IP-MS of human YKT6 upon mutation of possible ULK1-phosphorylation sites

Autophagy is a catabolic process during which cytosolic material is enwrapped in a newly formed double membrane structure called the autophagosome, and subsequently targeted for degradation in the lytic compartment of the cell. The fusion of autophagosomes with the lytic compartment is a tightly regulated step and involves membrane-bound SNARE proteins. These play a crucial role as they promote lipid mixing and fusion of the opposing membranes. Among the SNARE proteins implicated in autophagy, the essential SNARE protein YKT6 is the only evolutionary conserved SNARE protein from yeast to human. Alterations in YKT6 function, in both mammalian cells and nematodes, produces early and late autophagy defects that result in reduced survival. Moreover, mammalian autophagosomal YKT6 is phospho-regulated by the ULK1 kinase, preventing premature bundling with the lysosomal SNARE proteins and autophagosome-lysosome fusion. Together, our findings reveal that timely regulation of theYKT6 phosphostatus is crucial throughout autophagy progression and cell survival.