Endocytosis: Biochemical Analyses

Timothy E. McGraw1, Agathe Subtil1

1 Weill Medical School of Cornell University, New York, New York
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 15.3
DOI:  10.1002/0471143030.cb1503s03
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 the Steady‐State Surface‐To‐Internal Distribution of the Transferrin Receptor
  • Alternate Protocol 1: Measuring the Steady‐State Surface‐To‐Internal Distribution of Other Membrane Proteins
  • Basic Protocol 2: Measuring the Kinetics of Transferrin Internalization
  • Alternate Protocol 2: Measuring the Kinetics of Membrane Protein Internalization Using 125I‐Labeled Antibodies
  • Alternate Protocol 3: Measuring the Kinetics of Transferrin Internalization in Cells Grown in Suspension
  • Basic Protocol 3: Measuring the Kinetics of Transferrin Receptor Recycling
  • Support Protocol 1: Iron‐Loading Transferrin
  • Support Protocol 2: Radio‐Iodination of Diferric Transferrin
  • Basic Protocol 4: Time Course for Steady‐State Occupancy of Membrane Protein with Antibody
  • Basic Protocol 5: Detecting Degradation of Internalized Ligands
  • Basic Protocol 6: Measuring Fluid‐Phase Uptake
  • Support Protocol 3: Inhibition of Clathrin‐Mediated Endocytosis by Potassium Depletion
  • Support Protocol 4: Inhibition of Clathrin‐Mediated Endocytosis by Cytosol Acidification
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measuring the Steady‐State Surface‐To‐Internal Distribution of the Transferrin Receptor

  Materials
  • Cells of interest
  • SF medium (see recipe)
  • 125I‐labeled human diferric transferrin (∼500 cpm/ng; see protocol 8)
  • Unlabeled (nonradioactive) human diferric transferrin
  • Neutral pH buffer (see recipe), ice cold
  • pH 2.0 buffer (see recipe), ice cold
  • Solubilization solution: 1% (w/v) Triton X‐100 in 0.1 N NaOH
  • 10 mg/ml BSA
  • 6‐well tissue culture plates
  • γ counter and tubes

Alternate Protocol 1: Measuring the Steady‐State Surface‐To‐Internal Distribution of Other Membrane Proteins

  Materials
  • Cells of interest
  • SF medium (see recipe)
  • 125I‐labeled human diferric transferrin (∼500 cpm/ng; see protocol 8)
  • Unlabeled (nonradioactive) human diferric transferrin
  • Neutral pH buffer (see recipe), ice cold
  • pH 2.0 buffer (see recipe), ice cold
  • Solubilization buffer: 1% (w/v) Triton X‐100 in 0.1 N NaOH
  • 10 mg/ml BSA
  • 6‐well tissue culture plates
  • γ counter and tubes

Basic Protocol 2: Measuring the Kinetics of Transferrin Internalization

  • Suspension culture of interest
  • HEPES‐buffered SF medium (see recipe)
  • pH 2.0 medium, ice cold: medium formulation used to grow cells (e.g., RPMI, DMEM) with 25 mM sodium acetate, adjusted to pH 2.0 with HCl
  • pH 11.0 medium, ice cold: medium formulation used to grow cells (e.g., RPMI, DMEM) with 25 mM Tris base, adjusted to pH 11.0 with NaOH

Alternate Protocol 2: Measuring the Kinetics of Membrane Protein Internalization Using 125I‐Labeled Antibodies

  Materials
  • Cells of interest
  • SF medium (see recipe)
  • 125I‐labeled human transferrin (see protocol 8)
  • Unlabeled (nonradioactive) human diferric transferrin
  • pH 5.0 buffer (see recipe), prewarmed to 37°C
  • Efflux medium (see recipe), prewarmed to 37°C
  • Solubilization solution: 1% (w/v) Triton X‐100 in 0.1 N NaOH
  • 6‐well tissue culture plates
  • γ counter and tubes

