THE YEASTS SACCHAROMYCES CEREVISIAE AND SCHIZOSACCHAROMYCES POMBE: MODELS FOR CELL BIOLOGY RESEARCH Susan L Forsburg University of Southern California, Los Angeles, CA ABSTRACT Yeast species provide excellent models for fundamental biological research. In this review, I will describe characteristics of the two most common laboratory systems: the fission yeast Schizosaccharomyces pombe, and the budding yeast Saccharomyces cerevisiae. They have substantial similarities that make them powerful as research tools, and also striking biological differences that make them complementary experimental models. Each provides unique tools for understanding environmental effects on cellular systems.
INTRODUCTION Yeast is a general term, covering a wide range of very different single-celled fungi. In the molecular biology laboratory, two species are commonly employed as models for biomedical research: the budding or brewer’s yeast Saccharomyces cerevisiae, and the fission yeast Schizosaccharomyces pombe. Although they share the designation “yeast”, these two species are evolutionarily very distinct from one another, with estimates of up to 1000 million years (MY) since they diverged from a common ancestor (Heckman et al., 2001; Hedges, 2002). Although single cells, they are true eukaryotes, and share fundamental cell processes with metazoan systems. Each offers unique tools to the cell biologist, providing complementary approaches and insights into functions of larger eukaryotes (e.g., (Forsburg, 1999; Forsburg and Nurse, 1991; Lew et al., 1997; MacNeill and Nurse, 1997; Russell and Nurse, 1986)). Both yeasts are harmless, tractable genetic systems, easily manipulated in the laboratory using superb molecular tools (Forsburg, 2001).
HISTORY AND CHARACTERISTICS S. cerevisiae was adopted as a model system for laboratory study in the 1930s, as investigators developed genetic tools to understand its life cycle and differentiation (Hall and Linder, 1993). It provided an important tool to understand recombination and the transmission of genetic material, and launched into greater prominence with the molecular era in the 70s, when it became identified as a sort of eukaryotic E. coli. With potent genetic tools and a typical eukaryotic cell ____________________ * Correspondence to: Susan L Forsburg Molecular & Computational Biology Section University of Southern California 8335 W 37th St SHS172 Los Angeles CA 90089-1340 Email:
[email protected] Phone: 213-740-7342; Fax: 213-740-8631
organization, budding yeast became a favorite system to tackle cell biology questions. It was the first eukaryote to be sequenced (Goffeau et al., 1996), which has sparked a whole new era developing genomics tools. S. pombe lagged behind as an experimental system. It was isolated as late as the 1890s from East African millet beer (pombe means beer in Swahili), but limited genetic studies only began in the late 40s. It was picked up further in the 60s for studies of growth control, which were facilitated by its regular rod shaped morphology (Leupold, 1993; Mitchison, 1990). For some time, fission yeast was a model primarily for cell division and sexual differentiation (meiosis). At first, the molecular tools developed for S. cerevisiae were adapted to S. pombe, but were subsequently replaced with pombespecific methods. In recent years, more investigators have chosen to work on S. pombe, and additional areas of research have opened up, although the community is still smaller than that working on budding yeast. The complete genome sequence was published in 2002 (Wood et al., 2002). In the laboratory, both species are typically maintained as haploid cells. Cells are tolerant of cold and can be stored frozen at –70°C. Generation time varies with media and temperature, but is generally in the 2-4 hour range. In response to nutrient limitations, yeast cells exit the cell cycle and enter stationary phase; this a period of dormancy, more severe than the G0 phase in mammalian cells. A particular strength of both systems is that they also have a diploid sexual cycle: haploid cells of opposite mating types can mate, resulting in cell and nuclear fusion. Diploids can be maintained in the laboratory or induced to enter meiosis and sporulate. The four spores packaged in the yeast ascus are the fungal equivalent of human gametes. Thus, the entire life cycle of yeast cells provides a simple model for events occurring in human cells. Both cell types have highly organized internal structures with the membrane-delimited compartments typical of eukaryotic cells, including a nucleus, mitochondria, Golgi and other structures. S. cerevisiae grows by budding, which requires highly targeted cell growth and mechanisms for spatial coordination with nuclear division (Pruyne et al., 2004). S. pombe is a linear, rod shaped cell that grows at its ends and divides by medial fission. This likewise requires significant positional awareness (Hayles and Nurse, 2001). The maintenance of cellular organization can be perturbed with drugs or mutants, and provides an interesting problem for effects of microgravity.
