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Published online 27 November 2000. doi:10.1083/jcb.151.5.1025
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© The Rockefeller University Press, 0021-9525/2000//1025 $5.00
The Journal of Cell Biology, Volume 151, Number 5, , 2000 1025-1034

Original Article

Dissection of Autophagosome Biogenesis into Distinct Nucleation and Expansion Steps



Hagai Abeliovicha, William A. Dunn, Jr.b, John Kima, and Daniel J. Klionskya

a University of Michigan, Department of Biology, Ann Arbor, Michigan 48109
b University of Florida College of Medicine, Department of Anatomy and Cell Biology, Gainesville, Florida 32610
University of Michigan Department of Biology, Ann Arbor, MI 48109.(734) 647-0884(734) 615-6556

Rapamycin, an antifungal macrolide antibiotic, mimics starvation conditions in Saccharomyces cerevisiae through activation of a general G0 program that includes widespread effects on translation and transcription. Macroautophagy, a catabolic membrane trafficking phenomenon, is a prominent part of this response. Two views of the induction of autophagy may be considered. In one, up-regulation of proteins involved in autophagy causes its induction, implying that autophagy is the result of a signal transduction mechanism leading from Tor to the transcriptional and translational machinery. An alternative hypothesis postulates the existence of a dedicated signal transduction mechanism that induces autophagy directly. We tested these possibilities by assaying the effects of cycloheximide and specific mutations on the induction of autophagy. We find that induction of autophagy takes place in the absence of de novo protein synthesis, including that of specific autophagy-related proteins that are up-regulated in response to rapamycin. We also find that dephosphorylation of Apg13p, a signal transduction event that correlates with the onset of autophagy, is also independent of new protein synthesis. Finally, our data indicate that autophagosomes that form in the absence of protein synthesis are significantly smaller than normal, indicating a role for de novo protein synthesis in the regulation of autophagosome expansion. Our results define the existence of a signal transduction-dependent nucleation step and a separate autophagosome expansion step that together coordinate autophagosome biogenesis.

Key Words: autophagy • Tor • protein synthesis • vacuole • yeast

© 2000 The Rockefeller University Press

Abbreviations used in this paper: API, aminopeptidase I; Cvt, cytoplasm to vacuole targeting; ECL, enhanced chemiluminescence; FKBP, FK506 binding protein; PP2A, protein phosphatase 2A; RBP, rapamycin binding protein.


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