In Vitro Analysis of Chloroplast Protein Import

Matthew D. Smith1, Danny J. Schnell1, Lynda Fitzpatrick2, Kenneth Keegstra2

1 University of Massachussets, Amherst, Massachussets, 2 Michigan State University, East Lansing, Michigan
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 11.16
DOI:  10.1002/0471143030.cb1116s17
Online Posting Date:  February, 2003
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This unit describes protocols for isolating chloroplasts from pea (Pisum sativum) and Arabidopsis thaliana for the study of nuclear‐encoded plastid precursor proteins. Chloroplasts from both preparations are competent for the in vitro import of recombinant preproteins synthesized using in vitro translation systems derived from reticulocyte or wheat germ lysates. These assays can be used to test whether a particular protein is targeted to chloroplasts, for analyzing the suborganellar location of newly imported preproteins, or to study the mechanism of import itself.

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Table of Contents

  • Basic Protocol 1: In Vitro Chloroplast Protein Import Assay
  • Alternate Protocol 1: Fractionation of Re‐Isolated Choloroplasts Following Import
  • Support Protocol 1: Isolation of Intact Chloroplasts from Pea
  • Support Protocol 2: Isolation of Intact Chloroplasts from Arabidopsis Thaliana
  • Support Protocol 3: Production of [35S]Methionine‐Labeled Import Substrate by in Vitro Translation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
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Basic Protocol 1: In Vitro Chloroplast Protein Import Assay

  • Freshly isolated, intact chloroplasts (see protocol 3 and protocol 4)
  • HEPES‐sorbitol buffer, pH 7.5, (if using pea chloroplasts, see recipe) or pH 8.0 (if using Arabidopsis chloroplasts, see recipe)
  • Chloroplast import master mix (see recipe)
  • 0.1 M dithiothreitol (DTT)
  • 0.1 M ATP
  • In vitro—translated [35S]methionine‐labeled chloroplast protein (see protocol 5 or unit 11.2)
  • 40% (v/v) Percoll (see recipe)
  • 2 mg/ml thermolysin (see recipe)
  • 0.5 M EDTA
  • SDS‐PAGE sample buffer (see recipe)
  • Destain solution (see recipe)
  • 26°C water bath
  • 1.5‐ml microcentrifuge tubes
  • Refrigerated microcentrifuge
  • Enlightening fluorography enhancer (NEN)
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and fluorography or phosphorimagery (unit 6.3)

Alternate Protocol 1: Fractionation of Re‐Isolated Choloroplasts Following Import

  • Chloroplast pellet from the import reaction (see protocol 1)
  • HEPES‐sorbitol buffer (see recipe), ice cold
  • 2 mM EDTA, ice cold
  • 4 M NaCl
  • 0.5% and 100% (w/v) trichloroacetic acid (TCA; see recipe)
  • SDS‐PAGE sample buffer (see recipe)
  • 0.1 M NaCO 3, pH 11.5, ice cold
  • TE/DTT buffer (see recipe) containing 0.6 M sucrose and buffer containing 0.2 M sucrose
  • TE/DTT buffer (see recipe)
  • 1 M, 0.8 M, and 0.46 M sucrose
  • 3‐ml Beckman polyallomer thick‐walled ultracentrifuge tubes
  • Ultracentrifuge and rotor (Beckman TL 100 and TLA 100.3 rotor, or equivalent)
  • Dounce homogenizer or Potter homogenizer and pestle
  • Swinging‐bucket rotor (e.g., Beckman SW 50.1)
  • 5‐ml polyallomer tubes (e.g., for Beckman SW 50.1)
  • 1‐ml micropipettor
  • Gradient former (e.g., Buchler DensiFlow gradient former)
  • Gradient fractionater (e.g., ISCO gradient fractionater)
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and fluorography or phosphorimagery (unit 6.3)