Alternate Protocol 3: Measuring the Kinetics of Transferrin Internalization in Cells Grown in Suspension

  Materials
  • Apo‐transferrin (iron‐free transferrin; available from a number of commercial sources)
  • PBS (see recipe)
  • 1 mg/ml ferric ammonium citrate solution in 10 mM NaHCO 3/20 mM HEPES acid, pH 7.7
  • PD‐10 columns (pre‐packed Sephadex G‐25 columns; Pharmacia Biotech) or any other desalting column
  • 0.2‐µm sterile syringe filter

Basic Protocol 3: Measuring the Kinetics of Transferrin Receptor Recycling

  • PBS (see recipe)
  • Human diferric transferrin (see protocol 7)
  • 37 MBq/ml Na125I (100 mCi/ml; NEN Life Science Products)
  • 4 mg/ml chloramine T in recipePBS (prepare fresh)
  • 8 mg/ml sodium bisulfite in recipePBS (prepare fresh)
  • 1 mg/ml BSA
  • 100% (w/v) trichloroacetic acid (TCA)
  • 5 × 105 cells/well plated in 6‐well tissue culture plates (see protocol 1, step )
  • PD‐10 column (pre‐packed Sephadex G‐25 column; Pharmacia Biotech) or any other desalting column

Support Protocol 1: Iron‐Loading Transferrin

  Materials
  • Cells of interest
  • SF medium (see recipe)
  • 125I‐labeled antibody (Fab fragments; 2 to 10 µCi/µg)
  • Unlabeled (nonradioactive) antibody
  • Neutral pH buffer (see recipe), room temperature
  • Solubilization solution: 1% (w/v) Triton X‐100 in 0.1 N NaOH
  • 6‐well tissue culture plates
  • γ counter and tubes

Support Protocol 2: Radio‐Iodination of Diferric Transferrin

  Materials
  • Cells of interest
  • SF medium (see recipe)
  • Neutral pH buffer (see recipe), ice cold
  • 125I‐labeled ligand of interest ( protocol 8)
  • Unlabeled (nonradioactive) ligand
  • 10 mg/ml BSA
  • 100% (w/v) trichloroacetic acid (TCA) solution
  • 6‐well tissue culture plate
  • γ counter and tubes

Basic Protocol 4: Time Course for Steady‐State Occupancy of Membrane Protein with Antibody

  Materials
  • Cells of interest
  • SF medium (see recipe)
  • 5 mg/ml horseradish peroxidase (HRP) in recipeSF medium
  • Neutral pH buffer (see recipe), ice cold
  • 0.01% (w/v) Triton X‐100
  • 6‐well tissue culture plates
  • Additional reagents and equipment for colorimetric assay (see Steinman et al., ) and protein assay ( appendix 3A)

Basic Protocol 5: Detecting Degradation of Internalized Ligands

  Materials
  • Cells of interest
  • SF medium (see recipe)
  • Hypotonic medium: 1:1 (v/v) SF medium/water
  • K+‐depleted buffer: 100 mM NaCl, 50 mM HEPES acid, pH 7.4
  • 6‐well tissue culture plates
  • Additional reagents and equipment for measuring internalization (see protocol 3 or protocol 4)

Basic Protocol 6: Measuring Fluid‐Phase Uptake

  Materials
  • Cells of interest
  • SF medium (see recipe)
  • recipeSF medium supplemented with 25 mM NH 4Cl
  • 140 mM KCl/1 mM amiloride/40 mM HEPES acid, pH 7.0
  • 6‐well tissue culture plates
  • Additional reagents and equipment for measuring internalization (see protocol 3 or protocol 4)
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Figures

Videos

Literature Cited

Literature Cited
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   Brown, M.S. and Goldstein, J.L. 1976. Analysis of a mutant strain of human fibroblasts with a defect in the internalization of receptor‐bound low density lipoprotein. Cell 9:663‐674.
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