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.

[1]  R. Kessin,et al.  Macroautophagy is dispensable for intracellular replication of Legionella pneumophila in Dictyostelium discoideum , 2003, Molecular microbiology.

[2]  Daniel J Klionsky,et al.  A unified nomenclature for yeast autophagy-related genes. , 2003, Developmental cell.

[3]  D. Hall,et al.  Autophagy Genes Are Essential for Dauer Development and Life-Span Extension in C. elegans , 2003, Science.

[4]  G. Juhász,et al.  The Drosophila homolog of Aut1 is essential for autophagy and development , 2003, FEBS letters.

[5]  R. Kessin,et al.  Macroautophagy Is Required for Multicellular Development of the Social Amoeba Dictyostelium discoideum * , 2003, The Journal of Biological Chemistry.

[6]  T. Natsume,et al.  Mouse Apg16L, a novel WD-repeat protein, targets to the autophagic isolation membrane with the Apg12-Apg5 conjugate , 2003, Journal of Cell Science.

[7]  Chao Zhang,et al.  Chemical genetic analysis of Apg1 reveals a non-kinase role in the induction of autophagy. , 2003, Molecular biology of the cell.

[8]  M. Paoletti,et al.  Autophagy is induced during cell death by incompatibility and is essential for differentiation in the filamentous fungus Podospora anserina , 2003, Molecular microbiology.

[9]  Robert S. Ledley,et al.  The Protein Information Resource , 2003, Nucleic Acids Res..

[10]  D. Klionsky,et al.  Autophagy in yeast: a review of the molecular machinery. , 2002, Cell structure and function.

[11]  D. Shibata,et al.  Leaf Senescence and Starvation-Induced Chlorosis Are Accelerated by the Disruption of an Arabidopsis Autophagy Gene1 , 2002, Plant Physiology.

[12]  D. Klionsky,et al.  Convergence of Multiple Autophagy and Cytoplasm to Vacuole Targeting Components to a Perivacuolar Membrane Compartment Prior tode Novo Vesicle Formation* , 2002, The Journal of Biological Chemistry.

[13]  K Suzuki,et al.  The pre‐autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation , 2001, The EMBO journal.

[14]  D. Klionsky,et al.  Apg2 Is a Novel Protein Required for the Cytoplasm to Vacuole Targeting, Autophagy, and Pexophagy Pathways* , 2001, The Journal of Biological Chemistry.

[15]  A. Gingras,et al.  The target of rapamycin (TOR) proteins , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Y. Ohsumi,et al.  Beclin–phosphatidylinositol 3‐kinase complex functions at the trans‐Golgi network , 2001, EMBO reports.

[17]  Takeshi Tokuhisa,et al.  Dissection of Autophagosome Formation Using Apg5-Deficient Mouse Embryonic Stem Cells , 2001, The Journal of cell biology.

[18]  Takeshi Noda,et al.  Two Distinct Vps34 Phosphatidylinositol 3–Kinase Complexes Function in Autophagy and Carboxypeptidase Y Sorting inSaccharomyces cerevisiae , 2001, The Journal of cell biology.

[19]  T. Ueno,et al.  The Human Homolog of Saccharomyces cerevisiae Apg7p Is a Protein-activating Enzyme for Multiple Substrates Including Human Apg12p, GATE-16, GABARAP, and MAP-LC3* , 2001, The Journal of Biological Chemistry.

[20]  R. Kessin.  Dictyostelium: Evolution, Cell Biology, and the Development of Multicellularity , 2001 .

[21]  R. Kessin.  Dictyostelium: The Transition from Growth to Development: From Starvation to Self-Sustaining cAMP Signal Relay , 2001 .

[22]  D. Klionsky,et al.  Dissection of Autophagosome Biogenesis into Distinct Nucleation and Expansion Steps , 2000, The Journal of cell biology.

[23]  Takeshi Noda,et al.  A ubiquitin-like system mediates protein lipidation , 2000, Nature.

[24]  T. Egelhoff,et al.  Green fluorescent protein and epitope tag fusion vectors for Dictyostelium discoideum. , 2000, Plasmid.

[25]  Takeshi Noda,et al.  The Reversible Modification Regulates the Membrane-Binding State of Apg8/Aut7 Essential for Autophagy and the Cytoplasm to Vacuole Targeting Pathway , 2000, The Journal of cell biology.

