Analysis of Disulfide Bond Formation

Ineke Braakman1, Lydia Lamriben2, Guus van Zadelhoff1, Daniel N. Hebert2

1 Cellular Protein Chemistry, Bijvoet Center for Biomolecular Research, Faculty of Science, Utrecht University, Utrecht, 2 Department of Biochemistry and Molecular Biology, Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 14.1
DOI:  10.1002/cpps.43
Online Posting Date:  November, 2017
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Abstract

In this unit, protocols are provided for detection of disulfide bond formation in cultures of intact cells and in an in vitro translation system containing isolated microsomes or semi‐permeabilized cells. First, the newly synthesized protein of interest is biosynthetically labeled with radioactive amino acids in a short pulse. The labeled protein then is chased with unlabeled amino acids. At different times during the chase, a sample is collected, membranes are lysed with detergent, and the protein is isolated by immunoprecipitation, as described. A support protocol is provided for analysis of disulfide bonds in the immunoprecipitates by SDS‐PAGE with and without prior reduction. The difference in mobility observed between the gels with nonreduced and reduced samples is due to disulfide bonds in the nonreduced protein. An additional support protocol is included that uses PEG‐maleimide to modify free thiols and follow disulfide‐bond formation by SDS‐PAGE. © 2017 by John Wiley & Sons, Inc.

Keywords: disulfide bonds; endoplasmic reticulum; protein folding; secretory pathway; pulse‐chase; radiolabeling

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Analysis of Disulfide Bond Formation in Intact Monolayer Cells
  • Alternate Protocol 1: Analysis of Disulfide Bond Formation in Cells in Suspension
  • Alternate Protocol 2: Analysis of Postponed Post‐Translational Disulfide Bond Formation in Intact Cells
  • Basic Protocol 2: Analysis of Disulfide Bond Formation in Rough Endoplasmic Reticulum–Derived Microsomes
  • Alternate Protocol 3: Analysis of Disulfide‐Bond Formation in Intact Cells Without Starvation
  • Alternate Protocol 4: Analysis of Post‐Translational Disulfide Bond Formation in Rough Endoplasmic Reticulum–Derived Microsomes
  • Alternate Protocol 5: In Vitro Translation in the Presence of Semi‐Permeabilized Cells
  • Support Protocol 1: Immunoprecipitation of Lysates
  • Support Protocol 2: Nonreducing and Reducing SDS‐Page
  • Support Protocol 3: Analysis of Disulfide and Free Thiol by PEG‐Maleimide Modification
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Analysis of Disulfide Bond Formation in Intact Monolayer Cells

  Materials
  • Adherent cells
  • Cell culture medium containing methionine, 37°C
  • Wash buffer (see recipe), 37°C
  • Depletion medium (see recipe), 37°C
  • Labeling medium (containing 125 to 250 μCi/ml [35S]methionine; see recipe), 37°C
  • Chase medium (see recipe), 37°C
  • Stop buffer (see recipe), 0°C
  • Lysis buffer (see recipe), 0°C
  • 60‐mm cell culture dishes
  • 37°C humidified 5% CO 2 incubator
  • 37°C water bath with rack to hold cell‐culture dishes (e.g., Unwire racks for 15‐ and 50‐ml tubes, Nalgene)
  • Liquid aspiration system for radioactive waste
  • Large laboratory ice pan with fitted metal plate (e.g., VWR International)
  • Cell scraper
  • Additional reagents and equipment for immunoprecipitation (see protocol 8) and nonreducing and reducing SDS‐PAGE (see protocol 9)
NOTE: The volumes described here are for a 60‐mm dish of cells. Volumes must be adjusted, based on the surface area of the dish, for other sizes (double volumes for 100‐mm dishes, half‐volumes for 35‐mm dishes).NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 1: Analysis of Disulfide Bond Formation in Cells in Suspension

  Additional Materials (also see protocol 1)
  • Culture of suspension cells
  • 11 mCi/ml [35S]methionine (>1,000 Ci/mmol; Perkin Elmer or Amersham)
  • 2× lysis buffer (see recipe)
  • Concentrated chase medium (see recipe)
  • 50‐ml polystyrene tube with cap, sterile
  • Cell centrifuge
  • Additional reagents and equipment for immunoprecipitation (see protocol 8) and nonreducing SDS‐PAGE (see protocol 9)
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.NOTE: Keep cells in suspension during incubations by gently swirling the tube at regular intervals.

