Open and closed HORMAs regulate autophagy initiation

The Atg1/ULK complex functions as the most upstream factor among Atg proteins to initiate autophagy. ATG101 is a constitutive component of the Atg1/ULK complex in most eukaryotes except for budding yeast, and plays an essential role in autophagy; however, the structure and functions of ATG101 were largely unknown. Recently, we determined the crystal structure of fission yeast Atg101 in complex with the closed HORMA domain of Atg13, revealing that Atg101 is also a HORMA protein with an open conformation. These 2 HORMA proteins play essential roles in autophagy initiation through recruiting downstream factors to the autophagosome formation site.     

Dissecting the localization and function of Atg18, Atg21 and Ygr223c

Atg18p and Atg21p are two highly homologous yeast autophagy proteins. Atg18p functions in both autophagy and the selective Cvt-pathway, while the function of Atg21p is restricted to the Cvt-pathway. The yeast genome encodes with Ygr223cp (Hsv2p) a third member of this protein family. So far no function has been assigned to Ygr223cp. By colocalization with the endosomal marker Snf7-RFP and an RFP-tagged FYVE domain, we here identify the localization of a pool of Atg18p, Atg21p and Ygr223cp at endosomes. Endosomal recruitment of all three proteins depends on PtdIns3P generated by the Vps34-complex II containing Vps38p, but not on the function of the Vps34-complex I. Since only the Vps34-complex I is essential for autophagy, we expect that at endosomes Atg18p, Atg21p and Ygr223cp have a function distinct from autophagy. Some Vps Class D mutants involved in Golgi-to-endosome transport are required for the endosomal recruitment of GFP-Atg18p, -Atg21p and –Ygr223cp. These include the Qa-SNARE Pep12p, its SM protein Vps45p, the Rab GTPase Vps21p and the Rab effector Vac1p. Deletion of ATG18, ATG21 and YGR223c, alone or simultaneously has no obvious function on the MVB-pathway and CPY-sorting. However, overexpression of ATG21 leads to CPY secretion. We further show, to our knowledge for the first time that Ygr223cp affects an autophagic process, namely micronucleophagy.     

Global Analysis of Fission Yeast Mating Genes Reveals New Autophagy Factors

Macroautophagy (autophagy) is crucial for cell survival during starvation and plays important roles in animal development and human diseases. Molecular understanding of autophagy has mainly come from the budding yeast Saccharomyces cerevisiae, and it remains unclear to what extent the mechanisms are the same in other organisms. Here, through screening the mating phenotype of a genome-wide deletion collection of the fission yeast Schizosaccharomyces pombe, we obtained a comprehensive catalog of autophagy genes in this highly tractable organism, including genes encoding three heretofore unidentified core Atg proteins, Atg10, Atg14, and Atg16, and two novel factors, Ctl1 and Fsc1. We systematically examined the subcellular localization of fission yeast autophagy factors for the first time and characterized the phenotypes of their mutants, thereby uncovering both similarities and differences between the two yeasts. Unlike budding yeast, all three Atg18/WIPI proteins in fission yeast are essential for autophagy, and we found that they play different roles, with Atg18a uniquely required for the targeting of the Atg12–Atg5·Atg16 complex. Our investigation of the two novel factors revealed unforeseen autophagy mechanisms. The choline transporter-like protein Ctl1 interacts with Atg9 and is required for autophagosome formation. The fasciclin domain protein Fsc1 localizes to the vacuole membrane and is required for autophagosome-vacuole fusion but not other vacuolar fusion events. Our study sheds new light on the evolutionary diversity of the autophagy machinery and establishes the fission yeast as a useful model for dissecting the mechanisms of autophagy.     

Improved Blue,Green, and Red Fluorescent Protein Tagging Vectors for S. cerevisiae

Fluorescent protein fusions are a powerful tool to monitor the localization and trafficking of proteins. Such studies are particularly easy to carry out in the budding yeast Saccharomyces cerevisiae due to the ease with which tags can be introduced into the genome by homologous recombination. However, the available yeast tagging plasmids have not kept pace with the development of new and improved fluorescent proteins. Here, we have constructed yeast optimized versions of 19 different fluorescent proteins and tested them for use as fusion tags in yeast. These include two blue, seven green, and seven red fluorescent proteins, which we have assessed for brightness, photostability and perturbation of tagged proteins. We find that EGFP remains the best performing green fluorescent protein, that TagRFP-T and mRuby2 outperform mCherry as red fluorescent proteins, and that mTagBFP2 can be used as a blue fluorescent protein tag. Together, the new tagging vectors we have constructed provide improved blue and red fluorescent proteins for yeast tagging and three color imaging.     

