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Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-.
Introduction
The endoplasmic reticulum (ER) is the organelle wherein secretory and membrane proteins are synthesized. Most of the polypeptides synthesized in the ER are covalently modified with N-linked oligosaccharides. The folding of these glycoproteins is monitored and assisted by chaperone proteins and lectins in the ER. Correctly folded proteins are sorted out of the ER into secretory compartments, whereas misfolded polypeptides or unassembled subunits are retained in the ER. After several attempts to correctly fold these polypeptides, terminally misfolded proteins are retro-translocated out of the ER and degraded by the cytoplasmic proteasome. This mechanism is termed ER-associated degradation (ERAD) (1–4) (Figure 1).
Many steps are required for ERAD, including recognition of misfolded ERAD substrates, extraction of these proteins from the ER to the cytosol, polyubiquitination, deglycosylation, and finally protein degradation by the proteasome. To analyze the function of molecules involved in these processes, the degradation of ERAD substrates transfected into mammalian cells is monitored.
Protocol
Here, two kinds of protocol will be described to detect the degradation of ERAD substrates in mammalian cells: 1) pulse-chase experiment and 2) cycloheximide (CHX)-chase experiment. It is useful to add proteasome inhibitors to confirm the degradation of the substrate by ERAD pathway. The degradation kinetics estimated by pulse-chase and CHX-chase experiments of the same substrate sometimes differ (5). In pulse-chase experiments, the degradation kinetics of proteins synthesized during the pulse-labeling period are monitored, whereas in CHX-chase experiments, all ERAD substrates retained in the cells are detected. Furthermore, all protein synthesis is halted by the addition of CHX, including secretory proteins newly synthesized in the ER, which may affect the ER protein quality control machinery.
Materials
- 1.
Transfection reagents (Lipofectamine 2000 [Invitrogen, Waltham, MA, USA], PEI [polyethylenimine] [Sigma-Aldrich, St. Louise, MO, USA], and others)
- 2.
[35S]-methionine or [35S]-protein labeling mixture
- 3.
Dulbecco’s modified eagle medium (DMEM) lacking methionine/cysteine
- 4.
Dialyzed fetal bovine serum (FBS)
- 5.
Dulbecco’s phosphate-buffered saline (PBS)
- 6.
Protein A- or Protein G-Sepharose beads (GE Healthcare Life Science, Uppsala, Sweden and others)
- 7.
Cycloheximide
- 8.
Proteasome inhibitors (lactacystin, MG-132, epoxomicin, and others)
- 9.
Lysis buffer (1% NP-40, 150 mM of NaCl, and 50 mM of Tris-HCl [pH 7.5] supplemented with protease inhibitors)
- 10.
Protease inhibitors (2 mM of N-Ethylmaleimide (NEM), 0.2 mM of AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride), 1 μg/mL of pepstatin, and 1 μg/mL of leupeptin)
- 11.
High ionic wash buffer (1% NP-40, 400 mM of NaCl, and 50 mM of Tris-HCl [pH 7.5])
- 12.
Imaging plate
Instruments
- 1.
CO2 incubator
- 2.
Radioisotope experiment facility
- 3.
Rotator
- 4.
Phosphorimager
- 5.
Luminoimage analyzer
Methods
- 1.
Pulse-chase experiment
- a.
Plate cells on a 3.5-cm tissue culture dish.
- b.
Transfect plasmids encoding an ERAD substrate and proteins of interest (Note 1).
- c.
After washing once with prewarmed PBS, incubate cells in DMEM lacking methionine/cysteine supplemented with dialyzed FBS for 15–30 min.
- d.
Pulse-label cells with [35S]-methionine/cysteine for 15 min in DMEM lacking methionine/cysteine supplemented with dialyzed FBS (Note 2).
- e.
Remove the medium, and incubate cells in a normal growth medium for the time required.
- f.
After washing cells twice with PBS, harvest and extract cells by adding a buffer containing an appropriate detergent to the dish and keep on ice for 20 min (Note 3).
- g.
Collect cell lysate using a rubber policeman, centrifuge at 13,000 ×g at 4°C for 20 min, and transfer the supernatant to a new microtube.
- h.
Add ~30% (v/v) of glycerol and antibodies, and incubate at 4°C for the time required (Note 4).
- i.
Add Protein A- or Protein G-Sepharose beads (bed volume of 10–20 μL).
- j.
Rotate at 4°C for 1–2 h.
- k.
Collect beads by centrifugation at 1,500 ×g at 4°C for 3 min.
- l.
Wash beads twice with high ionic wash buffer and collect beads as in Step k.
- m.
Elute immunoprecipitates with Laemmli sample buffer.
- n.
Separate proteins by SDS-PAGE.
- o.
Dry the gel and expose to an imaging plate (Note 5).
- p.
Analyze using a phosphorimager (Figure 2).
- 2.
CHX-chase experiment
- a.
Plate cells on a 3.5-cm tissue culture dish.
