Initial observations that the budding yeast Saccharomyces cerevisiae can be induced to undergo a form of cell death exhibiting typical markers of apoptosis has led to the emergence of a thriving new field of research. Since this discovery, a number of conserved pro- and antiapoptotic proteins have been identified in yeast. Indeed, early experiments have successfully validated yeasts as a powerful genetic tool with which to investigate mechanisms of apoptosis. However, we still have little understanding as to why programmes of cell suicide exist in unicellular organisms and how they may be benefit such organisms. Recent research has begun to elucidate pathways that regulate yeast apoptosis in response to environmental stimuli. These reports strengthen the idea that physiologically relevant mechanisms of programmed cell death are present, and that these function as important regulators of yeast cell populations.
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Mechanisms of regulated or programmed cell death (PCD) are essential for development and maintenance among metazoans. PCD itself can occur via a number of distinct mechanisms, and the consequential subcategorization has given rise to an array of terminology, including: apoptosis (caspase dependent or independent), apoptosis-like PCD, necrosis, necrosis-like PCD, accidental necrosis, autophagic cell death and mitotic catastrophe. The completion of genomic sequencing projects has convincingly revealed that a dramatic increase in the complexity of at least one family of proteins involved in apoptosis, the caspases, arose with multicellularity (Aravind et al., 2001). However, data from a wide range of single-celled organisms, both pro- and eukaryotic, suggest that important components of PCD and apoptotic pathways have ancient origins (Koonin and Aravind, 2002). For example, so-called ‘apoptosis domains’ are prevalent in a number of unicellular organisms, including bacteria, and a central role for the mitochondria in apoptosis has been highly conserved among eukaryotes. This has led to the suggestion that some mechanisms of apoptosis arose from early prokaryotic host/pathogen relationships, an example of this being the capture, and ‘taming’, of a pathogenic mitochondrial precursor (Ameisen, 2002). Such a theory proposes that the prokaryotic mitochondrial precursor, widely believed to have been a proteobacterium, had evolved to possess a battery of molecules capable of killing its host. Under conditions whereby the host environment was no longer desirable, for example, starvation, the invading organism could release an arsenal capable of destroying the parasitized organism. In order to avoid such a fate the host cell would have to develop ways of suppressing this programme of cell death, while maintaining the pathogenic mitochondrial precursor and it's valuable metabolic potential. Evidence to support such a theory comes from the fact that factors secreted or associated with the mitochondria, such as cytochrome c, HtrA/OMI and apoptosis-inducing factor (AIF), have been found to play a role in apoptosis that is conserved throughout the eukaryotes. Another weapon employed by the pathogen may have been the production and release of damaging reactive oxygen species (ROS) via a membrane bound electron transport system. Again, ROS production by the mitochondria is central to a broad range of apoptotic mechanisms throughout eukaryotes. The hypothesis that subsequent appropriation of the pathogen followed by its adaption to participate in programmed and regulated suicide provides a tantalizing hypothesis as to the origins of the apoptotic machinery. The discovery that the model eukaryote Saccharomyces cerevisiae can undergo apoptosis allows us to examine such a hypothesis in more detail. For example, is the ability to undergo apoptosis beneficial to a unicellular organism, or does it simply represent an inability to suppress the mitochondria's latent killer instinct? To address this we must first understand the molecules involved, then identify physiologically relevant pathways that regulate their use. Finally, we must reveal circumstances under which apoptosis becomes beneficial to the organism involved.
Apoptosis in yeast
Yeast apoptosis was first described in cells carrying a mutation in the AAA-ATPase CDC48 gene, which has roles in cell division and vesicle trafficking (Madeo et al., 1997). These mutant cells displayed markers of apoptosis commonly observed in higher eukaryotes such as DNA fragmentation, chromatin condensation, plasma membrane exposure of phosphatidylserine and ROS production. Since that time a number of proteins with homology to apoptotic regulators in higher eukaryotes have been identified in yeast. For example, the metacaspase Yca1p/Mca1p (Madeo et al., 2002), a HtrA2/Omi homologue, Nma111p (Fahrenkrog et al., 2004), an inhibitor-of-apoptosis (IAP) (Walter et al., 2006), protein Bir1p and two AIF/AMID homologues, Aif1p and Ndi1p (Wissing et al., 2004; Li et al., 2006), have been described. However, although each of these proteins has been shown to function in yeast apoptosis under certain conditions, pathways in which they are involved remain to be elucidated, and targets for the presumed proteolytic activity of the metacaspase Yca1p have yet to be described.
