Evaluation of the role of autophagy in fungal development and pathogenesis 2

CHAPTER I: INTRODUCTION 1.1 General introduction of autophagy 1.1.1 Process and classification of autophagy Autophagy is a cellular mechanism for bulk degradation of long-lived cytosolic or short-lived damaged proteins and organelles within vacuoles/lysosomes. Autophagy is induced in response to environmental stress or developmental signals during cellular differentiation (Besteiro et al., 2006; Liu et al., 2005; Noda and Ohsumi, 1998; PinanLucarre et al., 2003b; Pinan-Lucarre et al., 2005). Take non-selective macroautophagy as example, when autophagy is induced, cytoplasmic constituents, including organelles, are sequestered by a unique membrane called the phagophore or isolation membrane. The complete sequestration by the elongating phagophore results in formation of the autophagosome, a double-membraned organelle (300-900 nm in diameter). In the next step, autophagosomes fuse with lysosomes (in metazoan cells) or vacuoles (in yeast and plant cells). Once macromolecules have been degraded in the lysosome/vacuole, monomeric units (e.g., amino acids) are exported to the cytosol for reuse. Besides macroautophagy, non-selective autophagy includes microautophagy, which involves the direct engulfment of cytoplasm at the surface of the vacuole (Noda et al., 1995). Eukaryotic cells also exert a highly selective process to deliver specific cytosolic proteins into the vacuole, which is called cytoplasm-to-vacuole targeting (Cvt) pathway (Scott et al., 1997). A selective autopahgy that is specific for cytosolic glycogen was identified in new-born animals and was named as glycogen autophagy. Autophagy can also target specific organelles for degradation, such as ER (reticulophagy) (Bernales et al., 2007) mitochondria (mitophagy) (Tolkovsky, 2009) and peroxisomes (pexophagy) (Sakai et al., 2006) (Figure 1). 1.1.1.1 Glycogen autophagy In newborn animals, a well-defined role for autophagy is the breakdown of intracellular glycogen reserves within autophagic vacuoles, namely glycogen autophagy, which is a strategy to cope with a sudden demand for ample energy substrates to confront metabolic requirements, before gluconeogenesis is initiated (Kotoulas et al., 2004, 2006). Glycogen autophagy can be induced by glucagons, and be suppressed by insulin, which abolishes glucagon secretion (Kalamidas and Kotoulas, 2000b; Kotoulas et al., 2006). Glucagon action is activated by the cAMP / protein kinase A (which in turn activates glycogen autophagy) and suppressed by phosphoinositides / mTOR pathways (which in turn surpresses glycogen autophagy) (Kalamidas et al., 1994; Kotoulas et al., 2004). That glycogen autophagy can be induced by rapamycin in newborn rat hepatocytes also suggests a TOR-dependent regulation on glycogen autophagy (Kalamidas and Kotoulas, 2000a, b). 1.1.1.2 The Cvt pathway The Cvt, cytosol-to-vacuole targeting pathway is a selective type of autophagy that is responsible for the sequestration of at least two resident vacuolar hydrolases, aminopeptidase I (Ape1) and α-mannosidase (Ams1), as specific cargos. The Cvt vesicles (140-160 nm in diameter) are also double-membrane bound but distinct from autophagosomes in cargo selectivity and size. However, Cvt and autophagy pathways are topologically and mechanistically similar and share most of the Atg (autophagy- Figure 1. Schematic diagram of selective and non-selective autophagy. Depending on the specificity of the cargos, autophagy can be a selective or a nonselective process. During nonselective autophagy, a portion of the cytoplasm is sequestered into a double-membrane autophagosome, which then fuses with the vacuole (macroautophagy). A biosynthetic cytoplasm to vacuole targeting (Cvt) pathway in yeast also shares similar morphological features and viewed as a selective type of autophagy.In contrast, the specific degradation of peroxisomes in certain conditions can be achieved by a macro- or microautophagy-like mode, termed macropexophagy and micropexophagy, respectively. The specific degradation of mitochondria, termed mitophagy also takes place. related) components (Hutchins and Klionsky, 2001). The Cvt pathway was identified in the unicellular yeasts (Baba et al., 1997), however, its existence in higher eukaryotes, including filamentous fungi, remains controversial. 