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.


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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]