Alternate Protocol 2: Analysis of Postponed Post‐Translational Disulfide Bond Formation in Intact Cells

  Materials
  • 1 equivalent/μl nuclease‐treated canine pancreas microsomes (Promega)
  • Rabbit reticulocyte lysate treated with ATP‐regenerating system and nucleases (Promega)
  • 1 mM amino acid mixture lacking methionine (Promega)
  • 10 mCi/ml [35S]methionine (1,000 Ci/mmol, Amersham or Perkin Elmer)
  • 100 mM dithiothreitol (DTT)
  • RNase‐free H 2O (e.g., DEPC‐treated; see recipe)
  • 23 U/ml RNase inhibitor (e.g., RNasin, Promega)
  • 1 μg/μl mRNA for the protein of interest
  • 100 mM oxidized glutathione (GSSG; appendix 3A), titrated to neutrality with KOH
  • 120 mM N‐ethylmaleimide (NEM) in 100% ethanol (prepare from 1 M stock; see recipe)
  • Lysis buffer (see recipe)
  • 2× SDS sample buffer (unit 10.1; optional)
  • 27°C water bath
  • 1.5‐ml microcentrifuge tubes, RNase‐free
  • Ice bath
  • Additional reagents and equipment for immunoprecipitation (see protocol 8) and nonreducing SDS‐PAGE (see protocol 9)
NOTE: The major source of failure in the in vitro translation/translocation of proteins is contamination with ribonucleases (RNases) that degrade mRNA. To avoid contamination, water and salt solutions should be treated with diethylpyrocarbonate (DEPC) to chemically inactivate RNases; glass and plasticware should be treated with DEPC‐treated water or otherwise treated to remove RNase activity (see recipe for DEPC treatment). Freshly opened plasticware that has not been touched by unprotected hands is also acceptable. It is also helpful to keep a set of solutions for RNA work alone to ensure that "dirty" pipets do not contaminate them. In addition, gloves should be worn at all times.

Basic Protocol 2: Analysis of Disulfide Bond Formation in Rough Endoplasmic Reticulum–Derived Microsomes

  Materials
  • MEF, COS7, HT1080, HEK293, HeLa, B cells, or CHO cell lines with appropriate medium grown in 75‐cm2 flask to near confluence (∼5 × 107 cells)
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • Trypsin/EDTA (0.25%) solution, tissue culture grade
  • KHM‐STI: KHM buffer (see recipe) containing 0.1 mg/ml soybean trypsin inhibitor (STI; add 16 µl of 50 mg/ml STI stock)
  • KHM buffer (see recipe)
  • 20 mg/ml digitonin
  • Resuspension buffer (see recipe)
  • Micrococcal nuclease (15,000 U/ml; Sigma‐Aldrich, cat. no. 10107921001), thawed immediately before use
  • 0.1 M CaCl 2
  • 250 mM EGTA
  • 15‐ml conical tubes
  • Refrigerated centrifuge
  • Additional reagents and equipment for cell culture, including trypsinization and counting of cells ( appendix 3C; Phelan, )

Alternate Protocol 3: Analysis of Disulfide‐Bond Formation in Intact Cells Without Starvation

