Skip to main content

Advertisement

SpringerLink
Log in

A genetic approach to identifying mitochondrial proteins

  • Article
  • Published:

From Nature Biotechnology

View current issue Submit your manuscript

Abstract

The control of intricate networks within eukaryotic cells relies on differential compartmentalization of proteins. We have developed a method that allows rapid identification of novel proteins compartmentalized in mitochondria by screening large-scale cDNA libraries. The principle is based on reconstitution of split-enhanced green fluorescent protein (EGFP) by protein splicing of DnaE derived from Synechocystis sp. PCC6803. The cDNA libraries are expressed in mammalian cells following infection with retrovirus. If a test protein contains a functional mitochondrial targeting signal (MTS), it translocates into the mitochondrial matrix, where EGFP is then formed by protein splicing. The cells harboring this reconstituted EGFP are screened rapidly by fluorescence-activated cell sorting, and the cDNAs are isolated and identified from the cells. The analysis of 258 cDNAs revealed various MTSs, among which we identified new transcripts corresponding to mitochondrial proteins. This method should provide a means to map proteins distributed within intracellular organelles in a broad range of different tissues and disease states.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1: Schematic of RING-MITO technology.
Figure 2: Selective and sensitive detection of mitochondrial proteins.
Figure 3: Selection of fluorescent cells by FACS.
Figure 4: Flow cytometry profiles and fluorescent images of representative cloned cells.
Figure 5: (A) Expression of EGFP-tagged cDNA clones (clones no. 16, 33, and 59) and their localization to mitochondria.

Similar content being viewed by others

References

  1. Lopez, M.F. & Melov, S. Applied proteomics. Mitochondrial proteins and effect on function. Circ. Res. 90, 380–389 (2002).

    Article  CAS  Google Scholar 

  2. Bell, A.W. et al. Proteomics characterization of abundant Golgi membrane proteins. J. Biol. Chem. 276, 5152–5165 (2001).

    Article  CAS  Google Scholar 

  3. Kruft, V., Eubel, E., Jansch, L., Werhahn, W. & Braun, H.P. Proteomic approach to identify novel mitochondrial proteins in Arabidopsis. Plant Physiol. 127, 1694–1710 (2001).

    Article  CAS  Google Scholar 

  4. Maltman, D.J. et al. Proteomic analysis of the endoplasmic reticulum from developing and germinating seed of castor (Ricinus communis). Electrophoresis 23, 626–639 (2002).

    Article  CAS  Google Scholar 

  5. Wijk, K. Proteomics of the chloroplast: experimentation and prediction. Trends Plant Sci. 5, 420–425 (2000).

    Article  Google Scholar 

  6. Ueki, N. et al. Selection system for genes encoding nuclear-targeted proteins. Nat. Biotechnol. 16, 1338–1342 (1998).

    Article  CAS  Google Scholar 

  7. Rhee, Y., Gurel, F., Gafni, Y., Dingwall, C. & Citovsky, V. A genetic system for detection of protein nuclear import and export. Nat. Biotechnol. 18, 433–437 (2000).

    Article  CAS  Google Scholar 

  8. Bejarano, L.A. & González, C. Motif trap: a rapid method to clone motifs that can target proteins to defined subcellular localisations. J. Cell Sci. 112, 4207–4211 (1999).

    CAS  PubMed  Google Scholar 

  9. Misawa, K. et al. A method to identify cDNAs based on localization of green fluorescent protein fusion products. Proc. Natl. Acad. Sci. USA 97, 3062–3066 (2000).

    Article  CAS  Google Scholar 

  10. Simpson, J.C., Wellenreuther, R., Poustka, A., Pepperkok, R. & Wiemann, S. Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing. EMBO Report 1, 287–292 (2000).

    Article  CAS  Google Scholar 

  11. Kumar, A. et al. Subcellular localization of the yeast proteome. Genes & Dev. 16, 707–719 (2002).

    Article  CAS  Google Scholar 

  12. Ozawa, T. & Umezawa, Y. Peptide assemblies in living cells. Methods for detecting protein–protein interactions. Supramol. Chem. 14, 271–280 (2002).

    Article  CAS  Google Scholar 

  13. Ozawa, T., Takeuchi, M., Kaihara, A., Sato, M. & Umezawa, Y. Protein splicing-based reconstitution of split green fluorescent protein for monitoring protein–protein interactions in bacteria: improved sensitivity and reduced screening time. Anal. Chem. 73, 5866–5874 (2001).

    Article  CAS  Google Scholar 

  14. Ozawa, T. & Umezawa, Y. Detection of protein-protein interactions in vivo based on protein splicing. Curr. Opin. Chem. Biol. 5, 578–583 (2001).

    Article  CAS  Google Scholar 

  15. Evans, T.C.J. et al. Protein trans-splicing and cyclization by a naturally split intein from the dnaE gene of Synechocystis species PCC6803. J. Biol. Chem. 275, 9091–9094 (2000).

    Article  CAS  Google Scholar 

  16. Roise, D. et al. Amphiphilicity is essential for mitochondrial presequence function. EMBO J. 7, 649–653 (1988).

    Article  CAS  Google Scholar 

  17. Heijne, G. Mitochondrial targeting sequences may form amphiphilic helices. EMBO J. 5, 1335–1342 (1986).

    Article  Google Scholar 

  18. Morita, S., Kojima, T. & Kitamura, T. Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Therapy 7, 1063–1066 (2000).

    Article  CAS  Google Scholar 

  19. Hayashizaki, Y. et al. Functional annotation of a full-length mouse cDNA collection. Nature 409, 685–690 (2001).

    Article  Google Scholar 

  20. Carninci, P. et al. Normalization and subtraction of cap-trapper-selected cDNAs to prepare full-length cDNA libraries for rapid discovery of new genes. Genome Res. 10, 1617–1630 (2000).

    Article  CAS  Google Scholar 

  21. Lee, C.M., Sedman, J., Neupert, W. & Stuart, R.A. The DNA helicase, Hmi1p, is transported into mitochondria by a C-terminal cleavable targeting signal. J. Biol. Chem. 274, 20937–20942 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work has been supported by Core Research for Evolutional Science and Technology of Japan Science and Technology and by grants to Y.U. from the Ministry of Education, Science and Culture, Japan.

Author information

Authors and Affiliations

  1. Department of Chemistry, School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Takeaki Ozawa, Yusuke Sako, Moritoshi Sato & Yoshio Umezawa

  2. Japan Science and Technology Corporation, Tokyo, Japan

    Takeaki Ozawa, Yusuke Sako, Moritoshi Sato & Yoshio Umezawa

  3. Department of Hematopoietic Factors, Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan

    Toshio Kitamura

Authors
  1. Takeaki Ozawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yusuke Sako
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Moritoshi Sato
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Toshio Kitamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yoshio Umezawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yoshio Umezawa.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ozawa, T., Sako, Y., Sato, M. et al. A genetic approach to identifying mitochondrial proteins. Nat Biotechnol 21, 287–293 (2003). https://doi.org/10.1038/nbt791

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt791

  • Springer Nature America, Inc.

This article is cited by

Search

Navigation