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
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

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.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: In Vitro Chloroplast Protein Import Assay

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Arnon, D.I. 1949. Copper enzymes in isolated chloroplasts. Polyphenooxidase in Beta vulgaris. Plant Physiol. 24:1‐15.
   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.
   Chen, K., Chen, X., and Schnell, D.J. 2000. Initial binding of preproteins involving the Toc159 receptor can be bypassed during protein import into chloroplasts. Plant Physiol. 122:813‐822.
   Chua, N.H. and Schmidt, G.W. 1978. Post‐translational transport into intact chloroplasts of a precursor to the small subunit of ribulose‐1,5‐bisphosphate carboxylase. Proc. Natl. Acad. Sci. U.S.A. 75:6110‐6114.
   Cline, K. 1986. Import of proteins into chloroplasts: Membrane integration of a thylakoid precursor protein reconstituted in chloroplast lysates. J. Biol. Chem. 261:14804‐14810.
   Cline, K., Werner‐Washburne, M., Andrews, J., and Keegstra, K. 1984. Thermolysin is a suitable protease for probing the surface of intact pea chloroplasts. Plant Physiol 74:675‐678.
   Dahlin, C. and Cline, K. 1991. Developmental regulation of the plastid protein import apparatus. Plant Cell 3:1131‐1140.
   Emanuelsson, O., Nielsen, H., and von Heijne, G. 1999. ChloroP, a neural network‐based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci. 8:978‐984.
   Emmanuelsson, O., Nielson, H., Brunak, S., and von Heijne, G. 2000. Prediciting subcellular localization of proteins based on their N‐terminal amino acid sequence. J. Mol. Biol. 300:1005‐1016.
   Fitzpatrick, L.M. and Keegstra, K. 2001. A method for isolating a high yield of Arabidopsis chloroplasts capable of efficient import of precursor proteins. Plant J. 27:59‐65.
   Froehlich, J.E., Benning, C., and Dormann, P. 2001. The digalactosyldiacylglycerol (DGDG) synthase DGD1 is inserted into the outer envelope membrane of chloroplasts in a manner independent of the general import pathway and does not depend on direct interaction with monogalactosyldiacylglycerol synthase for DGDG biosynthesis. J. Biol. Chem. 276:31806‐31812.
   Jackson‐Constan, D. and Keegstra, K. 2001. Arabidopsis genes encoding components of the chloroplastic protein import apparatus. Plant Physiol 125:1567‐1576.
   Joyard, J., Grossman, A., Bartlett, S.G., Douce, R., and Chua, N.‐H. 1982. Characterization of envelope membrane polypeptides from spinach chloroplasts. J. Biol. Chem. 257:1095‐1101.
   Keegstra, K. and Cline, K. 1999. Protein import and routing systems of chloroplasts. Plant Cell 11:557‐570.
   Keegstra, K. and Yousif, A.E. 1986. Isolation and characterization of chloroplast envelope membranes. Methods Enzymol. 118:316‐325.
   Mishkind, M.L., Wessler, S.R., and Schmidt, G.W. 1985. Functional determinants in transit sequences: Import and partial maturation by vascular plant chloroplasts of the ribulose‐1,5‐bisphosphate carboxylase small subunit of Chlamydomonas. J. Cell Biol. 100:226‐234.
   Muckel, E. and Soll, J. 1996. A protein import receptor of chloroplasts is inserted into the outer envelope membrane by a novel pathway. J. Biol. Chem. 271:23846‐23852.
   Pain, D. and Blobel, G. 1987. Protein import in chloroplasts requires a chloroplast ATPase. Proc. Natl. Acad. Sci. U.S.A. 84:3288‐3292.
   Pilon, M., De Boer, A.D., Knols, S.L., Koppelman, M.H.G.M., Van der Graaf, R.M., de Kruijff, B., and Weisbeek, P.J. 1990. Expression in Escherichia coli and purification of a translocation‐competent precursor of the chloroplast protein ferredoxin. J. Biol. Chem. 265:3358‐3361.
   Schnell, D.J. and Blobel, G. 1993. Identification of intermediates in the pathway of protein import into chloroplasts and their localization to envelope contact sites. J. Cell Biol. 120:103‐115.
   Su, Q. and Boschetti, A. 1994. Substrate‐ and species‐specific processing enzymes for chloroplast precursor proteins. Biochem. J. 300:787‐792.
   Theg, S.M., Bauerle, C., Olsen, L.J., Selman, B.R., and Keegstra, K. 1989. Internal ATP is the only energy requirement for the translocation of precursor proteins across chloroplastic membranes. J. Biol. Chem. 264:6730‐6736.
   Tu, S.L. and Li, H.M. 2000. Insertion of OEP14 into the outer envelope membrane is mediated by proteinaceous components of chloroplasts. Plant Cell 12:1951‐1960.
   Yu, L.M., Merchant, S., Theg, S.M., and Selman, B.R. 1988. Isolation of a cDNA clone for the gamma subunit of the chloroplast ATP synthase of Chlamydomonas reinhardtii: Import and cleavage of the precursor protein. Proc. Natl. Acad. Sci. U.S.A. 85:1369‐1373.
   Yuan, J., Cline, K., and Theg, S.M. 1991. Cryopreservation of chloroplasts and thylakoids for studies of protein import and integration. Plant Physiol. 95:1259‐1264.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library