Final Report Summary - AS_ETHZ_IEF_2012 (The Role of Atg8 Posttranslational Modifications in Autophagy)
Autophagy is a cellular degradation pathway required for eukaryotic protein homeostasis, development, differentiation and cellular defence. Deregulation of autophagy has been shown to cause the development of various human pathologies, while an increased basal level of autophagy has been shown to correlate with improved health. Autophagy involves the formation of double-membrane vesicles, called autophagosomes, which sequester portions of the cytosol (bulk autophagy) or whole organelles, pathogens or aggregates (selective autophagy). Autophagosomes then fuse with the lysosome or vacuole (in yeast and plants), where their cargo is degraded.
Recent proteomics studies have reported various posttranslational modifications of autophagy related (Atg) proteins. However, their functional implications are currently unknown. In order to bridge the knowledge gap between large-scale proteomics data and essential functional and mechanistic understanding we proposed to study the functional significance of posttranslational modifications of Atg proteins with a particular focus on the ubiquitin-like protein Atg8. To this end I developed an approach, which uses the in vitro phosphorylation sites of recombinantly expressed and purified Atg proteins as determined by mass spectrometry as leads for the functional screen in vivo. By this means I showed that Atg8 is phosphorylated by Atg1 and that this is required for autophagy progression. In a major expansion of the scope of my project, I have discovered many more novel Atg1 substrates amongst all the known Atg proteins involved in macroautophagy, which I am currently investigating further. For example the Vps34 complex is a direct target of Atg1 dependent phosphorylation. Having expressed and purified this and other large multisubunit complexes for the Atg1 target discovery study opened up a second major scientific direction - to use these complexes for structural determination by electron microscopy (EM) and single particle analysis. The Vps34 complex turned out to be a promising sample for structural studies allowing us to determine the negative stain EM structure for its autophagy and endosome specific variants.
In summary, our highly sensitive and largely unbiased in vitro approach has already yielded novel insights into the currently unknown signaling transduction pathways involved in the onset and progression of autophagy and provided a starting point for structural studies of the major multisubunit complexes involved in autophagy. Moreover, the data generated during the course of this study have paved the way for a significant conceptual advancement of our mechanistic understanding of autophagy in the imminent future.
Recent proteomics studies have reported various posttranslational modifications of autophagy related (Atg) proteins. However, their functional implications are currently unknown. In order to bridge the knowledge gap between large-scale proteomics data and essential functional and mechanistic understanding we proposed to study the functional significance of posttranslational modifications of Atg proteins with a particular focus on the ubiquitin-like protein Atg8. To this end I developed an approach, which uses the in vitro phosphorylation sites of recombinantly expressed and purified Atg proteins as determined by mass spectrometry as leads for the functional screen in vivo. By this means I showed that Atg8 is phosphorylated by Atg1 and that this is required for autophagy progression. In a major expansion of the scope of my project, I have discovered many more novel Atg1 substrates amongst all the known Atg proteins involved in macroautophagy, which I am currently investigating further. For example the Vps34 complex is a direct target of Atg1 dependent phosphorylation. Having expressed and purified this and other large multisubunit complexes for the Atg1 target discovery study opened up a second major scientific direction - to use these complexes for structural determination by electron microscopy (EM) and single particle analysis. The Vps34 complex turned out to be a promising sample for structural studies allowing us to determine the negative stain EM structure for its autophagy and endosome specific variants.
In summary, our highly sensitive and largely unbiased in vitro approach has already yielded novel insights into the currently unknown signaling transduction pathways involved in the onset and progression of autophagy and provided a starting point for structural studies of the major multisubunit complexes involved in autophagy. Moreover, the data generated during the course of this study have paved the way for a significant conceptual advancement of our mechanistic understanding of autophagy in the imminent future.