below ''Flow Cytometry to analyze for restored membrane substrate, Hmg2-GFP, degradation'' for details on how to analyze cells for suppression). We typically see complete suppression by P10.
CRITICAL: It is important to passage cells immediately after culture reach saturation phase. Sustained growth in saturation phase makes it difficult for cells to recover after dilution. Refer to troubleshooting section below.
CRITICAL: For growth, minimal medium is used rather than YPD to reduce background fluorescence which will not interfere with flow cytometry fluorescent readouts. This section outlines how Hmg2-GFP steady-state levels can be measured by flow cytometry throughout different passaging stages of dfm1D-null cells.
Controls and samples used: Non-induced control: cells are passaged continuously in the presence of glucose Degraded Hmg2 control: WT strains + GAL pr -Hmg2-GFP are passaged in induced galactose condition. This is a control for what steady-state Hmg2 levels would be when undergoing ERAD degradation. Stabilized Hmg2 control: cdc48-2 + GAL pr -Hmg2-GFP passaged in induced galactose condition. This is a retrotranslocation-deficient control for what stabilized steady-state levels of Hmg2 would be. Sample 1: P0 dfm1D + GAL pr -Hmg2-GFP is non-passaged and non-suppressed sample, which should look like control #3. Sample 2: passaged dfm1D + GAL pr -Hmg2-GFP is suppressed strain, which should look like control #2.
Biological triplicates were used for each strain and flow cytometry analysis was performed as technical triplicates.
Day 1 9. Thaw, inoculate, and passage dfm1D-null yeast cells containing GALpr-Hmg2-GFP as indicated above. 10. For P0 analysis of Hmg2-GFP levels: Once dfm1D-null yeast cells containing GALpr-Hmg2-GFP have been induced with galactose for at least two hours and grown to OD 600 $0.4, take 300 mL of P0 cells and analyze for mean fluorescence levels using BD Biosciences FACS Calibur flow cytometer. 11. Adjust the following settings before analyzing samples on flow cytometer ( Figure 4A): Run Settings: Run with limits, 10,000 events Fluidics flow rate: Medium 12. Run samples by aliquoting 300 mL of suspended cells into sample tubes and placing the tube on sample collector. 13. Data acquisition: For each run, create two plots: density (side scatter SSC vs. forward scatter FSC) and histogram plots (Cell Count vs. 530 filter for GFP FITC-A). 14. Draw a gate around the population of intact yeast cells and display gated population as a histogram (Cell count vs. 530 filter for GFP FITC-A) to obtain the mean fluorescence of the gated population ( Figures 4B and 4C). 15. For analysis of other passages, once cells are passaged and diluted in fresh minimal media, allow cells to double to OD 600 $.4 and analyze for mean fluorescence via flow cytometer.
CRITICAL: Analyzing cells in saturated growth phase will yield large cellular debris and an abnormal distribution of fluorescence. Accordingly, it is important to analyze cells in the log-phase. Finally, all flow cytometry readings were performed directly from minimal media since it has negligible background fluorescence. Spot growth assay of non-suppressed and suppressed dfm1D-null cells

Timing: [3-4 weeks]
This section outlines how spot growth assay can be utilized to demonstrate normal growth for suppressed dfm1D-null cells vs. a growth defect in non-suppressed dfm1D -null cells ( Figure 5).

Controls used
Negative control: WT and cdc48-2 strains expressing both GAL pr -Hmg2-GFP and empty vector. Negative control: dfm1D expressing empty vector.
Biological triplicates were used for each strain and the growth assay was performed as technical triplicates. Day 5 20. Dilute cells to OD 600 $0.1 and incubate with rotation at 30 C for 4-5 h; allowing cells to double or grow to an early log-phase with OD 600 $0.2-0.3.

