Study of Cellular Processes in Higher Eukaryotes Using the Yeast Schizosaccharomyces pombe as a Model

Schizosaccharomyces pombe (Sz. pombe) , or fission yeast, is an ascomycete unicellular fungus that has been used as a model system for studying diverse biological processes of higher eukaryotic cells, such as the cell cycle and the maintenance of cell shape, apoptosis, and ageing. Sz. pombe is a rod-shaped cell that grows by apical extension; it divides along the long axis by medial fission and septation. The fission yeast has a doubling time of 2–4 hours, it is easy and inexpensive to grow in simple culture conditions, and can be maintained in the haploid or the diploid state. Sz. pombe can be genetically manipulated using methods such as mutagenesis or gene disruption by homologous recombination. Fission yeast was defined as a micro-mammal because it shares many molecular, genetic, and biochemical features with cells of higher eukaryotes in mRNA splicing, post-translational modifications as N-glycosylation protein, cell-cycle regulation, nutrient-sensing pathways as the target of rapamycin (TOR) network, cAMP-PKA pathway, and autophagy. This chapter uses Sz. pombe as a useful model for studying important cellular processes that support life such as autophagy, apoptosis, and the ageing process. Therefore, the molecular analysis of these processes in fission yeast has the potential to generate new knowledge that could be applied to higher eukaryotes.


General features of Schizosaccharomyces pombe
Schizosaccharomyces pombe (Sz. pombe) is a yeast found in sugar-containing fermentations of alcohol involved in the production of beer in subtropical regions; the yeast was first described in 1893 by P. Lindner who isolated yeast from millet beer of East Africa and gave it the name "pombe" from the Swahili word for beer. Sz. pombe is a free-living fungus, classified with ascomyctes such as S. cerevisiae (Table 1). However, fission yeast is taxonomically and as evolutionarily distinct from budding yeasts as it is from humans. Sz. pombe reproduces by fission, a process similar to that used by higher eukaryotic cells. Despite the variation in the predicted time of divergence, phylogenetic analysis, and experimental evidence, it can be shown that the gene sequences of Sz. pombe are often more similar to its counterpart in mammals than the equivalent of genes of S. cerevisiae [1].
Sz. pombe is described as a fission yeast because it grows by apical extension and it divides along the long axis by medial fission and septation. The fission yeast has a doubling time of 2-4 hours, and can be maintained in the haploid or the diploid state. The yeast has a cylindrical rod-shape 3-4 μm in diameter and 7-14 μm in length [2]. The cell wall of Sz. pombe provides mechanical strength and protection from environmental stresses. The cell wall determines cellular morphology during the different stages of the life cycle, and is continuously remodelled during the cell cycle to allow cellular growth. The cell wall of Sz. pombe is composed of 74-82% glucan and 9-14% galactomannan, and has a chemical composition of β-1, 3-glucan, β-1, 6-glucan, α-1, 3-glucan, and α-galactomannan. Viewed by electron microscopy, the cell wall is organized in a three-layered structure: the outer layer is electron-dense, the adjacent layer is less dense, and the third layer bordering the cell membrane is dense [4,5].

Genome organization of Sz. pombe
The whole genome of Sz. pombe, distributed over three chromosomes, is estimated at 13.8 Mb [6]. The genome of Sz. pombe was fully sequenced using 452 cosmids, 22 plasmids, 15 BAC clones, and 13 PCR products, in a project under the control of Schizosaccharomyces f European Sequencing Consortium (EUPOM), the Wellcome Trust Sanger Institute (United Kingdom), and Cold Spring Harbor Laboratory (USA). The collaborating bodies were Funding Agency Cancer Research UK (Cambridge Institute United Kingdom) and the European Union (EU) [7]. The genome is organized in the chromosomes I (5.7 Mb), II (4.6 Mb), and III (3.5 Mb), and a 20-kb mitochondrial genome. The three centromeres are 35, 65, and 110 kb long for chromosomes I, II, and III, respectively. On the centromeres are 174 tRNAs genes, 45 of which have introns. The genome of the yeast has at least 4, 940 protein-coding genes (including 11 mitochondrial genes) and 33 pseudogenes. Sz. pombe introns average only 81 nucleotides in length. The gene density for the complete genome is one gene every 2, 528 bp, which is similar in chromosomes II and I (one gene every 2, 483 and 2, 457 bp, respectively) and is less dense for chromosome III (one gene every 2, 790 bp). The spliceosomal RNAs (U1±U6) are located with 16 small nuclear RNA genes (snRNAs) and 33 small nucleolar RNAs (sno-RNAs). The 5.8S, 18S, and 26S ribosomal RNA genes are grouped as 100±120 tandem repeats in two arrays on chromosome III, with the 5S ribosomal RNA genes being distributed throughout the genome [7]. In silico analysis of the Sz. pombe genome has made it known that there are genes highly conserved between the vegetable and animal kingdoms, some of which have been implicated in cancers in humans [8,9].