Atg9 proteins,not so different after all

Macroautophagy (hereafter autophagy) is a catabolic pathway present in all eukaryotic cells. The yeast Saccharomyces cerevisiae has been pivotal in the identification and characterization of the key autophagy-related (Atg) proteins, which play a central role in the generation of autophagosomes. The components of the core Atg/ATG machinery and their functions are highly conserved among species, although mammalian cells also have isoforms and auxiliary factors. Atg9/ATG9 is the only transmembrane protein that is part of the core Atg/ATG machinery, but it appears to have divergent localizations and molecular roles in yeast and mammals. A recent experimental analysis of the yeast endo-lysosomal system by the laboratory of Benjamin Glick, however, suggests a more simple organization of this membrane system. Although this study has not examined yeast Atg9, its findings place this protein in the same compartments as its mammalian counterpart. Here, we will discuss the implications of this conceptual change on the trafficking of yeast Atg9 and its function in autophagy.     

Comparative analyses of ubiquitin-like ATG8 and cysteine protease ATG4 autophagy genes in the plant lineage and cross-kingdom processing of ATG8 by ATG4

Autophagy is important for degradation and recycling of intracellular components. In a diversity of genera and species, orthologs and paralogs of the yeast Atg4 and Atg8 proteins are crucial in the biogenesis of double-membrane autophagosomes that carry the cellular cargoes to vacuoles and lysosomes. Although many plant genome sequences are available, the ATG4 and ATG8 sequence analysis is limited to some model plants. We identified 28 ATG4 and 116 ATG8 genes from the available 18 different plant genome sequences. Gene structures and protein domain sequences of ATG4 and ATG8 are conserved in plant lineages. Phylogenetic analyses classified ATG8s into 3 subgroups suggesting divergence from the common ancestor. The ATG8 expansion in plants might be attributed to whole genome duplication, segmental and dispersed duplication, and purifying selection. Our results revealed that the yeast Atg4 processes Arabidopsis ATG8 but not human LC3A (HsLC3A). In contrast, HsATG4B can process yeast and plant ATG8s in vitro but yeast and plant ATG4s cannot process HsLC3A. Interestingly, in Nicotiana benthamiana plants the yeast Atg8 is processed compared to HsLC3A. However, HsLC3A is processed when coexpressed with HsATG4B in plants. Molecular modeling indicates that lack of processing of HsLC3A by plant and yeast ATG4 is not due to lack of interaction with HsLC3A. Our in-depth analyses of ATG4 and ATG8 in the plant lineage combined with results of cross-kingdom ATG8 processing by ATG4 further support the evolutionarily conserved maturation of ATG8. Broad ATG8 processing by HsATG4B and lack of processing of HsLC3A by yeast and plant ATG4s suggest that the cross-kingdom ATG8 processing is determined by ATG8 sequence rather than ATG4.     

Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants

A series of versatile cloning vectors has been constructed that facilitate the expression of protein fusions to the Aequorea victoria green fluorescent protein (GFP) in plant cells. Amino-terminal- and carboxy-terminal protein fusions have been created and visualized by epifluorescence microscopy, both in transgenic Arabidopsis thaliana and after transient expression in onion epidermal cells. Using tandem dimers and other protein fusions to GFP, we found that the previously described localization of wild-type GFP to the cell nucleus is most likely due to diffusion of GFP across the nuclear envelope rather than to a cryptic nuclear localization signal. A fluorescence-based, quantitative assay for nuclear localization signals is described. In addition, we have employed the previously characterized mutants GFP–S65T and GFP–Y66H in order to allow for the expression of red-shifted and blue fluorescent proteins, respectively, which are suitable for double-labeling studies. Expression of GFP-fusions was controlled by a cauliflower mosaic virus 35S promoter. Using the Arabidopsis COP1 protein as a model, we confirmed a close similarity in the subcellular localization of native COP1 and the GFP-tagged COP1 protein. We demonstrated that COP1 was localized to discrete subnuclear particles and further confirmed that fusion to GFP did not compromise the activity of the wild-type COP1 protein.     