Day 6
21. Pellet 0.12 OD of cells by spinning at 14,000 3 g for 2 min at room temperature and resuspending pellets in 1 mL of sterilized dH 2 O. 22. Transfer 250 mL of each sample to a 96-well plate and perform a five-fold serial dilution in dH 2 O of each sample to obtain a gradient of 0.03-0.0000096 OD cells ( Figure 5). 8. Pin the cells using the 8312 pinning apparatus onto synthetic complete (-His) agar plates supplemented with 2% dextrose or 2% galactose. 23. Air-dry the pinned droplets of cells under the flame in sterile conditions, seal the plates with parafilm and incubate at 30 C.
Note: To make the droplets absorb faster and avoid droplet from running along the plate, it is important to pre-dry the plates with lids off in the Biosafety Cabinet or under a flame before spotting.
24. Remove the plates from the incubator for imaging with the ChemiDoc Imager (Setting: UV-Trans) or a camera as an alternative on Day 3 and 7. In vivo retrotranslocation assay of suppressed and non-suppressed dfm1D-null cells
Biological replicates were used for each strain and the in vivo assay was performed as technical triplicates.
Day 5: 30. Dilute cells to OD 600 $0.1 in fresh minimal media supplemented with 2% raffinose (total vol-ume= 50 mL in 250 mL Erlenmeyer flask) and incubate with shaking at 30 C for 4 h; allowing cells to double or grow to an early log-phase (OD 600 $0.2-0.3). Figure 6. Strongly expressed integral membrane substrates cause a growth defect in P0 nonsuppressed dfm1D cells and restored growth in P11 suppressed dfm1D cells Non-passaged dfm1D cells (P0) or cells passaged to suppression (P11) were assessed for growth defect in the dilution assay by spotting 5-fold dilutions of cells on galactose-containing plates to drive Hmg2-GFP overexpression, and plates were incubated at 30 C.

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31. Once cells have grown to early-log phase (OD 600 $0.2-0.3), remove 3 mL of cells suspension and add to culture tube. Continue passaging cells as outlined in major step above ''Culturing and passaging dfm1D-null +GAL pr -Hmg2-GFP cells to suppression'' (Steps 6-8) until cells are completely suppressed. 32. Non-suppressed, non-passaged cells: For the rest of the culture ($47 mL), add MG132 at a final concentration of 25 mg/mL and incubate for 1 h with shaking for 2 h.
Note: MG132 is a proteasome inhibitor that allows for accumulation of retrotranslocated HMG2 in cytosol and easier detection on western blot. We typically obtain efficient inhibition by MG132 with 1-2 hours of incubation.
33. After incubation with MG132 pellet 15 OD of cells in 50 mL falcon tubes by centrifuging at 1,000 3 g for 5 min in room temperature. 34. Discard supernatant and resuspend the pellets in sterile deionized water and centrifuge at 1,000 3 g for 5 min in room temperature. Pause point: At this point, cells can be stored in À80 C freezer and for future use until suppressed passaged cells are ready for analysis in the retrotranslocation assay.
35. Passaged suppressed cells: Once cells are suppressed through continued galactose induction during passaging, dilute cells to OD 600 $0.1 in fresh minimal media supplemented with 1.8% raffinose/ 0.2% galactose (total volume= 50 mL in 250 mL Erlenmeyer flask) and incubate with shaking at 30 C for 4 h; allowing cells to double or grow to an early log-phase (OD 600 $0.2-0.3). Follow steps 43-45. 36. Resuspend passaged and non-passaged cell pellets in 400 mL of sterile deionized water and prepared for bead lysis by dividing samples into 432 mL Eppendorf tubes ($100 mL of cell suspension per tube). Spin tubes at 10,000 3 g rpm for 2 min at room temperature and aspirate supernatant. 37. Resuspend each pellet with 100 mL of MF buffer supplemented with the following protease inhibitors 1 mM PMSF, 260 mM AEBSF, 100 mM leupeptin, 76 mM pepstatin, 5 mM aminocaproic acid, 5 mM benzamidine, and 142 mM TPCK.
Note: phenylmethylsulphonyl fluoride (PMSF) is prepared fresh for each experiment.
38. Add 0.5 mM silicone beads to meniscus of cell suspension. Vortex samples on multi-vortexer set at top speed for 6 3 1-min intervals with 10 min intervals on ice between each vortexing. 39. Check cells under the microscope (20 3 magnification) for lysis efficiency.
Note: Lysed cells are distinguishable by their fragmented shape and for optimal yield it is critical to achieve $80-90% lysis efficiency. See troubleshooting section below for more details on lysing.
CRITICAL: At the point, samples and all solutions should be kept on ice.
40. Add 100 mL of chilled MF buffer with PIs to each eppendorf tube and combine lysates by transferring to a new 1.5 mL Eppendorf tube with a 1 mL pipette. Centrifuge at 2,500 3 g for 5 min at 4 C to remove cell debris. 41. Transfer clarified supernatant to ultracentrifugation tube. 42. Ultracentrifuge the clarified lysate at 100,000 3 g for 15 min at 4 C to separate the pellet microsome fraction (P) and cytosolic supernatant fraction (S). 43. Resuspend pellet in 200 mL SUME buffer with PIs and NEM. 44. Add 600 mL of IPB with PIs and NEM to the (S) fraction and resuspended (P) fraction. 45. For immunoprecipitation of Hmg2-GFP add 15 mL of rabbit polyclonal anti-GFP antisera to the (P) and (S) samples. 46. Incubate the samples on ice for 5 min, spin at 14,000 3 g for 5 min, and remove the supernatant to a new 1.5 mL eppendorf tube and incubate overnight with gentle mixing using a nutator at 4 C.
Note: For equilibrating Protein A-Sepharose, do this before you begin the retrotranslocation assay: Add Protein-A Sepharose to 50 mL Falcon tube. Fill tubes with $40 mL of deionized water and place tube on ice. Once beads settle to the bottom, carefully pour out water (it is ok to leave residual water). Repeat these 6 times with deionized water. After final rinse with water, add 6 mL of IPB. This solution is suitable for storage at 4 C for future use.
47. Add 100 mL of equilibrated Protein A-Sepharose to the samples and incubate for 2 h at 4 C with gentle mixing using a nutator. 48. Wash Protein A-Sepharose beads twice by adding 900 mL of IPB followed by brief spin of 1,000 3 g for 30 s at room temperature and aspirating supernatant with an 18-gauge syringe needle. Repeat again with addition of IPB to beads. 49. Wash beads once more by adding 900 mL of IPW followed by brief spin of 1,000 3 g for 30 s at room temperature and aspirating beads to dryness using a 30-gauge syringe needle.

