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.

In response to amino acid supply, mTORC1, a master regulator of cell growth, is recruited to the lysosome and activated by the small GTPase Rheb. However, the intracellular localization of Rheb is controversial. In this study, we showed that a significant portion of Rheb is localized on the Golgi but not on the lysosome. GFP-Rheb could activate mTORC1, even when forced to exclusively localize to the Golgi. Likewise, artificial recruitment of mTORC1 to the Golgi allowed its activation. Accordingly, the Golgi was in contact with the lysosome at an newly discovered area of the cell that we term the Golgi-lysosome contact site (GLCS). The number of GLCSs increased in response to amino acid supply, whereas GLCS perturbation suppressed mTORC1 activation. These results suggest that inter-organelle communication between the Golgi and lysosome is important for mTORC1 regulation and the Golgi-localized Rheb may activate mTORC1 at GLCSs.

Macroautophagy is a major intracellular degradation system. We previously reported that overexpression of phosphatase‐deficient MTMR3, a member of the myotubularin phosphatidylinositol (PI) 3‐phosphatase family, leads to induction of autophagy. In this study, we found that MTMR3 interacted with mTORC1, an evolutionarily conserved serine/threonine kinase complex, which regulates cell growth and autophagy in response to environmental stimuli. Furthermore, overexpression of MTMR3 inhibited mTORC1 activity. The N‐terminal half of MTMR3, including the PH‐G and phosphatase domains, was necessary and sufficient for these effects. Phosphatase‐deficient MTMR3 provided more robust suppression of mTORC1 activity than wild‐type MTMR3. Furthermore, phosphatase‐deficient full length MTMR3 and the phosphatase domain alone were localized to the Golgi. These results suggest a new regulatory mechanism of mTORC1 in association with PI3P.

Kampo, a system of traditional Japanese therapy utilizing mixtures of herbal medicine, is widely accepted in the Japanese medical system. Kampo originated from traditional Chinese medicine, and was gradually adopted into a Japanese style. Although its effects on a variety of diseases are appreciated, the underlying mechanisms remain mostly unclear. Using a quantitative tf-LC3 system, we conducted a high-throughput screen of 128 kinds of Kampo to evaluate the effects on autophagy. The results revealed a suppressive effect of Shigyakusan/TJ-35 on autophagic activity. TJ-35 specifically suppressed dephosphorylation of ULK1 and TFEB, among several TORC1 substrates, in response to nutrient deprivation. TFEB was dephosphorylated by calcineurin in a Ca 2+ dependent manner. Cytosolic Ca 2+ concentration was increased in response to nutrient starvation, and TJ-35 suppressed this increase. Thus, TJ-35 prevents the starvation-induced Ca 2+ increase, thereby suppressing induction of autophagy.

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