Mating-type locus, heterothallic, and homothallic phenotype
Sz. pombe has been studied extensively since the 1950s. Urs Leupold isolated the Sz. pombe strains widely used in research from a culture of Sz. pombe Lindner str. liquefaciens obtained by Osterwalder [10] from grape juice in France, in 1921. In its normal life cycle, Sz. pombe cells are haploids, although they can grow in a diploid state. Leupold described the haploid character and the complexity of the mating-type genetics of Sz. pombe, by culturing the haploid and diploid yeast [11,12,13], which were both homothallic and heterothallic (h) strains. The mating of Sz. pombe works by controlling mat genes located on the mating-type locus in the chromosome II [14], which is organized by one expression locus (mat1) and two donor loci mat2 and mat3 that encode the mat P and mat M genes, respectively. There are three alleles mat for the mating-type: h + (strain 975) or P (Plus, +), h − (strain 972) or M (Minus, -), and h 90 (strain 968). The mating-type is determined by the mat1 locus, cells that present the segment called mat1-P determine the sexual type P or h + via the two genes mat-Pi and mat-Pc [15]. Cells with the segment mat1-M define the sexual type M or h − by two genes mat-Mi and mat-Mc. The P and M alleles determine the mating reactions of the heterothallic strain. The homothallic strains with cells of both the P and M mating type can mate and form asci with four ascospores.
The heterothallic strain with only one mating type, are able to mate with heterothallic Sz. pombe strains of the opposite mating type. The allele h 90 determine the homothallism [11], derived from P and M by recombination and mutation by the crossing of heterothallic strains. Homologous sequences flank the mating-type locus support site-specific recombination between the expression locus and the donor loci (mat1 and mat2, or mat1 and mat3) [16]. Heterothallic strains in Sz. pombe originated from strains having spontaneously rearranged their mating type loci. For homothallic segregants with the markers h + parent and recombination for two marker genes (his7 and his2) flanking the locus, two mating-type genes are generated and named initially as h1 and h2 and later renamed mat1 and mat2, respectively [17].

Cell cycle of Sz. pombe
Sz. pombe coordinate their growth with the cell cycle. Exponentially growing cells are of a similar cell size and double in mass before entering the next round of cell division. Cell length is a measure of the stage of the cell cycle that the cell has reached [18]. The cells are commonly cylindrical with rounded ends, but under conditions of starvation cells become shorter. Sz.
pombe is a single-celled, haploid organism with a cell cycle typical of a eukaryote, organized in the same stages as those of higher eukaryotes: interphase (G1, S, and G2 phases) and mitosis by mechanisms that are similar to those in multicellular animals. Sz. pombe usually proliferates in a haploid state. In a medium rich in nutrients, wild-type cells undergo a mitotic division approximately every two hours, with a short G1 phase taking less than 10% of the cell cycle. Next, the cells enters into the S phase following cytokinesis; the longer stage of the cell cycle is the G2 phase (70% of the cell cycle) [19]. Cells sense their size and use this information to coordinate cell division with cell growth to maintain a constant cell size by Pom1 protein and Nif1, localized to the cell ends of DNA, and inhibits mitotic activators in the middle of the cell acting as sensors of cell size controlling the onset of mitosis [20]. When starved of nitrogen, cells stop the division and arrest the cycle in G1, which promotes the sexual phase of the life cycle through conjugation of pairs of P and M cells to form diploid zygotes in a process known as karyogamy. In nitrogen-starved conditions, Sz. pombe haploid cells expressing the mat P and mat M genes enters into meiotic division after forming zygotes [21,22]. After mating and nuclear fusion, premeiotic replication occurs, DNA content is duplicated from 2N to 4N, there is pairing and recombination of homologous chromosomes, and ocurrs the reductional meiosis I and the meiosis II division for the production of four haploid nucleic (1N), which are separated into four spores in an ascus, where the ascospores of Sz. pombe are arranged linearly [13]. Sz. pombe is normally haploid, but it is possible to select diploid strains, which can then divide by vegetative mitotic growth until starved of nitrogen, when they undergo meiosis and form azygotic asci. The provision of a rich nutrient source induces new vegetative growth and mitotic cell division. In 1957, Mitchison measured changes in the volume of cells along the yeast cell cycle. He found that volume increased slightly in the interphase, then changed about an hour before the cell split and increased just after cleavage. Sz. pombe extended at one end only, usually the older one [23,24]. The cell cycle of Sz. pombe is described in Figure 1.

