In Vitro Analysis of Endoplasmic‐Reticulum‐to‐Golgi Transport in Mammalian Cells

Bernard B. Allan1, William E. Balch1

1 The Scripps Research Institute, La Jolla, California
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 11.3
DOI:  10.1002/0471143030.cb1103s00
Online Posting Date:  April, 2001
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Abstract

A temperature‐sensitive mutant of vesicular stomatitis G protein is used to follow the movement of that protein from the endoplasmic reticulum to transport vesicles to cis‐Golgi and finally medial/trans‐Golgi by assessing the maturation of two asparagine‐linked oligosaccharides. These assays can be used to identify the factors that are required for and regulate protein trafficking through these compartments.

     
 
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Table of Contents

  • Basic Protocol 1: Reconstitution of ER‐to‐Golgi Transport in Semi‐Intact Cells
  • Alternate Protocol 1: Reconstitution of ER‐to‐cis‐Golgi Transport in Semi‐Intact Cells
  • Basic Protocol 2: In Vitro Reconstitution of ER‐to‐Golgi Transport in Mammalian Microsomes
  • Basic Protocol 3: In Vitro Formation and Isolation of ER‐Derived Vesicles
  • Support Protocol 1: Preparation of Microsomal Membranes from NRK Cells
  • Support Protocol 2: Propagation of VSV ts045
  • Basic Protocol 4: Fusion of ER‐Derived Vesicles with Golgi Membranes
  • Support Protocol 3: Preparation of Rat Liver Cytosol
  • Support Protocol 4: Preparation of Golgi Membranes from Rat Liver
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Reconstitution of ER‐to‐Golgi Transport in Semi‐Intact Cells

  Materials
  • Normal rat kidney (NRK) cells
  • Alpha minimal essential medium (α‐MEM), serum‐free and with 5% (v/v) FBS ( appendix 2A)
  • VSV ts045 stock (∼2 × 109 pfu/ml; see protocol 6)
  • 1 mg/ml actinomycin D in ethanol
  • Methionine‐deficient labeling medium (see recipe)
  • [35S]Methionine (∼11 mCi/ml, 1175 Ci/mmol; Trans35S‐label, ICN Biomedicals)
  • 20 mM unlabeled methionine (tissue culture grade; Sigma)
  • Perforation buffer (see recipe), ice cold
  • Swelling buffer (see recipe), ice cold
  • 1% (w/v) trypan blue
  • Rat liver cytosol (see protocol 8)
  • 1 M HEPES acid, pH 7.4
  • 0.1 M magnesium acetate
  • 1 M potassium acetate
  • 10× Ca2+ buffer (see recipe)
  • 20× ATP‐regenerating system (see recipe)
  • 40 mM UDP‐N‐acetylglucosamine
  • Endo H buffer (see recipe)
  • 75 mU/ml endoglycosidase H (endo H; Boehringer Mannheim) in 0.1 M sodium acetate, pH 5.6
  • 4× SDS sample buffer ( appendix 2A)
  • Fluorographic enhancement solution: 125 mM salicylic acid (sodium salt), pH 7.0, in 30% (v/v) methanol
  • Culture incubator at 32°C
  • Water baths at 32°, 37°, and 39.5°C
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and for autoradiography and densitometry (unit 6.3)
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Alternate Protocol 1: Reconstitution of ER‐to‐cis‐Golgi Transport in Semi‐Intact Cells

  • Clone 15B chinese hamster ovary (CHO) cells (ATCC)
  • Endo D buffer (see recipe)
  • 0.5 mU/µl endoglycosidase D (endo D; Boehringer Mannheim)

Basic Protocol 2: In Vitro Reconstitution of ER‐to‐Golgi Transport in Mammalian Microsomes

