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.
Tekinay
Turgay
T
Department of Anatomy and Cell Biology, P&S 12-517, Columbia University, 630 West 168th St., New York, NY 10032, USA.
Wu
Mary Y
MY
Otto
Grant P
GP
Anderson
O Roger
OR
Kessin
Richard H
RH
D000595:Amino Acid Sequence; D000818:Animals; D001343:Autophagy / Q000502:physiology*; D015415:Biomarkers; D017124:Conserved Sequence; D004023:Dictyostelium / Q000166:cytology / Q000201:enzymology* / Q000254:growth & development* / Q000648:ultrastructure; D005508:Food Deprivation / Q000502:physiology; D005799:Genes, Dominant / Q000235:genetics; D009154:Mutation / Q000235:genetics; D010588:Phagosomes / Q000648:ultrastructure; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport; D015800:Protozoan Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D015415:Biomarkers; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases