Abstract
Mitochondria are dynamic organelles with activities that adjust to altering physiological conditions and variable metabolic demands. A conserved proteolytic system present within the organelle exerts essential functions during the biogenesis of mitochondria and ensures the maintenance of organellar activities under varying conditions. Proteases dependent on adenosine triphosphate, in concert with oligopeptidases, degrade nonassembled or damaged proteins in various subcompartments of mitochondria, preventing their accumulation and possibly deleterious effects on mitochondrial functions. Although an increasing number of mitochondrial peptidases are characterized and functionally linked to diverse cellular processes, only limited information is available on the stability of the mitochondrial proteome and the turnover rates of individual proteins. We describe experimental approaches in the yeast Saccharomyces cerevisiae and in mice, allowing analysis of the proteolytic breakdown of mitochondrial proteins individually or on a proteomewide scale.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Gakh, O., Cavadini, P., and Isaya, G. (2002) Mitochondrial processing peptidases. Biochim. Biophys. Acta 1592, 63–77.
Esser, K., Tursun, B., Ingenhoven, M., Michaelis, G., and Pratje, E. (2002) A novel two-step mechanism for removal of a mitochondrial signal sequence involves the m-AAA complex and the putative rhomboid protease Pcp1. J. Mol. Biol. 323, 835–843.
Herlan, M., Vogel, F., Bornhövd, C., Neupert, W., and Reichert, A.S. (2003) Processing of Mgm1 by the rhomboid-type protease Pcp1 is required for maintenance of mitochondrial morphology and of mitochondrial DNA. J. Biol. Chem. 278, 27,781–27,788.
McQuibban, G. A., Saurya, S., and Freeman, M. (2003) Mitochondrial membrane remodelling regulated by a conserved rhomboid protease. Nature 423, 537–541.
Van Dyck, L. and Langer, T. (1999) ATP-dependent proteases controlling mitochondrial function in the yeast Saccharomyces cerevisiae. Cell Mol. Life Sci. 55, 825–842.
Bota, D. A. and Davies, K. J. A. (2001) Protein degradation in mitochondria: implications for oxidative stress, aging and disease: a novel etiological classification of mitochondrial proteolytic disorders. Mitochondrion 1, 33–49.
Young, L., Leonhard, K., Tatsuta, T., Trowsdale, J., and Langer, T. (2001) Role of the ABC transporter Mdl1 in peptide export from mitochondria. Science 291, 2135–2138.
Augustin, S., Nolden, M., Müller, S., Hardt, O., Arnold, I., and Langer, T. (2005) Characterization of peptides released from mitochondria: evidence for constant proteolysis and peptide efflux. J. Biol. Chem. 280, 2691–2699.
Kambacheld, M., Augustin, S., Tatsuta, T., Müller, S., and Langer, T. (2005) Role of the novel metallopeptidase MOP112 and saccharolysin for the complete degradation of proteins residing in different subcompartments of mitochondria. J. Biol. Chem. 280, 20,132–20,139.
Daum, G., Gasser, S. M., and Schatz, G. (1982) Import of proteins into mitochondria. Energy-dependent, two-step processing of the intermembrane space enzyme cytochrome b2 by isolated yeast mitochondria. J. Biol. Chem. 257, 13,075–13,080.
Herrmann, J. M., Fölsch, H., Neupert, W., and Stuart, R. A. (1994) Isolation of yeast mitochondria and study of mitochondrial protein translation, in Cell Biology: A Laboratory Handbook, vol. 1 (Celis, D. E., ed.), Academic Press, San Diego, CA, pp. 538–544.
Meisinger, C., Sommer, T., and Pfanner, N. (2000) Purification of Saccharomcyes cerevisiae mitochondria devoid of microsomal and cytosolic contaminations. Anal. Biochem. 287, 339–342.
Brandt, A. (1991) Pulse labeling of yeast cells as a tool to study mitochondrial protein import. Methods Cell Biol. 34, 369–376.
Kozak, M. (1987) An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125–8148.
Black-Schaefer, C. L., McCourt, J. D., Poyton, R. O., and McKee, E. E. (1991) Mitochondrial gene expression in Saccharomyces cerevisiae. Proteolysis of nascent chains in isolated yeast mitochondria optimized for protein synthesis. Biochem. J. 274, 199–205.
Mattiazzi, M., D’Aurelio, M., Gajewski, C. D., et al. (2002) Mutated human SOD1 causes dysfunction of oxidative phosphorylation in mitochondria of transgenic mice. J. Biol. Chem. 277, 29,626–29,633.
Gancedo, J. M. (1998) Yeast carbon catabolite repression. Microbiol. Mol. Biol. Rev. 62, 334–361.
Käser, M., Kambacheld, M., Kisters-Woike, B., and Langer, T. (2003) Oma1, a novel membrane-bound metallopeptidase in mitochondria with activities overlapping with the m-AAA protease. J. Biol. Chem. 278, 46,414–46,423.
Leonhard, K., Guiard, B., Pellechia, G., Tzagoloff, A., Neupert, W., and Langer, T. (2000) Membrane protein degradation by AAA proteases in mitochondria: extraction of substrates from either membrane surface. Mol. Cell 5, 629–638.
Rottgers, K., Zufall, N., Guiard, B., and Voos, W. (2002) The ClpB homolog Hsp78 is required for the efficient degradation of proteins in the mitochondrial matrix. J. Biol. Chem. 277, 45,829–45,837.
Leonhard, K., Stiegler, A., Neupert, W., and Langer, T. (1999) Chaperone-like activity of the AAA domain of the yeast Yme1 AAA protease. Nature 398, 348–351.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Tatsuta, T., Langer, T. (2007). Studying Proteolysis Within Mitochondria. In: Leister, D., Herrmann, J.M. (eds) Mitochondria. Methods in Molecular Biology™, vol 372. Humana Press. https://doi.org/10.1007/978-1-59745-365-3_25
Download citation
DOI: https://doi.org/10.1007/978-1-59745-365-3_25
Publisher Name: Humana Press
Print ISBN: 978-1-58829-667-2
Online ISBN: 978-1-59745-365-3
eBook Packages: Springer Protocols