Supplementary Box 1 | Mechanistic details of the autophagy machinery



mTORC1 regulates autophagy via a multiprotein complex, composed of ULK1/2, Atg13, and
the scaffold protein AFIP200 (Figure 2).1,2 ULK1 and ULK2 are essential for autophagy induction.1,3,4 However, in animal models, a ULK1–/– mouse displayed normal autophagy in response to starvation, but showed delayed mitochondrial clearance during reticulocyte maturation, which suggests that ULK1 might be compensated for by ULK2 in certain tissues. 5 ULK3
has been identified and appears to have a role in oncogene-induced cell senescence.6 Atg13 is
crucial for autophagy, interacting directly with ULK1, ULK2, and FIP200, regardless of its
phosphorylation state.1,2 Under nutrient-rich conditions, mTORC1 is associated with the
ULK1 and ULK2 complexes, and phosphorylates ULK1, ULK2, and Atg13.2 Phosphorylated
ULK1, ULK2, and Atg13 provide negative feedback, inhibiting p70S6K activity.1 Conversely,
upon nutrient-deficient conditions, mTORC1 dissociates, Atg13, ULK1 and ULK2 are partially dephosphorylated, and activation of ULK1 and ULK2 promotes phosphorylation of
FIP200.2
Generating phosphatidylinositol(3)–phosphate (PIP3) at the phagophore assembly site (PAS)
is essential for the recruitment of Atg proteins. In humans, at least three distinct complexes
with PI3KCIII (also known as hVps34) complexes are involved in autophagosome formation
or clearance. The Atg14L complex (Atg14L–Beclin1–PI3KCIII–p150) and the UV radiation
resistance associated gene (UVRAG) complex (UVRAG–Beclin1–PI3KCIII–p150) are required for autophagy, whereas the Rubicon complex (Rubicon–UVRAG–Beclin1–PI3KCIII–
p150) negatively regulates autophagy.7–12 UVRAG competes with Atg14L for binding to Beclin1 and the interactions between Beclin1–PI3KCIII–p150 complex and Atg14L or UVRAG
are mutually exclusive.8 Ambra1 and Bif1 are essential for the induction of autophagy through
direct interaction with Beclin1 and UVRAG, respectively, whereas Bcl-2 binds to Beclin1 and
disrupts the Beclin1-associated PI3KCIII complex, thereby inhibiting autophagy.3,13 These
complexes function as core complexes in autophagosome nucleation and elongation, facilitating the recruitment of essential Atg proteins to the cell membrane. By synergizing Beclin1induced synthesis of phosphatidylinositol-3-phosphate (PIP3), Atg14L favors phagophore nucleation and elongation, whereas UVRAG, by decreasing PI3KCIII activity, might promote
autophagosome completion.10 PI3KCIII is also thought to activate mTORC1, protecting the
cell from a massive autophagic response leading to autophagic cell death.14
Atg9 and vacuole membrane protein 1 (VMP1) are transmembrane proteins necessary for autophagy. Atg9 localizes to the trans-Golgi network and late endosomes.15 Atg9 cycles between
the PAS and multiple peripheral sites, presumably carrying membrane components required
for phagophore expansion.16–19 Atg9 cycling is dependent on ULK1 and requires the kinase
activity of PI3KCIII.15 VMP1 localizes to the plasma membrane and, upon autophagy induction, it also co-localizes with Atg8/LC3 and Beclin1.20 Interestingly, ectopic overexpression of
VMP1 triggers autophagy even under nutrient-rich conditions, whereas depletion of VMP1
blocks autophagy even in the presence of starvation or mTOR inhibitors. VMP1 interaction
with Beclin1 is essential for VMP1 induced autophagy suggesting that VMP1 might function
as a transmembrane protein recruiting Beclin1 and other components of the PI3KCIII complex
to the phagophore.20 The origin of the phagophore membrane remains controversial. Data indicate that the endoplasmic reticulum membrane,21,22,23 the trans-golgi network,24 the mitochondrial outer membrane25 and the plasma membrane26 can contribute to autophagosome
formation, suggesting that a range of organelles can provide the required membrane components.27 Two ubiquitin-like proteins, Atg12, and Atg8/LC3 and their conjugation systems putatively play a key role in elongation and expansion of the phagophore membrane. Atg12 conjugates with Atg5, which is facilitated by Atg7 and Atg10. Then the Atg12–Atg5 conjugate
with Atg16L creates the Atg16L complex. Atg8/LC3 is cleaved at its C-terminus by Atg4 to
generate cytosolic LC3, in which the C-terminal glycine residue is conjugated to phosphatidylethanolamine in the presence of Atg7 and Atg3. The lipidated form of LC3 is attached to
both sides of phagophore membrane, but it is eventually removed from the outer membrane,
right before a fusion of the autophagosome with the lysosome.3 Recruitment of cargo from cytoplasm requires autophagy receptors such as p62, Nbr1, or NIX.28–30 Maturation results from
fusion of an autophagosome with endolysosomal compartments leading to the breakdown of
1
cargoes by acidic hydrolases. The fusion process requires the lysosomal membrane protein
LAMP-2 and the small GTPase Rab7.31,32
1. Jung, C. H. et al. ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery. Mol.
Biol. Cell 20, 1992–2003 (2009).
2. Hosokawa, N. et al. Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required
for autophagy. Mol. Biol. Cell 20, 1981–1991 (2009).