Cellular process studied using Sz. pombe
Sz. pombe is an excellent model for the study of common eukaryotic cellular processes. The yeast shares important biological aspects with metazoans, such as chromosomal structure and metabolism, relatively large chromosomes and centromeres, low-complexity replication origins, epigenetic mechanisms for regulation of genetic expression, G2/M cell cycle control, cytokinesis, mitosis and meiosis, DNA repair and recombination, the mitochondrial translation code, and spliceosome components, among others [2,25].
Fission yeast presents a short cell cycle and is easy to manipulate by genetic classical and molecular analysis. Sz. pombe is a particularly useful model for the study of the function and regulation of genes from more complex species [26]. A model organism is any species used for scientific research in order to answer a specific question that cannot be studied in any other way. A model must be amenable to experimental manipulation in the laboratory, require low costs of culture and maintenance, have a short generation time, be amenable to genetic manipulation to produce mutants and allow the study of their effects, and show high conservation of mechanisms or specific cellular process. A system model extensively used in microbiology, biochemistry, biotechnology, and molecular biology is Escherichia coli (E. coli).
Although this bacterium can be grown and be cultured easily and inexpensively in a laboratory setting, E. coli lacks the mechanisms for post-transcriptional on mRNA or post-translational modifications on proteins of eukaryotic origin. Saccharomyces cerevisiae (S. cerevisiae) is other model used due to its quick, inexpensive, and easy growth in control conditions of the laboratory. However, like E. coli, S. cerevisiae has a limited capability for performing posttranscriptional or post-translational modifications such as N-glycosylations. Drosophila melanogaster is other model system in the cellular biology field widely used for classical genetics analysis. Caenorhabditis elegans is the model of choice for understanding the genetic control of development, physiology, and the study of cellular process such as apoptosis and ageing. Mus musculus and Rattus norvegicus are two mammalian models used for the study of several diseases that affect humans; produced by highly conserved genes, its main disadvantage is slower reproduction than other models allow. In contrast, fission yeast is an ideal system for the analysis of cellular processes common in eukaryotic cells; since it shares many molecular, genetic, and biochemical characteristics with multicellular organisms -such as the splicing of mRNA, post-translational modifications, cell division, and cell cycle control [13,27,28]it has a high rate of reproduction in a short period.

Model for studying the eukaryotic cell cycle
Sz. pombe, the fission yeast, has been used as a model for the study of the eukaryote's cell cycle. Fission yeast is a powerful tool for the study of the cell cycle, since it has a typical cycle cell eucarionte G1, S, G2, and M. G2 occupies approximately 70% and the remaining stages on 30% of the total duration of the cell cycle [29,30]. Since it has a typical cell shape, it is a useful system for identifying genes involved in the cell cycle because mutants are easily identifiable when compared with the wild type [31]. In the 1970s, using Sz. pombe P. Nurse demonstrated the cyclin/cyclin-dependent kinase complex as the key regulator of cellular cycle. Sz. pombe has a single mitotic cyclin-dependent kinase (CDK), highly conserved, named cdc2p, homologous to CDC28 from S. cerevisiae and CDK1 from human cells [32]. Sz. pombe has been used for the molecular sexual differentiation analysis. In this system, it was discovered that a cluster of genes associated with the main biological events of the differentiation process are up-regulate: genes induced in response to environmental changes (starvation and pheromone-induced genes), early genes (pre-meiotic S phase and recombination), middle genes (meiotic divisions and early steps of spore formation), and late genes (spore maturation) [33]. In addition, the use of the fission yeast has contributed to the knowledge of other areas such as cytokinesis, the formation of microtubules, differentiation, cell morphogenesis and polarity, mechanisms of response to stress, and response to damage of the DNA [34,35].

Cellular ageing
The identification of evolutionarily conserved mechanisms that determine long lifespans has been of great interest, making the fission yeast Sz. pombe a novel model for the study of ageing.
