Measuring Retrograde Transport to the Trans‐Golgi Network

Mohamed Amessou1, Vincent Popoff1, Belèn Yelamos1, Agnès Saint‐Pol1, Ludger Johannes1

1 Institut Curie, Paris
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 15.10
DOI:  10.1002/0471143030.cb1510s32
Online Posting Date:  October, 2006
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The recently described retrograde transport route is a highly selective pathway that allows some internalized molecules to reach the trans‐Golgi network from early/recycling endosomes, bypassing the recycling route to the plasma membrane and the late endocytic pathway. The non‐toxic receptor‐binding B‐subunit of bacterial Shiga toxin has played an important role in the discovery and molecular dissection of membrane trafficking at the early/recycling endosomes–TGN interface. This unit describes several recent methods for quantitative biochemical and morphological analysis of retrograde transport. The sulfation assay permits the detection and quantification of cargo protein transport from endosomes to the TGN, describing how sulfation‐site peptides can be chemically coupled to cargo proteins. Furthermore, a variant of the sulfation assay on permeabilized cells is presented. The chemical crosslinking theme is extended to horseradish peroxidase for the ultrastructural study of the Shiga toxin–containing early/recycling endosomes by whole mount analysis. Finally, an endocytosis assay describes concomitant analysis of cellular uptake of Shiga toxin and transferrin.

Keywords: sulfation analysis; chemical coupling; endocytosis; Shiga toxin; Cholera toxin

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Table of Contents

  • Basic Protocol 1: Quantitave Assay to Measure Retrograde Transport of Cargo Proteins in Intact Cells
  • Alternate Protocol 1: Permeablized Cell Assay for Measuring Transport of Cargo Proteins From Early/Recycling Endosomes to the TGN
  • Support Protocol 1: Chemical Coupling of Proteins with Sulfation Sites into a Protein of Interest
  • Support Protocol 2: Preparing Tris‐Tricine Gels
  • Support Protocol 3: Chemical Coupling of HRP to STxB
  • Basic Protocol 2: STxB Internalization Assay
  • Support Protocol 4: Biotinylation of STxB‐K3
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Quantitave Assay to Measure Retrograde Transport of Cargo Proteins in Intact Cells

  • HeLa cells
  • DMEM3+ (see recipe)
  • DMEM without sulfate (Invitrogen), cold and 37°C
  • Sulfation substrate: e.g., STxB‐Sulf2, CTxB‐Sulf2, or anti‐GFP‐Sulf2 (see protocol 3)
  • Radioactive sulfate (35SO 42–; Amersham Pharmacia Biotech, specific activity of at least 50 µCi/µl)
  • RIPA buffer (see recipe)
  • Protease inhibitor cocktail (PIC; see recipe)
  • 13C4 monoclonal anti‐STxB antibody (hybridoma from ATCC #CRL 1794)
  • Protein G–Sepharose beads 4 fast flow (Amersham Pharmacia Biotech) equilibrated with RIPA buffer
  • Streptavidin agarose beads (Pierce) equilibrated with RIPA buffer
  • Washing buffer: 50 mM Tris·Cl, pH 8.0
  • 1.5× SDS sample buffer
  • Tris‐tricine mini‐gels (see protocol 4)
  • Fixation buffer: 40% ethanol, 10% acetic acid
  • 24‐well tissue culture dishes
  • 37°C, 5% CO 2 incubator
  • 1.5‐ml tubes
  • End‐over‐end rotator
  • Glass microfiber GF/C filters
  • Microbeta scintillation counter
  • Thermal cycler
  • Micropipettor
  • Gel dryer
  • PhosphorImager and appropriate screens

Alternate Protocol 1: Permeablized Cell Assay for Measuring Transport of Cargo Proteins From Early/Recycling Endosomes to the TGN

  • HeLa S3 cells for cytosol preparation
  • Phosphate‐buffered saline (PBS)
  • HK buffer (see recipe)
  • ICT/DTT buffer (see recipe), ice cold and at 37°C
  • Streptolysin O (SLO; see recipe)
  • ATP‐regenerating system (see recipe)
  • Potter‐Elvehjam homogenizer
  • Ultracentrifuge
  • 15‐ and 50‐ml tubes
  • Dialysis membrane (10 kDa MWCO)
  • 1.5‐ml microcentirifuge tubes

