Global Transcriptional Responses of Fission Yeast to Glucose Starvation Stress

Tesis , 2005 , 82 Páginas , Calificación: 1,1

Biología - Genética / Ingeniería genética

1. Introduction

1.1 Schizosaccharomyces pombe as a Model System

S. pombe functions as a suitable model system since it is easy and inexpensive to rear, has a convenient size, a short life cycle, and is genetically manipulable. As a unicellular eukaryote, the fission yeast S. pombe can exist either in a haploid or diploid state and possesses two different mating types (h+ and h-). The wild type, however, is h90, which means it can switch mating type.


Figure 1.01: Left, picture of S. pombe cells
At top are two dividing cells in late mitotic phase, showing the fission yeast typical septum at the point of cytoplasmic division. The lower cell is in early M phase, having its chromosomes already segregated.

Figure 1.02: Right, fission yeast cell cycle
Diagrammatic representation of the S. pombe cell cycles with the interchange between the two occurring in G1 phase (Figure obtained and used with permission from Trevor Pemberton, University of Sussex).

[...]

S. pombe can undergo two different life cycles, either the vegetative (mitotic) cycle or the sporulation (meiotic) cycle, depending on the environment it is living in. These two cycles are shown in figure 2 with the change between the two occurring in cells at the G1 stage of the mitotic cycle. Under laboratory conditions, given all nutrients required, S. pombe prefers the haploid state. This makes it a favorable organism for genetic research since it ensures that introduced mutations are not masked by another wild type allele. [...]

TABLE OF CONTENT

1. Introduction
1.1 Schizosaccharomyces pombe as a Model System
1.2 Why Investigating Signaling Cascades?
1.3 The SAPK Pathway
1.4 The cAMP Pathway
1.5 The Pheromone Pathway
1.6 Downstream the PKA and SAPK pathways
1.7 Transporters in Fission and Budding Yeast
1.8 Glycolysis, Gluconeogenesis and Glycerol Metabolism
1.9 Microarrays
1.10 Thesis Goal

2. Materials
2.1 Sources of Used Chemicals, Enzymes, and Kits
2.2 S. pombe Strains
2.3 Solutions and Yeast Media
2.4 Equipment

3. Methods
3.1 Experimental Design
3.2 Growth of S.pombe Strains
3.3 Harvesting Cells
3.4 RNA Extraction
3.5 Sample Preparation
3.6 Microarray

4. Results
4.1 Genes Up-Regulated upon Glucose Starvation
4.2 Highly Induced Genes of the Carbohydrate Metabolism
4.3 Genes Involved in Mating/Meiosis
4.4 Genes Involved in Global Transcriptional Regulation
4.5 Hexose Transporters
4.6 cAMP, SAPK, Pheromone Pathway Genes
4.7 Glucose Starvation vs. Oxidative Stress
4.8 Glucose starvation vs. Nitrogen starvation
4.9 Down-Regulated Processes
4.10 How does Gene Expression Change in a spc1 Deletion Mutant?
4.11 PombePerl

5. Discussion
5.1 Changes in Metabolic Pathways
5.2 Stress Activated Signaling Pathways
5.3 Changes of a Cell’s Global Processes
5.4 Effects of a spc1 Deletion on Signaling Pathways
5.5 Summary and Outlook

Acknowledgements

Figures and Tables

References

Appendices

Chapter 1

“Adversity has the effect of eliciting talents, which in

prosperous circumstances would have lain dormant.”

Horace (65 – 8 BC)

1. Introduction

1.1 Schizosaccharomyces pombe as a Model System

S. pombe functions as a suitable model system since it is easy and inexpensive to rear, has a convenient size, a short life cycle, and is genetically manipulable. As a unicellular eukaryote, the fission yeast S. pombe can exist either in a haploid or diploid state and possesses two different mating types (h+ and h-). The wild type, however, is h90, which means it can switch mating type.

Abbildung in dieser Leseprobe nicht enthalten

Abbildung in dieser Leseprobe nicht enthalten

Figure 1.01: Left, picture of S. pombe cells

At top are two dividing cells in late mitotic phase, showing the fission yeast typical septum at the point of cytoplasmic division. The lower cell is in early M phase, having its chromosomes already segregated.

Figure 1.02: Right, fission yeast cell cycle

Diagrammatic representation of the S. pombe cell cycles with the interchange between the two occurring in G1 phase (Figure obtained and used with permission from Trevor Pemberton, University of Sussex).

S. pombe can undergo two different life cycles, either the vegetative (mitotic) cycle or the sporulation (meiotic) cycle, depending on the environment it is living in. These two cycles are shown in figure 2 with the change between the two occurring in cells at the G1 stage of the mitotic cycle. Under laboratory conditions, given all nutrients required, S. pombe prefers the haploid state. This makes it a favorable organism for genetic research since it ensures that introduced mutations are not masked by another wild type allele.

