Recycling endosomes contribute to autophagosome formation

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.

Keywords: ATG13; ATG9; RAB11; Rab GTPases; RabGAPs; TBC1D14; ULK1; autophagosome formation; autophagy; recycling endosomes; transferrin receptor.

Figure 1. Recycling endosomes contribute to autophagosome formation. REs shuttle internalized cargo such as TF (not shown) with its receptor, TFR, back to the plasma membrane. Additionally, a pool of REs containing the transmembrane protein ATG9 and ULK1 are important in autophagosome formation. Blocking RE traffic by overexpressing the RAB11 effector protein TBC1D14 or inactive RAB11^N124I leads to tubulation of REs and inhibition of starvation-induced autophagy. While ULK1 remains associated with REs, ATG9 relocalizes upon starvation, possibly to recruit membrane from other sources. Meanwhile, ULK1-positive REs partially relocalize with the autophagosome marker LC3 (not shown) and the RE membrane can be integrated into forming autophagosomes leading to deposition of TF in the autophagosomal lumen.