  Materials
  • 10% (w/v) pre‐washed protein‐A‐Sepharose beads
  • Antibody against protein of interest
  • Lysate from pulse‐chase labeled cells or microsomes (see Basic Protocol protocol 11 or protocol 42 or Alternate Protocol protocol 21, protocol 32, or protocol 53)
  • Immunoprecipitation wash buffer (see recipe), 37°C
  • TE buffer, pH 6.8 (see recipe)
  • 2× nonreducing sample buffer (see recipe)
  • 200 mM dithiothreitol (DTT)
  • Refrigerated centrifuge
  • Microcentrifuge‐tube shaker
  • Rotator
  • 95°C heat block or water bath
  • Additional reagents and equipment for nonreducing and reducing SDS‐PAGE (see protocol 9)

Alternate Protocol 4: Analysis of Post‐Translational Disulfide Bond Formation in Rough Endoplasmic Reticulum–Derived Microsomes

  Materials
  • Samples in 1× sample buffer (see protocol 8)
  • 2× nonreducing sample buffer (see recipe)
  • Whatman 3MM filter paper
  • Additional reagents and equipment for SDS‐PAGE minigel with Laemmli buffers (unit 10.1; Gallagher, ), Coomassie blue staining and destaining (unit 10.5; Echan & Speicher, ), autoradiography (unit 10.11; Bundy, ), and immunoblotting (unit 10.10; Ni et al., )

Alternate Protocol 5: In Vitro Translation in the Presence of Semi‐Permeabilized Cells

  Materials
  • HEK293T cells (ATCC #CRL‐3216)
  • Complete DMEM medium (growth medium for HEK293T cells; see appendix 3C; Phelan, )
  • 1 M dithiothreitol (DTT)
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • Lysis buffer: 9% (w/v) SDS, 30 mM Tris·Cl pH 6.8 ( appendix 2E), 15% (v/v) glycerol (add 0.05% bromophenol blue for gel loading purposes)
  • Methoxypolyethylene glycol maleimide 5000 (PEG‐maleimide; Sigma‐Aldrich, cat. no. 63187)
  • 6‐cm tissue culture dishes
  • Cell scrapers
  • Probe sonicator
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1; Gallagher, ) and western blotting (immunoblotting; unit 10.10; Ni et al., )
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Figures