There are many similarities between relevant physiological processes in yeast and mammalian cells as supported by the cloning of the human homologs of yeast genes. Some physiological processes (e.g., mitosis, cell division) of S. pombe are more similar to those of human cells than those of the budding yeast Saccharomyces cerevisiae. Like human cells, S. pombe has a distinct G2 phase so a major checkpoint control is the decision to go from G2 to M (Russell and Nurse, 1986).

Assignment of distinct genes to different pathways has been obtained through epistasis analysis in which the phenotype of a double mutant strain is compared to the corresponding single mutant strain. Moreover, the genomes of S. cerevisiae and S. pombe were sequenced by an international group of laboratories. In addition, the availability of genome databases describing genes and predicted pathways of simple organisms could help in drug discovery programs. S.pombe is already used in a variety of applied researches, such as cancer and AIDS (Acquired Immuno Deficiency Syndrome) research (http://www.childrensmemorial.org/).

1.2 Why Investigating Signaling Cascades?

Since the major signaling pathways and cellular processes involved in a cell’s response to cytotoxic agents are conserved between yeasts and mammalian cells, simple eukaryotic systems can be valuable models for the identification of cellular/molecular mechanisms of sensitivity to drugs (Perego et al., 2000). The availability of the genomic sequence of these organisms as well as of new technologies (microarrays, proteomics) is expected to allow the identification of potential drug targets - the drug discovery process is already moving toward a genomic orientation. Signaling molecules regulate a variety of biological processes including cell cycle control, development, morphogenesis, and cellular response to environmental stress. Protein kinases are major signaling molecules found in eukaryotic cells. If scanning the entire fission yeast genome one finds the presence of 106 eukaryotic protein kinase domain-containing proteins. 44% (or 80%) of these known and putative protein kinases in fission yeast have orthologs (or nearest homologs) in mammalian systems. This suggests a conserved mechanism for signal transduction. Essential for vegetative growth are about 16% out of the total 106 protein kinases. Half of the dispensable (non-essential) protein kinases are important during growth under various stress conditions (Peng et al., 2004).

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Figure 1.03: Three signaling cascades

Three pathways relay stress related signals inS .pombe cells. The pheromone and SAPK pathway are signaling cascades with a MAP kinase as central element. The cAMP pathway mediates its signal via the second messenger cAMP. All three cascades have common downstream target genes which transcription is activated or inhibited, depending on extracellular signals.

Two well characterized signal transduction cascades which regulate virulence and fungal development are the MAP (Mitogen Activated Protein) kinase and cAMP (3’,5’-cyclic Adenosine MonoPhosphate) signaling cascades. While the processes regulated by these cascades in fungi are as diverse as the fungi themselves, the components involved in signal transduction are remarkably conserved (D'Souza and Heitman, 2001) (see figure 1.04). Therefore, learning about the mechanisms of signal transduction in S.pombe can help us to better understand and subsequently perhaps control the virulence of human pathogens such as Cryptococcus neoformans or the plant pathogen Ustilago maydis. Since it is reasoned that the cAMP and MAP kinase signaling pathways interact with each other (D'Souza and Heitman, 2001), it is important to look at the signaling network as a whole and not only investigate each pathway separately. Together with a third signaling pathway, a pheromone activated MAPK cascade, these two pathways are of central concern in this thesis.

1.3 The SAPK Pathway

As one of three well studied starvation triggered signaling pathways in S. pombe (see figure 1.03), the SAPK (Stress Activated Protein Kinase) signal transduction pathway is activated when a cell is starved for nutrients. The central element in this pathway is spc1, also known as sty1, which encodes the MAP kinase. The SAPK pathway counteracts the cAMP pathway in different ways: By positively regulating fbp1 (fructose-1,6-bisphosphatase) transcription, control of meiosis initiation, and sexual development of the cell. Most of the components of the SAPK signaling pathway have been identified in experiments looking for stress response to oxidative stress induced by H2O2. But there is also another form of stimulus that induces oxidative stress: glucose deprivation (Madrid et al., 2004).

The mak genes

The mak1, mak2, and mak3 gene products are among the first proteins in the stress activated SAPK pathway. Mak1p, Mak2p and Mak3p are three cytoplasmic histidine-kinases known to phosphorylate Mpr1p (Buck et al., 2001). However, it is not known how the Mak1-3p proteins get activated in the first place. It is possible, that the stress signal activates the histidine-kinase domains of Mak2p and/or Mak3p. In this manner, the response-regulator domains of Mak2p, Mak3p, or both would become phosphorylated and the phosphate group would, in turn, be transferred to Mpr1p. This has been shown in response to oxidative stress in S. pombe as well as C. albicans (Calera et al., 1998). The particular stress Mak1p acts upon could not be determined yet. It might be the sensor for nutritional stress, triggering the SAPK cascade. An intriguing possibility is that Mak1p is the link between the two main pathways of stress response, the cAMP and the SAPK pathways. It might pick up the cAMP signal and inhibit Mpr1p on the SAPK pathway. This inhibition could be shown upon oxidative stress, at least under some conditions (Quinn et al., 2002). Since in S. pombe the SAPK pathway is a commonly used pathway, triggered by various forms of stress, this might hold true for nutritional stress as well.

