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

Abstract

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

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

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

Materials

Basic Protocol 1: Modeling Pathogenic Mutations in a Conserved Non‐Essential Gene

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

Figures

Videos

Literature Cited

   Allikmets, R., Raskind, W.H., Hutchinson, A., Schueck, N.D., Dean, M., and Koeller, D.M. 1999. Mutation of a putative mitochondrial iron transporter gene (ABC7) in X‐linked sideroblastic anemia and ataxia (XLSA/A). Hum. Mol. Genet. 8:743‐749.
   Bach, M.L. and Lacroute, F. 1972. Direct selective techniques for the isolation of pyrimidine auxotrophs in yeast. Mol. Gen. Genet. 115:126‐130.
   Barrientos, A. 2003. Yeast models of human mitochondrial diseases. IUBMB Life 55:83‐95.
   Barrientos, A., Zambrano, A., and Tzagoloff, A. 2004. Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae. EMBO J. 23:3472‐3482.
   Barrientos, A., Gouget, K., Horn, D., Soto, I.C., and Fontanesi, F. 2009. Suppression mechanisms of COX assembly defects in yeast and human: Insights into the COX assembly process. Biochim. Biophys. Acta 1793:97‐107.
   Baruffini, E., Lodi, T., Dallabona, C., Puglisi, A., Zeviani, M., and Ferrero, I. 2006. Genetic and chemical rescue of the Saccharomyces cerevisiae phenotype induced by mitochondrial DNA polymerase mutations associated with progressive external ophthalmoplegia in humans. Hum. Mol. Genet. 15:2846‐2855.
   Baruffini, E., Lodi, T., Dallabona, C., and Foury, F. 2007. A single nucleotide polymorphism in the DNA polymerase gamma gene of Saccharomyces cerevisiae laboratory strains is responsible for increased mitochondrial DNA mutability. Genetics 177:1227‐1231.
   Becker, D.M. and Lundblad, V. 1993. Introduction of DNA into yeast cells. Curr. Protoc. Mol. Biol. 18:13.7.1‐13.7.10.
   Bennetzen, J.L. and Hall, B.D. 1982. Codon selection in yeast. J. Biol. Chem. 257:3026‐3031.
   Bloch, K.D. and Grossmann, B. 1995. Digestion of DNA with restriction endonucleases. Curr. Protoc. Mol. Biol. 31:3.1.1‐3.1.21.
   Boeke, J.D., LaCroute, F., and Fink, G.R. 1984. A positive selection for mutants lacking orotidine‐5′‐phosphate decarboxylase activity in yeast: 5‐Fluoro‐orotic acid resistance. Mol. Gen. Genet. 197:345‐346.
   Bourgeron, T., Rustin, P., Chretien, D., Birch‐Machin, M., Bourgeois, M., Viegas‐Pequignot, E., Munnich, A., and Rotig, A. 1995. Mutation of a nuclear succinate dehydrogenase gene results in mitochondrial respiratory chain deficiency. Nat. Genet. 11:144‐149.
   Butow, R.A., Henke, R.M., Moran, J.V., Belcher, S.M., and Perlman, P.S. 1996. Transformation of Saccharomyces cerevisiae mitochondria using the biolistic gun. Methods Enzymol. 264:265‐278.
   Cavadini, P., Gellera, C., Patel, P.I., and Isaya, G. 2000. Human frataxin maintains mitochondrial iron homeostasis in Saccharomyces cerevisiae. Hum. Mol. Genet. 9:2523‐2530.
   Chattoo, B.B., Sherman, F., Azubalis, D.A., Fjellstedt, T.A., Mehnert, D., and Ogur, M. 1979. Selection of lys2 mutants of the yeast Saccharomyces cerevisiae by the utilization of alpha‐aminoadipate. Genetics 93:51‐65.
   Claypool, S.M., McCaffery, J.M., and Koehler, C.M. 2006. Mitochondrial mislocalization and altered assembly of a cluster of Barth syndrome mutant tafazzins. J. Cell Biol. 174:379‐390.