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50. Resuspend beads in 60 mL of 23 urea sample buffer and solubilize samples by incubating at 55 C for 10 min. 51. Spin samples at 14,000 3 g for 5 min at room temperature. The eluted proteins are removed to a new tube. 52. Eluted proteins are resolved by SDS-PAGE using 8% gels, transferred to nitrocellulose membrane by electroblotting at 15 mAmp for 15 min using a TransBlot. 53. Immunoblot with monoclonal anti-ubiquitin (1:4,000 dilution) anti-GFP (1:10,000 dilution) along with Goat anti-mouse (Jackson ImmunoResearch, West Grove, PA) conjugated with horseradish peroxidase (HRP) recognized the primary antibodies. Immunoblotting was carried as described in (Neal et al., 2019).

EXPECTED OUTCOMES
Under ''Flow Cytometry to analyze for restored membrane substrate, Hmg2-GFP, degradation,'' Hmg2-GFP levels range from being stabilized to being degraded as dfm1-null cells are being suppressed. For example, P0 cells should have stabilized Hmg2-GFP levels with a mean fluorescence of $50K. P6 cells should have mixed population of cells with mean fluorescence of $50K and $20K. Finally, P10 cells and on should have majority of cells suppressed in which Hmg2-GFP levels are restored to degradation levels of $20K ( Figure 4D).
Under ''Spot growth assay of non-suppressed and suppressed dfm1D-null cells,'' no growth defect should be observed in passaged suppressed dfm1D cells (P11) in comparison to non-passaged nonsuppressed dfm1D cells (P0) (Figure 6) demonstrating that suppressed dfm1D null strains with alleviated retrotranslocation function have normal growth fitness.
Under ''In vivo retrotranslocation assay of suppressed and non-suppressed dfm1D-null cells,'' nonpassaged dfm1D P0 shows the typical buildup of ubiquitinated Hmg2-GFP in the pellet fraction in both untreated and MG132 treated cells (Figure 8). In striking contrast, suppressed P11 dfm1D cells shows normal Hmg2-GFP retrotranslocation, with buildup of ubiquitinated Hmg2-GFP is observed in both the pellet and supernatant fraction in MG132 treated cells.

LIMITATIONS
dfm1D-null strains are susceptible to suppression. Many factors that can trigger and influence the rate of suppression included multiple rounds of yeast transformations, growth at high temperatures and strong expression of ERAD membrane substrates. As such, experiments requiring use of temperature-sensitive mutants or involving multiple rounds of transformations will trigger suppression and will pose a challenge in studying Dfm1's contributions to ERAD. Below we outline tips, focused on handling dfm1-null strains. Figure 8. In vivo Hmg2-GFP retrotranslocation completely restored dfm1D suppressed cells Crude lysate was prepared from the indicated strains treated with vehicle or MG132 (25 mg/mL). Lysates were ultracentrifuged to discern ubiquitinated Hmg2-GFP that either has been retrotranslocated into the soluble fraction (S) or remained in the membrane (P). Following fractionation, Hmg2-GFP was immunoprecipitated from both fractions, resolved on 8% SDS-PAGE and immunoblotted with a-GFP and a-Ubi. Adapted from (Neal et al., 2018).