The mpr1 gene

The fission yeast mpr1 (spy1) gene encodes a protein with a histidine-containing phosphotransfer domain. It is required for an appropriate stress response within the SAPK pathway. Genetic screens have shown that spc1 activation upon stress is severely impaired in the Δmpr1 mutant. Together with its upstream activators, Mak1p, Mak2p, and Mak3p, Mpr1p is part of a multistep phosphorelay system (Aoyama et al., 2001). This system functions upstream the SAPK pathway and transmits a stress signal via phosphorylation to the MAPKKKs Wak1p and Win1p. Mpr1p in particular binds to the Mcs4p response regulator that functions upstream of the Spc1p cascade, suggesting that Mcs4p is a cognate response regulator for Mpr1p (Nguyen et al., 2000).

The mcs4 and prr1 genes

Two response regulators, named Mcs4p and Prr1p, have been identified and characterized in S. pombe. The Prr1p response regulator has a typical phospho-accepting receiver domain. It is not only responsible for the induction of some genes (e.g. ctt1, trr1) that are involved in response to oxidative stress, but also for regulating the critical gene for meiosis, ste11 (Aoyama et al., 2001). Mcs4p was identified as an upstream regulator of the Spc1p MAPK pathway (Shieh et al., 1997; Shiozaki et al., 1997). Mcs4p associates with Wak1p, one of two MAPKKKs that control phosphorylation of Spc1p. It interacts directly with the N terminus of the Wak1p MAPKKK both before and after stress stimulation. Phosphorylation of Mcs4p alters the activity of Wak1p (and probably also the Win1p MAPKKK), leading to sequential phosphorylation of the Wis1p MAPKK and Spc1p MAPK (Buck et al., 2001).

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Figure 1.04: Homology between the SAPK-pathways in mammals, S. pombe, and S. cerevisiae

These cascades are similarly regulated and control the activity of transcription factors that determine biological responses to various extracellular stress-induced conditions. In mammals they are involved in proliferation, differentiation, DNA repair, and apoptosis. In S. pombe and S. cerevisiae they may control response to oxidative stress, high osmolarity, heat shock, nutritional limitation and UV stress, as well as meiosis and resistance to drugs and heavy metals.

MAPKKKs

So far there have been identified two MAPKK kinases in S. pombe, named Wak1p (Wis4p or Wik1p) and Win1p, functioning in the Wis1p/Spc1p pathway. Both are targets for phosphorylation by Mcs4p and share substantial homology within the kinase domain with other MAPKKKs, like those in S. cerevisiae and human cells (Samejima et al., 1997). It has been shown that Wak1p phosphorylates Wis1p in vitro and activates it in vivo. Win1p is also required for full activation of Wis1p, and Win1p rather than Wak1p mediates the stress signal. The MAPKKKs are redundant in the way that they both can transduce a stress triggered signal to Wis1p. But there are differences in function of the two proteins. While Wak4p is required for basal Wis1p activity in vivo, it is not required for normal response to stress, which does, however, require function of the win1 gene product (shown for osmotic stress by (Samejima et al., 1997)).

The MAPKK

The next piece in the chain of signal transduction via the SAPK pathway is the product of wis1. Wis1p has been shown to be the bottle neck of this pathway, obtaining its signal from the branched upstream pathways, represented by the proteins of wak1 and win1. Wis1p is activated by phosphorylation at specific conserved serine/threonine residues (Alessi et al., 1994). As for the previous activators, it could be shown that replacement of the important serine/threonine residues with non-phosphorylatable alanine residues causes a reduction in the basal level of phophotyrosine on the MAPK substrate of Wis1p, Spc1p (Samejima et al., 1997). Spc1p MAPK is directly phosphorylated by Wis1p MAPKK in cells subjected to various forms of stress, and no Spc1p phosphorylation is detected in the absence of Wis1p under any stress condition (Degols and Russell, 1997; Degols et al., 1996; Millar et al., 1995; Shiozaki and Russell, 1995). It is therefore concluded that Wis1p is the only signal transducer for stress signals to Spc1p. This result also shows that the signal for various forms of stress is transduced via the Wis1p bottleneck.

The MAPK

The central element of SAPK cascade in S. pombe is the MAP kinase Spc1p (also know as Sty1p or Phh1p), which is highly homologous to mammalian p38 kinase (Degols and Russell, 1997; Degols et al., 1996; Millar et al., 1995; Shiozaki and Russell, 1995). Spc1p participates in several seemingly independent cellular events. In particular, cells lacking Spc1p are delayed in the timing of mitotic initiation, are defective in both long- and short-term responses to environmental stress, and are unable to undergo sexual differentiation. Spc1p has also been shown to control cell size at the point of cell division (Millar et al., 1995).

