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Eukaryotic Cell, October 2006, p. 1797-1806, Vol. 5, No. 10
1535-9778/06/$08.00+0     doi:10.1128/EC.00342-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Function of the Dictyostelium discoideum Atg1 Kinase during Autophagy and Development{dagger}

Turgay Tekinay,1 Mary Y. Wu,1,{ddagger} Grant P. Otto,1,{ddagger} O. Roger Anderson,2 and Richard H. Kessin1*

Department of Anatomy and Cell Biology, Columbia University, 630 West 168th Street, New York, New York 10032,1 Department of Biology, Lamont Doherty Earth Observatory, Columbia University, Palisades, New York 109642

Received 11 November 2005/ Accepted 11 July 2006

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.

* Corresponding author. Mailing address: Department of Anatomy and Cell Biology, P&S 12-517, Columbia University, 630 West 168th St., New York, NY 10032. Phone: (212) 305-5653. Fax: (212) 305-3970. E-mail: rhk2{at}columbia.edu.

{dagger} This is Lamont-Doherty contribution number 6949.

{ddagger} Present address: Developmental Signalling and Secretory Pathways Laboratories, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.

Eukaryotic Cell, October 2006, p. 1797-1806, Vol. 5, No. 10
1535-9778/06/$08.00+0     doi:10.1128/EC.00342-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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