1.1.1.3 Pexophagy Peroxisomes are single membrane-bound organelles in which lipid catabolism and hydrogen peroxide detoxification occurs. In Pichia pastoris, a species of methylotrophic yeast, peroxisome biogenesis is induced by growth on oleate, amine or methanol. P. pastoris has two alcohol oxidase (AOX)-encoding genes which allow it to use methanol as a carbon and energy source. The Aox protein resides in the peroxisomes and is induced along with the peroxisome biogenesis. Glucose or ethanol can suppress Aox expression and simultaneously induce pexophagy: the autophagic degradation of peroxisomes (for glucose, micropexophagy is induced whereas ethanol triggers macropexophagy) (Farre et al., 2007; Tuttle and Dunn, 1995). 1.1.2 Molecular basis of autophagy Autophagy was first identified by TEM imaging in S. cerevisiae and later studied extensively in the budding yeast and in animal cells. Thus far, 32 ATG genes have been characterized, which has led to a better understanding of the genetic and molecular regulation of autophagy (Kabeya et al., 2007; Klionsky et al., 2003), particularly the formation of autophagy-associated vesicular compartments, such as preautophagosomal structures (PAS), autophagosomes (cytosolic), and autophagic bodies (vacuolar) (Suzuki et al., 2001). Among the 32 ATG genes, 18 encode proteins involved in autophagosome formation. They are ATG1–10, ATG12–14, ATG16–18, ATG29, and ATG31 (Kabeya et al., 2007; Klionsky et al., 2003; Klionsky, 2005, 2007; Suzuki and Ohsumi, 2007). Atg1-Atg13 complex is required for autophagy induction (Funakoshi et al., 1997; Kamada et al., 2000). Atg17, Atg29 and Atg31 function together to form the scaffold for PAS organization (Cheong et al., 2005; Kabeya et al., 2007; Kawamata et al., 2005). Two unique ubiquitin-like conjugation systems, Atg8– phosphatidylethanolamine (Atg8–PE) and Atg12–Atg5, are involved in the biogenesis of autophagic vesicles (Ohsumi, 2001). Atg7 and Atg10 act as E1 ubiquitin-activating enzyme and E2 ubiquitin-conjugating enzyme, respectively, in Atg12-Atg5 conjugation system (Kim et al., 1999; Mizushima et al., 1998; Shintani et al., 1999). Atg12-Atg5 conjugate binds another protein, Atg16, to form a multimeric complex that is functionally important for autophagy (Mizushima et al., 1999). In Atg8-PE conjugation system, the cysteine protease Atg4 proteolytically removes a C-terminal arginine residue of Atg8, exposing a glycine that is now accessible to the E1-like Atg7, and another E2-like enzyme, Atg3, and eventually conjugated to PE through an amide bond (Ichimura et al., 2000). Atg6, Atg14 and several Vps proteins form PtdIns 3-kinase complex I that regulates membrane organization during autophagy and the Cvt pathway (Kihara et al., 2001). Atg18 is recruited to the PAS in a manner that is dependent on PtdIns 3-kinase complex I and is required for both autophagy and the Cvt pathway (Guan et al., 2001). Atg9 cycles between the PAS and the additional structures/organelles (Suzuki et al., 2001). Atg2, Atg18, and PtdIns 3-kinase complex I components are necessary for the retrieval of Atg9 (Shintani et al., 2001), which is triggered by Atg1-Atg13 complex (Reggiori et al., 2004). The following Atg proteins are specifically essential for the induction of the Cvt pathway: Atg11 is important for PAS organization (Kim et al., 2001; Suzuki and Ohsumi, 2007). Atg19 is the cargo receptor protein involved in the Cvt pathway (Scott et al., 2001). Atg20 and Atg24 bind PtdIns(3)P and belong to the sorting nexin family that functions in protein trafficking from the Golgi to the endosome (Hettema et al., 2003); and are involved in the Cvt pathway in S. cerevisiae (Nice DC et al., 2002). Like Atg18, Atg21 and Atg27 are also recruited to the PAS in PtdIns 3-kinase complex I-dependent manner. Atg21 and Atg27 are primarily required for the Cvt pathway (Stromhaug et al., 2004; Wurmser and Emr, 2002). Atg23 is needed for Cvt vesicle completion, and like Atg9, shows punctate localization which includes localization to the PAS (Tucker et al., 2003). Following the delivery to the vacuole, the outer membrane of the autophagosome is fused with the vacuolar membrane, which is mediated by the SNARE complex (Suzuki and Ohsumi, 2007; Wang et al., 2003). Subsequently, the degradation of autophagic body is dependent on two resident vacuolar proteases, Pep4 and Prb1, and the acidification of the vacuole (Nakamura et al., 1997; Takeshige et al., 1992). In addition to these factors, the transmembrane protein Atg15 is also required for lysis (Epple et al., 2001). Atg22 was identified as a putative amino acid effluxe r(Yang et al., 2006; Yang et al., 2007) that cooperates with other vacuolar permeases, such as Avt3 and Avt4, independent of these functions, in exporting the monomeric units (e.g. amino acids) derived