Analysis of the Association of Proteins with Membranes

T. Okamoto1, R.B. Schwab2, P.E. Scherer2, M.P. Lisanti2

1 Cleveland Clinic Foundation, Cleveland, Ohio, 2 Albert Einstein College of Medicine, Bronx, New York
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 5.4
DOI:  10.1002/0471143030.cb0101s05
Online Posting Date:  May, 2001
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Abstract

Proteins associate with membranes in a number of ways. Is the membrane an integral membrane protein? A peripheral protein? Does it have a glycosylphosphoinositol (GPI) tail? Is it associated with caveolae‐derived plasma membrane domains? This unit describes methods for determining how a protein of interest associates with the membrane including extraction with alkaline carbonate, urea, high salt, or Triton X‐114. GPI linkage is probed using PI‐PLC cleavage. There is a method for detergent solubilization of Triton‐X 100 insoluble integral and GPI‐linked proteins. Caveolae membranes can be isolated using Triton X‐100 or a detergent‐free method.

     
 
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Table of Contents

  • Basic Protocol 1: Alkaline Carbonate Extraction
  • Alternate Protocol 1: Urea Extraction
  • Alternate Protocol 2: High‐Salt Extraction
  • Alternate Protocol 3: Triton X‐114 Phase Separation
  • Support Protocol 1: Triton X‐114 Precondensation
  • Support Protocol 2: Preparation of 100× Protease Inhibitors Stock Solution
  • Basic Protocol 2: PI‐PLC Cleavage of GPI‐Linked Proteins
  • Basic Protocol 3: Detergent Solubilization of Triton X‐100 Insoluble Integral Membrane and GPI‐Linked Proteins
  • Basic Protocol 4: Triton‐Based Purification of Caveolae‐Derived Membranes
  • Alternate Protocol 4: Detergent‐Free Purification of Caveolae‐Derived Membranes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Alkaline Carbonate Extraction

  Materials
  • Cells of interest
  • Phosphate‐buffered saline (PBS; appendix 2A), ice‐cold
  • 100 mM NaCl
  • 100 mM sodium carbonate, pH 11.5, ice cold
  • 2× SDS‐PAGE sample buffer ( appendix 2A)
  • Triton X‐100
  • N‐octyl glucoside
  • Tris⋅Cl, pH 7.5 ( appendix 2A)
  • NaCl
  • 2‐ml Dounce homogenizer
  • Beckman TL‐100 centrifuge with TLA 100.2 rotor
  • 26‐G needles and 1‐ml syringes
  • 95°C water bath
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1), immunoblotting (unit 6.2), and immunoprecipitation (unit 7.2)

Alternate Protocol 1: Urea Extraction

  Materials
  • Cells of interest
  • Cell lysis buffer (see recipe)
  • Tris/NaCl/EDTA buffer (see recipe)
  • TNET‐OG buffer (see recipe)
  • 2× SDS‐PAGE sample buffer ( appendix 2A)
  • Additional reagents and equipment for immunoprecipitation (unit 7.2), SDS‐PAGE (unit 6.1), and immunoblotting (unit 6.2)

Alternate Protocol 2: High‐Salt Extraction

  Materials
  • Triton X‐114
  • Tris‐buffered saline (TBS): 10 mM Tris⋅Cl, pH 7.4 ( appendix 2A)/150 mM NaCl
  • Sodium dodecyl sulfate (SDS)
  • Triton X‐100

Alternate Protocol 3: Triton X‐114 Phase Separation

  Materials
  • Antipain
  • Pepstatin A
  • Leupeptin
  • Dimethylsulfoxide (DMSO)