  Materials
  • Microsomes (see protocol 5)
  • Rat liver cytosol (see protocol 8)
  • 1 M HEPES acid, pH 7.4
  • 0.1 M magnesium acetate
  • 1 M potassium acetate
  • 10× Ca2+ buffer (see recipe)
  • 20× ATP‐regenerating system (see recipe)
  • 2.5 M sorbitol
  • 40 mM UDP‐N‐acetylglucosamine
  • Endo H buffer (see recipe)
  • 75 mU/ml endoglycosidase H (endo H; Boehringer Mannheim) in 0.1 M sodium acetate, pH 5.6.
  • 4× SDS sample buffer ( appendix 2A)
  • Anti‐VSV‐G monoclonal antibody p5D4 (Kreis, )
  • Horseradish peroxidase (HRP)–conjugated secondary antibody
  • Water baths at 32° and 37°C
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1), immunoblotting (unit 6.2), and densitometry (unit 6.3)

Basic Protocol 3: In Vitro Formation and Isolation of ER‐Derived Vesicles

  Materials
  • Microsomes (see protocol 5)
  • Rat liver cytosol (see protocol 8)
  • 1 M HEPES acid, pH 7.4
  • 0.1 M magnesium acetate
  • 1 M potassium acetate
  • 10× Ca2+ buffer (see recipe)
  • 20× ATP‐regenerating system (see recipe)
  • 2.5 M sorbitol
  • Resuspension buffer (see recipe), ice cold
  • 1× SDS sample buffer ( appendix 2A)
  • Anti‐VSV‐G monoclonal antibody p5D4 (Kreis, )
  • Horseradish peroxidase (HRP)–conjugated secondary antibody
  • p5D4‐Dynabeads: p5D4 coupled to M‐500 Dynabeads (Dynal; see manufacturer's instructions)
  • Immunoprecipitation buffer (see recipe)
  • FBS ( appendix 2A)
  • 100 mM EDTA (adjust to pH 8.0 with KOH)
  • Transport buffer (see recipe)
  • Water bath at 32°C
  • Magnetic microcentrifuge tube holder
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1), immunoblotting (unit 6.2), and densitometry (unit 6.3)

Support Protocol 1: Preparation of Microsomal Membranes from NRK Cells

  • PBS ( appendix 2A), ice cold
  • Homogenization buffer I (see recipe)
  • 100× PIC (see recipe)
  • Potassium acetate buffer (see recipe)
  • Transport buffer (see recipe)
  • 1‐ml ball‐bearing homogenizer (Balch and Rothman, )
  • Culture incubator at 39.5°C
NOTE: The method described below is for a twelve‐dish microsome preparation.

Support Protocol 2: Propagation of VSV ts045

  Materials
  • Baby hamster kidney (BHK) cells (ATCC)
  • Glasgow minimal essential medium (G‐MEM; Life Technologies)
  • Tryptose phosphate broth (TPB; Sigma)
  • FBS ( appendix 2A)
  • TD buffer (see recipe)
  • Vesicular stomatitis virus (VSV) ts045 stock (Indiana serotype; multiplicity of infection = 0.1; ATCC)
  • Culture incubator at 32°C

Basic Protocol 4: Fusion of ER‐Derived Vesicles with Golgi Membranes

  Materials
  • ER‐derived vesicles (HSP; see protocol 4 step )
  • Desalted rat liver cytosol (see protocol 8)
  • Enriched rat liver Golgi membranes (see protocol 9)
  • 1 M HEPES acid, pH 7.4
  • 0.1 M magnesium acetate
  • 10× Ca2+ buffer (see recipe)
  • 20× ATP‐regenerating system (see recipe)
  • 40 mM UDP‐N‐acetylglucosamine
  • 2.5 M sorbitol
  • Endo H buffer (see recipe)
  • 75 mU/ml endoglycosidase H (endo H; Boehringer Mannheim) in 0.1 M sodium acetate, pH 5.6
  • 4× SDS sample buffer ( appendix 2A)
  • Anti‐VSV‐G monoclonal antibody p5D4 (Kreis, )
  • Horseradish peroxidase (HRP)–conjugated secondary antibody
  • Water bath at 37°C
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1), immunoblotting (unit 6.2), and densitometry (unit 6.3)