from macromolecule degradation. Some ATG genes are species-specific and only required for some selective autophagy. ATG25 encodes a novel coiled-coil protein involved in macropexophagy in Hansenula polymorpha (Monastyrska et al., 2005). ATG26 encodes a UDPglycosyltransferase that is essential for the selective autophagy of large (average peroxisome area > 0.10 µm2) peroxosomes in Pichia pastoris, while not required for pexophagy in S. cerevisiae (Monastyrska et al., 2005; Nazarko et al., 2007). Atg28 is important for both micro- and macropexophagy in P. pastoris (Stasyk et al., 2006). Atg30 is essential for pexophagy in P. pastoris, regardless of the size of the peroxisomes or the inducer of the peroxisome biogenesis (Farre et al., 2008). Atg32 is a membrane-anchored protein that is required for selective targeting of mitochondria for autophagic degradation in S. cerevisiae (Kanki et al., 2009). 1.1.3 Physiological function of autophagy Although well conserved in eukaryotes, autophagy plays pleiotropic roles including protein / carbohydrate / iron metabolism, cellular development, death or survival, and clearance of invasive pathogens, etc (Codogno and Meijer, 2005; Gannage and Munz, 2009; Kurz et al., 2008; Mizushima, 2005). Rapid progress has been made in research in the past decade and the biological functions of autophagy in various organisms are detailed here. 1.1.3.1 Yeasts Autophagy-deficient mutants were isolated and characterized in the budding yeast S. cerevisiae (Tsukada and Ohsumi, 1993). These mutants showed defects at different step of autophagy. Autophagy-defective budding yeast lost viability during nitrogen starvation and the homozygous diploids with atg mutation failed to sporulate. Increased pseudohyphal growth was commonly observed in several autophagydefective yeast (Cutler et al., 2001; Ma et al., 2007; Tsukada and Ohsumi, 1993). In contrast, autophagy has been less studied in the fission yeast Schizosaccharomyces pombe. A recent report showed that autophagy regulates sexual differentiation in S. pombe (Mukaiyama et al., 2009). 1.1.3.2 Filamentous fungi In Podospora anserina, autophagy is essential for sexual differentiation and cell death by incompatibility. It remains controversial whether autophagy executes a programmed cell death function or acts as a pro-survival response in P. anserina (Dementhon et al., 2003; Dementhon et al., 2004; Pinan-Lucarre et al., 2003a; PinanLucarre et al., 2005). It was initially thought that autophagy triggers cell death during incompatible interactions for it is induced when cells of unlike genotypes fuse in P. anserina (Dementhon et al., 2004; Pinan-Lucarre et al., 2003a). However, a recent study suggests that autophagy serves a pro-survival role during incompatibility, as loss of autophagy results in accelerated cell death (Pinan-Lucarre et al., 2005). Autophagy-deficient mutants of M. oryzae are non-pathogenic and show highly reduced asexual development (Deng et al., 2009b; Liu et al., 2007b; VeneaultFourrey et al., 2006). Autophagy has been proposed to be essential for cell death of the conidial cells to ensure the successful penetration of the host cuticle (VeneaultFourrey et al., 2006). Autophagy is also involved in lipid body turnover and thus is suggested to be essential for turgor generation and appressorium function (Liu et al., 2007b). Similarly, infection structures/appressoria from a CLK1-deletion (an ortholog of ATG1) mutant in Colletotrichum lindemuthianum, are unable to penetrate the host cuticle (Dufresne et al., 1998). However, Colletotrichum gloeosporioides, with a related infection strategy as M. oryzae, does not require autophagic cell death for successful infection (Nesher et al., 2008). Autophagy is required for the differentiation of aerial hyphae and in conidial germination in Aspergillus oryzae (Kikuma et al., 2006). In contrast to its function in fungi mentioned previously, autophagy plays little or no role in the differentiation of the dimorphic yeast Candida albicans within the host tissue (Palmer et al., 2007). The atg9∆ mutant in C. albicans remains unaffected for yeast-hypha or chlamydospore differentiation, though it shows specific defects in autophagy and the Cvt pathway. 