Support Protocol 1: Triton X‐114 Precondensation

  Materials
  • Cells of interest
  • Tris/NaCl/EDTA buffer (see recipe)
  • PI‐PLC incubation buffer (see recipe)
  • 1000 U/ml PI‐PLC (phosphatidylinositol‐specific phospholipase C; Boehringer Mannheim)
  • Phenyl‐Sepharose 4B (Pharmacia Biotech)
  • Tris‐buffered saline (TBS): 20 mM Tris⋅Cl, pH 8.0 ( appendix 2A)/150 mM NaCl
  • 12.5 mg/ml (100×) sodium deoxycholate
  • Trichloracetic acid
  • 2× SDS‐PAGE sample buffer ( appendix 2A)
  • 1 M Tris⋅Cl, pH 8.0 ( appendix 2A)
  • 1% (w/v) SDS/200 mM Tris⋅Cl, pH 8.0 (see appendix 2A for Tris⋅Cl)
  • TNET buffer (see recipe)
  • Eppendorf vortex mixer
  • Boiling water bath
  • Additional reagents and equipment for Triton X‐114 phase separation (see protocol 4), SDS‐PAGE (unit 6.1), and immunoprecipitation (unit 7.2).

Support Protocol 2: Preparation of 100× Protease Inhibitors Stock Solution

  Materials
  • Cells of interest
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Detergent solution—one of the following:
  •  60 mM N‐octylglucoside in either TBS (20mM Tris⋅Cl, pH 8.0/150 mM NaCl) or MES‐buffered saline (25 mM MES, pH 6.5/150 mM NaCl)
  •  30 mM CHAPS in either TBS (20mM Tris⋅Cl, pH 8.0/150 mM NaCl) or MES‐buffered saline (25 mM MES, pH 6.5/150 mM NaCl)
  • Wash buffer containing Triton X‐100 (unit 7.2)
  • Additional reagents and equipment for immunoprecipitation (unit 7.2)

Basic Protocol 2: PI‐PLC Cleavage of GPI‐Linked Proteins

  Materials
  • MDCK (Madin‐Darby canine kidney) cells (or virtually any other nontransformed cell line)
  • DMEM medium containing 5% FBS (unit 1.2) with 100 IU/ml penicillin and 50 µg/ml streptomycin
  • Phosphate‐buffered saline (PBS; appendix 2A), ice‐cold
  • Lysis buffer for caveolae (see recipe)
  • 5%, 30%, and 80% sucrose solutions (see recipe)
  • MES‐buffered saline: 25 mM 2‐(N‐morpholino)ethanesulfonic acid (MES)/150 mM NaCl
  • 75‐cm3 tissue culture flasks
  • 150‐mm tissue culture dishes
  • Cell scrapers
  • 15‐ml tubes
  • Dounce homogenizer
  • Beckman L8 ultracentrifuge with SW 41 rotor and corresponding ultraclear centrifuge tubes
  • Additional reagents and equipment for cell culture and trypsinization of cells (unit 1.1), preparation of serum‐containing tissue culture medium (unit 1.2), and preparation of sucrose gradients (unit 5.3)

Basic Protocol 3: Detergent Solubilization of Triton X‐100 Insoluble Integral Membrane and GPI‐Linked Proteins

  Materials
  • MDCK (Madin‐Darby canine kidney) cells or any other cell
  • Phosphate‐buffered saline (PBS; appendix 2A), ice cold
  • 500 mM sodium carbonate, pH 11, ice cold
  • MES‐buffered saline: 25 mM MES, pH 6.5/0.15 M NaCl
  • MES‐buffered saline plus 1% (w/v) Triton X‐100 and PMSF
  • 5%, 35%, and 90% (w/v) sucrose (see recipe)
  • 3× SDS‐PAGE sample buffer (see recipe)
  • Cell scrapers
  • Polytron tissue grinder (Kinematica GmbH, Brinkmann Instruments)
  • Branson Sonifier 250 (Branson Ultrasonic)
  • Beckman ultracentrifuge with SW 41 rotor and corresponding (14 × 89–mm) ultraclear tubes
  • Additional reagents and equipment for immunoprecipitation (unit 7.2) and SDS‐PAGE (unit 6.1)
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Figures