Ageing is a genetically programmed event [36,37] and can be defined as a decline in biological functions due to the accumulation of molecular and cellular damage imposed by intrinsic and extrinsic factors on living organisms, having mortality as a consequence. Lifespans of humans are measured chronologically from birth to death. Ageing can considered from the perspectives of two types of cellular lifespans: the replicative lifespans (RLS) of continuously dividing cells and the chronological lifespans (CLS) of post-mitotic cells, from terminally differentiated cells. RLS is the number of mitotic divisions a cell can undergo before senescence, and CLS is the length of time a non-dividing cell can remain viable. CLS measures how long cells can remain viable in a non-dividing state, allowing a continuous decline in viability without detectable re-growth until all cells in the culture are dead. The complexity of human bodies and the length of the human lifespan make research on the biology of human ageing difficult. Model organisms such as yeast, worms, and flies have been extensively utilized because of their short lifespans and their accessibility to classical genetics techniques, as well as the high degree of conservation of genes, processes, and signalling pathways that they share with humans and other mammals [38]. Some aspects of ageing studied in fission yeast have been mainly focused on the effect of calorie restriction on ageing and on the detection of nutrient signalling pathways [39,40]. The protein kinase target of rapamycin (TOR) and insulin/insulinlike growth factor (IGF)-1 pathway are the well-studied nutrient sensing pathways that determine the length of lifespan [41]. TOR exerts its function through two distinct complexes: TOR complex 1 (TORC1) and TOR complex 2 (TORC2), which regulate translation, cell growth, and the organization of actin cytoskeleton [42,43]. Inactivation of TORC1 increases the lifespan in model organisms such as budding yeast, worms, flies, and mice [44,45]. The insulin/IGF-1 signalling pathway, and their components such as PI3 kinase and AKT kinase, affect the longevity of flies, worms, and mice [39]; interestingly, this pathway has also been found in yeasts with the AKT kinase. In S. cerevisiae, nutrient signalling via the kinases Sch9/Tor/PKA pathway has a strong pro-ageing effect. Down-regulation of this pathway is partially responsible for CLS increase. In fission yeast, there are two homologues of these kinases with a similar pro-ageing activity in Sz. pombe: pka1 y sck2 [46]. In this model, deletion of pka1 or sck2 extended CLS by diminishing the accumulation of ROS and increasing oxidative stress resistance [47,48]. AKT kinase orthologues of Sz. pombe regulate CLS: loss of sck1 extended the lifespan in the condition with the excess of nutrients and loss of both genes showed that sck1 and sck2 control different longevity pathways. In addition, there are four genes identified as regulators of ageing in Sz. pombe: 1. psp1+/sds23+/moc1+, a gene required for long-term survival in the stationary phase. Psp1 is phosphorylated in the stationary phase by the cyclin-dependent kinase complex Cdc2/Cdc13. 2. Casein kinase cka1+, an orthologue of the α-subunit of casein kinase II in mammals, whose function is essential but the details of the effect of its deletion are not known. 3. adh1+, encoding the alcohol dehydrogenase that converts acetaldehyde to ethanol. 4. rpb10+, a protein subunit shared by RNA polymerases I, II, and III. It is noteworthy that lifespan extension from TOR-inhibition, Sch9/Akt, or caloric restriction is an effect highly conserved between yeast, worms, and flies [39]. Research of ageing on fission yeast has greatly benefited the knowledge of molecular process since the data obtained can be extrapolated to mammalian cells.

Autophagy
Autophagy is a catabolic mechanism that regulates the intracellular turnover of unfolded/ misfolded, long-lived, or damaged proteins, lipids, and organelles (such as mitochondria and the endoplasmic reticulum (ER), through its sequestration within a double-membrane and delivery to lysosomes for degradation and recycling of biocomponents. Under basal conditions, autophagy is a housekeeping programme, but it can also be activated by nutrient starvation, low cellular energy levels, amino acid deprivation, growth factor withdrawal, ER stress, hypoxia, oxidative stress, and infection [50][51][52][53][54]. Autophagy deregulation in higher eukaryotes leads to muscle atrophy, myopathy, and cardiac and immune disease [52][53][54], and plays a dual role in the pathogenesis of cancer [50,52,55,56].
