In Vitro Analysis of Yeast Mitochondrial Protein Import

Rosemary A. Stuart1, Carla M. Koehler2

1 Marquette University, Milwaukee, Wisconsin, 2 UCLA, Los Angeles, California
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 11.19
DOI:  10.1002/0471143030.cb1119s34
Online Posting Date:  March, 2007
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Abstract

This unit describes methods for importing in vitro–translated or recombinant proteins into isolated yeast mitochondria and for exporting mitochondrial proteins translated in the yeast mitochondrial matrix into the inner mitochondrial membrane. The methods use mitochondria isolated from yeast cells and mitochondrial protein precursors derived from an in vitro transcription/translation reaction or purified from an E. coli recombinant protein expression system. The described translocation assays can be used to determine whether a protein is targeted to mitochondria and its location within the mitochondrion. It can also be used to study the import mechanism and to investigate mitochondrial matrix translation of proteins and their export.

Keywords: mitochondria; Saccharomyces cerevisiae; protein translocation; protein import; protein export; translocon; TOM; TIM

     
 
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Table of Contents

  • Basic Protocol 1: In Vitro Mitochondrial Protein Import Assay: Import of Labeled Proteins
  • Alternate Protocol 1: In Vitro Mitochondrial Import Assay: Mitochondrial Fractionation and Protein Localization
  • In Organello Translation of Mitochondrially Encoded Proteins
  • Basic Protocol 2: In Organello Mitochondrial Protein Assay: Translation and Translocation of Mitochondrially Encoded Proteins
  • Alternate Protocol 2: In Organello Mitochondrial Protein Assay: Protein Localization by Protease Treatment
  • Support Protocol 1: Preparation of Precursor Proteins
  • Support Protocol 2: Final Processing of Samples and Analysis by SDS‐Page
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: In Vitro Mitochondrial Protein Import Assay: Import of Labeled Proteins

  Materials
  • Freshly isolated or frozen, intact mitochondria (unit 3.8)
  • Buffer C (see recipe)
  • 2× import buffer (see recipe)
  • 0.1 M ATP (see recipe)
  • 0.5 M NADH (see recipe)
  • 1 mg/ml valinomycin in ethanol: store in 50‐µl aliquots up to 6 months at −20°C
  • In vitro–translated [35S]methionine‐labeled mitochondrial precursor protein ( protocol 5) (e.g., Neurospora crassa Su9‐DHFR)
  • 10 mg/ml trypsin in 20 mM potassium HEPES, pH 7.4 (see recipe): prepare 0.5 ml fresh
  • 20 mM potassium HEPES, pH 7.4 (see recipe)
  • 20 mg/ml trypsin inhibitor (see recipe), freshly prepared
  • SDS sample buffer ( appendix 2A)
  • 15‐ml centrifuge tubes
  • 25°C water bath
  • Refrigerated microcentrifuge

Alternate Protocol 1: In Vitro Mitochondrial Import Assay: Mitochondrial Fractionation and Protein Localization

  Materials
  • Mitochondrial pellet from the import reaction ( protocol 1, step 10)
  • 1× import buffer + ATP + NADH (see recipe)
  • 20 mM potassium HEPES, pH 7.4 (see recipe), cold
  • 100% trichloroacetic acid (TCA): add 227 ml H 2O directly to bottle containing 500 g solid TCA and shake to dissolve; store up to 2 months at 25°C.
  • SDS sample buffer ( appendix 2A)
  • 10 mg/ml proteinase K/20 mM potassium HEPES, pH 7.4 (see recipe): prepare 0.5 ml fresh using 5 mg proteinase K (Sigma)
  • 0.2 M phenylmethylsulfonyl fluoride (PMSF) in ethanol: prepare 1 ml fresh using 34.8 mg PMSF in 1 ml ethanol
  • 10% (v/v) Triton X‐100
  • Acetone
  • 0.1 M Na 2CO 3: store up to several weeks at 4°C
  • Refrigerated microcentrifuge

Basic Protocol 2: In Organello Mitochondrial Protein Assay: Translation and Translocation of Mitochondrially Encoded Proteins

