Identification of respiratory chain gene mutations that shorten replicative life span in yeast

Keywords

• Mitochondria;
• Electron transport chain (ETC);
• Reactive oxygen species (ROS);
• Aging;
• Replicative life span;
• Chronological aging;
• Longevity;
• Yeast

1. Introduction

Mitochondria are of great importance as being the site of energy production of eukaryotic cells (Schneider and Guarente, 1991). Oxidative phosphorylation that takes place in the mitochondria is crucial for the energy metabolism and implicated to be related with the aging process (Speakman, 2005 ;  Yang and Hekimi, 2010). The yeast Saccharomyces cerevisiae has the ability to grow by fermentation in the presence of glucose or by mitochondrial respiration in the presence of respiratory substrates such as glycerol. However, it primarily prefers fermentation and can inhibit aerobic respiration in the presence of glucose ( Skinner and Lin, 2010). Therefore, since S. cerevisiae can grow either by fermentation or respiration, it is a suitable model to study the effects of the loss of genes needed for respiration, which can be lethal in many other organisms.

The mitochondrial electron transport chain (ETC), couples electrons from oxidized metabolic fuels to ATP production (Dimroth et al., 2000). The ETC is composed of five enzyme complexes (complexes I–V) (Dibrov et al., 1998), of which are known to be highly regulated in yeast (Schneider and Guarente, 1991). The assembly of the complexes requires several proteins including chaperones that assist their formation. The electrons are transfered from NADH and succinate to oxygen by respiratory complexes I to IV and the electrochemical gradients of protons formed via this transfer are used by the complex V (ATP synthase) to produce ATP (Hamel et al., 1998). Yeasts do not have a multisubunit complex I, instead they have two NADH-dehydrogenases and one NADH-ubiquinone oxidoreductase (de Vries and Grivell, 1988 ;  Melo et al., 2004). Reactive oxygen species (ROS) are produced mainly by the ETC in the inner mitochondrial membrane, especially from redox sites of complexes I and III (Boveris, 1977; Turrens, 2003; Drose and Brandt, 2008; Murphy, 2009; Brand, 2010 ;  Cortes-Rojo et al., 2011 ).

Mitochondrial dysfunction is shown to be related with several pathological processes (Janssen et al., 2004; Seppet et al., 2009 ;  Perry et al., 2011). The roles of the ROS and mitochondria in aging have been studied in yeast and other eukaryotes, and results vary depending on the model organisms studied. There is a continous increase of ROS formation and an age-dependent decline in mitochondrial function throughout the aging process (Harman, 1956; Shigenaga et al., 1994; Beckman and Ames, 1998; Laun et al., 2001; Navarro and Boveris, 2007 ;  Lam et al., 2011). Calorie restriction seems to extend replicative life span (RLS) by decreasing the production of ROS and increasing the rate of the mitochondrial respiration (Barros et al., 2004). Dinitrophenol, an uncoupling agent, mimics the effect of calorie restriction on ROS production and life span modulation. On the other hand, antimycin A, an inhibitor of complex III, increases ROS production and shortens the chronological life span (CLS) in yeast (Barros et al., 2004). In C. elegans, impairment of mitochondrial functions or treatment with antimycin A increases the life span ( Lee et al., 2003; Anson and Hansford, 2004 ;  Ventura et al., 2005). Mutation in the iron sulfur protein Isp-1, which is a component of complex III, results in lower respiratory rate and longer life span (Feng et al., 2001). In Drosophila, complex IV inhibitors such as KCN and sodium azide increase the production of hydrogen peroxide and decrease cytochrome c oxidase activity. Flies with deficiency in complex II (sdhB) suffer from elevated oxidative stress and age faster ( Walker et al., 2006). Additionally, inactivation of five genes encoding components of respiratory complexes I, III, IV, and V by RNAi can prolong life span (Copeland et al., 2009). In human cell line 143B, administration of ETC inhibitors results in increased ROS production (Indo et al., 2007). Thus, the roles of the elecron transport chain components or chemicals that modulate electron transfer show contradictory results on life spans and ROS levels.