Duve [57] introduced the term autophagy from the Greek auto (itself) and phagy (eat) which means that cells degrade their cytoplasmic components through lysosomal degradation. Clark described the autophagy process in 1957 through electron microscopy of the kidneys of newborn mice [58]; he discovered that inside lysosomes there were mitochondria. Unfortunately, it was very difficult to study autophagy via electron microscopy and biochemical analysis; thanks to the introduction of molecular techniques, it was later discovered that the yeast S. cerevisiae was a tractable model of eukaryotes. Thirty Atg proteins (autophagy-related proteins) were also discovered later, and it was found that they possess mammalian orthologues [50-52, 54, 59-61]. Four types of autophagy have been described in mammals ( Figure  2): macroautophagy (hereafter referred to as autophagy), microautophagy, chaperonemediated autophagy (CMA), and the more recently described RNautophagy [61][62][63][64]. In autophagy, the cytoplasmic components (also known as cargo) are first encapsulated in a double-membrane structure synthesized de novo, which at the beginning is the phagophore and when it is completely formed is called autophagosome (AP), after which it is trafficked and fused to the lysosome. In microautophagy the cargo is selected through an invagination in the lysosome where it is degraded. In the CMA, specific cytosolic proteins containing the motif KFERQ are recognized by the heat shock cognate protein 70 (HSC70), and are then delivered to the lysosomal associated membrane protein (LAMP2) of type A receptor that is located in the lysosome membrane, where the proteins are translocated inside and degraded [62][63][64]. In 2013, Fijiwara [65] described RNautophagy, where several RNAs were recognized by the LAMP2C receptor and degraded in the lysosome. However, autophagy is a very complex process in metazoans, making it important to have simplest biomodels that share with mammals the core of the autophagic machinery, such as the budding yeast S. cerevisiae and the fission yeast. In the budding yeast autophagy, microautophagy and cytoplasm to vacuole targeting (CVT) have been outlined; likewise in the fission yeast, autophagy shares almost the same conserved core of autophagic machinery ( Figure 2). However, microautophagy, CVT, and RNautophagy have not been described and further investigation is needed in order to unravel the conservation of these mechanisms. In mammals (H. sapiens, M. musculus, and R. norvergicus), macroautophagy, microautophagy, cytoplasm to vacuole targeting (CVT), and RNautophagy have been described. In macroautophagy, cargo is sequestered by an invagination of a lipid bilayer which at the beginning is called phagophore, before it elongates and madurates, forming the autophagosome, then it fuses to the lysosome. In microautophagy, cargo is selected by an invagination of the lysosome. In CVT, proteins with the motif KFERQ are recognized by the HSC70, delivered, and translocated inside the lysosome. In RNautophagy (in Sz. pombe), macroautophagy is highly conserved; however, microautophagy, CVT, and RNautophagy (in the blue square) have not yet been elucidated.
The Atg machinery was classified into five groups according to function in each step of the autophagic process ( regulators that signal the autophagosome biogenesis, the phosphatidylinositol (PtdIns) 3kinase complex controls the nucleation step that recruitsother Atg proteins hierarchically, the elongation of the phagophore mediated by the Atg12 and LC3 conjugation systems, and a subgroup of proteins with unknown functions. In mammals under nutrient rich conditions, insulin or growth factors activate the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway. When PI3K is activated, it converts phosphatidylinositol (3, 4)-bis phosphate (PIP2) to phosphatidylinositol-3 phosphate (PIP3), then Akt is activated and inactivated by phosphorylation to TSC which leads to mTOR complex 1 (mTORC1) activation. mTORC1 negatively regulates ULK1 and Atg13, so when mTORC1 is inactivated by nutrient depletion, low concentration of insulin, deprivation of amino acids, or the addition of its antagonist rapamycin, the ULK1 kinase and Atg13 are then activated, forming the ULK1 kinase complex (Table 2), which signals the induction of the AP formation.
There is another inductor of autophagy, the AMP activated protein kinase (AMPK), that senses low intracellular energy status and directly activates by phosphorylation to ULK1, and by inhibiting mTORC1 via phosphorylation of Raptor (a subunit of mTORC1) [66][67][68][69]. ULK1 phosphorylates Beclin1 at serine 14, which is a component of the PtdIns 3-kinase complex ( Table 2) and enhances Vps34 activity -this step is crucial in the AP formation because Vps34 inhibition by 3-methyladenine (3-MA) or wortmannin disrupts the biogenesis. Vps34 synthesizes PIP3 at the sites where AP are assembled; it has been suggested that PIP3 recruits additional factors for AP formation, but its role remains unclear. Beclin1 binding proteins that activate or inhibit AP biogenesis have been identified: the UV radiation resistance-associated gene protein (UVRAG) and Bcl-2 or Bcl-X L , respectively. Beclin-UVRAG interacts with the class C Vps complex proteins, which are part of the endosomal fusion machinery, so this interaction induces AP fusion with lysosomes [50,53,[70][71].
