Determining Protein Transport to the Plasma Membrane

Jacques Neefjes1

1 The Netherlands Cancer Institute, Amsterdam
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 15.4
DOI:  10.1002/0471143030.cb1504s05
Online Posting Date:  May, 2001
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Abstract

Many integral membrane proteins synthesized in the endoplasmic reticulum ultimately arrive at the cell surface to contact the cell environment. During transit through the Golgi and trans‐Golgi network, proteins acquire post‐translational modifications that can be used to track the appearance of such modified proteins at the cell surface. Cellular proteins can be treated with enzymes–e.g., sialidase or protease–or antibodies, or biotinylated to identify molecules that have reached the cell surface. Some proteins first enter the endocytic pathway before appearing at the cell surface; this is detected by treating the cells at 4o and 37oC. Analysis of the number of sialic acids on proteins of cells treated at 4oC identifies proteins resident at the cell surface, while cells treated at 37oC internalize the sialidase, which can then act with proteins in the endocytic compartments.

     
 
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Table of Contents

  • Basic Protocol 1: Measuring Arrival at the Cell Surface by Digestion with Sialidase
  • Alternate Protocol 1: Measuring Arrival at the Cell Surface by Biotinylation of Cell‐Surface Molecules
  • Alternate Protocol 2: Measuring Arrival at the Cell Surface Using Protease Sensitivity
  • Alternate Protocol 3: Measuring Arrival at the Cell Surface Using Antibodies
  • Support Protocol 1: Preparation of Biotinylated Fab Fragments
  • Basic Protocol 2: Measuring Transport to Endosomes Before Plasma Membrane Appearance
  • Support Protocol 2: Separation of Proteins by Charge Using Denaturing One‐Dimensional Isoelectric Focusing
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Measuring Arrival at the Cell Surface by Digestion with Sialidase

  Materials
  • Cells
  • Amino acid–free medium containing 8% to 10% (v/v) FBS
  • Radiolabeled amino acid
  • PBS ( appendix 2A), ice cold
  • PBS containing 1 mM CaCl 2
  • Sialidase Type V from Clostridium perfringens (Sigma) dissolved in PBS and stored in 10‐µl 1 to 10 U/ml aliquots up to several years at −20°C
  • PBS containing 10% (v/v) fetal bovine serum (FBS)
  • Lysis mixture (see recipe)
  • Fetuin
  • Additional reagents and equipment for pulse‐labeling (unit 7.1), TCA precipitation (unit 7.1), immunoprecipitation (unit 7.2), SDS PAGE (unit 6.1) or one‐dimensional IEF (see protocol 7), and autoradiography (unit 6.3)

Alternate Protocol 1: Measuring Arrival at the Cell Surface by Biotinylation of Cell‐Surface Molecules

  Materials
  • Cells
  • Wash buffer: PBS ( appendix 2A) containing 1 mM MgCl 2 and 0.1 mM CaCl 2
  • 15 mg/ml sulfo‐N‐hydroxysuccinimde‐biotin (sulfo‐NHS‐biotin; Pierce) in PBS, prepared fresh prior to use
  • Wash buffer containing 25 mM lysine monohydrochloride
  • Lysis buffer containing 50 mM glycine
  • 0.5% (w/v) SDS
  • 50 mM Tris⋅Cl, pH 7.5 ( appendix 2A)
  • Antibody specific for protein of interest
  • Avidin‐ or streptavidin‐Sepharose beads
  • Additional reagents and equipment for pulse labeling (unit 7.1), immunoprecipitation (unit 7.2), and SDS‐PAGE (unit 6.1)

Alternate Protocol 2: Measuring Arrival at the Cell Surface Using Protease Sensitivity

  Materials
  • Cells
  • PBS ( appendix 2A)
  • Protease: fresh 10 mg/ml stock solutions of either trypsin, chymotrypsin, or pronase in PBS
  • PBS containing 5% (v/v) fetal bovine serum (FBS)
  • Protease inhibitors, freshly prepared stocks:
  •  for trypsin: 100 mM (1000×) phenylmethylsulfonyl fluoride (PMSF) in methanol and 1 mg/ml (100×) trypsin inhibitor in PBS
  •  for chymotrypsin: 100 mM PMSF and 2 mg/ml (1000×) aprotinin in PBS
  •  for pronase: 100 mM PMSF and 10 mM (1000×) N‐tosyl‐lysine chloromethyl ketone (TLCK) in PBS
  • Lysis mixture (see recipe)
  • Antibody specific for protein of interest
  • Additional reagents and equipment for biotinylation of cell‐surface proteins ( protocol 2), immunoprecipitation (unit 7.2), SDS‐PAGE (unit 6.1), immunoblotting (unit 6.2), pulse labeling (unit 7.1), and autoradiography (unit 6.3)