Stress induced activation of Spc1p is balanced by the inhibitory effect of two phosphatase pairs. The phosphorylation activity of Wis1p at the tyrosine residues on Spc1p is counteracted by the two tyrosine-specific phosphatases Pyp1p and Pyp2p. Also, dephosphorylation at threonine residues is achieved by another set of specific serine/threonine phosphatases, Ptc1p and Ptc3p. Simultaneous deletion of pyp1 and pyp2, brings about a lethal hyperactivation of Spc1p, which is similar to that achieved by Wis1p overexpression (Millar et al., 1995). These observations suggest that, in general terms, the function of MAPK Spc1p might be regulated either by the dual phosphorylation of Spc1p protein, achieved by Wis1p, or by dephosphorylation carried out by Pyp1p/Pyp2p. During osmostress and oxidative stress, the corresponding signals are mainly mediated through phosphorylation of Wis1p (Nguyen and Shiozaki, 1999). In other words, this indicates that these two stresses are channeled to Spc1p via the first mentioned strategy of control. Nevertheless, considering the lethal phenotype of pyp1/pyp2 double mutants, Pyp1p and Pyp2p have a balancing function for maintaining proper levels of Spc1p in the cell.

1.4 The cAMP Pathway

Variations in extracellular glucose concentration trigger various genetic responses in S. pombe cells. Those responses are partly mediated via the PKA pathway, regulating the expression of the often used reporter gene fbp1 and several genes involved in meiosis. One gene acting at the very top of this signaling cascade is git3.

The git3 gene

The git3 gene was extensively characterized by Welton and Hoffman (Welton and Hoffman, 2000). BLAST (Basic Local Alignment Search Tool) analysis revealed that git3 is distantly related to S. cerevisiae Gpr1p putative G protein-coupled receptor. The Git3p protein is predicted to possess seven transmembrane domains and displays additional features of a seven-transmembrane coupled G protein-coupled receptor

Abbildung in dieser Leseprobe nicht enthalten

(Baldwin, 1994; Noda et al., 1994). Further genetic evidence also supports the model of Git3p being the glucose receptor in S. pombe cells. The regulatory target of Git3p is the activation of adenylate cyclase through the Gα protein, encoded by gpa2. Mutational analysis shows, that an activated gpa2 allele (carrying a mutation in the coding region for the GTPase domain) (Isshiki et al., 1992) can suppress a git3 deletion. Consistent with a role upstream the glucose detection pathway is the observation of defects in glucose detection and failure of producing a cAMP response to glucose in cells lacking Git3p. The git3Δ phenotypes also include a germination delay, derepression of fbp1 transcription, a defect in glucose repression of gluconate uptake, and starvation-independent conjugation and sporulation. These are very distinct processes, but are all subject to inhibition by PKA. Also consistent with a role as a glucose receptor, the germination delay due to the loss of git3 is not additive with the delay due to the loss of PKA (Welton and Hoffman, 2000).

The heterotrimeric G proteins

The glucose receptor, encoded by git3 is tightly coupled to a heterotrimeric G protein complex encoded by git8, git5, and git11. This complex consists of α, ß, and γ subunits which relay the extracellular signal detected by the receptor to adenylate cyclase. G proteins are guanine-binding proteins which get activated by binding to GTP and take an inactive conformation when bound to GDP (Nocero et al., 1994). In S. pombe the git8 gene, also know as gpa2, encodes the Gα subunit which is the key regulatory element of adenylate cyclase further downstream the cAMP pathway. Gα itself is regulated as part of a heterotrimer by its partner subunits Gß and Gγ, which act as a Gßγ dimer. The Gßγ dimer has no other function but activating Gα by mediating its switch from the GDP to the GTP bound state (Landry and Hoffman, 2001).

The git2 gene

Adenylate cyclase is a messenger protein, well conserved from yeast to humans. The S. pombe gene that encodes adenylate cyclase is git2 (cyr1). Although it has been shown that Gα activates git2, it could not be determined if that activation is of direct or indirect nature (Welton and Hoffman, 2000). Nevertheless, three more git genes with yet unknown function within the cAMP pathway are required for glucose-triggered adenylate cyclase activation (git7 is known to function in cell division, septation, and kinetochore assembly) (Landry and Hoffman, 2001). The fact that mutations in these “upstream” git genes are suppressed by multicopy git2 but not git8 overexpression suggests that git1, git7, and git10 work in between the G proteins and adenylate cyclase. A second, independently working activator of adenylate cyclase is the Cap1p protein (adenylate cyclase-associated protein). It has also been shown that cap is important for organization of the actin cytoskeleton (Freeman et al., 1996). Ultimately, adenylate cyclase is responsible for triggering the cAMP signal in S. Pombe cells.