1.1.3.3 Plants In plants, autophagy has been shown to be induced to deal with abiotic stresses including nutrient starvation (Bassham, 2009), oxidative stress (Xiong et al., 2007a; Xiong et al., 2007b), high salt and osmotic stress conditions (Liu et al., 2009; Slavikova S et al., 2008). Autophagy contributes to programmed cell death in the unicellular green alga Micrasterias denticulata in response to the biotic and abiotic stress (Affenzeller et al., 2009). Autophagy is also necessary for the proper regulation of hypersensitive response (programmed cell death) during the plant innate immune response during pathogen invasion (Hofius et al., 2009). Recent studies showed that autophagy is involved in various aspects of plant development, including pollen germination (Harrison-Lowe and Olsen, 2008) and leaf senescence in Arabidopsis thaliana (Wada et al., 2009), and number-control of fertile florets in wheat (Ghiglione et al., 2008). 1.3.3.4 Animals Chapter V CONCLUSIONS In recent years, our understanding of the physiological roles of autophagy has increased dramatically. As a conserved bulk degradation system, autophagy serves for cellular protein, organelle, and membrane turnover. But more than that, autophagy is naturally induced and needed for a number of cellular differentiation processes and morphogenesis, such as metamorphosis in insects or synapse formation in the nervous system (Bamber and Rowland, 2006; Mizushima, 2005; Shen and Ganetzky, 2009). Autophagy plays two seemingly opposite roles under different circumstances: prosurvival functions in the host against invading viruses (Lin et al., ; Liu et al., 2005), or cell death in specific cell types during early stages of embryonic development in mammals (Shimizu et al., 2004; Yu et al., 2004). It is well documented that autophagy play an important role in fungal development and pathogenesis. Autophagy is induced during sporulation in S. cerevisiae (Schlumpberger et al., 1997), A. oryzae(Kikuma et al., 2006), and M. oryzae (Liu et al., 2007b; Veneault-Fourrey et al., 2006). Autophagosomes / autophagic vacuoles are frequently formed during critical developmental stages including conidia formation and germination (Kikuma et al., 2006), infection structure differentiation (for pathogens) (Liu et al., 2007b), or filamentous fusion (P. anserina sexual development) (Pinan-Lucarre et al., 2003a), etc. However, the exact function(s) of autophagy in such developmental processes has not been completely elucidated. This study attempted to investigate the physiological role(s) of autophagy during the pathogenic life cycle of Magnaporthe oryzae. Autophagy-deficient atg8∆ mutant showed pleiotropic defects which included a significant reduction in conidiation and a 120 complete loss of pathogenicity in Magnaporthe. Autophagy may target cytosolic contents in a non-selective manner, for vacuolar degradation, or it may target specific substrate(s), under some particular physiological condition, e.g. conidiation. In this study, it was found that glycogen may be a selective target for autophagy during Magnaporthe conidiation. Mass spectrometry-based identification of Gph1, a glycogen phosphorylase that showed elevated accumulation in atg8∆ mutant but not in the wild type during conidiation, indicated that autophagy strictly regulates the levels of glycogen at this stage. Further study showed that total glycogen level is significantly higher in atg8∆ than in the wild type and likely accounts for the conidiation defects. Exogenous supply of the downstream product of glycogen hydrolysis, e.g. glucose, or G6P, successfully restored conidiation in atg8∆. More interestingly, ectopic expression of Sga1, the vacuolar glucoamylase that acts following autophagy-based delivery of glycogen into the vacuole, in the cytosol, could bypass the requirement of autophagy in glycogen hydrolysis and significantly restore conidiation in the atg8∆. These experimental results support a model in which autopahgy delivers, and Sga1 catalyzes the breakdown of glycogen in the vacuole, to supply abundant glucose, as an energy source or an important precusor during cellular differentiation. Such a process is defined as glycogen autophagy in new-born mammals and it seems to play an important role in the “new-born” spores in Magnaporthe too. However, glycogen autophagy does not seem to be important for Magnaporthe pathogenicity. Conidiation-restored atg8∆ mutant, by exogenous glucose or rice leaf extract, or by ectopic expression of Sga1 in the cytosol, remained non-pathogenic. On the other hand, sga1∆ mutant showed a significant reduction in conidiation but sga1∆ 121 conidia were pathogenic, indicating that Sga1-catalyzed glycogen hydrolysis is dispensible for host infection. Given that autophagy can target more than glycogen for vacuolar degradation, it was proposed that the specific substrate(s) for autophagy during Magnaporthe pahogenicity is different from