   de Lonlay, P., Valnot, I., Barrientos, A., Gorbatyuk, M., Tzagoloff, A., Taanman, J.W., Benayoun, E., Chretien, D., Kadhom, N., Lombes, A., de Baulny, H.O., Niaudet, P., Munnich, A., Rustin, P., and Rotig, A. 2001. A mutant mitochondrial respiratory chain assembly protein causes complex III deficiency in patients with tubulopathy, encephalopathy and liver failure. Nat. Genet. 29:57‐60.
   Di Fonzo, A., Ronchi, D., Lodi, T., Fassone, E., Tigano, M., Lamperti, C., Corti, S., Bordoni, A., Fortunato, F., Nizzardo, M., Napoli, L., Donadoni, C., Salani, S., Saladino, F., Moggio, M., Bresolin, N., Ferrero, I., and Comi, G.P. 2009. The mitochondrial disulfide relay system protein GFER is mutated in autosomal‐recessive myopathy with cataract and combined respiratory‐chain deficiency. Am. J. Hum. Genet. 84:594‐604.
   Feuermann, M., Francisci, S., Rinaldi, T., De Luca, C., Rohou, H., Frontali, L., and Bolotin‐Fukuhara, M. 2003. The yeast counterparts of human ‘MELAS’ mutations cause mitochondrial dysfunction that can be rescued by overexpression of the mitochondrial translation factor EF‐Tu. EMBO Rep. 4:53‐58.
   Fisher, N., Castleden, C.K., Bourges, I., Brasseur, G., Dujardin, G., and Meunier, B. 2004. Human disease‐related mutations in cytochrome b studied in yeast. J. Biol. Chem. 279:12951‐12958.
   Fontanesi, F., Palmieri, L., Scarcia, P., Lodi, T., Donnini, C., Limongelli, A., Tiranti, V., Zeviani, M., Ferrero, I., and Viola, A.M. 2004. Mutations in AAC2, equivalent to human adPEO‐associated ANT1 mutations, lead to defective oxidative phosphorylation in Saccharomyces cerevisiae and affect mitochondrial DNA stability. Hum. Mol. Genet. 13:923‐934.
   Gaisne, M., Becam, A.M., Verdiere, J., and Herbert, C.J. 1999. A ‘natural’ mutation in Saccharomyces cerevisiae strains derived from S288c affects the complex regulatory gene HAP1 (CYP1). Curr. Genet. 36:195‐200.
   Ghezzi, D., Goffrini, P., Uziel, G., Horvath, R., Klopstock, T., Lochmuller, H., D'Adamo, P., Gasparini, P., Strom, T.M., Prokisch, H., Invernizzi, F., Ferrero, I., and Zeviani, M. 2009. SDHAF1, encoding a LYR complex‐II specific assembly factor, is mutated in SDH‐defective infantile leukoencephalopathy. Nat. Genet. Epub ahead of print.
   Goffrini, P., Ercolino, T., Panizza, E., Giache, V., Cavone, L., Chiarugi, A., Dima, V., Ferrero, I., and Mannelli, M. 2009. Functional study in a yeast model of a novel succinate dehydrogenase subunit B gene germline missense mutation (C191Y) diagnosed in a patient affected by a glomus tumor. Hum. Mol. Genet. 18:1860‐1868.
   Goto, J.J., Zhu, H., Sanchez, R.J., Nersissian, A., Gralla, E.B., Valentine, J.S., and Cabelli, D.E. 2000. Loss of in vitro metal ion binding specificity in mutant copper‐zinc superoxide dismutases associated with familial amyotrophic lateral sclerosis. J. Biol. Chem. 275:1007‐1014.
   Hao, H.X., Khalimonchuk, O., Schraders, M., Dephoure, N., Bayley, J.P., Kunst, H., Devilee, P., Cremers, C.W.R.J., Schiffman, J.D., Bentz, B.G., Gygi, S.P., Winge, D.R., Kremer, H., and Rutter, J. 2009. SDH5, a gene required for flavination of succinate dehydrogenase, is mutated in paraganglioma. Science 325:1139‐1142.
   Hatanaka, T., Takemoto, Y., Hashimoto, M., Majima, E., Shinohara, Y., and Terada, H. 2001. Significant expression of functional human type 1 mitochondrial ADP/ATP carrier in yeast mitochondria. Biol. Pharm. Bull. 24:595‐599.
   Herrmann, J.M., Stuart, R.A., Craig, E.A., and Neupert, W. 1994. Mitochondrial heat shock protein 70, a molecular chaperone for proteins encoded by mitochondrial DNA. J. Cell Biol. 127:893‐902.
