Versatile regulation of autophagy by the deubiquitinase USP11

Ubiquitination is a versatile post-translational modification known to regulate autophagy. Here, we show that loss of the deubiquitinase USP11 leads to an increase in the autophagic flux. To investigate the molecular details of how USP11 impacts autophagy, we determined interaction partners of USP11 using an affinity purification-mass spectrometry approach. GFP-tagged, catalytically inactive USP11(C318S) was overexpressed in 293 cells. After 4 hour treatment with Torin 1, an inhibitor of mTOR that induces autophagy, cells were lysed and samples further processed for analysis. Catalytically inactive USP11 was used to increase the chances of capturing both interaction partners and potential substrates. We identified many autophagy-related proteins that co-precipitated with USP11, suggesting that USP11 does not regulate autophagy via a single substrate, but that the observed phenotype is the sum of many interactions, each of which should be independently investigated in more detail. Our work focused on elucidating the regulation of autophagy via the lipid kinase complex PI3KC3 complex I, as one of its components (VPS15) was identified in our USP11 interactome data. PI3KC3 complex I is crucial for initiation of autophagosome formation at the endoplasmatic reticulum. USP11 knockout caused an increase in complex activity, which could explain enhanced autophagy in these cells.