[26]  Kazuya Nagano,et al.  Tor-Mediated Induction of Autophagy via an Apg1 Protein Kinase Complex , 2000, The Journal of cell biology.

[27]  H. Hibshoosh,et al.  Induction of autophagy and inhibition of tumorigenesis by beclin 1 , 1999, Nature.

[28]  Yo Suzuki,et al.  Mouse ULK2, a novel member of the UNC-51-like protein kinases: unique features of functional domains , 1999, Oncogene.

[29]  Takeshi Noda,et al.  Formation Process of Autophagosome Is Traced with Apg8/Aut7p in Yeast , 1999, The Journal of cell biology.

[30]  A. Matsuura,et al.  Apg10p, a novel protein‐conjugating enzyme essential for autophagy in yeast , 1999, The EMBO journal.

[31]  Takeshi Noda,et al.  Apg16p is required for the function of the Apg12p–Apg5p conjugate in the yeast autophagy pathway , 1999, The EMBO journal.

[32]  N. Mizushima,et al.  Apg7p/Cvt2p: A novel protein-activating enzyme essential for autophagy. , 1999, Molecular biology of the cell.

[33]  Weiping Yuan,et al.  Glucose-induced autophagy of peroxisomes in Pichia pastoris requires a unique E1-like protein. , 1999, Molecular biology of the cell.

[34]  James E. Goldman,et al.  Protection against Fatal Sindbis Virus Encephalitis by Beclin, a Novel Bcl-2-Interacting Protein , 1998, Journal of Virology.

[35]  S. Kametaka,et al.  Apg14p and Apg6/Vps30p Form a Protein Complex Essential for Autophagy in the Yeast, Saccharomyces cerevisiae * , 1998, The Journal of Biological Chemistry.

[36]  H. Tokumitsu,et al.  Identification of mouse ULK1, a novel protein kinase structurally related to C. elegans UNC-51. , 1998, Biochemical and biophysical research communications.

[37]  Takeshi Noda,et al.  Tor, a Phosphatidylinositol Kinase Homologue, Controls Autophagy in Yeast* , 1998, The Journal of Biological Chemistry.

[38]  M. Straub,et al.  AUT3, a serine/threonine kinase gene, is essential for autophagocytosis in Saccharomyces cerevisiae , 1997, Journal of bacteriology.

[39]  M. Schlumpberger,et al.  AUT1, a gene essential for autophagocytosis in the yeast Saccharomyces cerevisiae , 1997, Journal of bacteriology.

[40]  K. Zhou,et al.  A phosphatidylinositol (PI) kinase gene family in Dictyostelium discoideum: biological roles of putative mammalian p110 and yeast Vps34p PI 3-kinase homologs during growth and development , 1995, Molecular and cellular biology.

[41]  H. Schuster,et al.  Cloning vectors for the production of proteins in Dictyostelium discoideum. , 1995, Gene.

[42]  T. Noda,et al.  Novel system for monitoring autophagy in the yeast Saccharomyces cerevisiae. , 1995, Biochemical and biophysical research communications.

[43]  T. Hunter,et al.  The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification 1 , 1995, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[44]  M. Schlumpberger,et al.  Isolation of autophagocytosis mutants of Saccharomyces cerevisiae , 1994, FEBS letters.

[45]  O. Anderson.  Fine Structure of the Marine Amoeba Vexillifera telmathalassa Collected from a Coastal Site Near Barbados with a Description of Salinity Tolerance, Feeding Behavior and Prey , 1994 .

[46]  Y. Ohsumi,et al.  Isolation and characterization of autophagy‐defective mutants of Saccharomyces cerevisiae , 1993, FEBS letters.

[47]  A. Kuspa,et al.  Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[48]  A. Lupas,et al.  Predicting coiled coils from protein sequences , 1991, Science.

[49]  R. Kessin,et al.  The cyclic nucleotide phosphodiesterase gene of Dictyostelium discoideum utilizes alternate promoters and splicing for the synthesis of multiple mRNAs , 1989, Molecular and cellular biology.

[50]  M. Sussman,et al.  Cultivation and synchronous morphogenesis of Dictyostelium under controlled experimental conditions. , 1987, Methods in cell biology.

[51]  M. Sussman,et al.  Metabolism of major cell components during slime mold morphogenesis. , 1961, Biochimica et biophysica acta.