   Hofmann, S., Rothbauer, U., Muhlenbein, N., Neupert, W., Gerbitz, K.D., Brunner, M., and Bauer, M.F. 2002. The C66W mutation in the deafness dystonia peptide 1 (DDP1) affects the formation of functional DDP1.TIM13 complexes in the mitochondrial intermembrane space. J. Biol. Chem. 277:23287‐23293.
   Hu, J., Dong, L., and Outten, C.E. 2008. The redox environment in the mitochondrial intermembrane space is maintained separately from the cytosol and matrix. J. Biol. Chem. 283:29126‐29134.
   Kaukonen, J., Juselius, J.K., Tiranti, V., Kyttala, A., Zeviani, M., Comi, G.P., Keranen, S., Peltonen, L., and Suomalainen, A. 2000. Role of adenine nucleotide translocator 1 in mtDNA maintenance. Science. 289:782‐785.
   Kramer, M.F. and Coen, D.M. 2001. Enzymatic amplification of DNA by PCR: Standard procedures and optimization. Curr. Protoc. Mol. Biol. 56:15.1.1‐15.1.14.
   Krasilnikova, M.M. and Mirkin, S.M. 2004. Replication stalling at Friedreich's ataxia (GAA)n repeats in vivo. Mol. Cell Biol. 24:2286‐2295.
   Kucharczyk, R., Rak, M., and di Rago, J.P. 2009. Biochemical consequences in yeast of the human mitochondrial DNA 8993T>C mutation in the ATPase6 gene found in NARP/MILS patients. Biochim. Biophys. Acta 1793:817‐824.
   Lemesle‐Meunier, D., Brivet‐Chevillotte, P., di Rago, J.P., Slonimski, P.P., Bruel, C., Tron, T., and Forget, N. 1993. Cytochrome b‐deficient mutants of the ubiquinol‐cytochrome c oxidoreductase in Saccharomyces cerevisiae. Consequence for the functional and structural characteristics of the complex. J. Biol. Chem. 268:15626‐15632.
   Lodi, T., Fontanesi, F., and Guiard, B. 2002. Co‐ordinate regulation of lactate metabolism genes in yeast: The role of the lactate permease gene JEN1. Mol. Genet. Genomics 266:838‐847.
   Lodi, T., Bove, C., Fontanesi, F., Viola, A.M., and Ferrero, I. 2006. Mutation D104G in ANT1 gene: Complementation study in Saccharomyces cerevisiae as a model system. Biochem. Biophys. Res. Commun. 341:810‐815.
   Lundblad, V. 1992. Yeast cloning vectors and genes. Curr. Protoc. Mol. Biol. 18:13.4.1‐13.4.10.
   Massa, V., Fernandez‐Vizarra, E., Alshahwan, S., Bakhsh, E., Goffrini, P., Ferrero, I., Mereghetti, P., D'Adamo, P., Gasparini, P., and Zeviani, M. 2008. Severe infantile encephalomyopathy caused by a mutation in COX6B1, a nucleus‐encoded subunit of cytochrome c oxidase. Am. J. Hum. Genet. 82:1281‐1289.
   Mayr, J.A., Merkel, O., Kohlwein, S.D., Gebhardt, B.R., Bohles, H., Fotschl, U., Koch, J., Jaksch, M., Lochmuller, H., Horvath, R., Freisinger, P., and Sperl, W. 2007. Mitochondrial phosphate‐carrier deficiency: A novel disorder of oxidative phosphorylation. Am. J. Hum. Genet. 80:478‐484.
   Morizono, H., Woolston, J.E., Colombini, M., and Tuchman, M. 2005. The use of yeast mitochondria to study the properties of wild‐type and mutant human mitochondrial ornithine transporter. Mol. Genet. Metab. 86:431‐440.
   Myers, A.M., Pape, L.K., and Tzagoloff, A. 1985. Mitochondrial protein synthesis is required for maintenance of intact mitochondrial genomes in Saccharomyces cerevisiae. EMBO J. 4:2087‐2092.
   Rak, M., Tetaud, E., Godard, F., Sagot, I., Salin, B., Duvezin‐Caubet, S., Slonimski, P.P., Rytka, J., and di Rago, J.P. 2007. Yeast cells lacking the mitochondrial gene encoding the ATP synthase subunit 6 exhibit a selective loss of complex IV and unusual mitochondrial morphology. J. Biol. Chem. 282:10853‐10864.
