Macroautophagy is required for multicellular development of the social amoeba Dictyostelium discoideum

Macroautophagy is a mechanism employed by eukaryotic cells to recycle non-essential cellular components during starvation, differentiation, and development. Two conjugation reactions related to ubiquitination are essential for autophagy: Apg12p conjugation to Apg5p, and Apg8p conjugation to the lipid phosphatidylethanolamine. These reactions require the action of the E1-like enzyme, Apg7p, and the E2-like enzymes, Apg3p and Apg10p. In Dictyostelium, development is induced by starvation, conditions under which autophagy is required for survival in yeast and plants. We have identified Dictyostelium homologues of 10 budding yeast autophagy genes. We have generated mutations in apg5 and apg7 that produce defects typically associated with an abrogation of autophagy. Mutants are not grossly affected in growth, but survival during nitrogen starvation is severely reduced. Starved mutant cells show little turnover of cellular constituents by electron microscopy, whereas wild-type cells show significant cytoplasmic degradation and reduced organelle number. Bulk protein degradation during starvation-induced development is reduced in the autophagy mutants. Development is aberrant; the autophagy mutants do not aggregate in plaques on bacterial lawns, but they do proceed further in development on nitrocellulose filters, forming defective fruiting bodies. The autophagy mutations are cell autonomous, because wild-type cells in a chimaera do not rescue development of the autophagy mutants. We have complemented the mutant phenotypes by expression of the cognate gene fused to green fluorescent protein. A green fluorescent protein fusion of the autophagosome marker Apg8 mislocalizes in the two autophagy mutants. We show that the Apg5-Apg12 conjugation system is conserved in Dictyostelium.     

Characterization of a novel autophagy-specific gene, ATG29

Autophagy is a process whereby cytoplasmic proteins and organelles are sequestered for bulk degradation in the vacuole/lysosome. At present, 16 ATG genes have been found that are essential for autophagosome formation in the yeast Saccharomyces cerevisiae. Most of these genes are also involved in the cytoplasm to vacuole transport pathway, which shares machinery with autophagy. Most Atg proteins are colocalized at the pre-autophagosomal structure (PAS), from which the autophagosome is thought to originate, but the precise mechanism of autophagy remains poorly understood. During a genetic screen aimed to obtain novel gene(s) required for autophagy, we identified a novel ORF, ATG29/YPL166w. atg29Delta cells were sensitive to starvation and induction of autophagy was severely retarded. However, the Cvt pathway operated normally. Therefore, ATG29 is an ATG gene specifically required for autophagy. Additionally, an Atg29-GFP fusion protein was observed to localize to the PAS. From these results, we propose that Atg29 functions in autophagosome formation at the PAS in collaboration with other Atg proteins.     

Cheating on ubiquitin with Atg8

Macroautophagy sequesters superflous cytosol and organelles into double-membraned autophagosomes. Over 30 autophagy-related (ATG) genes have been identified without elucidating the molecular details of autophagosome biogenesis. All proposed models for autophagosome formation require membrane fusion events (Fig. 1). Previous studies assumed that the autophagic machinery mediates these membrane fusions in a SNARE-independent manner and identified the ubiquitin-like protein Atg8 as a key component especially for elongation of the forming autophagosome. However, if and how Atg8 mediates membrane fusion and why a ubiquitin-like protein is needed for autophagosome biogenesis remained open questions. Since nuclear envelope growth and fusion of Golgi fragments are topologically similar to autophagosome formation and depend on the AAA (+) ATPase p97/VCP and p47 we analyzed the involvement of their yeast homologues Cdc48 and Shp1 in macroautophagy.     