3. Yang, Z. & Klionsky, D. J. Mammalian autophagy: core molecular machinery and signaling regulation. Curr.
Opin. Cell Biol. 22, 124–131 (2010).
4. Lee, S. B. et al. ATG1, an autophagy regulator, inhibits cell growth by negatively regulating S6 kinase. EMBO
Rep. 8, 360–365 (2007).
5. Kundu, M. et al. Ulk1 plays a critical role in the autophagic clearance of mitochondria and ribosomes during
reticulocyte maturation. Blood 112, 1493–1502 (2008).
6. Young, A. R. et al. Autophagy mediates the mitotic senescence transition. Genes Dev. 23, 798–803 (2009).
7. Kihara, A., Noda, T., Ishihara, N. & Ohsumi, Y. Two distinct Vps34 phosphatidylinositol 3-kinase complexes
function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae. J. Cell Biol. 152, 519–530
(2001).
8. Itakura, E., Kishi, C., Inoue, K. & Mizushima, N. Beclin 1 forms two distinct phosphatidylinositol 3-kinase
complexes with mammalian Atg14 and UVRAG. Mol. Biol. Cell 19, 5360–5372 (2008).
9. Sun, Q. et al. Identification of Barkor as a mammalian autophagy-specific factor for Beclin 1 and class III
phosphatidylinositol 3-kinase. Proc. Natl Acad. Sci. USA 105, 19211–19216 (2008).
10. Zhong, Y. et al. Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin
1-phosphatidylinositol-3-kinase complex. Nat. Cell Biol. 11, 468–476 (2009).
11. Takahashi, Y. et al. Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis.
Nat. Cell Biol. 9, 1142–1151 (2007).
12. Matsunaga, K. et al. Two Beclin 1-binding proteins, Atg14L and Rubicon, reciprocally regulate autophagy at
different stages. Nat. Cell Biol. 11, 385–396 (2009).
13. Fimia, G. M. et al. Ambra1 regulates autophagy and development of the nervous system. Nature 447, 1121–
1125 (2007).
14. Backer, J. M. The regulation and function of class III PI3Ks: novel roles for Vps34. Biochem. J. 410, 1–17
(2008).
15. Young, A. R. et al. Starvation and ULK1-dependent cycling of mammalian Atg9 between the TGN and
endosomes. J. Cell Sci. 119, 3888–3900 (2006).
16. Yen, W. L., Legakis, J. E., Nair, U. & Klionsky, D. J. Atg27 is required for autophagy-dependent cycling of Atg9.
Mol. Biol. Cell 18, 581–593 (2007).
17. Legakis, J. E., Yen, W. L. & Klionsky, D. J. A cycling protein complex required for selective autophagy.
Autophagy 3, 422–432 (2007).
18. Tucker, K. A., Reggiori, F., Dunn, W. A., Jr & Klionsky, D. J. Atg23 is essential for the cytoplasm to vacuole
targeting pathway and efficient autophagy but not pexophagy. J. Biol. Chem. 278, 48445–48452 (2003).
19. Reggiori, F., Tucker, K. A., Stromhaug, P. E. & Klionsky, D. J. The Atg1-Atg13 complex regulates Atg9 and
Atg23 retrieval transport from the pre-autophagosomal structure. Dev. Cell 6, 79–90 (2004).
20. Ropolo, A. et al. The pancreatitis-induced vacuole membrane protein 1 triggers autophagy in mammalian cells.
J. Biol. Chem. 282, 37124–37133 (2007).
21. Hayashi-Nishino, M. et al. A subdomain of the endoplasmic reticulum forms a cradle for autophagosome
formation. Nat. Cell Biol. 11, 1433–1437 (2009).
22. Axe, E. L. et al. Autophagosome formation from membrane compartments enriched in phosphatidylinositol
3-phosphate and dynamically connected to the endoplasmic reticulum. J. Cell Biol. 182, 685–701 (2008).
23. Yla-Anttila, P., Vihinen, H., Jokitalo, E. & Eskelinen, E. L. 3D tomography reveals connections between the
phagophore and endoplasmic reticulum. Autophagy 5, 1180–1185 (2009).
24. Nishida, Y. et al. Discovery of Atg5/Atg7-independent alternative macroautophagy. Nature 461, 654–658
(2009).
25. Hailey, D. W. et al. Mitochondria supply membranes for autophagosome biogenesis during starvation. Cell
141, 656–667 (2010).
26. Ravikumar, B., Moreau, K., Jahreiss, L., Puri, C. & Rubinsztein, D. C. Plasma membrane contributes to the
formation of pre-autophagosomal structures. Nat. Cell Biol. 12, 747–757 (2010).
27. Tooze, S. A. & Yoshimori, T. The origin of the autophagosomal membrane. Nat. Cell Biol. 12, 831–835 (2010).
28. Pankiv, S. et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein
aggregates by autophagy. J. Biol. Chem. 282, 24131–24145 (2007).
29. Schweers, R. L. et al. NIX is required for programmed mitochondrial clearance during reticulocyte maturation.
Proc. Natl Acad. Sci. USA 104, 19500–19505 (2007).
30. Amaravadi, R. K. et al. Principles and current strategies for targeting autophagy for cancer treatment. Clin.
Cancer Res. 17, 654–666 (2011).
31. Jager, S. et al. Role for Rab7 in maturation of late autophagic vacuoles. J. Cell Sci. 117, 4837–4848 (2004).
32. Tanaka, Y. et al. Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice. Nature
406, 902–906 (2000).
2