Atg27 is a second transmembrane cycling protein

Autophagy is a degradative pathway conserved among all eukaryotic cells, and is responsible for the turnover of damaged organelles and long-lived proteins. The primary morphological feature of autophagy is the sequestration of cargo within a double-membrane cytosolic vesicle called an autophagosome. More than 25 AuTophaGy-related (ATG) genes that are essential for autophagy have been identified from the yeast Saccharomyces cerevisiae. Despite the identification and characterization of Atg proteins, it remains a mystery how the double-membrane vesicle is made, what the membrane source(s) are, and how the lipid is transported to the forming vesicle. Among Atg proteins, Atg9 was the only characterized transmembrane protein required for the formation of double-membrane vesicles. Evidence has been obtained in yeast and mammalian cells for Atg9 cycling between different peripheral compartments and the phagophore assembly site/preautophagosomal structure (PAS), the proposed site of organization for autophagosome formation. This cycling feature makes Atg9 a potential membrane carrier to deliver lipids that are used in the vesicle formation process. Recently, in our lab we characterized a second transmembrane protein, Atg27. The unique localization and cycling features of Atg27 suggest the involvement of the Golgi complex in the autophagy pathway. In this addendum, we discuss the trafficking of Atg27 in yeast and compare it with that of Atg9, and consider the possible meaning of Atg27 Golgi localization.     

Conserved and unique features of the fission yeast core Atg1 complex

Although the human ULK complex mediates phagophore initiation similar to the budding yeast Saccharomyces cerevisiae Atg1 complex, this complex contains ATG101 but not Atg29 and Atg31. Here, we analyzed the fission yeast Schizosaccharomyces pombe Atg1 complex, which has a subunit composition that resembles the human ULK complex. Our pairwise coprecipitation experiments showed that while the interactions between Atg1, Atg13, and Atg17 are conserved, Atg101 does not bind Atg17. Instead, Atg101 interacts with the HORMA domain of Atg13 and this enhances the stability of both proteins. We also found that S. pombe Atg17, the putative scaffold subunit, adopts a rod-shaped structure with no discernible curvature. Interestingly, S. pombe Atg17 binds S. cerevisiae Atg13, Atg29, and Atg31 in vitro, but it cannot complement the function of S. cerevisiae Atg17 in vivo. Furthermore, S. pombe Atg101 cannot substitute for the function of S. cerevisiae Atg29 and Atg31 in vivo. Collectively, our work generates new insights into the subunit organization and structural properties of an Atg101-containing Atg1/ULK complex.     

Atg27 is a Second Transmembrane Cycling Protein

Autophagy is a degradative pathway conserved among all eukaryotic cells, and is responsible for the turnover of damaged organelles and long-lived proteins. The primary morphological feature of autophagy is the sequestration of cargo within a double-membrane cytosolic vesicle called an autophagosome. More than 25 AuTophaGy-related (ATG) genes that are essential for autophagy have been identified from the yeast Saccharomyces cerevisiae. Despite the identification and characterization of Atg proteins, it remains a mystery how the double-membrane vesicle is made, what the membrane source(s) are, and how the lipid is transported to the forming vesicle. Among Atg proteins, Atg9 was the only characterized transmembrane protein required for the formation of double-membrane vesicles. Evidence has been obtained in yeast and mammalian cells for Atg9 cycling between different peripheral compartments and the phagophore assembly site/pre-autophagosomal structure (PAS), the proposed site of organization for autophagosome formation. This cycling feature makes Atg9 a potential membrane carrier to deliver lipids that are used in the vesicle formation process.2 Recently, in our lab we characterized a second transmembrane protein, Atg27. The unique localization and cycling features of Atg27 suggest the involvement of the Golgi complex in the autophagy pathway. In this addendum, we discuss the trafficking of Atg27 in yeast and compare it with that of Atg9, and consider the possible meaning of Atg27 Golgi localization.

