Mitochondria Protein Import: Methods


Mitochondria are prominent and essential organelles within eukaryotic cells. The majority of proteins residing within mitochondria are nuclear encoded and consequently synthesised as precursor proteins on cytosolic ribosomes. These precursors must be imported across one or both of the mitochondrial membranes to reach their final destination where they can acquire their functional state. The import proceeds owing to the presence of different pathways formed by sophisticated complexes known as the import complexes. The complexes are located in all mitochondrial compartments including both mitochondrial membranes. Currently, we have knowledge of at least four different pathways that govern the import and sorting of nuclear‐encoded mitochondrial precursors. The elucidation and characterisation of these pathways has been greatly facilitated by the in vitro mitochondrial import assay. The in vitro assay is the main biochemical approach for analysing protein import into mitochondria and is performed by incubating a radiolabelled precursor protein with isolated mitochondria.

Key Concepts

  • Most mitochondrial proteins are encoded by nuclear DNA and need to be imported into the organelle following translation.
  • Different pathways exist for the import of proteins into the various mitochondrial subcompartments.
  • In vitro translated precursors can be imported into isolated mitochondria.
  • The final residence of the imported precursor can be analysed using various treatments.
  • In vitro import reactions can be accompanied with monitoring the assembly of the precursor into complexes.

Keywords: in vitro import assay; TOM; TIM; MIA; MIM; TOB/SAM; Oxa1; precursor protein; translocation

Figure 1. Outline of the experiment concerning the precursor protein import into isolated mitochondria. Step 1 – mitochondria are incubated with a precursor containing presequence (p) in the (a) presence (+ΔΨ) and (b) absence (−ΔΨ) of the inner membrane potential. Step 2 – the samples are split into two, and one half is treated with proteinase K (PK) which digests protein from the cytosolic side of the membrane. Mitochondrial processing peptidase (MPP) cleaves presequence (blue box).
Figure 2. Import of preornithine transcarbamylase (pOTC) into isolated mitochondria. Lysate‐containing [35S] pOTC (lane 1) was incubated with HeLa cell mitochondria in the absence or presence of a membrane potential for 20 min. Samples were split into two, and one half was treated with proteinase K before mitochondrial reisolation and separation of proteins by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Radiolabelled proteins were detected by phosphorimage analysis. Precursor and matrix‐processed (mature) forms of pOTC are indicated.


Baker MJ, Webb CT, Stroud DA, et al. (2009) Structural and functional requirements for activity of the Tim9‐Tim10 complex in mitochondrial protein import. Molecular Biology of the Cell 20: 769–779.

Banerjee R, Gladkova C, Mapa K, Witte G and Mokranjac D (2015) Protein translocation channel of mitochondrial inner membrane and matrix‐exposed import motor communicate via two‐domain coupling protein. eLife 4: e11897.

Banci L, Bertini I, Cefaro C, et al. (2010) Molecular chaperone function of Mia40 triggers consecutive induced folding steps of the substrate in mitochondrial protein import. Proceedings of the National Academy of Sciences of the United States of America 107: 20190–20195.

Becker T, Böttinger L and Pfanner N (2012) Mitochondrial protein import: from transport pathways to an integrated network. Trends in Biochemical Sciences 37: 85–91.

Bolender N, Sickmann A, Wagner R, Meisinger C and Pfanner N (2008) Multiple pathways for sorting mitochondrial precursor proteins. EMBO Reports 9: 42–49.

Böttinger L, Guiard B, Oeljeklaus S, et al. (2013) A complex of Cox4 and mitochondrial Hsp70 plays an important role in the assembly of the cytochrome c oxidase. Molecular Biology of the Cell 24: 2609–2619.

Buczek D, Wojtkowska M, Suzuki Y, et al. (2016) Protein import complexes in the mitochondrial outer membrane of Amoebozoa representatives. BioMed Central Genomics 17: 99.

Budzińska M, Gałgańska H, Karachitos A, Wojtkowska M and Kmita H (2009) The TOM complex is involved in the release of superoxide anion from mitochondria. Journal of Bioenergetics and Biomembranes 41: 361–367.

Callegari S, Richter F, Chojnacka K, et al. (2016) TIM29 is a subunit of the human carrier translocase required for protein transport. FEBS Letters 590: 4147–4158.

Chacinska A, Pfannschmidt S, Wiedemann N, et al. (2004) Essential role of Mia40 in import and assembly of mitochondrial intermembrane space proteins. EMBO Journal 23: 3735–3746.

Chacinska A, Koehler CM, Milenkovic D, Lithgow T and Pfanner N (2009) Importing mitochondrial proteins: machineries and mechanisms. Cell 138: 628–644.

Dolezal P, Likic V, Tachezy J and Lithgow T (2006) Evolution of the molecular machines for protein import into mitochondria. Science 313: 314–318.

Gentle I, Gabriel K, Beech P, Waller R and Lithgow T (2004) The Omp85 family of proteins is essential for outer membrane biogenesis in mitochondria and bacteria. Journal of Cell Biology 164: 19–24.

Glick BS (1995) Pathways and energetics of mitochondrial protein import in Saccharomyces cerevisiae. Methods in Enzymology 260: 224–231.

Harbauer AB, Opalińska M, Gerbeth C, et al. (2014) Mitochondria. Cell cycle‐dependent regulation of mitochondrial preprotein translocase. Science 346: 1109–1113.

Harsman A and Schneider A (2017) Mitochondrial protein import in trypanosomes: expect the unexpected. Traffic 18 (2): 96–109.

Hewitt V, Alcock F and Lithgow T (2011) Minor modifications and major adaptations: the evolution of molecular machines driving mitochondrial protein import. Biochimica et Biophysica Acta 1808: 947–954.

Johnston AJ, Hoogenraad J, Dougan DA, et al. (2002) Insertion and assembly of human Tom7 into the preprotein translocase complex of the outer mitochondrial membrane. Journal of Biological Chemistry 277: 42197–42204.

Kang Y, Baker MJ, Liem M, et al. (2016) Tim29 is a novel subunit of the human TIM22 translocase and is involved in complex assembly and stability. eLife 5: e17463.

Kmita H and Budzińska M (2000) Involvement of the TOM complex in external NADH transport into yeast mitochondria depleted of mitochondrial porin1. Biochimica et Biophysica Acta 1509: 86–94.

Mani J, Meisinger C and Schneider A (2016) Peeping at TOMs‐diverse entry gates to mitochondria provide insights into the evolution of eukaryotes. Molecular Biology and Evolution 33: 337–351.

Makiuchi T and Nozaki T (2014) Highly divergent mitochondrion‐related organelles in anaerobic parasitic protozoa. Biochimie 100: 3–17.

Mesecke N, Terziyska N, Kozany C, et al. (2005) A disulfide relay system in the intermembrane space of mitochondria that mediates protein import. Cell 121: 1059–1069.

Model K, Meisinger C, Prinz T, et al. (2001) Multistep assembly of the protein import channel of the mitochondrial outer membrane. Nature Structural Biology 8: 361–370.

Mokranjac D and Neupert W (2015) Cell biology; Architecture of a protein entry gate. Nature 528: 201–202.

Murcha MW, Narsai R, Devenish J, Kubiszewski‐Jakubiak S and Whelan J (2015) MPIC: a mitochondrial protein import components database for plant and non‐plant species. Plant Cell Physiology 56: e10.

Neupert W and Herrmann JM (2007) Translocation of proteins into mitochondria. Annual Review of Biochemistry 76: 723–749.

Neupert W (2015) A perspective on transport of proteins into mitochondria: a myriad of open questions. Journal of Molecular Biology 427: 1135–1158.

Ott M and Herrmann JM (2010) Co‐translational membrane insertion of mitochondrially encoded proteins. Biochimica et Biophysica Acta 1803: 767–775.

Singha UK, Hamilton V and Chaudhuri M (2015) Tim62, a novel mitochondrial protein in trypanosoma brucei, is essential for assembly and stability of the TbTim17 protein complex. Journal of Biological Chemistry 290: 23226–23239.

Sokol AM, Sztolsztener ME, Wasilewski M, Heinz E and Chacinska A (2014) Mitochondrial protein translocases for survival and wellbeing. FEBS Letters 588: 2484–2495.

Stojanovski D, Pfanner N and Wiedemann N (2007) Import of proteins into mitochondria. Methods in Cell Biology 80: 783–806.

Stojanovski D, Milenkovic D, Müller JM, et al. (2008a) Mitochondrial protein import: precursor oxidation in a ternary complex with disulfide carrier and sulfhydryl oxidase. Journal of Cell Biology 183: 195–202.

Stojanovski D, Müller JM, Milenkovic D, et al. (2008b) The MIA system for protein import into the mitochondrial intermembrane space. Biochimica et Biophysica Acta 1783: 610–617.

Stroud DA, Becker T, Qiu J, et al. (2011) Biogenesis of mitochondrial β‐barrel proteins: the POTRA domain is involved in precursor release from the SAM complex. Molecular Biology of the Cell 22: 2823–2833.

Sztolsztener ME, Brewinska A, Guiard B and Chacinska A (2013) Disulfide bond formation: sulfhydryl oxidase ALR controls mitochondrial biogenesis of human MIA40. Traffic 14: 309–320.

Szyrach G, Ott M, Bonnefoy N, Neupert W and Herrmann JM (2003) Ribosome binding to the Oxa1 complex facilitates co‐translational protein insertion in mitochondria. EMBO Journal 22: 6448–6457.

Vögtle FN, Burkhart JM, Rao S, et al. (2012) Intermembrane space proteome of yeast mitochondria. Molecular & Cellular Proteomics 11: 1840–1852.

Webb CT, Gorman MA, Lazarou M, Ryan MT and Gulbis JM (2006) Crystal structure of the mitochondrial chaperone TIM9‐10 reveals a six‐bladed α‐propeller. Molecular Cell 21: 123–133.

Wenz LS, Opaliński Ł, Wiedemann N and Becker T (2015) Cooperation of protein machineries in mitochondrial protein sorting. Biochimica et Biophysica Acta 1853: 1119–1129.

Westermann B (2010) Mitochondrial fusion and fission in cell life and death. Molecular Cell. Biology 11: 872–884.

Wrobel L, Trojanowska A, Sztolsztener ME and Chacinska A (2013) Mitochondrial protein import: Mia40 facilitates Tim22 translocation into the inner membrane of mitochondria. Molecular Biology of the Cell 24: 543–554.

Wojtkowska M, Buczek D, Stobienia O, et al. (2015) The TOM complex of Amoebozoans: the cases of the amoeba Acanthamoeba castellanii and the slime mold Dictyostelium discoideum. Protist 166: 349–362.

Further Reading

Schägger H and von Jagow G (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Analytical Biochemistry 199: 223–231.

Sickmann A, Reinders J, Wagner Y, et al. (2003) The proteome of Saccharomyces cerevisiae mitochondria. Proceedings of the National Academy of Sciences of the United States of America 100: 13207–13212.

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Stojanovski, Diana, Ryan, Michael T, Wojtkowska, Małgorzata, and Kmita, Hanna(Jul 2017) Mitochondria Protein Import: Methods. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0002617.pub3]