Artemisinins, derived from the wormwood herb Artemisia annua, are the most potent antimalarial drugs currently available. Despite extensive research, the exact mode of action of artemisinins has not been established. Here we use yeast, Saccharamyces cerevisiae, to probe the core working mechanism of this class of antimalarial agents. We demonstrate that artemisinin's inhibitory effect is mediated by disrupting the normal function of mitochondria through depolarizing their membrane potential. Moreover, in a genetic study, we identify the electron transport chain as an important player in artemisinin's action: Deletion of NDE1 or NDI1, which encode mitochondrial NADH dehydrogenases, confers resistance to artemisinin, whereas overexpression of NDE1 or NDI1 dramatically increases sensitivity to artemisinin. Mutations or environmental conditions that affect electron transport also alter host's sensitivity to artemisinin. Sensitivity is partially restored when the Plasmodium falciparum NDI1 ortholog is expressed in yeast ndi1 strain. Finally, we showed that artemisinin's inhibitory effect is mediated by reactive oxygen species. Our results demonstrate that artemisinin's effect is primarily mediated through disruption of membrane potential by its interaction with the electron transport chain, resulting in dysfunctional mitochondria. We propose a dual role of mitochondria played during the action of artemisinin: the electron transport chain stimulates artemisinin's effect, most likely by activating it, and the mitochondria are subsequently damaged by the locally generated free radicals.

Apoptosis-inducing factor (AIF) and AIF-homologous mitochondrion-associated inducer of death (AMID) are both mitochondrial flavoproteins that trigger caspase-independent apoptosis. Phylogenetic analysis suggests that these two proteins evolutionarily diverge back from their common prokaryote ancestor. Compared with AIF, the proapoptotic nature of AMID and its mode of action are much less clarified. Here, we show that overexpression of yeast AMID homologue internal NADH dehydrogenase (NDI1), but not external NADH dehydrogenase (NDE1), can cause apoptosislike cell death, and this effect can be repressed by increased respiration on glucose-limited media. This result indicates that the regulatory network of energy metabolism, in particular the cross-talk between mitochondria and the rest of the cell, is involved in Ndi1p-induced yeast cell apoptosis. The apoptotic effect of NDI1 overexpression is associated with increased production of reactive oxygen species (ROS) in mitochondria. In addition, NDI1 overexpression in sod2 background causes cell lethality in both fermentable and semifermentable media. Interruption of certain components in the electron transport chain can suppress the growth inhibition from Ndi1p overexpression. We finally show that disruption of NDI1 or NDE1 decreases ROS production and elongates the chronological life span of yeast, accompanied by the loss of survival fitness. Implication of these findings for Ndi1p-induced apoptosis is discussed. INTRODUCTIONApoptosis is a form of programmed cell death essential to the development and tissue homeostasis of multicellular organisms. Although programmed cell death or apoptosis is widely accepted as an essential process in multicellular organisms, until a few years ago it was generally considered to be unnecessary for single cell organisms that do not form multicellular structures. Recent studies demonstrated that apoptosis also occurs in bacteria and yeast (Madeo et al., 1997;Engelberg-Kulka and Glaser, 1999;Wadskog et al., 2004). Like mammalian cells, yeast cells (Saccharomyces cerevisiae) undergoing apoptosis show characteristic markers such as DNA cleavage, apoptosis-typical chromatin condensation, externalization of phosphatidylserine, and cytochrome c release from mitochondria (Madeo et al., 1997;Manon et al., 1997;Ludovico et al., 2002). Reactive oxygen species (ROS), the central regulator in the metazoan apoptosis, has also been identified as essential in at least some yeast apoptosis, indicating that the similarity between the two processes is not restricted to the appearance (Madeo et al., 1999). The past several years have seen the discovery of several yeast orthologues of crucial apoptotic regulators (Laun et al., 2001;Ligr et al., 2001;Blanchard et al., 2002;Madeo et al., 2002;Chae et al., 2003;Fahrenkrog et al., 2004;Fannjiang et al., 2004;Herker et al., 2004;Sauder and Aebi, 2004;Ahn et al., 2005). These findings firmly established that yeast and metazoan apoptosis are in essence the same cellular program and lay the foundation of using ...

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