Support Protocol 1: Chemical Coupling of Proteins with Sulfation Sites into a Protein of Interest

  • N‐Succinimidyl S‐acetylthioacetate (SATA; Pierce)
  • Dimethyl sulfoxide (DMSO)
  • Protein of interest; e.g., CTxB and anti‐GFP antibody
  • 50 mM sodium phosphate/1 mM EDTA buffer, pH 7.5
  • Bromoacetylated sulfation site peptide with or without biotin group (see Fig. )
  • 10× deacetylation solution (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Dialysis cassettes: 10‐kDa molecular weight cutoff (MWCO; Pierce), optional
  • PD‐10 desalting columns (Amersham Pharmacia Biotech), optional
  • Additional reagents and equipment for measuring protein concentration using the Bradford assay ( appendix 3H) and SDS‐PAGE (unit 6.1)
NOTE: Should the cargo protein naturally contain free sulfhydryl groups, such as in STxB/Cys, start at step .

Support Protocol 2: Preparing Tris‐Tricine Gels

  • Acrylamide solution A (see recipe)
  • Tris‐tricine gel buffer (see recipe)
  • Glycerol
  • 10% (w/v) ammonium persulfate (APS) solution in water
  • Tetramethyl‐1‐,2‐diaminomethane (TEMED)
  • Acrylamide solution B (see recipe)
  • 10× cathode buffer (see recipe)
  • 10× anode buffer (see recipe)
  • Glass plates and electrophoresis apparatus (BioRad)
  • Pasteur pipets
  • Teflon combs
  • Glass microfiber GF/C filters (Whatman)

Support Protocol 3: Chemical Coupling of HRP to STxB

  • Horseradish peroxidase (HRP; Sigma‐Aldrich)
  • Water‐soluble crosslinker: sulfo‐MBS (Pierce)
  • 100 mM HEPES buffer, pH 7.4
  • PD‐10 desalting columns (Sephadex G‐25, Amersham‐Pharmacia Biotech)
  • PBS/10 mM EDTA buffer, pH 8
  • 100 µM STxB/Cys
  • 1 M NH 4Cl
  • Superdex 200 (GE Bioscience)
  • 13C4 monoclonal anti‐STxB antibody (hybridoma from ATCC #CRL 1794)
  • Glycine buffer, pH 2.2
  • Nanosep 10‐kDa microconcentration chambers for 0.1‐0.5 ml (PallFiltron)
  • Gel filtration chromatography columns

Basic Protocol 2: STxB Internalization Assay

  • HeLa cells
  • PBS++ (see recipe)
  • DMEM without serum, 37°C
  • Phospahte‐buffered saline (PBS; appendix 2A)
  • PBS/2mM EDTA
  • PBS++ supplemented with 5 mM glucose, room temperature and ice cold
  • STxB‐K3, biotinylated (STxB‐K3‐SSbiot; see protocol 7)
  • Transferrin, biotinylated (transferrin‐SSbiot)
  • MESNA buffer (see recipe)
  • 300 mM iodoacetamide (Sigma) in TNB buffer (see recipe for TNB buffer)
  • Blocking buffer (see recipe)
  • 13C4 monoclonal antibody anti‐STxB (hybridoma from ATCC #CRL 1794) or polyclonal anti‐transferrin antibody (Les Makin)
  • Saturation buffer (see recipe)
  • Streptavidin‐conjugated HRP (Roche)
  • Hydrogen peroxide solution (H 2O 2, Sigma)
  • O‐Phenylenediamine tablets (OPD, Sigma)
  • Citrate buffer (see recipe)
  • 6 N hydrogen sulfate (H 2SO 4)
  • 15‐cm petri dishes
  • 37°C, 5% CO 2 incubator
  • PD‐10 columns desalting (Sephadex G‐25, Pharmacia Biotech)
  • Centrifuge
  • 1.5‐ml microcentrifuge tubes
  • 96‐well ELISA dishes
  • Microtiter plate reader
  • Additional reagents and equipment for cell counting (unit 1.1)

Support Protocol 4: Biotinylation of STxB‐K3

  • STxB‐K3 protein
  • PBS ( appendix 2A)
  • 1 M carbonate buffer, pH 9.5
  • NHS‐SS‐biotin in DMSO
  • 1 M NH 4Cl
  • Dialysis membrane (10‐kDa)
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Literature Cited

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