Apoptosis in yeast has been shown to be triggered by defects in a variety of cellular processes. For example, impairment of the N-glycosylation pathway in cells lacking the yeast homologue of the mammalian defender of apoptosis-1 (DAD1) protein, Ost2p, leads to caspase-independent apoptosis (Hauptmann et al., 2006). A reduction in the maintenance of sister chromatid cohesion as a result of a mutated cohesin-like gene, PDS5, led to apoptosis induction during early meiosis (Ren et al., 2005). Apoptosis was also found to occur in mutant yeast displaying defective initiation of DNA replication (Weinberger et al., 2005), and in cells exhibiting increased mRNA stability as a result of disruption to the efficiency of pre-mRNA splicing and mRNA decapping (Mazzoni et al., 2005). Phosphorylation of histone H2B, by the kinase Mst1, has been linked to apoptosis-induced chromatin condensation in human HL-60 cells (Cheung et al., 2003). Remarkably, this modification was also observed in yeast induced to undergo apoptosis by addition of H2O2 (Ahn et al., 2005). The kinase Ste20p, a yeast homologue of human Mst1, was shown to be responsible for this modification and the histone H2B modification was directly implicated as part of a H2O2-induced apoptosis pathway. This was determined as a modified strain expressing a version of histone H2B that could not be phosphorylated was apoptosis resistant, while expression of a phosphomimetic H2B promoted apoptosis (Ahn et al., 2005). Apoptosis may also play a role in ageing as prominent markers were observed in both replicative (Laun et al., 2001) and chronologically (Fabrizio et al., 2004; Herker et al., 2004) aged cells. A large number of exogenous stimuli have also been described that induce apoptosis-like cell death in yeast (for a review see Madeo et al., 2004). These include exogenous stress stimuli, such as hydrogen peroxide, acetic acid, hyperosmotic stress (Silva et al., 2005), UV irradiation, mating pheromone exposure (see below), amino acid starvation and aspirin.
Recently a case for the involvement of PCD in microbial competition has been presented by studies demonstrating that certain viral encoded yeast killer toxins can invoke an apoptotic response. Killer toxins K1 (S. cerevisiae) and zygocin (Zygosaccharomyces bailii), both ionophores, and K28 (S. cerevisiae), which inhibits DNA synthesis causing cell cycle arrest, are encoded by cytoplasmic double-stranded RNA viruses that are both common and persistent in yeasts. The presence of a killer toxin-producing virus also confers resistance to that toxin and therefore represents a mechanism to combat microbial competitors. Moderate doses of K1, K28 or Zygocin were shown to induce an apoptotic response in S. cerevisiae that involved both ROS and metacaspase, Yca1, activity (Reiter et al., 2005). Yeast cells harbouring killer viruses were also shown to induce apoptosis when incubated with uninfected, or susceptible, yeast cells (Ivanovska and Hardwick, 2005). Interestingly, in this study killer toxin-induced apoptosis required the activity of the dynamin-like GTPase Dnm1, the yeast homologue of the mammalian mitochondrial fission factor, Drp1. This, and previous data from the same group (Fannjiang et al., 2004), suggest that mitochondrial integrity plays an important role in yeast apoptosis, in a manner that is analogous to that observed in higher eukaryotes. Mitochondrial function and ROS production were also required for effective induction of apoptosis in S. cerevisiae by the plasmid-encoded killer toxin from Pichia acaciae (Klassen and Meinhardt, 2005).
Apoptotic signalling pathways in yeast
The elucidation of pathways, which regulate apoptosis, is essential if we are to demonstrate that PCD has evolved to fulfil a physiologically relevant role in yeast. Although a variety of factors have been demonstrated to trigger apoptosis, the pathways that become activated remain largely undiscovered. Advances have been made in this area, and although some areas of controversy remain, many data indicate that PCD plays an important part in regulation of cell populations in response to environmental stimuli. To date two main signalling pathways have been implicated in yeast apoptosis – the Ras pathway and a MAP kinase pathway activated in response to pheromone (Fig. 1).
The Ras pathway of apoptosis
Regulation of Ras signalling is a major determinant of yeast longevity (Longo, 2004). This arises from the role that Ras activity plays in modulating cell growth and stress in response to environmental stimuli. Yeast Ras proteins are highly homologous, and functionally interchangeable with the mammalian Ras oncogene. However, the downstream signalling events differ between yeast and mammals. In yeast, Ras activation leads to cAMP production by the adenylyl cyclase/cyclase-associated protein (Srv2p/CAP) complex. Elevation of cAMP activates protein kinase-A, which has effects on a number of cellular processes including regulation of stress response, metabolic activity, carbohydrate storage and mitochondrial function. Interestingly, constitutive activation of Ras signalling in both Candida albicans (Phillips et al., 2006) and S. cerevisiae (Gourlay and Ayscough, 2006) has been shown to result in cell death displaying markers of apoptosis. So does the activation of Ras signalling function as a physiologically relevant trigger of yeast apoptosis? Recent evidence suggests that this may well be the case. Studies using the plant antifungal Osmotin, a member of the PR-5 family of plant defence proteins, have shown that it induces apoptosis via a Ras2p signalling mechanism (Narasimhan et al., 2001). Osmotin binds to G-protein-like Pho36p and causes inappropriate activation of the Ras signalling cascade culminating in ROS production and death. This raises the possibility that plant defence molecules have evolved which trigger apoptosis in fungi, and generates a further impetus for the study of yeast apoptosis.
