Regulation of cargo receptors by cargo and Atg8 during selective autophagy

Autophagy is an intracellular process that mediates the bulk degradation of cellular material. During autophagy double membrane-bound vesicles called autophagosomes are formed de novo. Autophagosomes initially appear as small membrane structures called isolation membranes that gradually expand and thereby sequester cellular material. After their formation autophagosomes fuse with lysosomes (in complex eukaryotes) or the vacuole (in yeast) wherein the cargo is degraded. Autophagy thereby protects cells and thus the organism from various stresses including periods of starvation, protein aggregates, intracellular pathogens and damaged organelles. Consequently autophagy has been linked to various human diseases. It is becoming increasingly clear that autophagy can be highly selective with regard to the cargo that is captures within autophagosomes. Cargo receptor proteins that specifically bind to the cargo and to Atg8-family proteins that localize to the isolation membrane mediate this selectivity. In yeast a special form of selective autophagy exists. This autophagic pathway is called cytoplasm-to-vacuole transport (Cvt) pathway and mediates the transport of cytosolic proteases (e.g. Ape1) into the vacuolar lumen. The cargo receptor Atg19 binds both Atg8 on the isolation membrane and the Ape1 cargo and thereby ensures its selective enclosure into the autophagosome-like Cvt vesicles. In this proposal we are planning to study how Atg19, and the related protein Atg34, mediate the specific uptake of cargo into autophagic vesicles. In particular we will focus on the interplay between the cargo, the cargo receptors and Atg8. To this end we will employ biochemical approaches including a reconstitution assay using giant unilamellar vesicles and structural biology approaches. The biochemical results will then be extensively tested in vivo by the study of the Cvt pathway and autophagy in the yeast Saccharomyces cerevisiae. With the experiments outlined in this proposal we hope to shed light on the fundamental question of how specificity can be ensured during autophagic processes.

In order for our cells to remain healthy, old and damaged parts must be constantly renewed. It is therefore important that this material is recognized by the cells and degraded. One of the central processes in our cells that mediate the clearance of this material is called autophagy. During autophagy, the substances destined for degradation are enwrapped by a membrane, which eventually fuses with cellular structures called lysosomes. Within the lysosomes the damaged material is degraded and its building blocks are recycled for reuse. To allow autophagy to serve as quality control pathway that selectively degraded only damaged cellular substances (the cargo), the cargo has to be specifically recognized by the autophagy machinery. This function is supplied by so-called cargo receptors that bind to the cargo material and tether it to the membrane. During this process, the membrane is not directly bound by the receptors. Instead, the receptors bind to small Atg8 proteins that decorate the membrane. In this project is was investigated how the cargo receptors are regulated by the presence of the cargo and Atg8 proteins. To this end, the process of selective autophagy was studied in the bakers yeast. It was found that the presence of the cargo material concentrates and activates the yeast cargo receptor Atg19. Atg19 then binds to multiple Atg8 molecules at the same time. This allows Atg19 to wrap the membrane tightly around the cargo enabling the cell to spare non-cargo material from degradation. This process in the bakers yeast functions in principle in the same manner in our cells. The results offer deeper mechanistic insights into the process of selective autophagy, which protects us from various, mostly age-related diseases.