Support Protocol 1: Isolation of Intact Chloroplasts from Pea

  • Pea seeds (Pisum sativum Green Arrow, Jung Seed)
  • Potting soil (e.g., Premier Brand Pro‐Mix BX or Scotts Brand Metro Mix)
  • 85% and 40% (v/v) PBF‐Percoll (see recipe)
  • Grinding buffer containing 0.25% (w/v) bovine serum albumin (BSA) and 0.1% (w/v) ascorbic acid (see recipe), ice cold
  • Protease inhibitor cocktail (optional, e.g., Sigma‐Aldrich, unit 3.4)
  • HEPES‐sorbitol, pH 7.5 buffer (see recipe)
  • 80% acetone
  • 40% (v/v) DMSO (see recipe), ice cold
  • Liquid nitrogen
  • Standard potting trays (21.5 × 11 × 2.5–in.)
  • 30‐ml glass centrifuge tubes (Corex)
  • Scissors
  • 2‐liter beakers
  • Rotary homogenizer (Polytron or comparable with a 30‐mm saw‐tooth generator)
  • 50‐cm Miracloth squares
  • Large funnel (180‐mm diameter)
  • 250‐ml centrifuge bottles with screw‐cap lids
  • Superspeed centrifuge (e.g., Sorvall RC‐5B)
  • Large rotor capable of holding 250‐ml bottles (e.g., Sorvall GSA)
  • Swinging‐bucket rotor capable of holding 50‐ml tubes (e.g., Sorvall HB‐4)
  • Large‐bore pipet
  • Cryotubes

Support Protocol 2: Isolation of Intact Chloroplasts from Arabidopsis Thaliana

  • Murashige and Skoog growth medium (see recipe)
  • Seed sterilizing solution (see recipe)
  • Arabidopsis thaliana seeds, 30 to 40 mg per plate
  • Sterile, autoclaved water
  • 0.1% (w/v) agarose (autoclaved, sterile)
  • Digestion buffer (see recipe)
  • Digestion enzyme solution (see recipe)
  • recipe40% and 85% (v/v) AT Percoll (see recipe)
  • Protoplast resuspension buffer (see recipe)
  • Protoplast breakage buffer (see recipe)
  • HEPES‐sorbitol buffer, pH 8.0 (see recipe)
  • Plastic petri plates, 20‐25 mm × 150–mm diameter, sterile
  • Sterile laminar‐flow hood
  • 1.5‐ml microcentrifuge tubes or 15‐ml tubes
  • Platform shaker
  • Parafilm
  • Growth room or chamber (16‐hr day, 70 to 120 µEm−2sec−1, 20° to 25°C)
  • Single‐edge razor blades
  • 100‐mm petri dishes
  • 500‐ml beakers (optional)
  • Plastic wrap (optional)
  • 60 W light source (optional)
  • 30‐ml glass centrifuge tubes (e.g., Corex)
  • 200‐µm nylon mesh (e.g., Sefar America), 100‐ to 120‐mm squares, fashioned into a cone and stapled to hold its shape
  • Small funnel
  • 50‐ml centrifuge tubes
  • Tabletop centrifuge with swinging‐bucket rotor, capable of slow acceleration and deceleration
  • Protoplast rupturing device (see recipe)
  • Superspeed centrifuge capable of 39,000 × g, with a 50‐ml tube swing‐out rotor
  • 23‐cm Pasteur pipet

Support Protocol 3: Production of [35S]Methionine‐Labeled Import Substrate by in Vitro Translation

  • Expression vector harboring the gene that encodes protein to be used as import substrate
  • Plasmid isolation kit (e.g., Qiagen Plasmid Midi kit)
  • In vitro translation kit (e.g., TNT Coupled Reticulocyte Lysate system, Promega)
  • Destain solution (see recipe)
  • Image acquisition software (e.g., Molecular Dynamics)
  • Additional reagents and equipment for SDS‐PAGE gel (unit 6.1)
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Literature Cited

Literature Cited
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   Bauer, J., Hiltbrunner, A., and Kessler, F. 2001. Molecular biology of chloroplast biogenesis: Gene expression, protein import and intraorganellar sorting. Cell Mol. Life Sci. 58:420‐433.
   Bolter, B., May, T., and Soll, J. 1998. A protein import receptor in pea chloroplasts, Toc86, is only a proteolytic fragment of a larger polypeptide. FEBS Lett. 441:59‐62.
   Bruce, B.D., Perry, S., Froehlich, J., and Keegstra, K. 1994. In vitro import of proteins into chloroplasts. In Plant Molecular Biology Manual (B.S. Gelvin and R.A. Schilperoort, eds.) pp. 1‐15. Kluwer Academic Publishers Group, Dordrecht, The Netherlands.
   Chen, X. and Schnell, D.J. 1999. Protein import into chloroplasts. Trends Cell Biol. 9:222‐227.
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