Atg17 functions in cooperation with Atg1 and Atg13 in yeast autophagy

In eukaryotic cells, nutrient starvation induces the bulk degradation of cellular materials; this process is called autophagy. In the yeast Saccharomyces cerevisiae, most of the ATG (autophagy) genes are involved in not only the process of degradative autophagy, but also a biosynthetic process, the cytoplasm to vacuole (Cvt) pathway. In contrast, the ATG17 gene is required specifically in autophagy. To better understand the function of Atg17, we have performed a biochemical characterization of the Atg17 protein. We found that the atg17delta mutant under starvation condition was largely impaired in autophagosome formation and only rarely contained small autophagosomes, whose size was less than one-half of normal autophagosomes in diameter. Two-hybrid analyses and coimmunoprecipitation experiments demonstrated that Atg17 physically associates with Atg1-Atg13 complex, and this binding was enhanced under starvation conditions. Atg17-Atg1 binding was not detected in atg13delta mutant cells, suggesting that Atg17 interacts with Atg1 through Atg13. A point mutant of Atg17, Atg17(C24R), showed reduced affinity for Atg13, resulting in impaired Atg1 kinase activity and significant defects in autophagy. Taken together, these results indicate that Atg17-Atg13 complex formation plays an important role in normal autophagosome formation via binding to and activating the Atg1 kinase.     

A role for Atg8–PE deconjugation in autophagosome biogenesis

Formation of the autophagosome is likely the most complex step of macroautophagy, and indeed it is the morphological and functional hallmark of this process; accordingly, it is critical to understand the corresponding molecular mechanism. Atg8 is the only known autophagy-related (Atg) protein required for autophagosome formation that remains associated with the completed sequestering vesicle. Approximately one-fourth of all of the characterized Atg proteins that participate in autophagosome biogenesis affect Atg8, regulating its conjugation to phosphatidylethanolamine (PE), localization to the phagophore assembly site and/or subsequent deconjugation. An unanswered question in the field regards the physiological role of the deconjugation of Atg8–PE. Using an Atg8 mutant that bypasses the initial Atg4-dependent processing, we demonstrate that Atg8 deconjugation is an important step required to facilitate multiple events during macroautophagy. The inability to deconjugate Atg8–PE results in the mislocalization of this protein to the vacuolar membrane. We also show that the deconjugation of Atg8–PE is required for efficient autophagosome biogenesis, the assembly of Atg9-containing tubulovesicular clusters into phagophores/autophagosomes, and for the disassembly of PAS-associated Atg components.     

A role for Atg8-PE deconjugation in autophagosome biogenesis

Formation of the autophagosome is likely the most complex step of macroautophagy, and indeed it is the morphological and functional hallmark of this process; accordingly, it is critical to understand the corresponding molecular mechanism. Atg8 is the only known autophagy-related (Atg) protein required for autophagosome formation that remains associated with the completed sequestering vesicle. Approximately one-fourth of all of the characterized Atg proteins that participate in autophagosome biogenesis affect Atg8, regulating its conjugation to phosphatidylethanolamine (PE), localization to the phagophore assembly site and/or subsequent deconjugation. An unanswered question in the field regards the physiological role of the deconjugation of Atg8-PE. Using an Atg8 mutant that bypasses the initial Atg4-dependent processing, we demonstrate that Atg8 deconjugation is an important step required to facilitate multiple events during macroautophagy. The inability to deconjugate Atg8-PE results in the mislocalization of this protein to the vacuolar membrane. We also show that the deconjugation of Atg8-PE is required for efficient autophagosome biogenesis, the assembly of Atg9-containing tubulovesicular clusters into phagophores/autophagosomes, and for the disassembly of PAS-associated Atg components.     

ATG5 Is Essential for ATG8-Dependent Autophagy and Mitochondrial Homeostasis in Leishmania major

Macroautophagy has been shown to be important for the cellular remodelling required for Leishmania differentiation. We now demonstrate that L. major contains a functional ATG12-ATG5 conjugation system, which is required for ATG8-dependent autophagosome formation. Nascent autophagosomes were found commonly associated with the mitochondrion. L. major mutants lacking ATG5 (Δatg5) were viable as promastigotes but were unable to form autophagosomes, had morphological abnormalities including a much reduced flagellum, were less able to differentiate and had greatly reduced virulence to macrophages and mice. Analyses of the lipid metabolome of Δatg5 revealed marked elevation of phosphatidylethanolamines (PE) in comparison to wild type parasites. The Δatg5 mutants also had increased mitochondrial mass but reduced mitochondrial membrane potential and higher levels of reactive oxygen species. These findings indicate that the lack of ATG5 and autophagy leads to perturbation of the phospholipid balance in the mitochondrion, possibly through ablation of membrane use and conjugation of mitochondrial PE to ATG8 for autophagosome biogenesis, resulting in a dysfunctional mitochondrion with impaired oxidative ability and energy generation. The overall result of this is reduced virulence.     

Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases

During macroautophagy/autophagy, mammalian Atg8-family proteins undergo 2 proteolytic processing events. The first exposes a COOH-terminal glycine used in the conjugation of these proteins to lipids on the phagophore, the precursor to the autophagosome, whereas the second releases the lipid. The ATG4 family of proteases drives both cleavages, but how ATG4 proteins distinguish between soluble and lipid-anchored Atg8 proteins is not well understood. In a fully reconstituted delipidation assay, we establish that the physical anchoring of mammalian Atg8-family proteins in the membrane dramatically shifts the way ATG4 proteases recognize these substrates. Thus, while ATG4B is orders of magnitude faster at processing a soluble unprimed protein, all 4 ATG4 proteases can be activated to similar enzymatic activities on lipid-attached substrates. The recognition of lipidated but not soluble substrates is sensitive to a COOH-terminal LIR motif both in vitro and in cells. We suggest a model whereby ATG4B drives very fast priming of mammalian Atg8 proteins, whereas delipidation is inherently slow and regulated by all ATG4 homologs.     

ATG2, an autophagy-related protein, negatively affects powdery mildew resistance and mildew-induced cell death in Arabidopsis

The molecular interactions between Arabidopsis and the pathogenic powdery mildew Golovinomyces cichoracearum were studied by characterizing a disease-resistant Arabidopsis mutant atg2-2. The atg2-2 mutant showed enhanced resistance to powdery mildew and dramatic mildew-induced cell death as well as early senescence phenotypes in the absence of pathogens. Defense-related genes were constitutively activated in atg2-2. In atg2-2 mutants, spontaneous cell death, early senescence and disease resistance required the salicylic acid (SA) pathway, but interestingly, mildew-induced cell death was not fully suppressed by inactivation of SA signaling. Thus, cell death could be uncoupled from disease resistance, suggesting that cell death is not sufficient for resistance to powdery mildew. ATG2 encodes autophagy-related 2, a protein known to be involved in the early steps of autophagosome biogenesis. The atg2-2 mutant exhibited typical autophagy defects in autophagosome formation. Furthermore, mutations in several other ATG genes, including ATG5, ATG7 and ATG10, exhibited similar powdery mildew resistance and mildew-induced cell death phenotypes. Taken together, our findings provide insights into the role of autophagy in cell death and disease resistance, and may indicate general links between autophagy, senescence, programmed cell death and defense responses in plants.     

Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation

Macroautophagy is primarily a degradative process that cells use to break down their own components to recycle macromolecules and provide energy under stress conditions, and defects in macroautophagy lead to a wide range of diseases. Atg9, conserved from yeast to mammals, is the only identified transmembrane protein in the yeast core macroautophagy machinery required for formation of the sequestering compartment termed the autophagosome. This protein undergoes dynamic movement between the phagophore assembly site (PAS), where the autophagosome precursor is nucleated, and peripheral sites that may provide donor membrane for expansion of the phagophore. Atg9 is a phosphoprotein that is regulated by the Atg1 kinase. We used stable isotope labeling by amino acids in cell culture (SILAC) to identify phosphorylation sites on this protein and identified an Atg1-independent phosphorylation site at serine 122. A nonphosphorylatable Atg9 mutant showed decreased autophagy activity, whereas the phosphomimetic mutant enhanced activity. Electron microscopy analysis suggests that the different levels of autophagy activity reflect differences in autophagosome formation, correlating with the delivery of Atg9 to the PAS. Finally, this phosphorylation regulates Atg9 interaction with Atg23 and Atg27.     

The ATG12-conjugating enzyme ATG10 Is essential for autophagic vesicle formation in Arabidopsis thaliana

