Review
Yeast apoptosis—From genes to pathways

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Abstract

Yeast are eukaryotic unicellular organisms that are easy to cultivate and offer a wide spectrum of genetic and cytological tools for research. Yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have successfully been used as models for human cell division cycle. Stress conditions, cellular ageing, failed mating, certain mutations or heterologous expression of proapoptotic genes induce yeast cell death with the characteristic markers of apoptosis. Several crucial regulators of apoptosis are conserved between metazoans and yeast. This simple model organism offers the possibility to identify conserved and new components of the apoptotic machinery and to elucidate the regulatory pathways beyond.

Introduction

Yeast is a collective term for unicellular ascomycetous and basidiomycetous fungi, containing organisms of relatively far relationship. Most yeasts (like Saccharomyces spp.) proliferate by budding, with a small “daughter cell” growing from the “mother cell” and gradually increasing in size. Some (like Schizosaccharomyces spp.) proliferate by fission, splitting a cell into two equal-sized cells by forming a septum in the middle. Most yeasts can also reproduce sexually. Meiosis (sporulation) results in formation of haploid spores (ascospores, respectively, basidiospores) from a diploid cell, which exhibit different mating types. Haploids of Saccharomyces cerevisiae secrete specific mating pheromones (a or α factor) that bind to receptors of cells of the other mating type (MTα or MTa, respectively) and induce the mating process, resulting in a diploid cell.

Yeasts are quite amenable organisms as a whole. The relatively few pathogenic yeast species are opportunistic, infecting only weakened hosts, or populating especially favourable niches (like the damp caves between the toes). In most cases they form superficial infections of skin or toe nails with minor nuisance for the patient, thrush caused by Candida albicans being the most common mycosis. However, yeast infections can become serious and even lethal when inner organs are affected. And the frequency and severity of yeast infections has increased in recent years due to the growing number of immunocompromised patients (HIV, immunosuppressive therapies) and long-term invasive medical treatments (catheters, artificial heart-valves).

S. cerevisiae is the best-researched yeast and probably the best-known eukaryotic organism. It was the first eukaryotic organism with a completely sequenced genome, in 1996 (see [1], [2]). Schizosaccharomyces pombe is the runner-up for basic research, also completely sequenced by now, and especially notable for cell division cycle research [3]. While these two model yeasts are both ascomycetous yeast, their evolutionary split probably occurred at least 1 billion years ago [4], not long after the separation of fungi and animals. As a result, they differ in several fundamental features (see PombeWeb at The Forsburg Lab [5] for comprehensive information). The centromeres of Sch. pombe resemble those of metazoans, being 40–100 kbp in length and containing many repeats, while the S. cerevisiae CEN element is only 150 bp, easily clonable, allowing construction of CEN vectors and YACS (yeast artificial chromosomes). Sch. pombe is a homothallic haploid, sporulating right after conjugation. Wild-type S. cerevisiae is homothallic diploid (with many industrial strains being poly- and aneuploid). Most laboratory strains of S. cerevisiae allow heterothallic growth as stable haploids or diploids. This is advantageous for research, as lethal mutations can be hidden in heterozygous diploids and recessive phenotypes can be investigated easily in haploids.

The differing features of the two yeasts are a strong point of the dual yeast models. S. cerevisiae proliferating by budding enabled Leland Hartwell to recognize cell division cycle mutants by the mother cell/bud ratio [6], [7], the fission mode of Sch. pombe cell division enabled Paul Nurse to recognize loss of cell cycle control by the small (wee) cells [8], [9]. The two researchers, together with Timothy Hunt (cyclins in sea urchins and Xenopus) have been awarded the 2001 Nobel price in Medicine for the investigation of the cell division cycle, emphasising the value of the yeast model. The large evolutionary distance of the two yeasts provides an additional advantage for research. The yeasts can serve as mutual controls, and similar results obtained in both promise relevance for higher eukaryotes. Another useful feature common to all fungi is the high degree of homologous recombination, allowing a systematic and targeted integration of transformed DNA into the genome. Also, all fungi have a closed mitosis, the nuclear envelope remains intact and a nuclear fission precedes the cellular fission.

In addition, S. cerevisiae is a facultative anaerobic yeast and tolerates even the complete loss of its mitochondrial DNA (ρ° strains). This is especially helpful for research on PCD, as mitochondria have a central and complex role in apoptosis.

Section snippets

The yeast toolbox—easily mastering “the power of yeast genetics”

The small genome, fast growth and easy handling have made S. cerevisiae a favourite tool for genome wide exploration. Through homologous recombination, disruptants of every yeast gene have been constructed (as heterozygous diploids for essential genes, as homozygous diploids and haploids for the others), and are available individually or as complete collections from EUROSCARF [10]. These collections have been used for numerous genome-wide screenings with drugs or other stress-inducing

Phenotype and assays of yeast apoptosis (Fig. 1)

The basic test for cell death in yeast is the clonogenic assay. Cells are counted in a Neubauer chamber at the microscope or by a Casy counter, a defined number of cells (usually 500) are plated on complete media, and resulting colonies are counted after 2–3 days of incubation [18]. In contrast to cultured mammalian cells, viable individual yeast cells reliably give rise to a colony. Stains based on metabolic activity like MTT [19] or phloxine B [20] can also be used, but usually the clonogenic

Heterologous expression of proapoptotic genes

Model yeasts (e.g. S. cerevisiae, Sch. pombe) as monocellular organisms have long been assumed to lack apoptotic processes. S. cerevisiae has been (and still often is) employed as “clean room” for investigating the interaction of proteins involved in apoptosis and PCD in general. Expression of proapoptotic bax resulted in cell death, which, as in metazoans, could be prevented by coexpression of bcl-2. The yeast system was used to identify the interaction-relevant domains of bcl-2 [22] and later

Pheromones and Ste20p

As the research field of yeast apoptosis is rather young, details about regulatory pathways are just beginning to emerge. The observation that exposure to high concentrations of mating pheromone induces cell death in yeast [73] links a well studied signalling pathway – the mating cascade – to cell death. Mating pheromones are short secreted peptides which bind to a G-protein coupled receptor to activate a well defined MAP – kinase signalling cascade resulting in induction of mating – specific

Aging yeast

Two forms of cellular ageing occur in yeast. Replicative ageing, also called mother cell specific ageing is based on how many progenies a single mother cell has produced. Chronological ageing, also called postmitotic ageing, is defined by survival rates during long-term cultivation. Both forms of ageing involve pathways related to those regulating in higher organisms cellular aging of proliferating or resting cells (e.g. stem cell ageing, ageing in differentiated organelles/tissues),

Cell death in Sch. pombe

Fewer groups are investigating apoptosis in “the other model yeast”, Sch. pombe (reviewed in detail in [98], [99]), but their focus is of special interest for medicine. In addition to the heterologous expression experiments described above (chapter “Heterologous expression of proapoptotic genes”), Sch. pombe research mainly contributed to two special fields of apoptosis. Defects in DNA replication result in the production of high levels of ROS and cell death. This has been studied in budding

Perspectives

The ease of a genome wide approach and the multiple options to study interactions (2-hybrid, synthetic lethality, TAP-tag) are currently used by several groups to get a complete picture of apoptotic members and underlying regulatory networks in yeast. Clinically relevant cytopathological scenarios like accumulation of protein fibrils (Alzheimer, Parkinson) or lipid deregulation can be investigated in yeast for their ability to trigger cell death and for their dependence on apoptotic regulators.

Acknowledgements

We are grateful to Dr. Frank Madeo for critical reading of the manuscript and for many discussions, and to Tobias Eisenberg for PS/PI staining pictures.

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