In this study, we analyzed the RLS of ETC gene mutants to elucidate the roles of these genes in the aging process of the budding yeast S. cerevisiae. Out of 73 yeast deletion mutants of non-essential ETC genes, deletion of NDE1, TCM62, RIP1, CYT1, QCR8, PET117, COX11, ATP11, and FMC1 were found to be important in replicative life span determination. Short living ETC gene mutants had lower rates of respiration and showed different levels of sensitivity to exogenous administration of hydrogen peroxide. Moreover, they did not have significant alterations in their mitochondrial membrane potentials. Thus, our results suggest that short living ETC mutants neither experience severe oxidative stress nor have significant differences in means of their membrane potentials compared to wild type cells.

2. Materials and methods

2.1. Yeast strains and growth

WT strain BY4741 (MATa his3 leu2 met15 ura3) and its isogenic deletion mutants were obtained from the yeast deletion library (Invitrogen). Cells were grown on YPD agar (1% yeast extract, 2% peptone, 2% dextrose and 2% agar) media, if otherwise not stated.

2.2. Life span analyses

WT strain BY4741 (MATa his3 leu2 met15 ura3) and its isogenic deletion mutants were obtained from the yeast deletion library (Invitrogen). Cells were grown on YPD agar (1% yeast extract, 2% peptone, 2% dextrose and 2% agar) media for 2 days before analysis. For each strain, 25 daughter cells (starter mothers) were collected and lined up by a micromanipulator on agar plates. New buds (daughters) from these virgin cells were removed and discarded as they formed. This process continued until cells ceased dividing. The life span was determined as the total number of daughter cells that each mother cell generated. For crude reduction of sample numbers, we analyzed the life span of five cells per strain for the initial screening and identified the mutants with possible short life spans. In the second and third rounds of aging analyses, 25 cells for each strain were followed. For further analyses of the respiratory mutants, 0.1% glucose containing YPD media was used. For the chronological life span analysis, yeast strains were grown in 2 ml YPD media overnight and were suspended in 25 ml fresh YPD in 250 ml flasks and incubated at 30 °C for 15 days at 180 rpm. The survival rate of cells was measured by counting the colony forming units every 72 h. Each analysis was performed three times.

2.3. Hydrogen peroxide sensitivity

A halo assay was performed to assess the sensitivity of the cells to hydrogen peroxide. Briefly, cells were grown in liquid YPD overnight and their OD₆₀₀ values were adjusted to 0.2. 400 μl aliquot from each culture was transferred to YPD plates and dried for 30 min. Then, 5 μl 8.8 M hydrogen peroxide was administered to the center of the plates and the plates were incubated at 30 °C overnight. Radius of the clear zone in the center of each plate was measured by a ruler. The assay was performed three times in duplicates for each strain.

2.4. Determination of respiratory deficient strains

Yeast strains with shorter replicative lifespans were grown in YPG (3% glycerol). Spotting assay was performed with cells with adjusted OD₆₀₀ values of 0.2. Serial dilution was performed to obtain OD₆₀₀ values of 0.02, 0.002, and 0.0002. 5 μl of cell solutions was dropped onto YPG-agar plates and incubated at 30 °C for 48 h.

2.5. Determination of intracellular superoxide levels

The intracellular superoxide production rates of the yeast strains were measured with the fluorometric dye MitoSOX™ red (Invitrogen) as described by the manufacturer. Briefly, yeast cells were grown in either 2% glucose or 0.1% glucose containing YPD up to a OD₆₀₀ value of 0.5. Cells were pelleted and resuspended in YPD containing 5 μM of MitoSOX red dye. After 1 h of incubation at 30 °C, cells were washed with PBS twice and analyzed by a fluorometric spectrometer (Thermo VarioScan).