Autophagy is an important intracellular recycling system in eukaryotes that utilizes small vesicles to traffic cytosolic proteins and organelles to the vacuole for breakdown. Vesicle formation requires the conjugation of the two ubiquitin-fold polypeptides ATG8 and ATG12 to phosphatidylethanolamine and the ATG5 protein, respectively. Using Arabidopsis thaliana mutants affecting the ATG5 target or the ATG7 E1 required to initiate ligation of both ATG8 and ATG12, we previously showed that the ATG8/12 conjugation pathways together are important when plants encounter nutrient stress and during senescence. To characterize the ATG12 conjugation pathway specifically, we characterized a null mutant eliminating the E2-conjugating enzyme ATG10 that, similar to plants missing ATG5 or ATG7, cannot form the ATG12-ATG5 conjugate. atg10-1 plants are hypersensitive to nitrogen and carbon starvation and initiate senescence and programmed cell death (PCD) more quickly than wild type, as indicated by elevated levels of senescence- and PCD-related mRNAs and proteins during carbon starvation. As detected with a GFP-ATG8a reporter, atg10-1 and atg5-1 mutant plants fail to accumulate autophagic bodies inside the vacuole. These results indicate that ATG10 is essential for ATG12 conjugation and that the ATG12-ATG5 conjugate is necessary to form autophagic vesicles and for the timely progression of senescence and PCD in plants.     

An Arabidopsis homolog of yeast ATG6/VPS30 is essential for pollen germination

Yeast (Saccharomyces cerevisiae) Atg6/Vps30 is required for autophagy and the sorting of vacuolar hydrolases, such as carboxypeptidase Y. In higher eukaryotes, however, roles for ATG6/VPS30 homologs in vesicle sorting have remained obscure. Here, we show that AtATG6, an Arabidopsis (Arabidopsis thaliana) homolog of yeast ATG6/VPS30, restored both autophagy and vacuolar sorting of carboxypeptidase Y in a yeast atg6/vps30 mutant. In Arabidopsis cells, green fluorescent protein-AtAtg6 protein localized to punctate structures and colocalized with AtAtg8, a marker protein of the preautophagosomal structure. Disruption of AtATG6 by T-DNA insertion resulted in male sterility that was confirmed by reciprocal crossing experiments. Microscopic analyses of AtATG6 heterozygous plants (AtATG6/atatg6) crossed with the quartet mutant revealed that AtATG6-deficient pollen developed normally, but did not germinate. Because other atatg mutants are fertile, AtAtg6 likely mediates pollen germination in a manner independent of autophagy. We propose that Arabidopsis Atg6/Vps30 functions not only in autophagy, but also plays a pivotal role in pollen germination.     

ATG4B contains a C-terminal LIR motif important for binding and efficient cleavage of mammalian orthologs of yeast Atg8

The cysteine protease ATG4B cleaves off one or more C-terminal residues of the inactive proform of proteins of the ortholog and paralog LC3 and GABARAP subfamilies of yeast Atg8 to expose a C-terminal glycine that is conjugated to phosphatidylethanolamine during autophagosome formation. We show that ATG4B contains a C-terminal LC3-interacting region (LIR) motif important for efficient binding to and cleavage of LC3 and GABARAP proteins. We solved the crystal structures of the GABARAPL1-ATG4B C-terminal LIR complex. Analyses of the structures and in vitro binding assays, using specific point mutants, clearly showed that the ATG4B LIR binds via electrostatic-, aromatic HP1 and hydrophobic HP2 pocket interactions. Both these interactions and the catalytic site-substrate interaction contribute to binding between LC3s or GABARAPs and ATG4B. We also reveal an unexpected role for ATG4B in stabilizing the unlipidated forms of GABARAP and GABARAPL1. In mouse embryonic fibroblast (MEF) atg4b knockout cells, GABARAP and GABARAPL1 were unstable and degraded by the proteasome. Strikingly, the LIR motif of ATG4B was required for stabilization of the unlipidated forms of GABARAP and GABARAPL1 in cells.     

Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways

Autophagy is an important mechanism for nonselective intracellular breakdown whereby cytosol and organelles are encapsulated in vesicles, which are then engulfed and digested by lytic vacuoles/lysosomes. In yeast, this encapsulation employs a set of autophagy (ATG) proteins that direct the conjugation of two ubiquitin-like protein tags, ATG8 and ATG12, to phosphatidylethanolamine and the ATG5 protein, respectively. Using an Arabidopsis (Arabidopsis thaliana) atg7 mutant unable to ligate either tag, we previously showed that the ATG8/12 conjugation system is important for survival under nitrogen-limiting growth conditions. By reverse-genetic analyses of the single Arabidopsis gene encoding ATG5, we show here that the subpathway that forms the ATG12-ATG5 conjugate also has an essential role in plant nutrient recycling. Similar to plants missing ATG7, those missing ATG5 display early senescence and are hypersensitive to either nitrogen or carbon starvation, which is accompanied by a more rapid loss of organellar and cytoplasmic proteins. Multiple ATG8 isoforms could be detected immunologically in seedling extracts. Their abundance was substantially elevated in both the atg5 and atg7 mutants, caused in part by an increase in abundance of several ATG8 mRNAs. Using a green fluorescent protein-ATG8a fusion in combination with concanamycin A, we also detected the accumulation of autophagic bodies inside the vacuole. This accumulation was substantially enhanced by starvation but blocked in the atg7 background. The use of this fusion in conjunction with atg mutants now provides an important marker to track autophagic vesicles in planta.     