Work from our own laboratory has revealed a role for the actin cytoskeleton in controlling the Ras apoptosis pathway. Mutations, or addition of drugs, which reduce actin dynamics lead to the formation of F-actin aggregates resulting in constitutive activation of Ras2p, ROS production and apoptosis (Gourlay and Ayscough, 2006). Actin-induced ROS production in this pathway requires the actin regulatory protein Srv2/CAP, which also binds adenylate cyclase and is required for the transduction of an active Ras signal. These data add to our previous data that regulation of actin dynamics has an important mitochondrial-dependent role in ageing and apoptosis (Gourlay et al., 2004). As a reduction in the dynamic state of filamentous actin (F-actin) and the consequent formation of F-actin aggregates is commonly observed in ageing yeast, an exciting possibility is that this apoptosis pathway provides a means to eliminate aged cells from a population. Data from multicellular eukaryotic model systems also suggest that actin plays an important role in the regulation and execution of apoptosis (Foger et al., 2006; Thomas et al., 2006; for a review see Gourlay and Ayscough, 2005a).
In both actin- and Osmotin-induced yeast apoptosis, ROS accumulation is thought to be the major killer, demonstrated by the fact that the addition of antioxidants, or the reduction of ROS at source can circumvent cell death (Narasimhan et al., 2001; Gourlay and Ayscough, 2005b). Although the precise mechanism by which ROS are produced in response to Ras activation is unknown, it is most likely to be the result of dysfunctional mitochondria. This is supported by the fact the actin-mediated ROS production and apoptosis can be prevented either by downregulating Ras signalling by overproduction of the cAMP phosphodiesterase PDE2, or by the inhibition of respiratory chain function (Gourlay and Ayscough, 2005b). Expression of the constitutively active rasala18val19 allele has been shown to lead to ROS accumulation in yeast by locking the mitochondria into a non-phosphorylating state of respiration (Hlavata et al., 2003). Further evidence comes from the fact that in S. cerevisiae, actin-stimulated apoptosis is dependent on the PKA subunit Tpk3p, which is known to regulate the enzymatic content of mitochondria (Chevtzoff et al., 2005). The discovery apoptosis can be triggered in yeast by constitutive activation of Ras signalling, through exposure to the antifungal Osmotin or actin aggregation, and provides strong evidence that apoptosis serves a physiologically relevant function.
Apoptosis and the pheromone response pathway
Sexual conjugation of haploid yeast cells of opposing mating types (Mat a and Mat α) is mediated by the secretion and detection of mating pheromones (a-factor and α-factor). These short peptide pheromones are detected by heterotrimeric G-protein coupled receptors (Ste2p and Ste3p) and trigger a well-defined MAP kinase signalling cascade. This leads to polarization of the cell towards the pheromone source, remodelling of the cell wall and the formation of a mating projection. In 2002, Severin and Hyman demonstrated that the exposure of haploid yeast to high concentrations of the appropriate mating pheromone leads to PCD exhibiting markers of apoptosis, including cytochrome c release from the inner mitochondrial space, ROS production and DNA fragmentation (Severin and Hyman, 2002). Work from the same laboratory characterized this phenomenon further and proposed a model in which pheromone-activated MAP kinase signalling led to the elevation of intracellular Ca2+ and an increase in respiratory function coupled to energy production (Pozniakovsky et al., 2005). This upregulation of mitochondrial activity was shown to precede production of ROS from complex III of the mitochondrial respiratory chain. The authors proposed that elevation of oxidative stress forms part of a commitment to apoptosis in cells which fail to find mating partners, so selecting against weak or sterile individuals. However, other recent studies contest this viewpoint, arguing that selection against non-mating cells in this way would be detrimental to a population of non-motile cells, the majority of which may fail to mate. Indeed, mixing cells of opposing mating types does not appear to lead to high levels of cell death (Zadrag et al., 2006). In addition, it has been proposed that the levels of pheromone required to induce apoptosis may be higher than are likely to be encountered under physiological conditions. It is possible, however, that pheromone hypersensitive, or unresponsive individuals may be removed from a population by this mechanism. A recent study by Zhang et al. (2006) proposes that pheromone-induced PCD can occur via three distinct mechanisms leading to slow, intermediate and fast modes of killing. All death modes involve an increase in ROS, but with high levels of pheromone, DNA fragmentation was not detected and dead cells displayed permeabilized plasma membranes indicating necrotic-like PCD, as opposed to apoptosis. Therefore, whether pheromone-induced PCD represents a physiologically relevant mechanism, and indeed apoptosis, remains controversial.