Alternate Protocol 3: Measuring Arrival at the Cell Surface Using Antibodies

  Materials
  • Cells
  • PBS ( appendix 2A), ice cold
  • Biotinylated Fab fragments of antibody specific for protein of interest (see protocol 5) or complete antibody
  • PBS containing 5% (v/v) fetal bovine serum (FBS)
  • Unlabeled cells or antigen
  • Protein A–Sepharose or avidin‐ or streptavidin‐coupled beads
  • Additional reagents and equipment for pulse labeling (unit 7.1), immunoprecipitation (unit 7.2), SDS‐PAGE (unit 6.1), and autoradiography (unit 6.3)

Support Protocol 1: Preparation of Biotinylated Fab Fragments

  Materials
  • 23 mg/ml papain in 70% ethanol
  • 25 and 40 mM sodium acetate buffer, pH 4.5 ( appendix 2A)
  • 2‐Mercaptoethanol
  • 100 mM sodium acetate buffer, pH 5.5 ( appendix 2A)
  • Protein A– or G–purified antibody (unit 16.3)
  • 150 mM NaCl/5 mM EDTA/100 mM Tris⋅Cl, pH 8.0
  • Dithiothreitol (DTT)
  • Iodoacetamide (store protected from light and moisture)
  • 50 mM NaHCO 3 buffer, pH 8.5
  • Sulfo‐N‐hydroxysuccinimidyl‐6‐biotinamido‐hexanoate (sulfo‐NHS‐SS‐biotin; Pierce)
  • 10 mM Na 3PO 4, pH 7.4/150 mM NaCl buffer
  • Sephadex G‐25 column
  • Centricon‐30 (Amicon)
  • Additional reagents and equipment for dialysis ( appendix 3C), SDS‐PAGE (unit 6.1), and standard anion or cation exchange chromatography (unit 16.4)

Basic Protocol 2: Measuring Transport to Endosomes Before Plasma Membrane Appearance

  Materials
  • Cells
  • Sialidase Type V from Clostridium perfringens (Sigma), dissolved in PBS and stored in aliquots at −20°C
  • Normal culture medium with 0.1% (w/v) BSA and no FBS
  • PBS ( appendix 2A) containing 10% (v/v) fetal bovine serum (FBS), ice‐cold
  • Lysis mixture (see recipe)
  • Fetuin
  • Additional reagents and equipment for pulse labeling and TCA precipitation (unit 7.1), immunoprecipitation (unit 7.2) one‐dimensional IEF (see protocol 7), SDS‐PAGE (unit 6.1), and autoradiography (unit 6.3)

Support Protocol 2: Separation of Proteins by Charge Using Denaturing One‐Dimensional Isoelectric Focusing

  Materials
  • 0.8% (w/v) agarose in water
  • Urea
  • 10% (v/v) Triton X‐100 or Igepal CA‐630 (Sigma)
  • 30% (w/v) acrylamide/1.6% (w/v) bisacrylamide solution
  • Ampholytes (Pharmacia Biotech)
  • 10% (w/v) ammonium persulfate
  • N,N,N′,N′‐tetramethylethylenediamine (TEMED)
  • Isoelectric focusing sample buffer (see recipe)
  • Immunoprecipitates (see protocol 1 or see protocol 6)
  • 20 mM H 3PO 4
  • Bromphenol blue
  • 50 mM NaOH
  • 30 × 19–cm glass plates
  • 1‐mm spacers
  • 20‐well Teflon comb
  • Vertical gel chamber
  • Additional reagents and equipment for gel electrophoresis (unit 6.1) and autoradiography (unit 6.3)
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Figures

Videos

Literature Cited

Literature Cited
   Burke, B., Matlin, K., Bause, E., Legier, G., Peyrieras, N., and Ploegh, H. 1984. Inhibition of N‐linked oligosaccharide trimming does not interfere with surface expression of certain integral membrane proteins. EMBO J. 3:551‐556.
   Marks, M.S., Woodruff, L., Ohno, H., and Bonifacino, J.S. 1996. Protein targeting by tyrosine‐ and di‐leucine‐based signals: Evidence for distinct saturable components. J. Cell Biol. 135:341‐354.
   Neefjes, J.J. and Ploegh, H.L. 1988. Allele and locus‐specific differences in cell surface expression and the association of HLA‐class I heavy chain with β2‐microglobulin: Differential effects of inhibition of glycosylation on class I subunit association. Eur. J. Immunol. 18:801‐810.
   Neefjes, J.J., Breur‐Vriesendorp, B.S., Van Seventer, G.A., Lvanyi, P., and Ploegh, H.L. 1986. An improved biochemical method for the analysis of HLA‐class I antigens. Definition of new HLA class I subtypes. Hum. Immunol. 16:169‐181.
   Neefjes, J.J., Stollorz, V., Peters, P.J., Geuze, H.L., and Ploegh, H.L. 1990. The biosynthetic pathway of MHC class II but not class I molecules intersects the endocytic route. Cell 61:171‐181.
   Warmerdam, P.A.M., Long, E.O., and Roche, P.A. 1996. lsoforms of the invariant chain regulate transport of MHC class II molecules to antigen processing compartments. J. Cell Biol. 133:281‐291.
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