that for conidiation, which remains unknown at present. Autophagy likely plays two contrasting roles in Magnaporthe: cell survival during conidiation (Deng et al., 2009a) and cell death during pathogenicity (Veneault-Fourrey et al., 2006). Another autophagy-related gene included in our study was ATG20, which was first identified as a gene encoding a PX-domain containing protein that is essential for the Cvt pathway in yeast (Yorimitsu and Klionsky, 2005). Atg20 was found to be involved in other membrane trafficking events, besides the Cvt pathway. Atg20 mediates protein retrieval transport from endosomes to the golgi complex, and a failure to so results in mis-targeting of proteins to the vacuole instead of the golgi (Hettema et al., 2003). Magnaporthe atg20∆ mutant displayed more severe defects in conidiation compared to the atg8∆. The atg20∆ mutant was non-conidiating, and such defects could not be restored by external supplied glucose, G6P, or rice leaf extract, indicating that the mechanism underlying atg20∆ conidiation defects is likely different from glycogen catabolism in the atg8∆. By systematic characterization of non-selective pathway, the Cvt pathway, and the pexophagy, by immunobloting, I have shown that the nonselective autophagy and the Cvt pathway were normal while pexophagy was significantly delayed in the atg20∆. However, further studies on other pexophagydeficient mutant (Pex1461-361) ruled out the possibility that pexophagy was required 122 for proper conidiaion or pathogenicity in Magnporthe. Besides, pexophagy was not naturally induced during Magnaporthe conidiation. The detailed study on subcellular localization of Atg20-GFP revealed that Atg20 is mostly localized to vesicular or tubular membrane structures at proximity of vacuole, probably representing late endosomes. Hence the role of Atg20 during Magnaporthe conidiation was proposed to be endosomal sorting and/or retrieval trafficking. Among the yeast proteins that depend on Snx4/Snx41/Snx42 complex for retrieval trafficking, Snc1 is an exocytic v-SNARE that is implicated in sporulation of S. cerevisiae (Morishita et al., 2007). Snc1-GFP in Magnaporthe localizes to multiple vesicular compartments including small puncta that may represent exomer or secretory vesicles or endosomes, filamentous membrane structures that may be the golgi complex, at plasma membranes and inside the vacuole. An increased vacuolar localization of Snc1-GFP was evident in atg20∆ mycelia. During conidiation, Snc1-GFP tends to associate with the plasma membrane or in spherical or round vacuoles in the aerial hyphae of atg20∆, while in wild type it is localized to numerous vesicles but were rarely seen in the vacuole of aerial hyphae. Furthermore, an SNC1 knockdown strain was generated and conidiation of this mutant was significantly reduced compared to that of the wild type. I propose that Snc1 may be one of the targets of Atg20-mediated retrieval trafficking that play a role in Magnaporthe conidiation. However the exact mechanism for Atg20-mediated protein sorting during Magnaporthe conidiation remains elusive. Study on co-localization of Atg20-GFP and RFP-Atg8 revealed that Atg20- or Atg8enriched compartments are relatively independent from each other, while occasionally 123 associated with each other. Loss of Atg8 does not interfere with Atg20 localization, and vise versa. Recent studies indicate that endosomes contribute to and regulate PAS membrane expansion and autophagosome formation at multiple steps (Razi et al., 2009; Rusten and Stenmark, 2009). The observation here may reflect such cross-talk between endosome and autophagosome / autophagic vacuole. In summary, functional study on Atg8 revealed pleiotropic roles of autophagy in Magnaporthe pathogenic life cycle, one of which is glycogen autophagy, as an executor of carbohydrate catabolism specifically for Magnaporthe asexual development. Autophagy is also required for Magnaporthe pathogenesis, but instead of glycogen, other cellular components may be targeted, which are not identified yet. Study on Atg20 ruled out the requirement of pexophagy for Magnaporthe pathogenic life cycle. Atg20-dependent (probably endosomal) trafficking pathway is essential for Magnaporthe asexual development, through an unidentified mechanism. 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(2008) Pex14 is the sole component of the peroxisomal translocon that is required for pexophagy. Autophagy 4: 63-66. 135 [...]