Videos

Literature Cited

Literature Cited
   Bickel, P.E., Scherer, P.E., Schnitzer, J.E., Oh, P., Lisanti, M.P., and Lodish, H.F. 1997. Flotillin and epidermal surface antigen define a new family of caveolae‐associated integral membrane proteins. J. Biol. Chem. 272:13793‐13802.
   Bordier, C. 1981. Phase separation of integral membrane proteins in Triton X‐114 solution. J. Biol. Chem. 256:1604‐1607.
   Brown, D.A. and London, E. 1997. Structure of detergent‐resistant membrane domains: Does phase separation occur in biological membranes. Biochem Biophys Res Commun. 240:1‐7.
   Chang, W.J., Ying, Y., Rothberg, K., Hooper, N., Turner, A., Gambliel, H., De Gunzburg, J., Mumby, S., Gilman, A., and Anderson, R.G.W. 1994. Purification and characterization of smooth muscle cell caveolae. J. Cell Biol. 126:127‐138.
   Engelman, J.A., Wycoff, C.C., Yasuhara, S., Song, K.S., Okamoto, T., and Lisanti, M.P. 1997. Recombinant expression of caveolin‐1 in oncogenically transformed cells abrogates anchorage‐independent growth. J. Biol. Chem. 272:16374‐16381.
   Fra, A.M., Masserini, M., Palestini, P., Sonnino, S., and Simons, K. 1995a. A photo‐reactive derivative of ganglioside GM1 specifically cross‐links VIP21‐caveolin on the cell surface. FEBS Lett. 375:11‐14.
   Fra, A.M., Williamson, E., Simons, K., and Parton, R.G. 1995b. De novo formation of caveolae in lymphocytes by expression of VIP21‐caveolin. Proc. Natl. Acad. Sci. U.S.A. 92:8655‐8659.
   Fujiki, Y., Hubbard, A.L., Fowler, S., and Lazarow, P.B. 1982a. Isolation of intracellular membranes by means of sodium carbonate treatment: Application to endoplasmic reticulum. J. Cell Biol. 93:97‐102.
   Fujiki, Y., Fowler, S., Shio, H., Hubbard, A.L., and Lazarow, P.B. 1982b. Polypeptide and phospholipid composition of the membrane of rat liver peroxisomes: Comparison with endoplasmic reticulum and mitochondrial membranes. J. Cell Biol. 93:103‐110.
   James, G. and Olson, E.N. 1989. Identification of a novel fatty acylated protein that partitions between the plasma membrane and cytosol and is deacylated in response to serum and growth factor stimulation. J. Biol. Chem. 264:20998‐21006.
   Kubler, E., Dohlman, H.G., and Lisanti, M.P. 1996. Identification of Triton‐X100 insoluble membrane domains in the yeast Saccharomyces cerevisiae: A model system for the molecular evolution of mammalian caveolin genes. J. Biol. Chem. 271:32975‐32980.
   Li, S., Song, K.S., and Lisanti, M.P. 1996a. Expression and characterization of recombinant caveolin: Purification by poly‐histidine tagging and cholesterol‐dependent incorporation into defined lipid membranes. J. Biol. Chem. 271:568‐573.
   Li, S., Song, K.S., Koh, S., and Lisanti, M.P. 1996b. Baculovirus‐based expression of mammalian caveolin in Sf21 insect cells: A model system for the biochemical and morphological study of caveolae biogenesis. J. Biol. Chem. 271:28647‐28654.
   Lisanti, M.P., Scherer, P.E., Vidugiriene, J., Tang, Z.‐L., Hermanoski‐Vosatka, A., Tu, Y.‐H., Cook, R.F., and Sargiacomo, M. 1994. Characterization of caveolin‐rich membrane domains isolated from an endothelial‐rich source: Implications for human disease. J. Cell Biol. 126:111‐126.
   Moldovan, N.I., Heltianu, C., Simionescu, N. and Simionescu, M. 1995. Ultrastructural evidence of differential solubility in Triton X‐100 of endothelial vesicles and plasma membrane. Exp. Cell Res. 219:309‐313.
