Assembly and function of membrane-scaffolds in autophagy

• Applicant: Dr. Thomas Wollert
• Subject Area: Biochemistry
• Term: from 2014 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 270641522

Autophagy is a fundamental catabolic process that delivers cytoplasmic material to lysosomal compartments for degradation. Cargo sequestration occurs either specifically or unspecifically by a de novo generated membrane structure, called phagophore. This cup-shaped membrane sack is nucleated through fusion of small donor vesicles. The phagophore expands to surround the cytoplasmic cargo entirely and is eventually sealed, giving rise to an autophagosome which fuses with the lysosome/vacuole. The dynamic membrane-remodeling events that drive phagophore-expansion are largely uncharacterized, but require a set of autophagy related (Atg) proteins. An ubiquitin (Ub)-like conjugation system is essential for autophagy and its action covalently attaches the small Ub-like protein Atg8 to the phagophore membrane. The amount of Atg8 on the phagophore regulates the size of the autophagosome. Our laboratory recently demonstrated that Atg8 forms together with its E3-like ligase complex Atg12-Atg5-Atg16 a membrane scaffold with mesh-like architecture. We now aim to understand how this scaffold is assembled and how scaffold formation influences membrane-shape and thereby phagophore-expansion. In vivo, a cascade of Atg-protein complexes drive the ordered recruitment of Atg12-Atg5-Atg16 to the phagophore. Autophagic membranes are rich in the signaling-lipid phosphatidylinositol-3-phosphate (PI3P), which is generated through the action of an autophagy specific PI3-kinase complex. The PI3P binding protein Atg18 acts immediately upstream of the Ub-like conjugation system and recruits Atg12-Atg5-Atg16 to the phagophore. Our first objective is to explore how Atg18 nucleates the membrane scaffold and how scaffold formation proceeds from this nucleation center. Therefore, we will reconstitute these processes in vitro from purified components and analyze scaffold dynamics by total internal reflection fluorescence microscopy (TIRFM) and scaffold organization by atomic force microscopy (AFM). Whereas Atg18 functions during stress-induced unspecific cargo sequestration, the second PI3P-binding protein, Atg21, is involved in specific cargo-sequestration, which is essential to remove superfluous and damaged cytoplasmic components during vegetative conditions. Our second aim is to study Atg21 function and in particular, whether it fulfills identical functions during specific autophagy as Atg18 does under stress-induced conditions. Although the localization patterns of Atg18 and Atg21 are apparently distinct, hetero-dimerization of both proteins have been observed. Our third aim involves studying the dynamic recruitment of Atg21 and Atg18 in yeast cells. We are particularly interested in their specific recruitment to cargo during vegetative and stress conditions. Taken together, our proposed work will reveal how scaffold assembly during vegetative and stress-conditions is initiated and coordinated. In addition, we will reveal how scaffold assembly impacts on phagophore expansion.

• DFG Programme: Research Grants