The next step in AP building is the elongation of the phagophore, which requires membrane input from organelles (such as ER, mitochondria, cytoplasmic membrane, or possibly from de novo synthesis); however, the exact membrane source is unknown. In mammals, Atg9 is a transmembrane protein that probably carries membrane expansion of the phagophore but its downstream effectors have not yet been elucidated. However, there are two ubiquitin-like complexes that are required for elongation; the first one is the Atg12 conjugation system (Table  2), in which the ubiquitin-like protein Atg12 is covalently joined to Atg5. This must first be activated by Atg7 (E1 ubiquitin activating enzyme-like or E1). Atg7 uncovers Atg12 glycine carboxyl-terminus, then Atg12 is transferred to Atg10 (E2 ubiquitin conjugation enzyme-like or E2), after which it is covalently attached by its terminal glycine to lysine 130 (Lys 130) of Atg5.
Finally, Atg12-Atg5 conjugates with Atg16L1 forming a tetramer; this tetramer is essential for the elongation but when AP has been completed it dissociates [76][77][78]. The second ubiquitinlike complex is the microtubule-associated protein 1 light chain 3 (LC3) conjugation system ( Table 2). LC3 is a precursor of LC3-I, it is obtained by the cleavage of the protease Atg4B to its carboxyl terminus. LC3-I is attached to phosphatidylethanolamine (PE) by the enzymes E1 and E2-ubiquitin-like Atg7 and Atg3, respectively, obtaining the LC3-II product which forms part of the AP. Notice that the LC3 complex lacks an E3 ubiquitin ligase-like enzyme that could facilitate LC3I-PE conjugation; however, a crosstalk between these two ubiquitin-like systems has been demonstrated by the tetramer Atg12-Atg5-Atg16L1 acting as E3-like enzyme. When AP is formed, LC3 can be recycled from the form LC3II; this is achieved through Atg4 which breaks apart LC3I from PE, and LC3-I and LC3-II assays are widely used for monitoring autophagy [60,77]. Finally AP moves bidirectionally along microtubules towards the microtubule organizing centre (MTOC) where lysosomes are enriched, then AP fuses with lysosomes forming autolysosomes where the content is degraded and the components are recycled [50]. Atg2 is required for the AP location and can possess a different role when binding to Atg18 in order to target the complex to autophagic membranes [79]. In Sz. pombe, depletion of nitrogen from the culture medium triggers autophagy. Unlike mammals, the addition of AMPK does not induce autophagy, nor can rapamycin induce it. It has been reported that rapamycin has a partially inhibitory effect on TOR activity, moreover, the addition of rapamycin plus caffeine prompts a starvation-specific gene expression pattern and autophagy [80][81]. Atg 1,2,3,4,5,6,7,8,9,11,12,13,14,15,16,18a, 18b, 18c, Vps34 homologues have been found ( Finally, Atg12-Atg5 conjugates with Atg16L1 forming a tetramer; this tetramer is essential for the elongation but when AP has been completed it dissociates [76][77][78]. The second ubiquitinlike complex is the microtubule-associated protein 1 light chain 3 (LC3) conjugation system ( Table 2). LC3 is a precursor of LC3-I, it is obtained by the cleavage of the protease Atg4B to its carboxyl terminus. LC3-I is attached to phosphatidylethanolamine (PE) by the enzymes E1 and E2-ubiquitin-like Atg7 and Atg3, respectively, obtaining the LC3-II product which forms part of the AP. Notice that the LC3 complex lacks an E3 ubiquitin ligase-like enzyme that could facilitate LC3I-PE conjugation; however, a crosstalk between these two ubiquitin-like systems has been demonstrated by the tetramer Atg12-Atg5-Atg16L1 acting as E3-like enzyme. When AP is formed, LC3 can be recycled from the form LC3II; this is achieved through Atg4 which breaks apart LC3I from PE, and LC3-I and LC3-II assays are widely used for monitoring autophagy [60,77].
AP moves bidirectionally along microtubules towards the microtubule organizing centre (MTOC) where lysosomes are enriched, then AP fuses with lysosomes forming autolysosomes where the content is degraded and the components are recycled [50]. Atg2 is required for the AP location and can possess a different role when binding to Atg18 in order to target the complex to autophagic membranes [79]. In Sz. pombe, depletion of nitrogen from the culture medium triggers autophagy. Unlike mammals, the addition of AMPK does not induce autophagy, nor can rapamycin induce it. It has been reported that rapamycin has a partially inhibitory effect on TOR activity, moreover, the addition of rapamycin plus caffeine prompts a starvation-specific gene expression pattern and autophagy [80][81]. Atg 1,2,3,4,5,6,7,8,9,11,12,13,14,15,16,18a, 18b, 18c, Vps34 homologues have been found (Table 2) [73][74][75], and their deletion inhibits AP formation.

