In Vitro Analysis of Peroxisomal Protein Import

Stanley R. Terlecky1

1 Wayne State University School of Medicine, Detroit, Michigan
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 11.15
DOI:  10.1002/0471143030.cb1115s14
Online Posting Date:  May, 2002
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Abstract

This unit describes a quantitative in vitro assay for peroxisomal protein import that accurately reconstitutes established properties of the pathway. The cell‐free system is ELISA based and employs semi‐permeabilized human cells with a biotinylated peroxisomal targeting signal‐containing substrate.

     
 
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Table of Contents

  • Basic Protocol 1: Peroxisomal Protein Import In Vitro
  • Alternate Protocol 1: Import into Organelle Pellets/Peroxisomes
  • Support Protocol 1: Luciferase Biotinlyation
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Peroxisomal Protein Import In Vitro

  Materials
  • Rabbit polyclonal appendix 2A anti‐luciferase antibodies (unit 16.2)
  • ELISA coating solution: 50 mM sodium carbonate, pH 9.0
  • ELISA blocking solution (EBS; see recipe), pH 7.4
  • Human epidermoid carcinoma (A431; ATCC CRL‐1555) cells or diploid lung (IMR90; ATCC CCL‐186) fibroblasts growing in tissue culture
  • Hanks balanced salt solution (HBSS, Life Technologies; appendix 2A)
  • Dulbecco's minimum essential medium containing 10% (defined) fetal bovine serum (FBS) (DMEM‐10)
  • Import reaction buffer (IRB, see recipe), pH 7.4
  • ATP and regenerating system (ATP, see recipe)
  • 800 µM ZnCl in recipeIRB
  • Biotinylated luciferase (B‐Luc, see protocol 3)
  • recipeIRB containing 1.25% or 0.2% (w/v) bovine serum albumin (IRB/1.25% BSA or IRB/0.2% BSA, respectively)
  • Avidin (Calbiochem)
  • Biocytin (Calbiochem‐Novabiochem)
  • Phosphate‐buffered saline (PBS; appendix 2A), pH 7.4
  • Streptavidin horseradish peroxidase (Roche Diagnostics)
  • 30% (v/v) hydrogen peroxide (H 2O 2)
  • o‐Phenylenediamine
  • ELISA development buffer: 27 mM citric acid/51 mM sodium phosphate, pH 5.0
  • 4 N sulfuric acid (H 2SO 4)
  • ELISA microtiter well strips (Maxisorp‐Immunomodule, Nunc)
  • 15‐cm cell culture dishes (Corning)
  • Rubber policeman
  • 50‐ml conical centrifuge tubes (Corning)
  • Platform rocker
  • 1.5‐ml microcentrifuge tubes (Eppendorf)
  • 37°C water bath
  • Microtiter plate washer (optional)
  • Microtiter plate reader

Alternate Protocol 1: Import into Organelle Pellets/Peroxisomes

  • Antibody‐coated microtiter well strips ( protocol 1, steps and )
  • Homogenization buffer (see recipe), pH 7.8
  • 15‐ml conical centrifuge tubes (Corning)
  • 27‐G needle
  • 1‐ml syringe
  • 2‐ml glass Dounce tissue grinder, with loose‐ and tight‐fitting pestles (Kontes)

Support Protocol 1: Luciferase Biotinlyation

  Materials
  • Firefly (Photinus pyralis) luciferase (Sigma‐Aldrich)
  • Phosphate‐buffered saline (PBS; appendix 2A) pH 7.4
  • 8.25 mM 6‐((6‐((biotinoyl)amino)hexanoyl)amino)hexanoic acid, succinimidylester (Molecular Probes) in dimethylsulfoxide (B‐XX‐SE)
  • PBS containing 0.2% (w/v) BSA (PBS/0.2% BSA)
  • Streptavidin alkaline‐phosphatase (KPL)
  • Rabbit polyclonal anti‐luciferase antibodies (unit 16.2)
  • Micro Bio‐Spin P‐6 chromatography column (spin column, Bio‐Rad)
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Figures

Videos

Literature Cited

Literature Cited
   Lazarow, P., Thieringer, R., Cohen, G., Imanaka, T., and Small, G. 1991. Protein import into peroxisomes in vitro. Methods Cell Biol. 34:303‐326.
   Smythe, E., Redelmeir, T.E., and Schmid, S.L. 1992. Receptor‐mediated endocytosis in semiintact cells. Methods Enzymol. 219:223‐234.
   Terlecky, S.R. and Fransen, M. 2000. How peroxisomes arise. Traffic 1:465‐473.
   Terlecky, S.R., Legakis, J.E., Hueni, S.E., and Subramani, S. 2001. Quantitative analysis of peroxisomal protein import in vitro. Exp. Cell Res. 263:98‐106.
   Walton, P.A., Gould, S.J., Feramisco, J.R., and Subramani, S. 1992. Transport of microinjected proteins into peroxisomes of mammalian cells: Inability of Zellweger cell lines to import proteins with the SKL tripeptide peroxisomal targeting signal. Mol. Cell. Biol. 12:531‐541.
   Wendland, M. 1994. A permeabilized cell system to study peroxisomal protein import. In Cell Biology: A Laboratory Handbook (J. Celis, ed.) pp. 140‐147. Academic Press. San Diego.
Key References
   Smythe et al., 1992. See above.
  Highly recommended first description of an ELISA‐based in vitro internalization assay using biotinylated substrates and semi‐intact cells. This method is the basis for the assay described in this unit.
   Terlecky et al., 2001. See above.
  First description of the use of the system outlined in the previous reference for assaying peroxisomal protein import.
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