The copper containing ferroxidase, Fet3p, has been shown to increase iron concentrations in yeast (Stearman et al., 1996
). The life span of fet3
mutants is not increased by either copper or iron supplementation, showing both that the extension by copper is due to the increase in iron supplied to the cell through Fet3p, and that life span extension by iron is dependent on Fet3p for entry into the cell. Even at high iron concentrations the iron responsible for life span extension is entering cells through the high affinity transporter Fet3p. While iron is still required, the low affinity transporter, Fet4p, is sufficient to provide iron for survival. Fet4p is a low affinity transporter and the amount of iron it imports does not change much over a range of iron concentrations (Dix et al., 1997
). In fact, Fet4p transport of iron is less than optimal even for growth since the fet3
strain displays a growth defect on non-fermentable carbon sources (Yuan et al., 1995
and our unpublished observation).
The extension of life span in cells supplied with iron or copper is possibly due to a reduction in oxidative stress, which is supported by our findings that strains with deletions of either SOD1 or SOD2 have greatly reduced life spans that are partially rescued by iron supplementation (), and that copper supplemented cells have reduced production of superoxide (). We did not directly measure superoxide production in cells supplemented with iron because the precipitate caused by the high iron concentration titrates out the DHE, resulting in poor staining. To remove this precipitate for the iron assay required extensive washing, which would likely result in metabolically inactive cells. Since copper supplementation increases cellular iron the decrease in superoxide production is likely due to the increased iron in cells.
Initially the idea that increasing iron concentration extends life span seems counter-intuitive due to the possibility of superoxide production by the often referenced Fenton reaction. Disorders that result in high levels of iron, such as hemochromatosis (Toyokuni, 2009
), or Friedreich’s ataxia (Armstrong, et al., 2010
) show increased oxidative stress due to the Fenton reaction, in which ferrous iron (Fe2+) catalytically generates hydoroxyl radicals (Wardman and Candeias, 1996
). However, evidence for the existence of free ferrous iron in non-diseased eukaryotic cells or organisms is rare due to a lack of methods to detect it (Pate, et al., 2006
; Toyokuni, 2009
). Anemia on the other hand is a confirmed problem in elderly humans (Vanasse and Berliner, 2010
). Therefore, the more realistic danger in normal aging may be a lack of iron that results in the inhibition of an efficient electron flow.
A variety of situations that inhibit electron transfer through the electron transport chain, such as disease (Esposito et al., 1999
; Calabrese et al., 2005
; Napoli et al., 2006
) or chemical inhibitors (St-Pierre et al., 2002
) are known to increase mitochondrial production of reactive oxygen species (ROS). Yeast cells in which electron transport is inhibited either chemically, or through mutation or loss of mitochondrial DNA display an increase in nuclear mutations due to increased ROS production (Rasmussen et al., 2003
). Iron deficiency, in the form of anemia in humans, has been shown to reduce heme content in mitochondrial and lead to the increase in ROS (Atamna et al., 2002
; Ames et al., 2005
). This may be the finding most similar to the results presented here. The yeast strains were grown on rich media containing yeast extract. Even without supplementation the iron concentration is far from limiting for growth, as colonies have no problem growing in these conditions and supplemented liquid cultures grow at the same rate as unsupplemented (not shown). However, the life span assay looks not for survival of the colony, but for survival of the individual. In order to produce exponential growth each cell only needs to bud twice. For maximal life span the iron requirements are higher than they are for basic survival, which is likely true for a variety of nutrients in all organisms given the opportunity to reach maximum longevity (Ames et al., 2005
). The reduced ability of cells to obtain and/or incorporate iron into the components of the electron transport chain could, in part, account for increased oxidative stress and damage found in tissue with increasing age (Moghaddas et al., 2003
; Choksi et al., 2008
). Lam et al. (2011)
have recently shown that oxidative stress begins to accumulate early in replicatively aging yeast. Even slightly increased levels of ROS at earlier ages due to less efficient electron transport could result in a shortening of replicative life span.
It has been known for some time that increased respiration leads to increased longevity in yeast. Lin et al. (2002)
showed that the shift in metabolism toward respiration is responsible for the increased replicative life span of yeast grown under conditions that mimic caloric restriction in yeast (0.5% glucose vs. the normal 2% glucose). Although copper or iron supplementation does not increase the life span of glucose grown yeast, we have preliminary evidence that copper or iron supplementation does increase the replicative life span of yeast grown on raffinose, although no to the same extent as on glycerol (not shown). This is likely due to the fact that on raffinose mitochondrial biogenesis is not repressed to the same extent as it is on glucose, resulting in increased respiratory metabolism (Szekely & Montgomery, 1984
). Further support comes from Barros et al. (2004)
who found that that increasing respiration by lowering glucose concentration or adding an uncoupling agent increases yeast chronological life span and lowers the production of ROS.
Veatch et al. (2009)
have shown that older yeast cells (>20 generations) produce buds that have lost heterozygosity (LOH). They propose that decreased iron-sulfur cluster (ISC) biogenesis results from the loss of mitochondrial DNA and alters the iron regulon in cells, leading to LOH. In our system, with haploid cells grown on a non-fermentable carbon source, the loss of either nuclear or mitochondrial chromosomes will result in cell death. Their system uses diploid cells grown on a fermentable carbon source, allowing viability and detection of both cells that have lost mitochondrial DNA or cells that have lost chromosomes. What initially causes the loss of mitochondrial DNA is an open question. Iron-sulfur clusters are known to be targets of ROS damage (Sideri et al., 2009
) and, when damaged can release free iron, leading to DNA damage (Keyer and Imlay, 1996
). A protein containing an iron-sulfer cluster, which is also known to be involved in mitochondrial DNA maintenance, is mitochondrial aconitase (Chen et al., 2005
). This role is independent of its catalytic activity in the Krebs cycle. Mitochondrial aconitase is part of a mitochondrial nucleoid that protects mitochondrial DNA from oxidative damage (Chen et al., 2007
) and is regulated by the retrograde response (Liu & Butow, 2006
), which coordinates metabolism and aging (Jazwinski, 2005
). Interestingly, mitochondrial aconitase has recently been shown to be asymmetrically inherited in yeast, with the oxidatively-damaged, inactive molecules remaining in the mother cell (Klinger, et al., 2010
). If increased iron counteracts the initial decrease in iron-sulfur cluster or heme production, thereby reducing oxidative stress, it would lead to the extension of life span seen in our system by decreasing the rate of damage to mitochondrial aconitase and mitochondrial DNA. These mechanisms clearly fit into the free radical theory of aging (Harman, 1973
) and the various “vicious cycle” theories that follow (Alexeyev, et al., 2004
Iron regulation and utilization is clearly a complex process. The Saccharomyces cerevisiae
genome database (www.yeastgenome.org
) lists 75 genes involved with iron. We are currently looking for the point at which iron supplementation influences life span. While it appears from our data that iron supplementation may act in the mitochondrion to reduce oxidative stress, the effect may be complex. The analysis is made more difficult by the fact that many of the genes involved cause respiratory deficiency and are therefore not assayable in our system. While this does present a problem, we continue to believe that analyses on media requiring yeast to respire will lead to new insights in cellular longevity, which is demonstrated by the fact that neither copper nor iron supplementation have any effect on life span when yeast cells are allowed to ferment.