Dissection of autophagosome biogenesis into distinct nucleation and expansion steps.
Rapamycin, an antifungal macrolide antibiotic, mimics starvation conditions in Saccharomyces cerevisiae through activation of a general G(0) 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.
Abeliovich
H
H
University of Michigan, Department of Biology, Ann Arbor, Michigan 48109, USA.
Dunn
W A
WA
Kim
J
J
Klionsky
D J
DJ
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D048868:Adaptor Proteins, Signal Transducing; D000626:Aminopeptidases / Q000378:metabolism; D000935:Antifungal Agents / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D000071190:Autophagy-Related Protein 8 Family; D000071183:Autophagy-Related Proteins; D018797:Cell Cycle Proteins; D003513:Cycloheximide / Q000494:pharmacology; D003593:Cytoplasm / Q000201:enzymology; D005656:Fungal Proteins / Q000096:biosynthesis / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects / Q000502:physiology; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000502:physiology; D009584:Nitrogen / Q000494:pharmacology; D010641:Phenotype; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction / Q000502:physiology*; D020123:Sirolimus / Q000494:pharmacology; D014617:Vacuoles / Q000201:enzymology*; D033921:Vesicular Transport Proteins
D048868:Adaptor Proteins, Signal Transducing; D000626:Aminopeptidases; D000935:Antifungal Agents; D000071190:Autophagy-Related Protein 8 Family; D000071183:Autophagy-Related Proteins; D018797:Cell Cycle Proteins; D003513:Cycloheximide; D005656:Fungal Proteins; D008074:Lipoproteins; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D009584:Nitrogen; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D017346:Protein Serine-Threonine Kinases; D029701:Saccharomyces cerevisiae Proteins; D020123:Sirolimus; D033921:Vesicular Transport Proteins; C413216:ATG13 protein, S cerevisiae; C113757:ATG8 protein, S cerevisiae; C111373:VAC8 protein, S cerevisiae; C083324:TOR1 protein, S cerevisiae; C081135:TOR2 protein, S cerevisiae; C019689:APE1 protein, S cerevisiae