Evaluation of the Mitochondrial Respiratory Chain and Oxidative Phosphorylation System Using Yeast Models of OXPHOS Deficiencies

Flavia Fontanesi1, Francisca Diaz1, Antoni Barrientos1

1 University of Miami Miller School of Medicine, Miami, Florida
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 19.5
DOI:  10.1002/0471142905.hg1905s63
Online Posting Date:  October, 2009
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The oxidative phosphorylation (OXPHOS) system consists of five multimeric complexes embedded in the mitochondrial inner membrane. They work in concert to drive the aerobic synthesis of ATP. Mitochondrial and nuclear DNA mutations affecting the accumulation and function of these enzymes are the most common cause of mitochondrial diseases and have also been associated with neurodegeneration and aging. Several approaches for the assessment of the OXPHOS system enzymes have been developed. Based on the methods described elsewhere, this unit describes the creation and study of yeast models of mitochondrial OXPHOS deficiencies. Curr. Protoc. Hum. Genet. 63:19.5.1‐19.5.20. © 2009 by John Wiley & Sons, Inc.

Keywords: electron transport chain; mitochondria; OXPHOS deficiencies; yeast model

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Modeling Pathogenic Mutations in a Conserved Non‐Essential Gene
  • Alternate Protocol 1: Modeling Pathogenic Mutations in a Nonessential Gene by Construction of Chimeric Proteins
  • Alternate Protocol 2: Modeling Pathogenic Mutations in a Non‐Essential Gene Lacking Heterologous Complementation
  • Alternate Protocol 3: Modeling Pathogenic Mutations in an Essential Gene
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Modeling Pathogenic Mutations in a Conserved Non‐Essential Gene

  • Primers
  • Plasmids containing disruptor gene or wild‐type genomic DNA
  • Tris⋅acetate/EDTA (TAE; see recipe)
  • DNA purification kit (Promega)
  • Wild‐type cells
  • Complete YP‐D solid and liquid medium (see recipe), sterile
  • Lithium acetate (TEL) solution (see recipe), sterile
  • 2 mg/ml salmon sperm carrier DNA (see recipe for DNA carrier solution)
  • 40% polyethylene glycol (PEG) solution (see recipe)
  • WO‐D solid and liquid media containing amino acids and nucleobases (see recipe for yeast media), sterile
  • Geneticin/G418 (Gibson)
  • Cell solubilization solution (see recipe)
  • 10% (w/v) sodium dodecyl sulfate (SDS; appendix 2D)
  • 8 M ammonium acetate ( appendix 2D)
  • Isopropanol
  • 80% (v/v) ethanol
  • Human cDNA clones (Open Biosystems)
  • Restriction enzymes and digestion buffers (New England Biolabs)
  • T4 DNA ligase and buffer (Promega)
  • E. coli competent cells and selective medium (Invitrogen)
  • DNA miniprep kit (Promega)
  • DNA maxiprep kit (Promega)
  • YP and WO containing E/G/L/P/A solid media and amino acids and nucleobases (see recipe for yeast media), sterile
  • QuickChange II Site‐Directed Mutagenesis kit (Stratagene)
  • 30°C incubator with shaker
  • 10‐ml glass culture flasks (Bellco)
  • Spectrophotometer
  • 2‐ml microcentrifuge tubes
  • 95°C boiling water bath or heating block
  • 42°C water bath or heating block
  • Additional reagents and equipment for PCR (Kramer and Coen, ), agarose gel electrophoresis (unit 2.7)

Alternate Protocol 1: Modeling Pathogenic Mutations in a Nonessential Gene by Construction of Chimeric Proteins

  • Two haploid wild‐type strains of opposite mating type (a and α) with same genetic background and both ura3 mutant
  • WO‐D solid media (see recipe for yeast media)
  • URA3 replicative (centromeric or episomal) vector
  • K‐acetate solid medium (see recipe)
  • β‐Glucuronidase (Sigma)
  • 5‐FOA solid medium
  • 15‐ml tubes (Falcon)
  • Flamed loops
  • Micromanipulator
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Internet Resources
  The Saccharomyces genome database is a scientific database of the molecular biology and genetics of the yeast Saccharomyces cerevisiae, containing the most current and accurate data relative to genomic sequences, gene annotation, protein function, and expression and yeast literature.
  The EUROSCARF (European Saccharomyces cerevisiae archive for functional analysis) collects yeast strains and plasmids that were generated during various yeast functional analysis projects, including the complete knockout collection of non‐essential genes. EUROSCARF is run by the Institute of Microbiology, University of Frankfurt.
  The Open Biosystems company sells the entire S. cerevisiae knockout collection of non‐essential genes and knockout inducible essential genes, yeast vectors, proteins tagged constructs, and human cDNA clones.
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