Kinetics comparisons of mammalian Atg4 homologues indicate selective preferences toward diverse Atg8 substrates

The Atg4 cysteine proteases are required for processing Atg8 for the latter to be conjugated to phosphatidylethanolamine on autophagosomal membranes, a key step in autophagosome biogenesis. Notably, whereas there are only one atg4 and one atg8 gene in the yeast, the mammals have four Atg4 homologues and six Atg8 homologues. The Atg8 homologues seem to play different roles in autophagosome biogenesis, and previous studies had indicated that they could be differentially processed by Atg4 homologues. The present study provided the first detailed kinetics analysis of all four Atg4 homologues against four representative Atg8 homologues. The data indicated that Atg4B possessed the broadest spectrum against all substrates, followed by Atg4A, whereas Atg4C and Atg4D had minimal activities as did the catalytic mutant of Atg4B (C74S). On the other hand, GATE-16 seemed to be the overall best substrate for Atg4 proteases. The kinetics parameters of Atg4B were also affected by its structure and that of the substrates, indicating a process of induced fit. The determination of the kinetics parameters of the various Atg4-Atg8 pairs provides a base for the understanding of the potential selective impact of the reaction on autophagosome biogenesis.     

Beyond Atg8 binding: The role of AIM/LIR motifs in autophagy

Selective macroautophagy/autophagy mediates the selective delivery of cytoplasmic cargo material via autophagosomes into the lytic compartment for degradation. This selectivity is mediated by cargo receptor molecules that link the cargo to the phagophore (the precursor of the autophagosome) membrane via their simultaneous interaction with the cargo and Atg8 proteins on the membrane. Atg8 proteins are attached to membrane in a conjugation reaction and the cargo receptors bind them via short peptide motifs called Atg8-interacting motifs/LC3-interacting regions (AIMs/LIRs). We have recently shown for the yeast Atg19 cargo receptor that the AIM/LIR motifs also serve to recruit the Atg12–Atg5-Atg16 complex, which stimulates Atg8 conjugation, to the cargo. We could further show in a reconstituted system that the recruitment of the Atg12–Atg5-Atg16 complex is sufficient for cargo-directed Atg8 conjugation. Our results suggest that AIM/LIR motifs could have more general roles in autophagy.     

Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure during the Cvt pathway

Atg21 and Atg18 are homologue yeast proteins. Whereas Atg18 is essential for the Cvt pathway and autophagy, a lack of Atg21 only blocks the Cvt pathway. Our proteinase protection experiments now demonstrate that growing atg21Delta cells fail to form proaminopeptidase I-containing Cvt vesicles. Quantitative measurement of autophagy in starving atg21Delta cells showed only 35% of the wild-type rate. This suggests that Atg21 plays a nonessential role in improving the fidelity of autophagy. The intracellular localization of Atg21 is unique among the Atg proteins. In cells containing multiple vacuoles, Atg21-yellow fluorescent protein clearly localizes to the vertices of the vacuole junctions. Cells with a single vacuole show most of the protein at few perivacuolar punctae. This distribution pattern is reminiscent to the Vps class C(HOPS) (homotypic fusion and vacuolar protein sorting) protein complex. In growing cells, Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure. Consistently, the covalent linkage of Atg8 to the lipid phosphatidylethanolamine is significantly retarded. Lipidated Atg8 is supposed to act during the elongation of autophagosome precursors. However, despite the reduced autophagic rate and the retardation of Atg8 lipidation, electron microscopy of starved atg21Delta ypt7Delta double mutant cells demonstrates the formation of normally sized autophagosomes with an average diameter of 450 nm.     