   Murata, M., Peranen, J., Schreiner, R., Weiland, F., Kurzchalia, T., and Simons, K. 1995. VIP21/caveolin is a cholesterol‐binding protein. Proc. Natl. Acad. Sci. U.S.A. 92:10339‐10343.
   Parolini, I., Sargiacomo, M., Lisanti, M.P., and Peschle, C. 1996. Signal transduction and GPI‐linked proteins (Lyn, Lck, CD4, CD45, G proteins, CD 55) selectively localize in Triton‐insoluble plasma membrane domains of human leukemic cell lines and normal granulocytes. Blood 87:3783‐3794.
   Puertollano, R., Li, S., Lisanti, M.P., and Alonso, M.A. 1997. Recombinant expression of the MAL proteolipid, a component of glycolipid‐enriched membrane microdomains, induces the formation of vesicular structures in insect cells. J. Biol. Chem. 272:18311‐18357.
   Sargiacomo, M., Sudol, M., Tang, Z.‐L., and Lisanti, M.P. 1993. Signal transducing molecules and GPI‐linked proteins form a caveolin‐ rich insoluble complex in MDCK cells. J. Cell Biol. 122:789‐807.
   Scherer, P.E., Manning‐Krieg, U.C., Jeno, P., Schatz, G., and Horst, M. 1992. Identification of a 45‐kDa protein at the protein import site of the yeast mitochondrial inner membrane. Proc. Natl. Acad. Sci. U.S.A. 89:11930‐11934.
   Scherer, P.E., Lederkremer, G.Z., Williams, S., Fogliano, M., Baldini, G., and Lodish, H. F. 1996. Cab45, a novel (Ca2+)‐binding protein localized to the Golgi lumen. J. Cell Biol. 133:257‐268.
   Scherer, P.E., Lewis, R.Y., Volonte, D., Engelman, J.A., Galbiati, F., Couet, J., Kohtz, D.S., van Donselaar, E., Peters, P. and Lisanti, M.P. 1997. Cell‐type and tissue‐specific expression of caveolin‐2. Caveolins 1 and 2 co‐localize and form a stable hetero‐oligomeric complex in vivo. J. Biol. Chem. 272:29337‐29346.
   Schnitzer, J.E., Liu, J., and Oh, P. 1995a. Endothelial caveolae have the molecular transport machinery for vesicle budding, docking, and fusion including VAMP, NSF, SNAP, annexins, and GTPases. J. Biol. Chem. 270:14399‐14404.
   Schnitzer, J.E., Oh, P., Jacobson, B.S., and Dvorak, A.M. 1995b. Caveolae from luminal plasmalemma of rat lung endothelium: Microdomains enriched in caveolin, Ca2+‐ATPase, and inositol triphosphate receptor. Proc. Natl. Acad. Sci. U.S.A. 92:1759‐1763.
   Schook, W., Puszkin, S., Bloom, W., Ores, C., and Kochwa, S. 1979. Mechanochemical properties of brain clathrin: Interactions with actin and alpha‐actinin and polymerization into basketlike structures or filaments. Proc Natl Acad Sci U.S.A. 76:116‐120.
   Sevinsky, J.R., Rao, L.V.M., and Ruf, W. 1996. Ligand‐induced protease receptor translocation into caveolae: A mechanism for regulating cell surface proteolysis of the tissue factor‐dependent coagulation pathway. J. Cell Biol. 133:293‐304.
   Simons, K. and Ikonen, E. 1997. Functional rafts in cell membranes. Nature 387:569‐572.
   Smart, E., Ying, Y.‐S., Conrad, P., and Anderson, R.G.W. 1994. Caveolin moves from caveolae to the Golgi apparatus in response to cholesterol oxidation. J. Cell Biol. 127:1185‐1197.
   Smart, E.J., Ying, Y., Mineo, C., and Anderson, R.G.W. 1995. A detergent free method for purifying caveolae membrane from tissue cultured cells. Proc. Natl. Acad. Sci. U.S.A. 92:10104‐10108.
   Song, K.S., Li, S., Okamoto, T., Quilliam, L., Sargiacomo, M., and Lisanti, M.P. 1996. Copurification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains. Detergent free purification of caveolae membranes. J. Biol. Chem. 271:9690‐9697.
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