Plant mitochondria contain about 1000 proteins, 90–99% of which in different plant species are nuclear encoded, synthesized on cytosolic polyribosomes, and imported into the organelle. Most of the nuclear-encoded proteins are synthesized as precursors containing an N-terminal extension called a presequence or targeting peptide that directs the protein to the mitochondria. Here we describe in vitro and in vivo methods to study mitochondrial protein import in plants. In vitro synthesized precursor proteins can be imported in vitro into isolated mitochondria (single organelle import). However, missorting of chloroplast precursors in vitro into isolated mitochondria has been observed. A novel dual import system for simultaneous import of proteins into isolated mitochondria and chloroplasts followed by reisolation of the organelles is superior over the single import system as it abolishes the mistargeting. Precursor proteins can also be imported into the mitochondria in vivo using an intact cellular system. In vivo approaches include import of transiently expressed fusion constructs containing a presequence or a full-length precursor protein fused to a reporter gene, most commonly the green fluorescence protein (GFP) in protoplasts or in an Agrobacterium-mediated system in intact tobacco leaves.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Truscott, K. N., Brandner, K., and Pfanner, N. (2003) Mechanisms of protein import into mitochondria. Curr. Biol. 13, R326–337.
Gray, M. W., Burger, G., and Lang, B. F. (1999) Mitochondrial evolution. Scienc, 283, 1476–1481.
Zhang, X. P. and Glaser, E. (2002) Interaction of plant mitochondrial and chloroplast signal peptides with Hsp70 molecular chaperone. Trends Plant Sci. 7, 14–21.
Glaser, E. and Soll, J. (2004) Targeting signals and import machinery of plastids and plant mitochondria. In Molecular Biology and Biotechnology of Plant Organelles: Chloroplasts and Mitochondria(Daniell, H. and Chase, C., eds.) Springer, Dordrecht, The Netherlands, pp. 385–418.
Marc, P., Margeot, A., Devaux, F., Blugeon, C., Corral-Debrinski, M., and Jacq, C. (2002). Genome-wide analysis of mRNAs targeted to yeast mitochondria. EMBO Rep. 3, 159–164.
Glaser, E. and Dessi, P. (1999) Integration of the mitochondrial processing peptidase into the bc1 complex of the respiratory chain in plants. J. Bioenerg. Biomembr. 31, 259–274.
Ståhl, A. Moberg, P., Ytterberg, J., et al. (2002) Isolation and identification of a novel mitochondrial metalloprotease (PreP) that degrades targeting presequences. J. Biol. Chem. 277, 41931–41939.
Moberg, P., Ståhl, A., Bhushan, S., et al. (2003) Characterization of a novel zinc metalloprotease involved in degrading signal peptides in mitochondria and chloroplasts. Plant J. 36, 616–628.
Bushan, S., Lefebvre, B., Ståhl, A., Boutry, M., and Glaser, E. (2003) Dual targeting and function of a protease in mitochondria and chloroplasts. EMBO Rep. 4, 1073–1078.
Ståhl, A., Nilsson, S., Lundberg, P., Bhushan, S., et al. (2005) Two novel targeting peptide degrading proteases, PrePs, in mitochondria and chloroplasts, so similar and still different. J. Mol. Biol. 349, 847–860.
Johnson, K. A., Bhushan, S., Ståhl, A., et al. (2006) The closed structure of presequence protease PreP forms a unique 10,000 Å3 chamber for proteolysis. EMBO J. 25, 1977–1986.
Creissen, G., Reynolds, H., Xue, Y., and Mullineaux, P. (1995) Simultaneous targeting of pea glutathione reductase and of a bacterial fusion protein to chloroplasts and mitochondria in transgenic tobacco. Plant J. 8, 167–175.
Small, I., Wintz, H., Akashi, K., and Mireau, H. (1998) Two birds with one stone: genes that encode products targeted to two or more compartments. Plant Mol. Biol. 38, 265–277.
Hedtke, B., Borner, T., and Weihe, A. (2000) One RNA polymerase serving two genomes. EMBO Rep. 1, 435–440.
Rudhe, C., Chew, O., Whelan, J., and Glaser, E. (2002) A novel in vitro system for simultaneous import of precursor proteins into chloroplast and mitochondria. Plant J. 30, 213–220.
