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Structural basis of selective autophagy mediated by cargo receptors

Final Report Summary - AUTOCARGO (Structural basis of selective autophagy mediated by cargo receptors)

The fellow was awarded the MC-IEF Fellowship to conduct research on dissecting the molecular mechanisms of cargo recognition by selective autophagy receptors using the structural techniques electron cryo-microscopy (cryo-EM) and X-ray crystallography to study the proteins involved. To achieve this, efforts were also required to develop technical approaches aimed at integrating these techniques.

Autophagy is a process in eukaryotic cells that employs a sophisticated molecular machinery to selectively degrade bulky and potentially hazardous structures in the cytosol, such as neurotoxic protein deposits, damaged organelles or invading pathogens. A diverse set of proteins known as autophagy receptors recognize cargo destined for degradation and link it to a nascent double-membrane vesicle decorated with membrane-bound proteins that bind After maturation the vesicle (autophagosome) fuses with the lysosomal compartment where it releases its cargo for degradation.

One intriguing property of all currently identified autophagy receptors is that they contain one or more oligomerisation motifs, which seem to be essential for their function as autophagy receptors. The molecular basis of why these oligomeric assembly states are functionally relevant and how they mechanistically relate to the process of selective autophagy is presently unknown. The AUTOCARGO project aimed to address these questions by structurally characterizing functionally relevant assembly states of selective autophagy receptors at atomic resolution. Oligomeric protein assemblies pose significant challenges to approaches attempting to solve the structure in atomic detail due to their size and complexity. This project therefore employed the unique capabilities of two advanced structural biology techniques, cryo-EM and X-ray crystallography. In addition, during the course of solving the respective autophagic protein structures significant effort had to be spent on devising suitable protocols for the cryo-EM based refinement of atomic models that are generally applicable for structure determination from cryo-EM maps. We employed and adapted tools originally developed for X-ray model refinement to address the specific challenges associated with cryo-EM data. The structures obtained reveal that a subset of autophagy receptors form filamentous assemblies and mutations in key interface residues abolish this property as well as the formation of punctate structures in cells, a hallmark of selective autophagy. Analogous to higher eukaryotes, also in the yeast Cvt pathway (a yeast model of selective autophagy) both cargo and receptor proteins form higher-order oligomers. Structural and biochemical studies on Cvt pathway components (led by two PhD students in the host lab) further highlighted the relevance of oligomeric assembly states of selective autophagy components in cargo aggregation and in mediating close supposition between cargo and the nascent vesicle membrane. The ability to form oligomeric structures thus seems to be a conserved property of selective autophagy receptors and our structural studies will help to reveal important mechanistic insight into these processes.