TROUBLESHOOTING Problem 1
Homologous recombination only occurs at <30% with 50 bp of homolog arm to the DFM1 gene. This can pose a challenge for labs that are new to yeast transformations as transformation efficiency can range widely across different laboratories.

Potential solution
To improve targeted gene efficiency, the homology arm can be increased from 50 bp to 100 bp. Alternatively, an existing dfm1D-null strain can be used from the yeast knockout collection (available by Dharmacon) and the deletion KanMx cassette can be amplified with 0.5-1 kb of homology arm to the Dfm1 gene via amplification by PCR of the genomic data. This method has improved homologous recombination efficiency to >70%. Alternatively, any other strain with Dfm1 knocked out can be used as opposed to the yeast knockout collection.

Problem 2
High background colonies are present in no DNA control transformation with antibiotic selection.

Potential solution
Check if the yeast strain already contains the antibiotic resistance marker. For growth on antibiotic selection plates, it is possible that incorrect amount of antibiotic stock was added for making plates.
Plates with the appropriate amount of antibiotics should be remade.

Problem 3
No colonies on plates after yeast transformation.

Potential solution
You can increase DNA amount to 2-3 mg per transformation, increase incubation time of competent cells with DNA, or remake PEG solution (barring the possibility that the solution was made incorrectly). For growth on antibiotic plates, it is suggested to increase recovery time incubation on YPD plates (up to 36 h) before being replica plated onto antibiotic selections plates.

Problem 4
For non-induced conditions in raffinose, you still get basal levels of GAL promoter activity; increasing the tendency for dfm1D-null strains to suppress.

Potential solution
For non-induced conditions, you can alternatively grow cultures in 2% glucose instead of 2% raffinose. In this case, glucose completely represses the GAL promoter. Prior to induction, rinse cells with sterile deionized water three times before transferring cells to minimal media supplemented with 1.8% raffinose/0.2% galactose.

Problem 5
In the in vivo retrotranslocation assay, low lysing efficiency of cells can yield low overall western blot signal with anti-GFP and anti-ubiquitin.

Potential solution
Lysis efficiency can be evaluated under the microscope using 203 magnification. Lysed cells are clearly distinguishable by their fragmented shape; for optimal yield it is critical to achieve $80%-90% lysis efficiency. If below this range, continue to vortex for up to three more 1-min cycles at 4 C. We use either 0.5 mm glass-based or silica-based beads for lysis (Biospec Products), but the lysis efficiency with silica-based beads appears to be somewhat higher.

Problem 6
Ubiquitin signal is low in in vivo retrotranslocation assay.

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Potential solution 1) Ubiquitination is reversible and this modification can therefore easily be eliminated by deubiquitinases (DUBs). For this reason, it is essential to include DUB inhibitors such as NEM in the buffers used during the long incubation times used for immunoprecipitation in order to preserve the state of substrate ubiquitination (most DUBs are cysteine proteases that are inhibited by NEM).
2) For anti-ubiquitin blots, it is important to note that ubiquitin is small and difficult to denature and the ubiquitin epitopes might not be accessible to antibodies due to insufficient denaturation during SDS-PAGE or renaturation on the membrane. Therefore, after transfer to nitrocellulose membranes, the signal strength of anti-ubiquitin antibodies can frequently be enhanced significantly if the membrane is subjected to a denaturing treatment prior to blocking. Accordingly, the membranes are rinsed with water, sandwiched between sheets of Whatman paper, and placed in a glass dish. Deionized water is added to the dish and the membrane is boiled in a microwave oven at 3 3 1-min intervals; periodically check in between intervals to ensure that the water has not evaporated. This brings about a remarkable increase in signal strength, presumably due to revealing of cryptic epitopes from the heat

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Sonya Neal(seneal@ucsd.edu).

Materials availability
Plasmids and yeast strains used in this study are available from our laboratory.

Data and code availability
Original/source data for figures in the paper is available upon request. Original data have been deposited to Mendeley Data: https://doi.org/10.17632/ym9mtgmrwh.1.