Boutry, M., Nagy, F., Poulsen, C., Aoyagi, K., and Chua, N.H. (1987) Targeting of bacterial chloramphenicol acetyltransferase to mitochondria in transgenic plants. Nature 328, 340–342.
Schmitz, U. K. and Lonsdale, D. M. (1989) A yeast mitochondrial presequence functions as a signal for targeting to plant mitochondria in-vivo. Plant Cell 1, 783–791.
Silva Filho, M. d. C., Wieers, M.-C., Flugge, U.-I., Chaumont, F., and Boutry, M. (1997) Different in vitro and in vivo targeting properties of the transit peptide of a chloroplast envelope inner membrane protein. J. Biol. Chem. 272, 15264–15269.
Whelan, J., Knorpp, C., and Glaser, E. (1990) Sorting of precursor proteins between isolated spinach leaf mitochondria and chloroplasts. Plant Mol. Biol. 14, 977–982.
Glaser, E., Sjoling, S., Tanudji, M., and Whelan, J. (1998) Mitochondrial protein import in plants. Plant Mol. Biol. 38, 311–338.
Soll, J. and Tien, R. (1998) Protein translocation into and across the chloroplastic envelope membranes. Plant Mol. Biol. 38, 191–207.
Hugosson, M., Nurani, G., Glaser, E. and Franzen, L.G. (1995) Peculiar properties of the PsaF photosystem I protein from the green alga Chlamydomonas reinhardtii: presequence independent import of the PsaF protein into both chloroplasts and mitochondria. Plant Mol. Biol. 28, 525–535.
Brink, S., Flugge, U. I., Chaumont, F., et al. (1994) Preproteins of chloroplast envelope inner membrane contain targeting information for receptor-dependent import into fungal mitochondria. J. Biol. Chem. 269, 16478–16485.
von Stedingk, E. (1999) Sorting and import of plant mitochondrial precursors. PhD thesis, Stockholm University, Stockholm.
Lister, R., Chew, O., Lee, M., and Whelan, J. (2001) Arabidopsis thaliana ferrochelatase-I and -II are not imported into Arabidopsis mitochondria. FEBS Lett. 506, 291–295.
Cleary, S. P., Tan, F.-C., Nakrieko, K.-A., et al. (2002) Isolated plant mitochondria import chloroplast precursor proteins in vitro with the same efficiency as chloroplasts. J. Biol. Chem. 277, 5562–5569.
Duby, G., Oufattole, M., and Boutry, M. (2001) Hydrophobic residues within the predicted N-terminal amphiphilic alpha-helix of a plant mitochondrial targeting presequence play a major role in in vivo import. Plant J. 27, 539–549.
Von Stedingk, E., Pavlov, P. F., Grinkevich, V.A., and Glaser, E. (1999) The precursor of F_1ß subunit of the ATP synthase is covalently modified upon binding to plant mitochondria. Plant Mol. Biol. 41, 505–515.
Datta, K. and Datta, S.K. (1999) Transformation of rice via PEG-mediated DNA uptake into protoplasts. Meth. Mol. Biol. 111, 335–347.
Chew, O., Rudhe, C., Glaser, E., and Whelan, J. (2003) Characterization of the targeting signal of dual-targeted pea glutathione reductase. Plant Mol. Biol. 53, 341–356.
Lukaszewicz, M., Jerouville, B. and Boutry, M. (1998) Signs of translational regulation within the transcript leader of a plant plasma membrane H(+)-ATPase gene. Plant J. 14, 413–423.
Acknowledgments
The authors would like to thank Prof. J. Whelan and Dr. O. Chew for a fruitful collaboration on the development of the dual import system and Prof. M. Boutry and Dr. B. Lefebvre for instructions and cooperation using in vivo import procedures. This work was supported by a grant from The Swedish Research Council to EG.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc.
About this protocol
Cite this protocol
Bhushan, S., Pavlov, P.F., Rudhe, C., Glaser, E. (2007). In Vitro and In Vivo Methods to Study Protein Import Into Plant Mitochondria. In: van der Giezen, M. (eds) Protein Targeting Protocols. Methods in Molecular Biology™, vol 390. Humana Press. https://doi.org/10.1007/978-1-59745-466-7_9
Download citation
DOI: https://doi.org/10.1007/978-1-59745-466-7_9
Publisher Name: Humana Press
Print ISBN: 978-1-58829-702-0
Online ISBN: 978-1-59745-466-7
eBook Packages: Springer Protocols