Videos

Literature Cited

  Blobel, G., & Dobberstein, B. (1975). Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. The Journal of Cell Biology, 67, 852–862. doi: 10.1083/jcb.67.3.852.
  Braakman, I., & Hebert, D. N. (2013). Protein folding in the endoplasmic reticulum. Cold Spring Harbor Perspectives in Biology, 5, a013201. doi: 10.1101/cshperspect.a013201.
  Braakman, I., Helenius, J., & Helenius, A. (1992a). Manipulating disulfide bond formation and protein folding in the endoplasmic reticulum. The EMBO Journal, 11, 1717–1722.
  Braakman, I., Helenius, J., & Helenius, A. (1992b). Role of ATP and disulphide bonds during protein folding in the endoplasmic reticulum. Nature, 356, 260–262. doi: 10.1038/356260a0.
  Braakman, I., Hoover‐Litty, H., Wagner, K. R., & Helenius, A. (1991). Folding of influenza hemagglutinin in the endoplasmic reticulum. The Journal of Cell Biology, 114, 401–411. doi: 10.1083/jcb.114.3.401.
  Bulleid, N. J., & Freedman, R. (1988). Defective cotranslational formation of disulphide bonds in protein disulphide‐isomerase‐deficient microsomes. Nature, 335, 649–651. doi: 10.1038/335649a0.
  Bundy, D. C. (2001). Autoradiography. Current Protocols in Protein Science, 10, 10.11.1–10.11.6. doi: 10.1002/0471140864.ps1011s10.
  Creighton, T. E. (1986). Disulfide bonds as probes of protein folding pathways. Methods Enzymology, 131, 83–106.
  Echan, L. A., & Speicher, D. W. (2002). Protein detection in gels using fixation. Current Protocols in Protein Science, 29, 10.5.1–10.5.18. doi: 10.1002/0471140864.ps1005s29.
  Francis, E., Wang, N., Parag, H., Halaban, R., & Hebert, D. N. (2003). Tyrosinase maturation and oligomerization in the endoplasmic reticulum requires a melanocyte‐specific factor. The Journal of Biological Chemistry, 278, 25607–25617. doi: 10.1074/jbc.M303411200.
  Gallagher, S. R. (2012). One‐dimensional SDS gel electrophoresis of proteins. Current Protocols in Protein Science, 68, 10.1.1–10.1.44. doi: 10.1002/0471140864.ps1001s68.
  Giclas, P. C. (2001). Alternative pathway evaluation. Current Protocols in Immunology, 9, 13.2.1–13.2.9. doi: 10.1002/0471142735.im1302s09.
  Hebert, D. N., Foellmer, B., & Helenius, A. (1995). Glucose trimming and reglycosylation determine glycoprotein association with calnexin in the endoplasmic reticulum. Cell, 81, 425–433. doi: 10.1016/0092‐8674(95)90395‐X.
  Jansens, A., & Braakman, I. (2003). Pulse‐chase labeling techniques for the analysis of protein maturation and degradation. Methods in Molecular Biology, 232, 133–145. doi: 10.1385/1‐59259‐394‐1:133.
  Kleizen, B., van Vlijmen, T., de Jonge, H. R., & Braakman, I. (2005). Folding of CFTR is predominantly cotranslational. Molecular and Cellular, 20, 277–287. doi: 10.1016/j.molcel.2005.09.007.
  Marquardt, T., Hebert, D. N., & Helenius, A. (1993). Post‐translational folding of influenza hemagglutinin in isolated endoplasmic reticulum‐derived microsomes. The Journal of Biological Chemistry, 268, 19618–19625.
  Ni, D., Xu, P., & Gallagher, S. (2017). Immunoblotting and immunodetection. Current Protocols in Protein Science, 88, 10.10.1–10.10.37. doi: 10.1002/cpps.32.
  Nicchitta, C. V., & Blobel, G. (1993). Lumenal proteins of the mammalian endoplasmic reticulum are required to complete protein translocation. Cell, 73, 989–998. doi: 10.1016/0092‐8674(93)90276‐V.
  Pearse, B. R., Gabriel, L., Wang, N., & Hebert, D. N. (2008). A cell‐based reglucosylation assay demonstrates the role of GT1 in the quality control of a maturing glycoprotein. The Journal of Cell Biology, 181, 309–320. doi: 10.1083/jcb.200712068.
  Phelan, M. C. (2006). Techniques for mammalian cell tissue culture. Current Protocols in Protein Science, 46, A.3C.1–A.3C.18. doi: 10.1002/0471140864.psa03cs46.
  Scheele, G., & Jacoby, R. (1982). Conformational changes associated with proteolytic processing of presecretory proteins allow glutathione‐catalyzed formation of native disulfide bonds. The Journal of Biological Chemistry, 257, 12277–12282.
  Wilson, R., Allen, A. J., Oliver, J., Brookman, J. L., High, S., & Bulleid, N. J. (1995). The translocation, folding, assembly and redox‐dependent degradation of secretory and membrane proteins in semi‐permeabilized mammalian cells. The Biochemical Journal, 307, 679–687. doi: 10.1042/bj3070679.
Key References
  Braakman et al., 1991. See above.
  Describes the protocol in intact cells, results with influenza hemagglutinin, and considerations for ultra‐short pulse times.
  Hebert et al., 1995. See above.
  Describes the use of rough‐ER‐derived microsomes to follow oxidation and chaperone binding of a maturing substrates.
  Marquardt et al., 1993. See above.
  Describes the protocol in microsomes.
  Jansens & Braakman, 2003. See above.
  Describes the pulse‐chase laboratory set up and protocols.
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