cAMP

The interaction of adenylate cyclase and cAMP is part of many eukaryotic signal transduction pathways. cAMP, also often referred to as second messenger, is produced from ATP by adenylate cyclase. Thus, adenylate cyclase is a positive regulator of cAMP levels in the cell. The cgs2 gene (pde1), encodes the cAMP phosphodiesterase that converts cAMP to AMP and is required for the maintenance of normal cAMP levels in S. Pombe (DeVoti et al., 1991). The rise of cAMP levels in S. Pombe cells triggered by glucose might be due to an increase in adenylate cyclase activation or the result of inhibition of cAMP phosphodiesterase activity. Assays of mutant defective in either phosphodiesterase or adenylate cyclase however indicate that rising cAMP levels are regulated by adenylate cyclase in response to glucose (Byrne and Hoffman, 1993). Exogenous cAMP represses mutations in all of the git genes but the git6 mutants with regard to the expression of the reporter gene fbp1 (Hoffman and Winston, 1991) . This observation hardens the model of cascade-like signal mediation by the git genes all down to cAMP, having only git6 remaining.

The PKA complex

The git6 gene, identical to pka1 (Maeda et al., 1994), encodes the catalytical subunit of PKA (Jin et al., 1995). The holoenzyme is completed by the encoding of its regulatory subunit by cgs1. At low cAMP levels, PKA activity is maintained silent in a Pka1p-Cgs1p complex. In contrast, at high cAMP levels, the regulatory subunit Cgs1p is sequestered from PKA, which acquires full catalytic phosphorylating activity upon dissociation from the blocking component (Gacto et al., 2003). By this mechanism, PKA mediates the cAMP signal and negatively regulates sexual development and gluconeogenesis by suppressing the transcription of ste11 for the former and the transcription of fbp1 for the latter. PKA does suppress fbp1 expression in two distinct ways. For one, it binds directly to a cis -acting element (UAS1) of fbp1 and inhibits activation at this upstream activation sequence. Secondly, a different cis -acting element (UAS2) of fpb1 is conquered by rst2. The rst2 gene, encoding a zinc finger protein that can bind to the upstream region of ste11 and fbp1, therefore mediates the activity of PKA to transcription of these genes. Nevertheless, there must be an additional pathway separating the expression of ste11 and fbp1 according to nitrogen starvation as trigger for the first and glucose starvation for the latter (Higuchi et al., 2002).

1.5 The Pheromone Pathway

Binding of the mating pheromones to their receptors on the surface of the target cell activates the signaling machinery that consists of a MAPK cascade and both heterotrimeric and monomeric G proteins (see figure 1.05).

Mating pheromone receptors

M-cells express the P-factor receptor (Mam2p) and therefore respond to P-factor (Kitamura and Shimoda, 1991), whereas P-cells express the M-factor receptor (Map3p) (Tanaka et al., 1993) and respond to M-factor. The pheromone receptors are the only mating-type-specific parts of the signaling machinery whereas all other components are the same in both cell types.

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Figure 1.06: The pheromone response pathway

Binding of the pheromones to their receptors on the surface of the target cell triggers intracellular machinery that brings about the changes necessary for fusion. P-factor binds to Mam2p, which is exposed on the surface of M-cells, and M-factor binds to Map3p on P-cells. The stimulated receptor interacts with a heterotrimeric G protein and the released Gα-GTP (Gpa1p) is one of the components required to activate the MAP kinase cascade of Byr2p, Byr1p, and Spk1p. Activation of Byr2p also requires Ras1p, a small GTPase with similarity to the mammalian Ras onco-protein. Interconversion of the active and inactive forms of Ras1p is controlled by the guanine nucleotide exchange factor, Ste6p, and the GTPase activating protein, Gap1p. Shk1p, a PAK-like kinase that lies downstream of Ras1p (see text for details), and Ste4p are also required for activation of Byr2p. The targets immediately downstream of Spk1p are not yet defined but pheromone stimulation leads to activation of the Ste11p transcription factor (Davey, 1998)).

A heterotrimeric G protein

The pheromone receptors couple to a heterotrimeric guanine nucleotide-binding protein (G protein) located on the cytoplasmic side of the membrane. On the basis of models established in other systems, binding of the pheromone is thought to induce a conformational change in the receptor that is transmitted to the G protein (Bukusoglu and Jenness, 1996). The Gα subunit, encoded by the gpa1 gene, is most responsible for bringing about the intracellular responses usually associated with pheromone stimulation. The S. pombe G protein is therefore functionally very different from that used in the S. cerevisiae pheromone response pathway, where the Gβγ subunits are responsible for propagating the intracellular signal (Nakayama et al., 1988).