Apoptosis
The maintenance of homeostasis in pluricellular and unicellular organisms is achieved through lots of mechanisms, one of the most important of which is related to the death of the cell itself.
In this way, cell death regulates the number of cells in a tissue or a colony, and the removal of damaged cells. In 1963, Lockshin introduced the term Programmed Cell Death (PCD) in order to define the programmed and controlled self-destruction process in a local and temporal way [83][84]. There are many ways for a cell to die; one of them is via apoptosis. The term apoptosis was introduced by Kerr and colleagues in 1972, which is defined as a highly coordinated cellular suicide programme controlled principally by zymogens (i.e., caspases and metacaspases), proteins of the Bcl-2 family, and mitochondria [85]. The Nomenclature Committee on Cell Death established the morphological and biochemical changes in the apoptotic cell, such as membrane blebbing, cell shrinkage (pyknosis), chromatin condensation plus fragmentation (karyorrhexis), formation of membrane bound cell fragments (apoptotic bodies), decrease in mitochondrial inner transmembrane potential, selective cleavage of various cellular proteins, and the translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane [86].
The importance of Bcl-2 proteins relies on Cyt C, which in basal conditions works at the respiratory chain but plays a different role in the cytoplasmic space: Cyt C oligomerizes with the adaptor protein Apoptosis protease activating factor-1 (Apaf-1), forming the complex known as apoptosome [98,99]. The apoptosome activates by proteolytic cleavage to caspase-9 which is one of the cascade cysteine-aspartate proteases (caspases); in other words, caspases are liberated from their inhibitory prodomain [98,99]. Once caspase-9 is activated, the downstream zymogens procaspase 3/7 are activated as caspase 3/7, then they attach to specific substrates in the cell leading to cell dismantling but they must first be released from their endogenous inhibitor X-linked inhibitor of apoptosis (XIAP). XIAP activity is inhibited by SMAC/ DIABLO and HtrA2/Omi, which should be remembered as mitochondrial proteins that were released along Cyt C [94,95,100]. In C. elegans the mitochondrial permeabilization and the release of Cyt C have no role in CED-3 activation, unlike its mammalian counterparts. Another difference between the nematode and mammals is that CED-4 lacks a Cyt C interaction motif, this means that some of the apoptotic machinery are conserved among them. However, some protein-protein interactions differ from each other. On the other hand, the extrinsic pathway start when death ligands such as tumour necrosis factor (TNF), Fas ligand (FasL), TNF-related apoptosis-inducing ligand (TRAIL), among others, join and activate their respective transmembrane death receptor, such as Fas. The interaction of death ligands with death receptors result in the recruitment of adaptor proteins like the Fas-associated death domain protein (FADD), which recruits, aggregates, and promotes procaspase-8 activation. Caspase-8 switch on procaspase 3/7 and the Bid protein through is proteolysis. At this point, a crosstalk between the extrinsic and intrinsic pathways are mediated by Bid activation since truncated Bid (tBid) promotes Cyt-C by interacting with Bax, leading to Bax insertion in the mitochondrial membrane as an oligomer pore [93][94][95].
Study of apoptosis is difficult in higher eukaryotes due to the complexity of the phenomena itself, which is why it was thought until recently that unicellular organisms could not have PCD machinery because it would mean that the organism could orchestrate its suicide, so yeast were employed as purely naïve backgrounds for studying proteins' interaction. In 1997, B. Ink and JM. Jurgensmeier independently performed a yeast two-hybrid system in Sz. pombe to study the proteins of the apoptotic cascade. It was found that the expression of the proteins Bax and Bak lead to the induction of chromatin condensation and DNA cleavage [101][102]. Moreover, the lethal phenotype was obliterated by the co-expression of the antiapoptic Bcl-XL and Bcl-2, therefore, yeasts undergo mammalian-like apoptosis. Likewise, C. James proved that CED-4 expression in the fission yeast induced chromatin condensation and it was abolished by CED-9 interaction [103]. Induction of a yeast apoptotic programme can be triggered by exogenous and intrinsic stresses, such as low doses of H 2 O 2 , acetic acid, valproic acid, HCl, and NaOH in the media, cell ageing, and temperature stress [104][105][106][107]; these stimuli induce ROS that also enable an apoptosis programme. Additionally, DNA damage-responsive proteins are highly conserved among fission yeast and metazoans; hence, defects in DNA replication promote high levels of ROS, promoting apoptosis. Interestingly, SpRad9 -the orthologue of the human Rad9 -controls cell cycle checkpoints, and also possess a BH3 domain which enables binding with Bcl-2 and Bcl-XL in mammal cells, prompting cell death. This proves that the Bcl-2 family of proteins may be evolutionarily conserved from fission yeast to mammals [108][109][110][111][112].