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S. Forsburg — Models for Cell Biology Research GENOME STUDIES AND SPECIES CONSERVATION Comparison of the genomes reveals significant differences between the species (Goffeau et al., 1996; Wood et al., 2002; Wood et al., 2001). Although both have a haploid genome with over 12 megabases (Mb) of DNA, haploid S. cerevisaie has 16 chromosomes while S. pombe has only 3. There is no synteny (conserved gene order), not surprising given their long period of divergence. S. cerevisiae has about 5800 likely proteinencoding genes. A significant fraction of these are paralogues (genes of related sequence that are not exact functional homologues). Evidence suggests that the budding yeast genome has undergone several large scale duplications through evolution , and following gene loss, the remaining duplicates may have diverged in expression or function (Dietrich et al., 2004; Kellis et al., 2004; Wolfe and Shields, 1997). Thus, it has proven a good model for genome evolution. In contrast, the fission yeast, with a similar total genome size has about 4800 genes, with no evidence for large scale duplication (Wood et al., 2002). Genome organization also differs: over 40% of fission yeast genes have introns (non-coding sequences that interrupt the gene), while a scant 5% of budding yeast genes are interrupted. S. pombe has proportionally more genes conserved in metazoans than does S. cerevisiae, although each yeast species shares genes with metazoans that the other yeast lacks (Aravind et al., 2000; Wood et al., 2002; Wood et al., 2001). Interestingly, these often fall into functional groups (Aravind et al., 2000). For example, budding yeast has lost many of the genes associated with the signalosome, a proteosome-related complex required for diverse signaling pathways that is present in fission yeast (Aravind et al., 2000). S. cerevisiae lacks a number of genes required for the spliceosome, that is responsible for removing introns: again, these are genes that are preserved in S. pombe (Aravind et al., 2000). The proteins required for RNA interference (Dcr1, Ago1, Rdp1; (Hall et al., 2002; Schramke and Allshire, 2003; Volpe et al., 2002) are absent from budding yeast, but present in fission yeast. Finally, a significant number of chromosome associated proteins are absent in budding yeast but shared between fission yeast and metazoa, including the conserved Clr4/SuVar3-9 histone methyltransferase (Bannister et al., 2001; Nakayama et al., 2001), and the Swi6 and Chp2 HP1-heterochromatin proteins (Eissenberg and Elgin, 2000; Ekwall et al., 1995; Halverson et al., 2000; Thon and Verhein-Hansen, 2000), telomere proteins Taz1/TRF2 (Cooper et al., 1997; Nimmo et al., 1998)and Pot1 (Baumann and Cech, 2001) and the centromere associated CENP-B proteins (Baum and Clarke, 2000; Irelan et al., 2001; Nakagawa et al., 2002). This difference extends beyond gene sequences to chromosomal elements. Typically, chromosome structures in budding yeast are very small and streamlined, in contrast to fission yeast and metazoan
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which have large, degenerate, and diffuse structures. For example, while the budding yeast origin of replication is a 100bp element with a short, highly conserved consensus sequence, the fission yeast origin is 10 times larger (rev. in (Clyne and Kelly, 1995; Newlon and Theis, 1993; Zhu et al., 1994)). There is no consensus sequence in the fission yeast origin, and no single mutation abolishes its function (Clyne and Kelly, 1995; Dubey et al., 1996; Kim and Huberman, 1998). Importantly, a large, diffuse structure appears to be characteristic of metazoan replicators as well (rev. in Gilbert, 2001), suggesting the fission yeast origin is particularly appropriate as a model for function of higher eukaryotic origins. Similarly, the centromeres of the two species are very different in size and structure (reviewed in Clarke, 1998; Hegemann and Fleig, 1993; Sullivan et al., 2001). In budding yeast, the centromere is a small element (about 100 bp) with a discrete consensus sequence. In contrast, the fission yeast centromere is a large, degenerate element of between 40100 kilobases (kb) with numerous simple repeats, very similar to the centromere structure reported for Drosophila (reviewed in Clarke, 1998; Hegemann and Fleig, 