MAPK cascade

The pheromone response pathway in S. Pombe uses a MAPK module that consists of Byr2p (MAPKKK), Byr1p (MAPKK) and Spk1p (MAPK) and genetic analysis suggests that these enzymes function sequentially (Neiman et al., 1993). Disruption of either byr2 or byr1 inhibits the pheromone response and thus prevents haploid cells from mating and diploid cells from sporulating. Spk1p is also essential for sexual development in both haploid and diploid strains. A key event in the stimulation of the MAPK cascade is likely to be the activation of Byr2p. Like other MAPKKKs, Byr2p is thought to be maintained in an inactive conformation through an inhibitory interaction between its N-terminal regulatory domain and the C-terminal catalytic domain (Tu et al., 1997). Pheromone-induced disruption of this interaction could trigger activation of the MAPK cascade. Genetic analyses implicate Gap1p, Ras1p, Shk1p, and Ste4p in the activation process (Davey, 1998).

Targets for Spk1p

Spk1p is essential for propagating the pheromone signal (Neiman et al., 1993) and presumably phosphorylates one or more target proteins. These targets have not been identified but the transcriptional changes observed following pheromone stimulation suggest that Spk1p, either directly or indirectly, affects the activity of at least one transcription factor. Consistent with this suggestion is the observation that Spk1p is localized to the cell nucleus (Toda et al., 1991). The best candidate for a pheromone-responsive transcription factor is Ste11p.

Meiosis

Conjugation of two haploid cells produces a diploid zygote. This can be maintained as a diploid if transferred immediately to rich medium, but zygotes normally enter meiosis soon after conjugation. Premeiotic DNA synthesis doubles the DNA content of each zygote to 4C and the fused nucleus begins to move from one end of the cell to the other. Two consecutive meiotic divisions generate four 1C nuclei which are then encapsulated to form haploid spores. Germination of the spores completes the mating process. Entry into meiosis is mainly controlled by the interaction between Mei2p and Pat1p. Mei2p is essential to enter meiosis. It is not produced during mitotic growth and its induction is dependent upon the Ste11p transcription factor (Sugimoto et al., 1991).

1.6 Downstream the PKA and SAPK pathways

Several proteins have been identified to work downstream the two signaling pathways cAMP and SAPK. Interestingly, the cAMP pathway and the SAPK pathway regulate transcription of ste11 and fbp1 in an opposite manner. Activation of the cAMP pathway results in the inhibition of expression of both genes, whereas activation of Spc1p MAPK stimulates their transcription. This situation exemplifies the existence of crosstalk between regulatory pathways.

The bZIP transcriptional activator Atf1p is a target for phosphorylation by Spc1p. It also physically associates to Spc1p in vivo. In fact, Spc1p is the only known kinase involved in Atf1p phosphorylation during stress. Together with Pcr1p, it forms a heterodimer complex which is involved in response to osmotic stress, high-level oxidative stress, and nutrient starvation (Takeda et al., 1995; Watanabe and Yamamoto, 1996; Wilkinson et al., 1996). Downstream this transcription factor, several genes are switched on and off in a stress dependent manner. These genes include gpx1 (coding for glutathione peroxidase), ntp1 (neutral trehalase), ctt1 (cytoplasmic catalase), fbp1 or ste11. Atf1p is not the only transcription factor known to be controlled by Spc1p. pap1 has been isolated as required for survival to oxidative stress, but not to osmotic stress or nutrient deprivation.

1.7 Transporters in Fission and Budding Yeast

Glucose is the preferred carbon and energy source for most cells. In addition to being a major nutrient, glucose can act as a “growth hormone” to regulate several aspects of cell growth, metabolism, and development. How a eukaryotic cell senses glucose and signals its presence, how this signal affects cellular processes, and how optimal utilization of the sugar is achieved are fundamental, largely unanswered questions. Defects in glucose sensing, signaling, and metabolism cause the severe and prevalent metabolic disorder in mammals known as diabetes. Thus, it is of major interest to understand these processes. The first and limiting step of glucose metabolism is its transport across the plasma membrane. Thus, it is not surprising that in many different kinds of cells glucose ensures its own efficient metabolism by serving as an environmental stimulus that regulates the quantity, types, and activity of glucose transporters, both at the transcriptional and post-translational levels (Boles and Hollenberg, 1997).

Hexose transporters comprise a family of proteins involved in cellular sugar uptake. They have been well described for a variety of organisms, including bacteria, yeasts, plants, and humans. Regarding sugar metabolism, fission yeast shares a number of characteristic properties with the budding yeast Saccharomyces cerevisiae. Both species grow as facultative aerobes and use aerobic alcoholic fermentation in the presence of an excess of sugar (de Jong-Gubbels et al., 1996). Among the utilized carbon sources, distinct differences are present. D-Glucose, D-fructose, glycerol, and maltose are metabolized by both yeast species, with D-glucose being the preferred substrate. S. pombe cells can grow on the monosaccharide D-gluconate (Milbradt and Hofer, 1994), whereas S. cerevisiae cells can utilize D-galactose and disaccharides such as sucrose (de Jong-Gubbels et al., 1996). The narrow spectrum of carbon sources accepted by S. pombe is attributed to corresponding differences in carbon metabolism. The carbon metabolism of S. pombe does not involve the glyoxylate cycle, and furthermore, some enzymes of ethanol metabolism and gluconeogenesis are not constitutively expressed (Tsai et al., 1989).