Addendum to:

Atg27 is Required for Autophagy-Dependent Cycling of Atg9

W.-L. Yen, J.E. Legakis, U. Nair and D.J. Klionsky

Mol Biol Cell 2006; In press     

Dictyostelium macroautophagy mutants vary in the severity of their developmental defects

Macroautophagy is the major mechanism that eukaryotes use to recycle cellular components during stressful conditions. We have shown previously that the Atg12-Atg5 conjugation system, required for autophagosome formation in yeast, is necessary for Dictyostelium development. A second conjugation reaction, Aut7/Atg8 lipidation with phosphatidylethanolamine, as well as a protein kinase complex and a phosphatidylinositol 3-kinase complex are also required for macroautophagy in yeast. In this study, we characterize mutations in the putative Dictyostelium discoideum orthologues of budding yeast genes that are involved in one of each of these functions, ATG1, ATG6, and ATG8. All three genes are required for macroautophagy in Dictyostelium. Mutant amoebae display reduced survival during nitrogen starvation and reduced protein degradation during development. Mutations in the three genes produce aberrant development with defects of varying severity. As with other Dictyostelium macroautophagy mutants, development of atg1-1, atg6(-), and atg8(-) is more aberrant in plaques on bacterial lawns than on nitrocellulose filters. The most severe defect is observed in the atg1-1 mutant, which does not aggregate on bacterial lawns and arrests as loose mounds on nitrocellulose filters. The atg6(-) and atg8(-) mutants display almost normal development on nitrocellulose filters, producing multi-tipped aggregates that mature into small fruiting bodies. The distribution of a green fluorescent protein fusion of the autophagosome marker, Atg8, is aberrant in both atg1-1 and atg6(-) mutants.     

Human hAtg2A protein expressed in yeast is recruited to preautophagosomal structure but does not complement autophagy defects of atg2Δ strain

Yeast Atg2, an autophagy-related protein, is highly conserved in other fungi and has two homologues in humans, one of which is hAtg2A encoded by the hATG2A/KIAA0404 gene. Region of homology between Atg2 and hAtg2A proteins comprises the C-terminal domain. We used yeast atg2D strain to express the GFP-KIAA0404 gene, its fragment or fusions with yeast ATG2, and study their effects on autophagy. The GFP-hAtg2A protein localized to punctate structures, some of which colocalized with Ape1-RFP-marked preautophagosomal structure (PAS), but it did not restore autophagy in atg2Δ cells. N-terminal fragment of Atg2 and N-terminal fragment of hAtg2A were sufficient for PAS recruitment but were not sufficient to function in autophagy. Neither a fusion of the N-terminal fragment of hAtg2A with C-terminal domain of Atg2 nor a reciprocal fusion were functional in autophagy. hAtg2A, in contrast to yeast Atg2, did not show interaction with the yeast autophagy protein Atg9 but both Atg2 proteins showed interaction with Atg18, a phospholipid-binding protein, in two-hybrid system. Moreover, deletion of ATG18 abrogated PAS recruitment of hAtg2A. Our results show that human hAtg2A can not function in autophagy in yeast, however, it is recruited to the PAS, possibly due to the interaction with Atg18.     

Phosphatidylinositol-3-phosphate regulates response of cells to proteotoxic stress

Human Nedd4 ubiquitin ligase, or its variants, inhibit yeast cell growth by disturbing the actin cytoskeleton organization and dynamics, and lead to an increase in levels of ubiquitinated proteins. In a screen for multicopy suppressors which rescue growth of yeast cells producing Nedd4 ligase with an inactive WW4 domain (Nedd4w4), we identified a fragment of ATG2 gene encoding part of the Atg2 core autophagy protein. Expression of the Atg2-C1 fragment (aa 1074-1447) improved growth, actin cytoskeleton organization, but did not significantly change the levels of ubiquitinated proteins in these cells. The GFP-Atg2-C1 protein in Nedd4w4-producing cells primarily localized to a single defined structure adjacent to the vacuole, surrounded by an actin filament ring, containing Hsp42 and Hsp104 chaperones. This localization was not affected in several atg deletion mutants, suggesting that it might be distinct from the phagophore assembly site (PAS). However, deletion of ATG18 encoding a phosphatidylinositol-3-phosphate (PI3P)-binding protein affected the morphology of the GFP-Atg2-C1 structure while deletion of ATG14 encoding a subunit of PI3 kinase suppressed toxicity of Nedd4w4 independently of GFP-Atg2-C1. Further analysis of the Atg2-C1 revealed that it contains an APT1 domain of previously uncharacterized function. Most importantly, we showed that this domain is able to bind phosphatidylinositol phosphates, especially PI3P, which is abundant in the PAS and endosomes. Together our results suggest that human Nedd4 ubiquitinates proteins in yeast and causes proteotoxic stress and, with some Atg proteins, leads to formation of a perivacuolar structure, which may be involved in sequestration, aggregation or degradation of proteins.     

