ULK 1-mediated phosphorylation of ATG 16 L 1 promotes xenophagy , but destabilises the 1 ATG 16 L 1 Crohn ’ s mutant 2

22 Autophagy is a highly regulated catabolic pathway that is potently induced by stressors including 23 starvation and infection. An essential component of the autophagy pathway is an ATG16L1-containing 24 E3-like enzyme, which is responsible for lipidating LC3B and driving autophagosome formation. 25 ATG16L1 polymorphisms have been linked to the development of Crohn’s disease (CD) and 26 phosphorylation of CD-associated ATG16L1 T300A (caATG16L1) has been hypothesised to contribute to 27 cleavage and autophagy dysfunction. Here we show that ULK1 kinase directly phosphorylates ATG16L1 28 in response to infection and starvation. Phosphorylated ATG16L1 localises to the site of internalised 29 bacteria and stable cell lines harbouring a phospho-dead mutant of ATG16L1 have impaired xenophagy, 30 indicating a role for ATG16L1 phosphorylation in the promotion of anti-bacterial autophagy. In contrast 31 to wild-type ATG16L1, ULK1-mediated phosphorylation of caATG16L1 drives its destabilization in 32 response to stress. In summary, our results show that ATG16L1 is a novel target of ULK1 kinase and that 33 ULK1-signalling to ATG16L1 is a double-edged sword, enhancing the function of the wildtype ATG16L1, 34 but promoting degradation of caATG16L1. 35