Yeast apoptosis – accident, altruism or self-preservation?
The evolutionary rationale for unicellular organisms to possess PCD pathways is currently a topic of much debate. However the observation that both chronologically (Herker et al., 2004) and replicatively (Laun et al., 2001) aged yeast cells undergo apoptosis argues that PCD may play an important physiological role, particularly in ageing. It has been hypothesized that apoptosis in unicellular organisms serves an altruistic purpose, whereby old and sick cells self-sacrifice for the good of the population. However, evidence from the chlorophyte Dunaliella tertiolecta indicates that this need not always be the case (Segovia et al., 2003). D. tertiolecta is an obligate phototroph that cannot use dissolved organic compounds (and so benefit from lysis of surrounding cells), and which does not sexually reproduce in culture. It does, however, undergo darkness induced apoptosis-like cell death, and this death does not depend on the age of the cells. As no single organism benefits from the death of its neighbour, then apoptosis cannot be occurring for an altruistic purpose. Although the molecules involved in killing were not identified, the authors speculate that proteins with a housekeeping function may become activated to have an unregulated proteolytic function under conditions of prolonged darkness. Thus, in the case of D. tertiolecta apoptosis may occur as an accidental form of cell death. In contrast, yeast cells are able to utilize the lysed remains of their neighbours, and may therefore use this energy to reproduce, thus a theory of altruism seems more feasible. However, it should be considered that although many of the accumulated yeast apoptosis data have come from cells grown in liquid suspension, budding yeast do in fact grow as multicellular colonies which are capable of simple differentiation. Each colony arises from a single yeast cell which has undergone many rounds of growth and division, thus sacrifice of an aged cell containing high levels of ROS and potentially damaged DNA not only releases nutrients for other cells, but in fact protects the integrity of the original cell's genome. Thus, in the case of budding yeast we should consider it as a multicellular entity in which apoptosis is acting as a self-preservation mechanism for the colony as a whole. Importantly, apoptosis has been shown to occur in a regulated fashion in yeast colonies (Fig. 2). In an elegant study, Vachova and Palkova (2005) demonstrated that a zone enriched for differentially chronologically aged cells develops in S. cerevisiae colonies grown for prolonged periods. In addition to this, a central zone that is enriched for aged cells continuously displayed several markers of apoptosis including ROS production, DNA fragmentation and chromatin shrinkage. In contrast, peripheral regions that are enriched for younger cells do not display markers of apoptosis in colonies grown longer than 12 days on solid medium-containing glucose. The establishment of these zones required a functional SOK2 gene product, which functions in the ammonia signalling pathway. Sok2p regulates the transition from an acidic to alkali growth phase that is regulated by ammonia and which results in an alteration of metabolism and stress response that is required for the long-term survival of a colony (Vachova et al., 2004). Deletion of SOK2 causes apoptosis to occur throughout the ageing yeast colony, implicating ammonia signalling as a central regulator in establishing both a dead zone and peripheral region of protected youth within the colony (Fig. 2). Interestingly, the physical removal of the dead zone from growing colonies caused a reduction in growth at the colony periphery strongly suggesting that apoptosis within the central dead zone benefits the population and thereby acts to preserve the whole colony.
Concluding remarks and future questions
The field of yeast apoptosis has grown rapidly and our increased understanding will be key, both in terms of increasing our knowledge of apoptotic mechanisms and also as a tool for development of useful compounds such as fungicides and antifungal therapeutics. However, while many triggers of apoptosis have been identified, critical questions remain concerning the pathways and mechanisms that culminate in this programmed form of cell death. Of particular importance will be the identification of specific cellular targets critical to the execution phase of apoptosis, the characterization of mitochondrial states prone to ROS production and the further elucidation of natural processes that use apoptosis to regulate populations of yeast.
We would like to thank Steve Winder, Dana Gheorghe and Jeelan Moghraby for critical reading of this manuscript. K.R.A. is supported by a Medical Research Council (MRC) Senior Research Fellowship (G117/394) and Campbell Gourlay by a Wellcome Trust Value in People award.