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

Bernard B. Allan1,  William E. Balch1

1The 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

  • Unit Introduction
  • Basic Protocol 1: Reconstitution of ER-to-Golgi Transport in Semi-Intact Cells
  • Alternate Protocol: 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 (-MEM), serum-free and with 5% (v/v) FBS (appendix 2A)
  • VSV ts045 stock (~2 × 109 pfu/ml; see Support Protocol 2)
  • 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 Support Protocol 3)
  • 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: Reconstitution of ER-to-cis-Golgi Transport in Semi-Intact Cells

 Additional Materials (also see Basic Protocol 1)
  • 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 Support Protocol 1)
  • Rat liver cytosol (see Support Protocol 3)
  • 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, 1986)
  • 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 Support Protocol 1)
  • Rat liver cytosol (see Support Protocol 3)
  • 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, 1986)
  • 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

 Additional Materials (also see Basic Protocol 1)
  • 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, 1985)
  • 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 Basic Protocol 3 step )
  • Desalted rat liver cytosol (see Support Protocol 3)
  • Enriched rat liver Golgi membranes (see Support Protocol 4)
  • 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, 1986)
  • 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
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 11.3.1
    (A) Time course of 35S-labeled VSV-G transport in semi-intact NRK cells, analyzed by endo H digestion. At the early time points, only GH0 is detected. GH1 appears as a transient intermediate and is replaced by GH2 as transport proceeds. These bands reflect discrete intermediates in VSV-G processing (see Background Information and Fig. 11.3.3).(B) Kinetics of VSV-G transport in semi-intact NRK cells as measured by the appearance of the GH1 and GH2 forms of VSV-G. Reprinted from Schwaninger et al. (1991) with permission from the American Society for Biochemistry and Molecular Biology.

    View Image
  • Figure 11.3.2
    (A) Time course of 35S-labeled VSV-G transport in semi-intact Clone 15B CHO cells, analyzed by endo D digestion. Clone 15B CHO cells are missing the enzyme GlcNAc T I, which is required for development of endo H resistance. At the early time points only GH0 is detected. Only GD2 is detected under standard SDS-PAGE conditions, although GD1, which is produced when the first of the N-linked oligosaccharides is processed to the endo D–sensitive form, can sometimes be seen as an intermediate by running the dye front off the end of the gel to improve resolution. Reprinted from Schwaninger et al. (1991) with permission from the American Society for Biochemistry and Molecular Biology. (B) Kinetics of VSV-G transport as measured by the appearance of the GD1 and GD2 forms of VSV-G.

    View Image
  • Figure 11.3.3
    A schematic representation of the different oligosaccharide side chain processing forms of VSV-G. GH0 is the ER Man9 form, which is endo H sensitive (endo Hs) and endo D resistant (endo Dr). GD is formed in the cis-Golgi by trimming GH0 to the Man5 form, which is endo D sensitive (endo Ds). This is the only intermediate in the oligosaccharide processing of VSV-G that is sensitive to endo D and cannot normally be detected in wild-type cells due to its transient formation. Further processing of GD in the cis-Golgi generates GH1, which is endo H resistant (endo Hr). In the medial Golgi, GH1 is processed to the GH2 form, which is also endo H resistant (endo Hr), but which migrates more slowly than GH1in SDS/polyacrylamide gels. TGN, trans-Golgi network.

    View Image
  • Figure 11.3.4
    Comparison of the rates and extent of VSV-G transport from the ER to the Golgi apparatus measured either in vivo, in SIC preparations, or reconstituted using the microsome-based protocol.

    View Image

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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