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.     

Cdc14 Phosphatase Promotes TORC1-Regulated Autophagy in Yeast

Cdc14 protein phosphatase is critical for late mitosis progression in budding yeast, although its orthologs in other organisms, including mammalian cells, function as stress-responsive phosphatases. We found herein unexpected roles of Cdc14 in autophagy induction after nutrient starvation and target of rapamycin complex 1 (TORC1) kinase inactivation. TORC1 kinase phosphorylates Atg13 to repress autophagy under nutrient-rich conditions, but if TORC1 becomes inactive upon nutrient starvation or rapamycin treatment, Atg13 is rapidly dephosphorylated and autophagy is induced. Cdc14 phosphatase was required for optimal Atg13 dephosphorylation, pre-autophagosomal structure formation, and autophagy induction after TORC1 inactivation. In addition, Cdc14 was required for sufficient induction of ATG8 and ATG13 expression. Moreover, Cdc14 activation provoked autophagy even under normal conditions. This study identified a novel role of Cdc14 as the stress-responsive phosphatase for autophagy induction in budding yeast.     

Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells

Clathrin-mediated endocytosis (CME) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. Previous live-cell imaging studies using ectopically overexpressed fluorescent fusions of endocytic proteins indicated that mammalian CME is a highly dynamic but inefficient and heterogeneous process. In contrast, studies of endocytosis in budding yeast using fluorescent protein fusions expressed at physiological levels from native genomic loci have revealed a process that is very regular and efficient. To analyse endocytic dynamics in mammalian cells in which endogenous protein stoichiometry is preserved, we targeted zinc finger nucleases (ZFNs) to the clathrin light chain A and dynamin-2 genomic loci and generated cell lines expressing fluorescent protein fusions from each locus. The genome-edited cells exhibited enhanced endocytic function, dynamics and efficiency when compared with previously studied cells, indicating that CME is highly sensitive to the levels of its protein components. Our study establishes that ZFN-mediated genome editing is a robust tool for expressing protein fusions at endogenous levels to faithfully report subcellular localization and dynamics.     

Function of the Dictyostelium discoideum Atg1 kinase during autophagy and development

When starved, the amoebae of Dictyostelium discoideum initiate a developmental process that results in the formation of fruiting bodies in which stalks support balls of spores. The nutrients and energy necessary for development are provided by autophagy. Atg1 is a protein kinase that regulates the induction of autophagy in the budding yeast Saccharomyces cerevisiae. In addition to a conserved kinase domain, Dictyostelium Atg1 has a C-terminal region that has significant homology to the Caenorhabditis elegans and mammalian Atg1 homologues but not to the budding yeast Atg1. We investigated the function of the kinase and conserved C-terminal domains of D. discoideum Atg1 (DdAtg1) and showed that these domains are essential for autophagy and development. Kinase-negative DdAtg1 acts in a dominant-negative fashion, resulting in a mutant phenotype when expressed in the wild-type cells. Green fluorescent protein-tagged kinase-negative DdAtg1 colocalizes with red fluorescent protein (RFP)-tagged DdAtg8, a marker of preautophagosomal structures and autophagosomes. The conserved C-terminal region is essential for localization of kinase-negative DdAtg1 to autophagosomes labeled with RFP-tagged Dictyostelium Atg8. The dominant-negative effect of the kinase-defective mutant also depends on the C-terminal domain. In cells expressing dominant-negative DdAtg1, autophagosomes are formed and accumulate but seem not to be functional. By using a temperature-sensitive DdAtg1, we showed that DdAtg1 is required throughout development; development halts when the cells are shifted to the restrictive temperature, but resumes when cells are returned to the permissive temperature.     

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 the genomes of Arabidopsis thaliana and Homo sapiens were 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. Remarkably, the A. thaliana and human genomes encode multiple proteins highly similar to specific Atg proteins (paralogs), the function of which is unknown. 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, the receptor of Cvt cargo, Atg19, was only detected in S. cerevisiae. Nevertheless, Atg11, a protein that links receptor-bound cargo (peroxisomes, Cvt bodies) 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.