Considering transport into the cells as the first step of the utilization of sugar, both yeast species express specific transporters on the basis of related functions. In S. cerevisiae, D-glucose and D-fructose uptake is mediated by the hexose transport (HXT) proteins (Bisson et al., 1993; Tsai et al., 1989), although more than 20 genes encode proteins related to hexose transporters. In the fission yeast S. pombe, glucose uptake is realized via ght1 through ght6, which encode monosaccharide transporters. Those transporters are not only involved in glucose transport, but also, to a different extent, in D-gluconate, maltose, and glycerol transport (Heiland et al., 2000).

The utilization of a carbon source in fission yeast was described to be energy dependent, driven by the plasma membrane ATPase-generated electrochemical gradient (Höfer and Nassar, 1987). In S. cerevisiae, however, HXT proteins transport their substrates by passive, energy-independent facilitated diffusion, with glucose moving down a concentration gradient (Bisson et al., 1993). This fact, plus a comparison of the predicted Ght protein topologies and the amino acid alignments with those of the hexose transporters in budding yeast suggest, that fission yeast ght1 to ght6 belong to a distinct family of transporters.

Two uptake systems were described in S. cerevisiae: a constitutive, low-affinity system (high Km, 15 to 20 mM) and a glucose-repressed, high-affinity system (low Km, 1 to 2 mM) (Ramos et al., 1988). None of these transporters are essential for growth on glucose, indicating their functional redundancy. Just as in S. cerevisiae, kinetic experiments in fission yeast showed different affinities of the 6 ght genes for glucose (Heiland et al., 2000). The presence of multiple hexose transporters with different affinities for glucose in both yeasts is not surprising, given the fact that they grow well on a broad range of glucose concentrations (from a few µM to 2 M). Indeed, the amount of glucose available dictates the expression of the appropriate glucose transporters by closely regulating HXT / ght gene expression. So are high-affinity transporters in budding yeast only expressed at low external glucose concentrations and low-affinity ones at high concentrations (Reifenberger et al., 1997).

Although, the S. pombe glucose transport system is not yet as well investigated as the S. cerevisiae one, it seems to be less complex. Fission yeast employs six characterized hexose transporters with different affinities for glucose, plus two putative hexose transporters which are highly induced upon glucose starvation (see chapter 4.6).

This table shows homologs of human and yeast hexose transport proteins, based on sequence similarities. Sequence similarities were derived from an alignment of some representative amino acid sequences of the hexose transporters of S. pombe, S. cerevisiae, and Homo sapiens (Heiland et al., 2000). Proteins are listed in the order of similarity to each other, Ght3p and Ght4p being most closely related. All transporter proteins of the hexose family belong to the 12-transmembrane sugar porter subfamily of the major facilitator superfamily of proteins.

Table 1.01: Human and yeast hexose transport proteins

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1.8 Glycolysis, Gluconeogenesis and Glycerol Metabolism

When plenty of sugar is available, fermentation is the preferred pathway by which the fission yeast obtains its energy, even in the presence of oxygen. This is called the Crabtree effect. In the absence of sugar, yeast can also grow on nonfermentable carbon sources, such as glycerol, ethanol or lactate. In this case, gluconeogenesis provides metabolites for biosynthesis, and energy is obtained by respiration.

Activation of yeast glycolysis

A common biological pathway for the fermentation of glucose is glycolysis. In stage one, glucose is phosphorylated by ATP yielding glucose-6-phosphate; this is then converted to an isomeric form, fructose-6-phosphate, and a second phosphorylation leads to the production of fructose-1,6-bisphosphate, which is a key intermediate product of glycolysis. The enzyme aldolase splits fructose-1,6-bisphosphate into two three-carbon molecules, glyceraldehydes-3-phosphate and its isomer, dihydroxyacetone phosphate (DHAP). In stage two, the first redox reaction occurs during the conversion of glyceraldehydes-3-phosphate to 1,3-bisphosphoglyceric acid. The synthesis of ATP occurs when each molecule of 1,3-phosphoglyceric acid is converted to 3-phosphoglyceric acid. Subsequently, 2-phosphoglyceric acid, phosphoenolpyruvate (PEP), and pyruvate are formed by the action of various enzymes (Madigan et al., 2000). Enzymes catalyzing each step in S. pombe are shown in figure 4.03.

S. pombe is able to grow on different non-carbohydrate carbon sources. In order to synthesize the sugar phosphates necessary for the synthesis of several cell components, many glycolytic steps, or all of them, need to be reversed depending on the non-sugar carbon source used. Most of the glycolytic reactions are reversible under physiological conditions; however, two reactions cannot be reversed due to the unfavorable concentrations of substrates found in the cell. These are the reactions catalyzed by phosphofructokinase and pyruvate kinase. Therefore, to synthesize fructose 6-phosphate and phospho-enol-pyruvate during gluconeogenesis, specific `gluconeogenic' enzymes catalyze reactions that are different from a simple reversal of those catalyzed by phosphofructokinase or pyruvate kinase.