1993; Sullivan et al., 2001). For this reason, S. pombe has become a particularly powerful system for understanding chromosome dynamics. S. cerevisiae proves to be a better model for other cellular functions. For example, S. cerevisiae has been used to study the peroxisome, a metabolic organelle that functions in oxidation of fatty acids and oxidative stress (Brown and Baker, 2003; Lazarow, 2003); in contrast, S. pombe lacks many of the conserved proteins involved in peroxisomal biogenesis and function ((Wood et al., 2002), and V. Wood, pers. comm.). Even where functions are superficially similar, study in the two yeasts provides unique and complementary information. For example, both species were studied intensely to determine mechanisms of cell division control (rev. in Forsburg and Nurse, 1991). These studies allowed identification and characterization of many genes that are required to regulate normal events in the cell cycle progression and respond to any defects. However, at first it appeared that they used a different regulatory logic: S. cerevisiae regulates its cell cycle at the G1/S phase transition, when DNA replication occurs; in contrast, data suggested that S. pombe primarily regulates the decision to initiate mitosis (chromosome segregation). Only by combining the data using a “compare and contrast” approach was it apparent that both species used the same regulatory molecules, and in fact, both control points were present in both species, with the balance shifting between them due to growth conditions. This resulted in a powerful experimental synergy that significantly expanded our understanding of cell cycle regulation and provided unique insights into mammalian cell function. Neither yeast was sufficient alone to explain cell cycle dynamics in all larger eukaryotes: both yeasts contributed essential information.
S. Forsburg — Models for Cell Biology Research These cell cycle genes are conserved throughout metazoa, showing that they define fundamental pathways common to eukaryotes. Defects in their function are implicated in many forms of human cancer (reviewed in (Evan and Vousden, 2001; Fodde and Smits, 2002; Ford and Pardee, 1999; Maser and DePinho, 2002; Vessey et al., 2000; Wassmann and Benezra, 2001; Willers et al., 2002)). The significance of these studies in both yeasts was recognized by the 2001 Nobel prize that S. cerevisiae geneticist Lee Hartwell and S. pombe geneticist Paul Nurse shared with biochemist Tim Hunt. DAMAGE RESPONSE Further studies have examined the response of normal and mutant cells to specific genome insults caused by ionizing or ultraviolet radiation, alkylating agents, nucleotide starvation, or replication defects, thus providing a fingerprint of cellular responses that can be used to identify specific forms of genetic damage. These studies are well-developed and have provided significant insights into the maintenance of genome integrity. This wealth of information provides important tools to study the effects of space radiation on living systems, and moreover, provides a clear context to relate results to known effects on Earth. When DNA is damaged or DNA synthesis is blocked, cells activate checkpoint pathways (rev. in Boddy and Russell, 2001; Carr, 2002; Caspari and Carr, 2002; Osborn et al., 2002). These pathways recognize a damage signal, transmit the signal and respond appropriately. They ensure that cells arrest S phase progression, protect replication structures, repair any defects, and finally restart the replication process and recover. Checkpoints also ensure that the cell chooses the correct mechanism of repair for the lesion it encounters. The response to DNA damage in the yeasts is mediated by kinase response pathways (reviewed in (Carr, 1998; Foiani et al., 1998; Huberman, 1999; McGowan, 2002; Murakami and Nurse, 2000; Nyberg et al., 2002; O'Connell et al., 2000; Rhind and Russell, 1998; Rhind and Russell, 2000; Walworth, 2000)). The type of damage determines the response: replication fork stalling and exposed single stranded DNA activates one arm of the pathway, and reparable lesions such as double strand breaks activates the other. A common group of proteins activates the response. Damage is recognized by a ring-shaped structure related to the replication protein PCNA, called the 9-1-1 complex (Sp Rad9-Hus1-Rad1, or ScDdc1-Mec3-Rad17). Just as PCNA is loaded onto the DNA by the clamp loader RFC, the 9-1-1 complex is loaded by an RFC variant in which the RFC1 subunit is replaced by a related, checkpoint specific protein called SpRad17 (ScRad24). This complex contributes to activation of the ATR homologue SpRad3/ScMec1 and its associated activator ATRIP (SpRad26/ScDdc2) which in turn activates the appropriate downstream pathway (Caspari and Carr, 2002; Cortez et al., 2001; Murakami and Nurse, 2000; O'Connell et al., 2000; Rhind and Russell, 2000; Zhou