- b.
Transfect plasmids encoding an ERAD substrate and proteins of interest.
- c.
Add 50–100 μg/mL of CHX to the medium (Note 6).
- d.
After washing cells twice with PBS, harvest and extract cells by adding a buffer containing an appropriate detergent to the dish and keep on ice for 20 min.
- e.
Collect cell lysate using a rubber policeman, centrifuge at 13,000 ×g at 4°C for 20 min, and transfer the supernatant to a new microtube.
- f.
Add 2× Laemmli sample buffer to cell extracts (final; in 1× Laemmli sample buffer), and separate proteins by SDS-PAGE.
- g.
Transfer the separated proteins in the gel to a nitrocellulose or a polyvinylidene fluoride membrane.
- h.
Detect specific proteins using appropriate antibodies (Western blotting) (Figure 3).
Notes
- 1.
Approximately 1 μg of plasmid encoding an ERAD substrate is used. For the co-transfection with other plasmids, the amount of each plasmid should be adjusted to obtain the appropriate expression level of each protein. The confluency of cells at the time of transfection should be modified as recommended for transfection reagents.
- 2.
Normally 8.2 MBq/mL of [35S]-protein labeling mixture is used for pulse labeling.
- 3.
Other detergents, such as CHAPS and digitonin, are used.
- 4.
The amount of antibodies used and the period of incubation required to immunoprecipitate proteins vary and depend on the antibodies. Glycerol was added to increase the stability of proteins in the lysate.
- 5.
The period required for exposure depends on the radioactivity incorporated, the quality of imaging plate, and the sensitivity of the phosphorimager.
- 6.
The concentration of cycloheximide added should be optimized depending on the cell lines used based on previous publications.
References
- 1.
- Helenius A, Aebi M. Roles of N-Linked Glycans in the Endoplasmic Reticulum. Annu Rev Biochem. 2004;73:1019–49. [PubMed: 15189166]
- 2.
- Smith MH, Ploegh HL, Weissman JS. Road to ruin: targeting proteins for degradation in the endoplasmic reticulum. Science. 2011;334(6059):1086–90. [PMC free article: PMC3864754] [PubMed: 22116878]
- 3.
- Preston GM, Brodsky JL. The evolving role of ubiquitin modification in endoplasmic reticulum-associated degradation. Biochem J. 2017;474(4):445–69. [PMC free article: PMC5425155] [PubMed: 28159894] [CrossRef]
- 4.
- Amm I, Sommer T, Wolf DH. Protein quality control and elimination of protein waste: the role of the ubiquitin-proteasome system. Biochim Biophys Acta. 2014;1843(1):182–96. [PubMed: 23850760] [CrossRef]
- 5.
- Wu Y, Termine DJ, Swulius MT, Moremen KW, Sifers RN. Human endoplasmic reticulum mannosidase I is subject to regulated proteolysis. J Biol Chem. 2007;282(7):4841–9. [PMC free article: PMC3969733] [PubMed: 17166854]
- 2.
- Hirao K, Natsuka Y, Tamura T, Wada I, Morito D, Natsuka S, Romero P, Sleno B, Tremblay LO, Herscovics A, Nagata K, Hosokawa N. EDEM3, a soluble EDEM homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming. J Biol Chem. 2006;281(14):9650–8. [PubMed: 16431915]
- 3.
- Hattori T, Hanafusa K, Wada I, Hosokawa N. SEL1L degradation intermediates stimulate cytosolic aggregation of polyglutamine-expanded protein. FEBS J. 2021;288(15):4637–54. [PubMed: 33576152] [CrossRef]
Footnotes
The authors declare no competing or financial interests.
Figures
Figure 1:
Scheme of endoplasmic reticulum-associated degradation (ERAD).
Figure 2:
Pulse-chase experiment of an endoplasmic reticulum-associated degradation (ERAD) substrate NHK (α1-antitrypsin variant null Hong Kong). HEK (human embryonic kidney) 293 cells were transfected with plasmids encoding NHK, together with an empty vector or EDEM3-HA, and labeled with [35S]-protein labeling mixture for 15 min. NHK and EDEM3 were immunoprecipitated with an anti-α1-antitrypsin antibody (lanes 1–6) or with an anti-HA-tag antibody (lanes 7–9). Immunoprecipitates were separated on a 10% SDS-PAGE gel (A), and radioactive signal was quantified (B). Cells were treated with 20 μM of lactacystin for 3 h before pulse labeling (lanes 4–6, indicated as +) or were untreated (lanes 1–3, indicated as −) (C). This figure was originally published in (6).
Figure 3:
CHX-chase experiment of NHK. HEK293 cells co-transfected with NHK, HRD1-myc, and S-SEL1L were chased for the indicated period after the addition of CHX. The cell lysate was separated by 10% SDS-PAGE and analyzed by Western blotting. The signal intensity of NHK is quantified in the graph. This figure was originally published in (7).