Caspases are the main components of the apoptotic pathway, and are not found in Sz. Pombe; however, it possess an orthologue in the metacaspase PcaI ( Figure 4). Unlike mammalian caspase activation, PcaI undergoes auto proteolytic cleavage activation just as in metazoan caspases; however, until now there has been no evidence of any substrate of PcaI [104,105,113]. Notwithstanding, overexpressing PcaI in Sz. pombe does not induce an apparent cell death phenotype, moreover it appears to stimulate growth. Also, PcaI expression was up-regulated by oxidative and heavy metal toxicity, heat, hyperosmotic stress and DNA damage conditions, H 2 O 2 , sodium nitroprusside, and CdCl, giving cytoprotection; this evidence suggests that the metacaspase could be involved in growth and stress adaptation of Sz. pombe. It has also been demonstrated that the level of oxidized proteins is proportional to the presence of the metacaspase, possibly due to a cleaning function within the population. Apparently, PcaI has prosurvival and pro-apoptotic roles and further investigation is needed to understand its function [105,113,114]. The Htra2/Omi-like protein Nma11p (Nuclear mediator of apoptosis) is found in S. cerevisiae, which is a serine protease, having a pro-apoptotic function, and which aggregates in the nucleus after cellular stress conditions; in Sz. Pombe, its putative homologue has been found [115]. In mammals, an apoptotic marker is the release of CytC; as in yeasts, its presence in the cytoplasm is found after the induction of the apoptotic programme. Just as in mammals, the release of CytC decreases mitochondrial inner transmembrane potential, but unlike metazoans the upstream and downstream effectors of PcaI have not been elucidated. The release of CytC has been correlated with the induction of cell death in S. cerevisiae [116], which correlates highly with the mammalian intrinsic pathway; nevertheless, CytC release has yet to be investigated in Sz. pombe.

Sz. pombe: Heterologous expression systems of proteins
Fission yeast is a useful system for studying the function and regulation of genes, and an excellent host for heterologous expression of molecules derived particularly from higher eukaryotes because it produces high cell densities, short fermentation times, and the use of chemically defined media [117]. Difficulties in the production and purification of heterologous proteins are related mainly to proteolytic degradation of gene products by specific proteases of the host [118]. This makes it highly important to obtain strains that have specific mutations eliminating the activity of proteases. Success of this expression is based on the use of genomereduced strains with the deletion of unnecessary genes. Gene-deletion technology used for this purpose is the LATOUR (latency to universal rescue) method, which eliminates a DNA segment of more than 100 kb [119,120]. This strategy of gene deletion is useful for identifying genes that are essential to the yeast [121]. The Sz. pombe proteolytic system has been seldom studied. Currently, two proteolytic systems have been identified with different subcellular localizations: a vacuolar one (where can be found the Proteinase yspA, Proteinase yspB, Carboxipeptidasa Y1p, Aminopeptidase yspI, Dipeptidyl aminopeptidase DPK, and DPC) and another Cytosolic level (Leucin Aminopeptidase yspII, peptidase multicorn, and proteasome 26S) [122,123]. From full-genome sequence data which are deposited in the MEROPS database, an output of 121 peptidases and probable peptidases, being non-peptidase homologues 39 proteases, has been identified [124].

Conclusions
This chapter discussed some topics related to the employment of Sz. pombe as a model system for the study of the cellular processes of eukaryotes. Despite the complexity of mammalian cells with respect to single-celled yeasts, many intracellular processes are highly conserved, such as cell cycle, autophagy, and ageing; there are even similarities at the level of the mechanisms of the processes carried out in the cell. In some cases, there is only the sharing of some genes, as in the apoptosis process. However, their genetic regulation could be similar in triggering the specific cellular responses. The use of yeast Sz. pombe as a model of study allows the generation of new knowledge with direct application to mammalian cells such as human ones.