and Elledge, 2000). Recent data suggest that exposed single strand DNA (ssDNA) coated by replication protein A (RPA) as damaged regions are exposed or resected may be an activating signal for Rad3-family kinases (Zou and Elledge, 2003). Although the checkpoint proteins are largely in common between S. pombe and S. cerevisiae, the details of their responses differ. Unusually, in S. cerevisiae the G2, or damage checkpoint operates by controlling the metaphase to anaphase transition, while in fission yeast and metazoans, it operates by controlling the Cdc2 kinase (reviewed in Nyberg et al., 2002). In addition, several of the checkpoint proteins are essential for viability in normal vegetative S. cerevisiae, due to an additional role in regulating nucleotide synthesis; while they are not essential in S. pombe (Liu et al., 2003; Zhao et al., 1998). These differences provide a further example of the complementary data obtained by studying both yeasts. Many repair and replication genes interact with these checkpoint kinases providing the cell with multiple mechanisms to respond to genotoxic insults (rev in (Caspari and Carr, 2002; O'Connell et al., 2000; Rhind and Russell, 2000)). There are also differences in the repair of DNA damage. Fission yeast is significantly more resistant to UV or ionizing radiation than budding yeast, which suggests additional repair pathways are operating (discussed in (McCready et al., 2000; Murray et al., 1994)). It is known that fission yeast has an additional UV-repair system (the Uve1 endonuclease) not found in budding yeast (Yasui and McCready, 1998). Both species, however, show significantly more radioresistance than metazoan cells. Mutants defective in various repair pathways can be used to categorize the damage that the cells have suffered. Probably the most threatening form of damage that a cell can suffer is double strand breaks (DSBs) in the genome. Therefore, checkpoint and repair pathways operate to minimize the conditions where DSBs can occur and maximize efficient repair. Ionizing radiation induces DSBs which also occurs following treatment with the alkylating agent MMS, radiomimetic drugs such as bleomycin, or collapse of unprotected replication forks (Kostrub et al., 1997; Memisoglu and Samson, 2000; Rhinds and Russell, 2001). DSBs are repaired by two broad pathways (reviewed in Haber, 2000; Krejci et al., 2003). The first is recombination, either error-free homologous recombination (HR) or error-generating single strand annealing (SSA). The second is nonhomologous end joining (NHEJ), which is likely to generate errors, rearrangements and translocations. HR and NHEJ may be distinguished by the enzymes they employ. Mutants lacking these functions are not surprisingly very sensitive to ionizing radiation and other DSB related challenges. UV radiation produces a variety of lesions including pyrimidine dimers and other photoproducts. These are generally repaired by photolyases (light-activated repair Gravitational and Space Biology 18(2) June 2005
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S. Forsburg — Models for Cell Biology Research enzymes), or by recombination and in some species, including fission yeast, there is an additional excision repair pathway termed UVDE (rev. in Yasui and McCready, 1998). Interestingly, recombination mutants in S. pombe are generally also sensitive to UV radiation, suggested a role for HR proteins in processing UV damage in this species (McCready et al., 2000). Alkylating agents such as MMS have a range of effects. They typically generate abnormal bases, which are substrates for repair by base or nucleotide excision repair (BER and NER). In addition, MMS damage ultimately results in DSBs (discussed in (Memisoglu and Samson, 2000)). DNA damage associated with UV and MMS often is associated with mutagenic repair, in which