Intracellular activation of zymogen granules triggers pancreas self-digestion and  leads to acute pancreatitis. Most cases of acute pancreatitis are self-limited, highlighting the importance of stress-response programs in acinar cells. VMP1 is an autophagy protein, essential for autophagosome biogenesis, and which is induced during pancreatitis. VMP1 mediates zymophagy, a selective type of autophagymediated degradation of zymogen granules that prevents the spreading of activated enzymes within pancreas tissue. Previously, we demonstrated that the deubiquitinase USP9x interacts with VMP1 during zymophagy. The aim of this work is to elucidate the role of USP9x in autophagosome formation. By immunofluorescence, USP9x presents a cytoplasmic diffuse pattern at basal conditions. USP9x respond to starvation (as a physiological induction of autophagy) with a relocation to perinuclear area as early as 15 min after treatment (48.0% ± 1.2 vs 40.5% ± 1.9 p<0.01). By western blot, we observed that this response is not accompanied by an increase of USP9x protein levels, confirming a protein movement instead of an induction. During the starvation-induced autophagy, USP9x co-localizes with autophagy-related proteins such as DFCP1 (a marker of omegasome), Wipi1 (a marker of isolation membrane) and Beclin 1 (a marker of PI3k complex), suggesting the participation in autophagosome biogenesis.
Moreover, we demonstrate that VMP1 is necessary for USP9x relocation since this relocation is significantly reduced in cells depleted of VMP1 (41.3%±2.2 vs 48.0%± 1.2 p<0.01) and it is increased in cells overexpressing VMP1 (47.5%± 2.2 vs 40.5% ± 1.0 p<0.01). Finally, shRNA-mediated depletion of USP9x significantly reduces
autophagy evaluated by LC3-RFP dots per cell in starved HeLa cells (6.8±4.2 vs 33.6±2.4 p<0.05), indicating that USP9x is required for autophagosome formation. In conclusion, we demonstrate for the first time that the deubiquitinase USP9x is a novel autophagy-related protein. In response to starvation, USP9x relocates to a perinuclear area and participates of the initial steps in the autophagosome biogenesis. Hierarchically, USP9x acts downstream of VMP1, since VMP1 expression induces USP9x relocation during autophagosome formation. Finally, USP9x may plays a relevant role in the cell response to acute pancreatitis, being part in the early nucleation steps of the VMP1-mediated selective autophagy.