Cloning and Multi-Subunit Expression of Mitochondrial Membrane Protein Complexes in Saccharomyces cerevisiae

  • Porsha L. R. Shaw
  • Kathryn A. Diederichs
  • Ashley Pitt
  • Sarah E. Rollauer
  • Susan K. BuchananEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2127)


Saccharomyces cerevisiae is a useful eukaryotic expression system for mitochondrial membrane proteins due to its ease of growth and ability to provide a native membrane environment. The development of the pBEVY vector system has further increased the potential of S. cerevisiae as an expression system by creating a method for expressing multiple proteins simultaneously. This vector system is amenable to the expression and purification of multi-subunit protein complexes. Here we describe the cloning, yeast transformation, and co-expression of multi-subunit outer mitochondrial membrane complexes using the pBEVY vector system.

Key words

pBEVY Saccharomyces cerevisiae Multi-subunit expression Mitochondrial membrane protein LiAc/SS transformation Membrane protein complexes 



The authors would like to thank Dr. Edmund Kunji (Cambridge, UK) for the yeast expression strain and the pBEVY vectors. This work was supported by the Intramural Research Program of the NIH, the National Institute of Diabetes & Digestive & Kidney Diseases.


  1. 1.
    Routledge SJ, Mikaliunaite L, Patel A, Clare M, Cartwright SP, Bawa Z, Wilks MD, Low F, Hardy D, Rothnie AJ, Bill RM (2016) The synthesis of recombinant membrane proteins in yeast for structural studies. Methods 95:26–37. Scholar
  2. 2.
    Daum G, PC B, Schatz G (1982) Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermemembrane space of yeast mitochondria. J Biol Chem 257(21):13028–13033PubMedGoogle Scholar
  3. 3.
    Glick BS, Pon LA (1995) Isolation of highly purified mitochondria from Saccharomyces cerevisiae. Methods Enzymol 260(1):213–223CrossRefGoogle Scholar
  4. 4.
    Meisinger C, Sommer T, Pfanner N (2000) Purification of Saccharomcyes cerevisiae mitochondria devoid of microsomal and cytosolic contaminations. Anal Biochem 287(2):339–342. Scholar
  5. 5.
    Miller CA, Martinat MA, Hyman LE (1998) Assessment of aryl hydrocarbon receptor complex interactions using pBEVY plasmids expression vectors with bi-directional promoters for use in Saccharomyces cerevisiae. Nucleic Acids Res 26(15):3577–3583. Scholar
  6. 6.
    Gietz RD, Schiestl RH (2007a) Frozen competent yeast cells that can be transformed with high efficiency using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2(1):1–4. Scholar
  7. 7.
    Gietz RD, Schiestl RH (2007b) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2(1):31–34. Scholar
  8. 8.
    Gietz RD, Schiestl RH (2007c) Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2(1):35–37. Scholar
  9. 9.
    Deters D, Müller U, Homberger H (1976) Breakage of yeast cells Large scale isolation of yeast mitochondria with a continuous-flow disintegration. Anal Biochem 70(1):262–267CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Porsha L. R. Shaw
    • 1
  • Kathryn A. Diederichs
    • 1
  • Ashley Pitt
    • 1
  • Sarah E. Rollauer
    • 1
    • 2
  • Susan K. Buchanan
    • 1
    Email author
  1. 1.Laboratory of Molecular BiologyNational Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of HealthBethesdaUSA
  2. 2.Vertex PharmaceuticalsBostonUSA

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