ATG8 lipidation and ATG8‐mediated autophagy in Arabidopsis require ATG12 expressed from the differentially controlled ATG12A AND ATG12B loci

Autophagic recycling of intracellular plant constituents is maintained at a basal level under normal growth conditions but can be induced in response to nutritional demand, biotic stress, and senescence. One route requires the ubiquitin‐fold proteins Autophagy‐related (ATG)‐8 and ATG12, which become attached to the lipid phosphatidylethanolamine (PE) and the ATG5 protein, respectively, during formation of the engulfing vesicle and delivery of its cargo to the vacuole for breakdown. Here, we genetically analyzed the conjugation machinery required for ATG8/12 modification in Arabidopsis thaliana with a focus on the two loci encoding ATG12. Whereas single atg12a and atg12b mutants lack phenotypic consequences, atg12a atg12b double mutants senesce prematurely, are hypersensitive to nitrogen and fixed carbon starvation, and fail to accumulate autophagic bodies in the vacuole. By combining mutants eliminating ATG12a/b, ATG5, or the ATG10 E2 required for their condensation with a method that unequivocally detects the ATG8‐PE adduct, we also show that ATG8 lipidation requires the ATG12–ATG5 conjugate. Unlike ATG8, ATG12 does not associate with autophagic bodies, implying that its role(s) during autophagy is restricted to events before the vacuolar deposition of vesicles. The expression patterns of the ATG12a and ATG12b genes and the effects of single atg12a and atg12b mutants on forming the ATG12–ATG5 conjugate reveal that the ATG12b locus is more important during basal autophagy while the ATG12a locus is more important during induced autophagy. Taken together, we conclude that the formation of the ATG12–ATG5 adduct is essential for ATG8‐mediated autophagy in plants by promoting ATG8 lipidation.     

The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy. 'Protein modifications: beyond the usual suspects' review series

As a lysosomal/vacuolar degradative pathway that is conserved in eukaryotic organisms, autophagy mediates the turnover of long-lived proteins and excess or aberrant organelles. The main characteristic of autophagy is the formation of a double-membrane vesicle, the autophagosome, which envelops part of the cytoplasm and delivers it to the lysosome/vacuole for breakdown and eventual recycling of the degradation products. Among the approximately 30 autophagy-related (Atg) genes identified so far, there are two ubiquitin-like proteins, Atg12 and Atg8. Analogous to ubiquitination, Atg12 is conjugated to Atg5 by Atg7--an E1-like protein--and Atg10--an E2-like protein. Similarly, Atg7 and Atg3 are the respective E1-like and E2-like proteins that mediate the conjugation of Atg8 to phosphatidylethanolamine. Both Atg12-Atg5 and Atg8 localize to the developing autophagosome. The Atg12-Atg5 conjugate facilitates the lipidation of Atg8 and directs its correct subcellular localization. Atg8-phosphatidylethanolamine is probably a scaffold protein that supports membrane expansion and the amount present correlates with the size of autophagosomes.     

ATG genes involved in non-selective autophagy are conserved from yeast to man, but the selective Cvt and pexophagy pathways also require organism-specific genes

ATG genes encode proteins that are required for macroautophagy, the Cvt pathway and/or pexophagy. Using the published Atg protein sequences, we have screened protein and DNA databases to identify putative functional homologs (orthologs) in 21 fungal species (yeast and filamentous fungi) of which the genome sequences were available. For comparison with Atg proteins in higher eukaryotes, also an analysis of Arabidopsis thaliana and Homo sapiens databases was included. This analysis demonstrated that Atg proteins required for non-selective macroautophagy are conserved from yeast to man, stressing the importance of this process in cell survival and viability. The A. thaliana and human genomes encode multiple proteins highly similar to specific fungal Atg proteins (paralogs), possibly representing cell type-specific isoforms. The Atg proteins specifically involved in the Cvt pathway and/or pexophagy showed poor conservation, and were generally not present in A. thaliana and man. Furthermore, Atg19, the receptor of Cvt cargo, was only detected in Saccharomyces cerevisiae. Nevertheless, Atg11, a protein that links receptor-bound cargo (peroxisomes, the Cvt complex) to the autophagic machinery was identified in all yeast species and filamentous fungi under study. This suggests that in fungi an organism-specific form of selective autophagy may occur, for which specialized Atg proteins have evolved.