Analysis of the role of TFG in autophagosome formation and trafficking

TFG (TRK-fused gene) is a protein ubiquitously expressed in human cells, and its main role is played in the early secretory export. TFG, indeed, acts to facilitate the export of COPII (coat protein II complex) vesicles following their scission from the ER (Endoplasmic Reticulum). TFG regulates the distribution of COPII transport carriers promoting their uncoating and retaining these vesicles at the region between the ER and the ERGIC (ER Golgi Intermediate Compartment), in order to facilitate their fusion with the compartment. Although growing evidence suggest that COPII vesicles are important for autophagosome biogenesis, the molecular regulatory network controlling this role is still vastly unveiled. One key event is that, during autophagy induction and upon PI3K activation, COPII vesicles are relocated to the ERGIC, where they guarantee LC3 lipidation-active vesicle budding, a critical step for autophagosome formation. In this work, by mass spectrometry analysis, we identified TFG as a putative ULK1 interacting protein. Given the tight connection between COPII vesicles and autophagy, we decided to investigate the involvement of TFG in autophagy. First, we confirmed that TFG and ULK1 interact and colocalize in semi-endogenous conditions. We found that TFG is a positive regulator of autophagy; indeed, its depletion leads to an impairment of autophagy, characterized by inhibition of LC3B lipidation with concomitantly accumulation of early pro-autophagy proteins. Most importantly, we found that TFG downregulation affects the proper dynamic of associations between LC3B and ATG proteins involved in the isolation membrane formation, suggesting that TFG is important for the correct maturation of early autophagosomes structures when autophagy is induced. Further, we noticed that ULK1 accumulates to the ERGIC upon TFG depletion in starved cells, this indicating that TFG could be critical for ULK1 and LC3 distribution within cells. Finally, we found that TFG interacts with LC3C by a canonical LIR (LC3 interacting) motif, and that this binding could be crucial for the role that TFG plays in autophagosome maturation. Altogether our results uncover the link between TFG and autophagy and further identify TFG-LC3 interaction as a novel regulatory step of autophagosome formation.