genetic information is lost or changed. The genetic requirements for these different repair pathways tend to merge as the pathways feed into common mechanisms of resolution, so that they share various components. Damage sensitivity of different mutants, and the activation of different forms of repair can distinguish the nature of the damage suffered by cells. Overall viability in response to damage can be determined by cell viability. Because some repair mechanisms are mutagenic, rates of mutation provide an additional metric to determine the cellular response. If cells can be fixed in ethanol or formaldehyde, cytological methods that distinguish particular forms of damage can be employed. For example, in mammals, ATM/ATR-dependent phosphorylation of the H2AX histone variant marks regions of DSBs and undergoes ATM/ATR dependent phosphorylation; in fission yeast, the same role is served by phosphorylation of “regular” H2A (Burma et al., 2001; Nakamura et al., 2004; Redon et al., 2002; Shroff et al., 2004; Ward and chen, 2001). Similarly, recruitment of repair proteins such as the recombination proteins Rad52 (SpRad22) or Rad51 (SpRhp51) can be used to generate a variety of DNA lesions that include single strand DNA and DSBs (e.g., (Caspari et al., 2002; Du et al., 2003; Grishchuk et al., 2004; Kim et al., 2000; Noguchi et al., 2003)) . Thus, analysis of the biological responses of wild type and mutant yeast cells to space travel is likely to provide significant insights into the sorts of damage suffered by living systems in space. Since the two species have different response pathways, they provide complementary information to one another. CONCLUSION The yeasts S. pombe and S. cerevisiae are the workhorses of modern cell biology. Their study has provided significant insights not only into cell cycle control and damage responses, but to every aspect of cell behavior from chromosome segregation to protein secretion. These two organisms provide sophisticated genetic and molecular tools, as well as genome-level strategies to examine gene regulation and cellular responses. Despite superficial similarities, these species are significantly diverged from one another. Studies over many years,
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particularly in the cell cycle field, has shown that there is a terrific synergy to examining phenomena in both species. If the goal is insight into the responses of metazoa, particularly human cells, long experience shows that these yeasts provide complementary information, which has led to important advances in our understanding of mechanisms of cell growth and regulation. REFERENCES Aravind, L., Watanabe, H., Lipman, D. J., and Koonin, E. V. (2000). Lineage-specific loss and divergence of functionally linked genes in eukaryotes. Proc Natl Acad Sci USA 97, 11319-11324. Bannister, A. J., Zegerman, P., Partridge, J. F., Miska, E. A., Thomas, J. O., Allshire, R. C., and Kouzarides, T. (2001). Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 410, 120124. Baum, M., and Clarke, L. (2000). Fission yeast homologs of human CENP-B have redundant functions affecting cell growth and chromosome segregation. Mol Cell Biol 20, 2852-2864. Baumann, P., and Cech, T. R. (2001). Pot1, the putative telomere end-binding protein in fission yeast and humans. Science 292, 1171-1175. Boddy, M. N., and Russell, P. (2001). DNA replication checkpoint. Curr Biol 11, R953-956. Brown, L. A., and Baker, A. (2003). Peroxisome biogenesis and the role of protein import. J Cell Mol Med 7, 388-400. Burma, S., Chen, B. P., Murphy, M., Kurimasa, A., and Chen, D. J. (2001). ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J Biol Chem 276, 42462-42467. Carr, A. M. (1998). Analysis of fission yeast DNA structure checkpoints. Microbiology 144, 5-11. Carr, A. M. (2002). DNA structure dependent checkpoints as regulators of DNA repair. DNA Repair 1, 983-994. Caspari, T., and Carr, A. M. (2002). Checkpoints: how to flag up double-strand breaks. Curr Biol 12, R105-R107. Caspari, T., Murray, J. M., and Carr, A. M. (2002). Cdc2cyclin B kinase activity links Crb2 amd Rqh1topoisomerase III. Genes Dev 16, 1195-1208. Clarke, L. (1998). Centromeres: proteins, protein complexes, and repeated domains at centromeres of simple eukaryotes. Curr Opin Genet Dev 8, 212-218.
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