Fructose-1,6-bisphosphatase (Fbp1p) catalyzes the hydrolysis of fructose-1-6-bisphosphate to fructose-6-P. This reaction is the terminal step of gluconeogenesis and therefore the corresponding enzyme is required for metabolism of every non-sugar carbon source. The expression of the gene is repressed in the presence of glucose (see table 4.01 chapter 4). This catabolite repression of the fbp1 gene is dependent on a functional cAMP signaling pathway (Flores et al., 2000).

Glycerol may be utilized as a carbon source under aerobic conditions by many types of yeast. In S. pombe dehydrogenation of glycerol by a NAD-dependent glycerol dehydrogenase (SPAC13F5.03c) forms dihydroxyacetone (DHA) which is then phosphorylated by a dihydroxyacetone kinase (Dak1p) (see figure 4.03 for details).

1.9 Microarrays

It is widely believed that thousands of genes and their products (i.e., RNA and proteins) in a given living organism function in a complicated and orchestrated way that creates the mystery of life. However, traditional methods in molecular biology generally work on a "one gene in one experiment" basis, which means that the throughput is very limited and the "whole picture" of gene function is hard to obtain.

Array-based technologies allow one to study expression levels in parallel (Lockhart et al., 1996; Schena et al., 1995; Schena et al., 1996), thus providing static information about gene expression (that is, in which (mutant) cell colony the gene is expressed) and dynamic information (that is, how the expression pattern of one gene relates to those of others). Now that the genome of many model organisms is completely sequenced, the microarray technology comes into play.

How do microarrays work?

Gene array experiments are sometimes referred to as "reverse Northerns". As for Northerns, base-pairing (i.e., A-T and G-C for DNA; A-U and G-C for RNA) or hybridization is the underlining principle of DNA microarray.

In Northern blots, RNA is blotted onto a filter and hybridized with a target to detect a particular species of mRNA as a distinct band or spot. In gene array hybridization, cDNAs (probes) are spotted onto a slide and hybridized with a target made from an mRNA population. Targets are made by reverse-transcribing mRNA into single-stranded cDNA in the presence of labeled nucleotides. The labeled target, therefore, is a population of cDNA molecules representing the original mRNA population. The amount of hybridization to a given clone represents the amount of mRNA present for the corresponding gene.

cDNA arrays are produced by spotting PCR products (of approximately 0.6–2.4 kb) representing specific genes onto a matrix. Each array element is generated by the deposition of a few nanoliters of purified PCR product. Printing is carried out by a robot that spots a sample of each gene product onto a number of matrices in a serial operation. The sample spot sizes in microarrays are less than 200 microns in diameter and these arrays contain almost 5000 spots.

Gene array experiments attempt to compare gene expression levels at different times after a treatment. RNA is extracted from each condition and added to a reaction mix containing oligodT primers, which can base pair with the polyA tail on mRNA, Reverse Transcriptase (RNA-dependent DNA polymerase), and labeled nucleotides. Labeled nucleotides are tagged with a chemical tail that fluorescent dyes such as Cy3 and Cy5 can bind to and which can be detected using chemiluminescent detection. In principle, for every mRNA molecule in the original RNA population, a single-stranded labeled cDNA will be produced, complementary to the mRNA. The higher the concentration of a particular mRNA, the more cDNA will be present.

In an array experiment, many gene–specific polynucleotides derived from the 3´ end of RNA transcripts are individually arrayed on a single matrix. This matrix is then simultaneously probed with fluorescently tagged cDNA representations of total RNA pools from test and reference cells, allowing one to determine the relative amount of transcript present in the pool by the type of fluorescent signal generated. Relative message abundance is inherently based on a direct comparison between a 'test' cell state and a 'reference' cell state; an internal control is thus provided for each measurement.

What are we trying to learn from gene arrays?

The primary goal of gene array experiments is to generate expression information for every gene in the array, under some set of conditions. The raw data consists of a series of expression curves, where different environmental conditions are compared. The goal is to find which groups of genes have the most similar expression patterns. It seems reasonable to hypothesize that genes with similar expression profiles, i.e., genes that are co-expressed, may share something common in their regulatory mechanisms, i.e. may be co-regulated. Therefore, by clustering together genes with similar expression profiles one can find groups of potentially co-regulated genes allowing one to search for putative regulatory signals.

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• Universidad: TU Bergakademie Freiberg
• Calificación: 1,1
• Autor: Michael Sassen (Autor)
• Año de publicación: 2005
• Páginas: 82
• No. de catálogo: V41887
• ISBN (Ebook): 9783638400572
• ISBN (Libro): 9783638848558
• Idioma: Inglés
• Etiqueta: Global, Transcriptional, Responses, Fission, Yeast, Glucose, Starvation, Stress