| Abstract: | Autophagosome formation is a complex cellular process, which requires major membrane rearrangements leading to the creation of a relatively large double-membrane vesicle that directs its contents to the lysosome for degradation. Although various membrane compartments have been identified as sources for autophagosomal membranes, the molecular mechanism underlying these membrane trafficking steps remains elusive. To address this question we performed a systematic analysis testing all known Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins for their ability to inhibit autophagosome formation by disrupting a specific membrane trafficking step. TBC proteins are thought to act as inhibitors of Rab GTPases, which regulate membrane trafficking events. Up to 11 TBC proteins inhibit autophagy when overexpressed and one of these, TBC1D14, acts at an early stage during autophagosome formation and is involved in regulating recycling endosomal traffic. We found that the early acting autophagy proteins ATG9 and ULK1 localize to transferrin receptor (TFR)-positive recycling endosomes (RE), which are tubulated by excess TBC1D14 leading to an inhibition of autophagosome formation. Finally, transferrin (TF)-containing recycling endosomal membranes can be incorporated into newly forming autophagosomes, although it is likely that most of the autophagosome membrane is subsequently acquired from other sources. |