  Materials
  • Frozen, intact mitochondria (see unit 3.8)
  • Buffer C (see recipe)
  • 20 mM potassium HEPES, pH 7.4 (see recipe)
  • 0.2 M ATP (see recipe)
  • 1.5× translation buffer (see recipe), freshly prepared
  • 0.5 mg/ml pyruvate kinase: prepare fresh using 2.5 mg pyruvate kinase (Roche) and 5 ml sterile H 2O
  • 0.2 M NADH: prepare fresh using 14.2 mg NADH and 100 µl sterile H 2O
  • [35S]methionine (15 mCi/ml >1000 Ci/mmol in vitro–translation grade; GE Healthcare)
  • 0.2 M methionine: prepare using 30 mg methionine and 1 ml sterile H 2O; store up to 1 year at −20°C
  • 1 mg/ml puromycin: prepare 1 ml fresh using sterile H 2O
  • SDS sample buffer ( appendix 2A)
  • Heating block set at 25°C
  • Refrigerated microcentrifuge
  • Rotary shaker
  • Additional reagents and equipment for hypotonic swelling of mitochondria ( protocol 4; optional), SDS‐PAGE (unit 6.1)

Alternate Protocol 2: In Organello Mitochondrial Protein Assay: Protein Localization by Protease Treatment

  Materials
  • Labeled mitoplasts ( protocol 3 step 8)
  • 20 mM potassium HEPES, pH 7.4 (see recipe)
  • 0.2 M ATP (see recipe)
  • Buffer C (see recipe)
  • 0.5 mg/ml pyruvate kinase: prepare fresh using 2.5 mg pyruvate kinase (Roche) and 5 ml sterile H 2O
  • 1 mg/ml proteinase K in 20 mM potassium HEPES, pH 7.4 (see recipe), cold: prepare 0.5 ml fresh using 0.5 mg proteinase K (Sigma)
  • 0.2 M phenylmethylsulfonyl fluoride (PMSF): prepare fresh using 34.8 mg PMSF and 1 ml alcohol
  • SDS sample buffer ( appendix 2A)
  • Refrigerated microcentrifuge
  • Rotary shaker

Support Protocol 1: Preparation of Precursor Proteins

  Materials
  • Expression vector (e.g., pET vector using the SP6 or T7 promoter; Novagen) carrying the gene encoding the protein of interest
  • Plasmid isolation kit (e.g., Qiagen or Promega)
  • In vitro translation kit (e.g., TNT coupled reticulocyte lysate system; Promega)
  • [35S]methionine (10 mCi/ml; >1000 Ci/mmol in vitro translation grade; GE Healthcare)
  • Ammonium sulfate solution, saturated (see recipe)
  • 8 M urea solution (see recipe)
  • Refrigerated microcentrifuge
  • Addition reagents and equipment for performing SDS‐PAGE (unit 6.1)

Support Protocol 2: Final Processing of Samples and Analysis by SDS‐Page

  Materials
  • Labeled protein samples (from protocol 1, protocol 2, protocol 3, or protocol 4)
  • 25% (w/v) ammonium hydroxide solution
  • Labeled precursor proteins ( protocol 5)
  • 10 mg/ml heat‐denatured mitochondria: heat mitochondria (see unit 3.8 and protocol 1) 5 min at 95°C
  • SDS sample buffer ( appendix 2A)
  • Antibodies against endogenous mitochondrial markers (e.g., Hsp60, ADP/ATP carrier, and cytochrome b 2), optional (not commercially available; contact authors at or for more information)
  • 5% (w/v) trichloroacetic acid (TCA) solution
  • 1 M Tris base (store up to 2 months at 4°C)
  • 1 M sodium salicylate (store up to 6 months at 25°C)
  • 65°C water bath
  • Rotary shaker
  • Nitrocellulose membrane, optional
  • Gel vacuum, optional
  • Storage phosphor screen and image acquisition software and instrumentation (e.g., PhosphorImager; GE Healthcare) or
  • X‐ray film and cassette and densitometer
  • Additional reagents and equipment for performing SDS‐PAGE (unit 6.1), immunoblotting (unit 6.2), and detecting and quantitating radiolabeled proteins (unit 6.3)
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Figures

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Literature Cited

Literature Cited
   Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D, Seidman, J.G., Smith, J.A., and Struhl, K. (eds.) 2007. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, N.J.
   Barrientos, A., Zambrano, A., and Tzagoloff, A. 2004. Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae. EMBO J. 23:3472‐3482.
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   Pfanner, N., Muller, H.K., Harmey, M.A., and Neupert, W. 1987. Mitochondrial protein import: Involvement of the mature part of a cleavable precursor protein in the binding to receptor sites. EMBO J. 6:3449‐3454.
   Rojo, E.E., Guiard, B., Neupert, W., and Stuart, R.A. 1999. N‐terminal tail export from the mitochondrial matrix. Adherence to the prokaryotic “positive‐inside” rule of membrane protein topology. J. Biol. Chem. 274:19617‐19622.
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