Molecular mechanisms of cargo-driven autophagosome formation

Autophagy is a conserved degradation pathway which serves the clearance of protein aggregates or damaged organelles, and is vitally important for cell and tissue homeostasis. During autophagy the surplus cellular material is recognized and selectively removed from the cytoplasm. The selectivity is conferred by cargo receptors that specifically recognize the targeted cargo. The autophagic cargo is sequestered in a double-membrane vesicle called autophagosome and delivered to the vacuole/lysosome for degradation. Upon autophagy induction the autophagosomal vesicles are formed de novo in the cytoplasm. One of the crucial steps in the process is to generate an Atg8- positive isolation membrane that subsequently expands to enclose a cargo. The ubiquitin-like Atg8 is conjugated to the lipid phosphatidylethanolamine (PE) of the isolation membrane via a sequential action of enzymes constituting the so-called Atg8 conjugation machinery. The overall scope of the proposed project is to better understand the molecular basis for autophagosome biogenesis by detailed examination of the interaction between the cargo receptor Atg19 and the Atg8 conjugation machinery using a combination of in vitro and in vivo approaches. In particular, I plan to employ a fully reconstituted in vitro system containing purified recombinant autophagic proteins. In parallel, I will translate the results from the cell-free system to the cell by investigating the cytoplasm-to-vacuole targeting (Cvt) pathway as a model of selective autophagy in Saccharomyces cerevisiae.

FWF Summaries for Public Relations Hertha-Firnberg Project T-724 Autophagosome: under (re)construction Each living cell must remove harmful or excess material from its interior in order to remain healthy and operational. Cells pack this waste into "garbage bags" called autophagosomes in a process called autophagy. Autophagosomes are double-membrane organelles that enwrap the unwanted material and transport it for degradation. Remarkably, they are formed in the cells de novo and a cell has to provide all necessary factors to the site of their biogenesis. Those factors are so-called autophagy proteins that make sure that only the unwanted material is eliminated. Although there is a very good knowledge about which proteins participate in building autophagosomes, still little is known how they come together to initiate the formation of these organelles from scratch in the right place of the cell. To address the puzzling question how cell's waste ends up in the autophagosome, Justyna Sawa-Makarska together with her colleagues in Max Perutz Labs reconstructed the initial steps of the autophagosome formation. They did it in vitro which means outside of the cell. To understand what really is behind a certain physiological process, it often helps to take it apart and rebuild it. With this approach scientists are able to reconstitute certain processes in a controlled manner, which allows them to dissect functions of individual factors and understand how each of the individual pieces comes together to conduct a complicated cellular process. The biogenesis of autophagosome involves numerous proteins. By isolating and characterizing 21 of these components, the scientists were able to rebuild parts of the autophagy machinery in the 'test tube'. They found that tiny vesicles loaded with the protein Atg9 act as the seed from which the autophagosome emerges. A number of the tested autophagy proteins assembled on those vesicles and decorated them with a small protein Atg8, while others bound to the waste material. The concerted action of all the components resulted in targeting the Atg9 vesicles to the waste material. Moreover, one of the proteins namely Atg2, transferred lipids that are building blocks required for the growth of the autophagosomal membrane. A strong interaction between Atg8 on the Atg9 vesicles and the waste material keeps the growing membrane tightly bound to the cargo destined for degradation. What still remains to be addressed is to get a detailed picture of the membrane growth and sealing of the autphagosome.