Mitochondrial Physiology Network 10.9: 122-123 (2005) - download pdf

Yeast as a model for investigating mitochondrial oxidative damage.

Helena M Cochemé, MP Murphy

MRC Dunn Human Nutrition Unit, Cambridge, UK. - hmc@mrc-dunn.cam.ac.uk

    The mitochondrial respiratory chain is an important source of ROS (reactive oxygen species), which cause protein damage, lipid peroxidation and mitochondrial DNA (mtDNA) mutations. This oxidative stress has been implicated in fundamental processes such as ageing, degenerative diseases and apoptosis. However many aspects of ROS production by mitochondria and its physiological implications for cell death and ageing are unknown. We are studying how ROS production contributes to mitochondrial dysfunction, in particular how levels of oxidative stress correlate with mtDNA damage and fixation of mutations. These processes are being investigated in the budding yeast Saccharomyces cerevisiae, which is an excellent eukaryotic model amenable to genetic manipulation and extensive colony scoring.

    Superoxide (O₂^•-) is the proximal ROS produced by the mitochondrial respiratory chain via single-electron reduction of O₂, and is therefore of primary interest and significance to the study of oxidative damage. As little was previously known about ROS production by yeast mitochondria, it was first necessary to understand the sources of ROS in yeast, before focusing on the genetic consequences. We therefore optimised a range of biochemical assays for O₂^•- measurement, to establish the sites, topology and magnitude of O₂^•- production by the yeast respiratory chain. Levels of O₂^•- were inferred from assaying: the rate of aconitase inactivation (an enzyme of the Krebs cycle, located in the mitochondrial matrix which contains an iron-sulphur cluster at its active site, susceptible to attack by O₂^•-) [1], coelenterazine chemiluminescence (a compound involved in the bioluminescence of marine organisms, which reacts specifically with O₂^•- in vitro leading to quantifiable light emission) [2], and hydrogen peroxide efflux (a more stable conversion product of O₂^•-, which is able to diffuse across biological membranes and can be detected with fluorometric probes).

    The above techniques have being applied to isolated yeast mitochondria, energised with various respiratory substrates, and incubated with compounds such respiratory inhibitors, redox cycler and uncoupler. From these experiments we have identified the conditions of O₂^•- production by yeast mitochondria, which provides support for current studies into the relationship between ROS production and oxidative damage to mitochondria, with particular focus on mtDNA. We are exploring various methods to monitor levels of mtDNA damage and mutations rates, including quantitative PCR [3] and HPLC detection of 8-hydroxydeoxyguanosine (8OHdG), a DNA lesion formed by attack with the hydroxyl radical (ŸOH) and widely used as a biomarker for oxidative damage [4]. This research aims to clarify how ROS production leads to mtDNA damage.

1.  Gardner PR (2002) Aconitase: sensitive target and measure of superoxide. Methods Enzymol. 349: 9-23.

2.  Teranishi K, Shimomura O (1997) Coelenterazine analogs as chemiluminescent probe for superoxide anion. Anal. Biochem. 249: 37-43.

3.  Santos JH et al. (2002) Measuring oxidative mtDNA damage and repair using quantitative PCR. Methods Mol. Biol. 197: 159-176.

4.  Beckman KB, Ames BN (1999) Endogenous oxidative damage of mtDNA. Mutat. Res. 424: 51-58.