   Rothstein, R.J. 1983. One‐step gene disruption in yeast. Methods Enzymol. 101:202‐211.
   Schiestl, R.H. and Gietz, R.D. 1989. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr. Genet. 16:339‐346.
   Sikorski, R.S. and Boeke, J.D. 1991. In vitro mutagenesis and plasmid shuffling: From cloned gene to mutant yeast. Methods Enzymol. 194:302‐318.
   Singh, A. and Sherman, F. 1974. Characteristics and relationships of mercury‐resistant mutants and methionine auxotrophs of yeast. J. Bacteriol. 118:911‐918.
   Spinazzola, A., Viscomi, C., Fernandez‐Vizarra, E., Carrara, F., D'Adamo, P., Calvo, S., Marsano, R.M., Donnini, C., Weiher, H., Strisciuglio, P., Parini, R., Sarzi, E., Chan, A., DiMauro, S., Rotig, A., Gasparini, P., Ferrero, I., Mootha, V.K., Tiranti, V., and Zeviani, M. 2006. MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion. Nat. Genet. 38:570‐575.
   Steele, D.F., Butler, C.A., and Fox, T.D. 1996. Expression of a recoded nuclear gene inserted into yeast mitochondrial DNA is limited by mRNA‐specific translational activation. Proc. Natl. Acad. Sci. U.S.A. 93:5253‐5257.
   Stuart, G.R., Santos, J.H., Strand, M.K., Van Houten, B., and Copeland, W.C. 2006. Mitochondrial and nuclear DNA defects in Saccharomyces cerevisiae with mutations in DNA polymerase gamma associated with progressive external ophthalmoplegia. Hum. Mol. Genet. 15:363‐374.
   Sturtz, L.A., Diekert, K., Jensen, L.T., Lill, R., and Culotta, V.C. 2001. A fraction of yeast Cu, Zn‐superoxide dismutase and its metallochaperone, CCS, localize to the intermembrane space of mitochondria. A physiological role for SOD1 in guarding against mitochondrial oxidative damage. J. Biol. Chem. 276:38084‐38089.
   Thompson, J.D., Gibson, T.J., and Higgins, D.G. 2003. Multiple sequence alignment using ClustalW and ClustalX. Curr. Protoc. Bioinform. 00:2.3.1‐2.3.22
   Tzagoloff, A., Akai, A., Needleman, R.B., and Zulch, G. 1975. Assembly of the mitochondrial membrane system. Cytoplasmic mutants of Saccharomyces cerevisiae with lesions in enzymes of the respiratory chain and in the mitochondrial ATPase. J. Biol. Chem. 250:8236‐8242.
   Valente, L., Tiranti, V., Marsano, R.M., Malfatti, E., Fernandez‐Vizarra, E., Donnini, C., Mereghetti, P., De Gioia, L., Burlina, A., Castellan, C., Comi, G.P., Savasta, S., Ferrero, I., and Zeviani, M. 2007. Infantile encephalopathy and defective mitochondrial DNA translation in patients with mutations of mitochondrial elongation factors EFG1 and EFTu. Am. J. Hum. Genet. 80:44‐58.
   Valnot, I., von Kleist‐Retzow, J.C., Barrientos, A., Gorbatyuk, M., Taanman, J.W., Mehaye, B., Rustin, P., Tzagoloff, A., Munnich, A., and Rotig, A. 2000. A mutation in the human heme A:farnesyltransferase gene (COX10) causes cytochrome c oxidase deficiency. Hum. Mol. Genet. 9:1245‐1249.
   Young, M.J. and Court, D.A. 2008. Effects of the S288c genetic background and common auxotrophic markers on mitochondrial DNA function in Saccharomyces cerevisiae. Yeast 25:903‐912.
   Zitomer, R.S. and Lowry, C.V. 1992. Regulation of gene expression by oxygen in Saccharomyces cerevisiae. Microbiol. Rev. 56:1‐11.
Internet Resources
  http://www.yeastgenome.org/
  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.
   http://web.uni‐frankfurt.de/fb15/mikro/euroscarf/index.html
  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.
   http://www.openbiosystems.com
  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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library