Support Protocol 3: Preparation of Rat Liver Cytosol

  Materials
  • Male Sprague‐Dawley rats
  • PBS ( appendix 2A), ice cold
  • Cytosol buffer (see recipe), ice cold
  • 100 mM ATP
  • 100× PIC (see recipe)
  • 40‐ml Dounce homogenizer with type A (tight‐fitting) and type B (loose‐fitting) glass pestles
  • Cheesecloth
  • Sephadex G‐25M/PD‐10 column (Pharmacia Biotech)

Support Protocol 4: Preparation of Golgi Membranes from Rat Liver

  Materials
  • Male Sprague‐Dawley rats
  • Homogenization buffer II (see recipe), ice cold
  • 100× PIC (see recipe)
  • Sucrose solutions in 10 mM Tris⋅Cl, pH 7.4 ( appendix 2A): 0.5 M (refractive index 1.3575), 1.0 M (1.3815), 1.1 M (1.3865), 1.25 M (1.3939), and 2.35 M (1.4464)
  • Dilution buffer (see recipe)
  • Transport buffer (see recipe)
  • Homogenizer (Potter‐Elvehjem tissue grinder, size C) and Teflon pestles with 0.026‐in. (66‐mm) and 0.012‐in. (30‐mm) clearance
  • Cheesecloth
  • Refractometer
  • 18‐G needle
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Figures

Videos

Literature Cited

Literature Cited
   Balch, W.E. and Rothman, J.E. 1985. Characterization of protein transport between successive compartments of the Golgi apparatus: Asymmetric properties of donor and acceptor activities in a cell‐free system. Arch. Biochem. Biophys. 240:413‐425.
   Beckers, C.J.M., Keller, D.S., and Balch, W.E. 1987. Semi‐intact cells permeable to macromolecules: Use in reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex. Cell 50:523‐534.
   Conradt, B., Haas, A., and Wickner, W. 1994. Determination of four biochemically distinct, sequential stages during vacuole inheritance in vitro. J. Cell Biol. 126:99‐110.
   Davidson, H.W. and Balch, W.E. 1993. Differential inhibition of multiple vesicular transport steps between the endoplasmic reticulum and trans Golgi network. J. Biol. Chem. 268:4216‐4226.
   Gottlieb, C., Baenziger, J., and Kornfeld, S. 1975. Deficient uridine diphosphate‐N‐acetylglucosamine: Glycoprotein N‐acetylglucosaminyltransferase activity in a clone of Chinese hamster ovary cells with altered surface glycoproteins. J. Biol. Chem. 250:3303‐3309.
   Kreis, T.E. 1986. Microinjected antibodies against the cytoplasmic domain of vesicular stomatitis virus glycoprotein block its transport to the cell surface. EMBO J. 5:931‐941.
   Lafay, F. 1974. Envelope viruses of vesicular stomatitis virus: Effect of temperature‐sensitive mutations in complementation groups III and V. J. Virol. 14:1220‐1228.
   Palade, G.E. 1975. Intracellular aspects of the process of protein transport. Science 189:347‐354.
   Rexach, M.F. and Schekman, R.W. 1991. Distinct biochemical requirements for the budding, targeting, and fusion of ER‐derived transport vesicles. J. Cell Biol. 114:219‐229.
   Rowe, T., Aridor, M., McCaffery, J.M., Plutner, H., and Balch, W.E. 1996. COPII vesicles derived from mammalian endoplasmic reticulum (ER) microsomes recruit COPI. J. Cell Biol. 135:895‐911.
   Schwaninger, R., Beckers, C.M.J., and Balch, W.E. 1991. Sequential transport of protein between the endoplasmic reticulum and successive Golgi compartments in semi‐intact cells. J. Biol. Chem. 266:13055‐13063.
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