Autophagy is an intracellular degradation pathway conserved in eukaryotes. Among core autophagy-related (Atg) proteins, mammalian Atg9A is the sole multi-spanning transmembrane protein, and both of its N- and C-terminal domains are exposed to the cytoplasm. It is known that Atg9A travels through the trans-Golgi network (TGN) and the endosomal system under nutrient-rich conditions, and transiently localizes to the autophagosome upon autophagy induction. However, the significance of Atg9A trafficking for autophagosome formation remains elusive. Here, we identified sorting motifs in the N-terminal cytosolic stretch of Atg9A that interact with the adaptor protein AP-2. Atg9A with mutations in the sorting motifs could not execute autophagy and was abnormally accumulated at the recycling endosomes. The combination of defects in autophagy and Atg9A accumulation in the recycling endosomes was also found upon the knockdown of TRAPPC8, a specific subunit of the TRAPPIII complex. These results show directly that the trafficking of Atg9A through the recycling endosomes is an essential step for autophagosome formation.
The authors declare no competing or financial interests.
K.I. performed most of the experiments. Y.T. analyzed the deletion mutant of Atg9A. F.H. helped a knock down experiments of TRAPPC8 and performed IP experiment with Y.A. N.F. and M.H. contributed valuable discussion. T.Y. and T.N. designed the study and wrote the manuscript.
This work was supported by Special Coordination Funds for Promoting Science and Technology of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan [grant numbers 25111002 to T.Y., and 26111512 and 25293372 to T.N.].
Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.196196.supplemental
- Received August 1, 2016.
- Accepted August 17, 2016.
- © 2016. Published by The Company of Biologists Ltd