Caloric restriction improves efficiency and capacity of the mitochondrial electron transport chain in Saccharomyces cerevisiae

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Abstract

Caloric restriction (CR) is known to extend lifespan in a variety of species; however, the mechanism remains unclear. In this study, we found that CR potentiated the mitochondrial electron transport chain (ETC) at both the transcriptional and translational levels. Indeed, mitochondrial membrane potential (MMP) was increased by CR, and, regardless of ages, overall reactive oxygen species (ROS) generation was decreased by CR. With these changes, overall growth rate of cells was maintained under various CR conditions, just like cells under a non-restricted condition. All of these data support increased efficiency and capacity of the ETC by CR, and this change might lead to extension of lifespan.

Highlights

Calorie restriction (CR) increases electron transport chain (ETC) at both RNA and protein level. ► CR enhances mitochondrial membrane potential, and, regardless of ages, reduces reactive oxygen species. ► CR increases both efficiency and capacity of the ETC. ► CR induces intensive modulation at mitochondrial ETC where might be a major site leading to extension of lifespan.

Introduction

Limitation of caloric intake, termed CR, is a regimen known to extend lifespan in a variety of species, from yeast to mammals. Yeast has been used as a model for study of this highly conserved phenomenon. CR can be demonstrated in yeast through lowering of glucose concentration in culture media; therefore, CR accompanies release from glucose repression, in part [1]. Due to the pluripotent traits of CR, the mechanism responsible for the beneficial effects of CR is still unclear.

However, recent studies have demonstrated activation of mitochondrial function by CR. It is hypothesized that increased mitochondrial activity is related to the life-prolonging effect of CR in diverse species [2], [3], [4], [5]. Mitochondria play multiple roles, including energy, anabolic, and catabolic metabolism [6], [7], [8], [9]. They are also a significant source of cellular ROS, which cause damage to macromolecules, leading to decline of cellular function [10]. In addition, cytoplasmic ROS accumulation is associated with a reduced MMP in yeast [11]. Several studies have reported the requirement of mitochondrial function for extension of chronological lifespan (CLS), while CLS was decreased upon treatment with antimycin A, an inhibitor of oxidative phosphorylation [12]. In addition, cellular ROS level was increased, and CLS was shortened in a yeast strain mutated in mitochondrial RNA polymerase [13]. The yeast mutant known as HsTnII shows certain characteristics, including shortened CLS with remarkably fragmented and dysfunctional mitochondria [14].

In our previous report, we found several biological process terms, including oxidative phosphorylation, and a cellular component term, mitochondrion, which is the only subcellular organelle showing a significant relationship with different CR conditions [15]. In addition, CR yeast showed higher oxygen consumption than non-CR cells; however, ATP levels of CR yeast were similar to those of non-CR yeast, despite their limitation as an energy source [16]. Based on this evidence, mitochondria, particularly the ETC, would be an important place for understanding the mechanism of lifespan extension by CR in yeast. In this study, to further evaluate the role of ETC in lifespan extension, we measured transcriptional activities of mitochondrial DNA, protein levels of most components for ETC, MMP, total ROS level, and growth rate under different CR conditions.

Section snippets

Yeast strains and culture

Saccharomyces cerevisiae BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and Tandem Affinity Purification (TAP)-tagged strains for immunoblot were purchased from Open Biosystems (Huntsville, AL, USA). Each genotype-confirmed colony was cultured as previously described [15], except that starting OD600 was 0.05 into 200 ml YPD. Depending on the study purpose, cultured cells were harvested at the early-, mid-, and late-point of the exponential phase.

Protein extraction, immunoblot, and microarray data

For preparation of whole cell extracts, yeast cells

CR potentiated efficiency and capacity of mitochondrial ETC.

Previously, we selected 160 nuclear genes as transcriptional markers of CR [15]. Among them, 11 genes encode ETC components, and showed positive correlation with different CR conditions. Using the Q-PCR method, we measured expression of ETC genes encoded in mitochondrial DNA, and relative copy number of mitochondrial DNA to nuclear DNA. The results consistently indicated that CR resulted in increased expression of ETC genes in mitochondrial DNA (Fig. 1A) without change of relative copy number

Discussion

CR is the most effective intervention for extension of lifespan in diverse species. In our previous study, we mimicked the mammalian CR condition in a yeast batch culture system and obtained results indicating that mitochondria-related genes were gradually increased at the transcriptional level according to CR strength [15]. Data from microarray analysis clearly showed beyond glucose derepression. For example, genes regulated by Mig1p, a major transcription factor for release of glucose

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MEST) (Nos. 2008-0061063 and 2009-0084110) to C.K.L., as well as by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. 351-2009-1-C00063) to J.S.C.

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These authors contributed equally for this work.

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