... M oryzae to tag GFP at the C-terminal of SNC1 gene in its locus The primers used for RFP and GFP tagging were listed in Table 2 All the plasmid vectors created for gene deletion, genetic complementation, and RFP and GFP tagging are given in Table 3 (specifying the backbone vector chosen and the corresponding fungal selection marker) 2. 2 .2 DNA techniques 2. 2 .2. 1 DNA extraction The DNA samples from PCR... PstI and NdeI) and the RFP ORF (digested with NdeI and XcaI) in one step, so that the newly created plasmid contained an in- frame insertion of RFP ORF at the translational start site within the ATG8 coding sequence while retaining the requisite native regulatory sequences This plasmid was named as pRFP-ATG8 and introduced as a single-copy insertion in the atg8∆ strain For ATG8-RFP construct, the 1... pathway (Thines et al., 20 00) Gph1 and Sga1 are also present in M.oryzae and their activities are conserved 1.5 Aims and objectives of this study Conidiation is an important step in M oryzae pathogenic life cycle It provides suitable inoculum for the pathogen and determines the severity of the disease However, the genetic and biochemical control of the onset of conidiation, and the regulation of proper... in Table 2 The fragment was ligated to pFGL44 and randomly inserted into the genome of the wild-type strain Transformants with multiple copies of GPH1 (examined by Southern blot) were selected for checking the overexpression of GPH1 (by reverse transcriptase PCR) and further characterization For expression of RFP-ATG8, the promoter fragment of the ATG8 gene was PCR amplified from genomic DNA from the. .. stages of embryonic development in mammals (Shimizu et al., 20 04; Yu et al., 20 04) Autophagy also plays a key role in host defense against viral and intracellular bacterial pathogens in animals Overexpression of mammalian Beclin 1 / ATG6 promotes immunity against Sindbis virus infection in mice (Liang et al., 1998) Induction of autophagy by starvation or rapamycin treatment promotes the degradation of. .. compartments and their resident proteins in eukaryotic cells Endosomes also play a role in cellular signaling The G protein signaling occurring on the endosome, executed by Gpa1, Vps34 and Vps15, ensures the yeast responsive to the pheromone and triggers mating (Slessareva et al., 20 06) Vps34 activation by TOR signal, and the subsequent PI3P production, is critical for autophagy induction and completion and. .. defence system (in addition to the ubiquitin-proteasome system) against toxic build-up of misfolded proteins (Chin et al., 20 10; Matsuda and Tanaka, 20 09) Recent studies showed that the Parkinson's disease (PD)-linked E3 ligase, parkin, regulates specific induction of autophagy for selective clearance of misfolded and aggregated proteins during proteotoxic stress Dysfunction of Parkin promotes neurodegenerative... during Drosophila melanogaster development Autophagy genes are induced in dying salivary glands and are essential for the PCD Autophagic PCD is likely regulated by apoptopsis genes and members of the Rho, Rac, and Rab families of small guanosine triphosphatases (GTPases) (Baehrecke, 20 03; Martin and Baehrecke, 20 04) Likewise, autophagy is essential for promoting cell death in specific cell types during... Cat.740609 .25 0) 2. 2 .2. 2 Recombinant DNA techniques Restriction and modifying enzymes were from New England Biolabs or Roche Diagnostics and used according to the manufacturer’s instructions 32 Table 3 Plasmids used in this study Name pFGL44 Plasmid description pCAMBIA1300, removing HPH from XhoI site and re-ligating into SalI site pFGL97 pCAMBIA, removing HPH from XhoI site and re-ligating into BAR BamHI-PstI... with BamHI and then end-filled with Klenow enzyme and ligated in frame to the Sga146-655 coding sequence at the N-terminus The SpeI-XbaI fragment from this plasmid, containing the MPG1 promoter-GFP-SGA146-655-TrpC terminator was released and then ligated to pFGL44 and transformed into the wild type or an atg8∆ strain, respectively For ATG20-GFP construct, the 1 Kb fragment just proximal to the translation . protein involved in the Cvt pathway 6 (Scott et al., 20 01). Atg20 and Atg24 bind PtdIns(3)P and belong to the sorting nexin family that functions in protein trafficking from the Golgi to the. (Hettema et al., 20 03); and are involved in the Cvt pathway in S. cerevisiae (Nice DC et al., 20 02) . Like Atg18, Atg21 and Atg27 are also recruited to the PAS in PtdIns 3-kinase complex I-dependent. al., 20 07; Tuttle and Dunn, 1995). 1.1 .2 Molecular basis of autophagy Autophagy was first identified by TEM imaging in S. cerevisiae and later studied extensively in the budding yeast and in