Dissection of Autophagosome Biogenesis into Distinct Nucleation and Expansion Steps

Abstract

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.

Autophagy and signaling: their role in cell survival and cell death

“…The upregulation of Atg proteins is not a common trait during starvation-induced autophagy. 84 The initiation of autophagy may not in fact depend on protein synthesis (for a discussion, see Abeliovich et al 85 ).…”

A novel, human Atg13 binding protein, Atg101, interacts with ULK1 and is essential for macroautophagy

“…10,12,35 Therefore, we were interested in determining if human Atg13, like its yeast counterpart, is a phosphoprotein. We expressed HA-Atg13 in HEK293 cells and metabolically labeled the cells with 32 PO 4 .…”

“…[6][7][8][9][10] The Atg1-interacting proteins are important, in part, because of their effect on Atg1 kinase activity, which is regulated by the phosphorylation state and association of Atg13, and by the interaction between Atg17 and Atg13, in a Target of Rapamycin (TOR) dependent manner. [10][11][12] In yeast, macroautophagy (nonselective autophagy) and the cytoplasmic-to-vacuole (Cvt) pathway (selective autophagy) are similar in that they both require many of the same Atg proteins for cargo sequestration and delivery. The two pathways differ in the size of the sequestering membrane and in the nutrient conditions under which they are active.…”

Recycle or die: The role of autophagy in cardioprotection

“…Atg12 is then covalently conjugated to Atg5 at a lysine residue and Atg10 is released [22]. Finally, the Atg12-Atg5 complex interacts non-covalently with Atg16, and this complex then initiat es the elongation of the membrane by recruiting LC3-PE [23][24][25]. LC3 is synthesized as a larger precursor and is cleaved by the cysteine protease Atg4 to LC3-I which exposes a C-terminal glycine residue [26].…”