*4.1.2 Expansion*

*Cell Growth*

**Figure 2.**

autophagosome precursors called phagophores. In mammalian cells, phagophores can be formed at PAS sites proximal to ER, mitochondria, or plasma membrane [13, 14]. In yeast however, PAS structures are perivacuolar in nature, leading phagophores to originate at locations proximal to the vacuole with membrane contributions with other cellular locations like discussed below. While mammalian cells can display many PAS structures at steady state, yeast often display one of these struc-

*Lipid biosynthesis and trafficking during autophagosome biogenesis and fusion. The relative lipid contributions via lipid trafficking or lipid biosynthesis from each organelle are shown. (A) upon autophagy induction, membrane is sequestered from pools of lipid donors such as the ER, mitochondria, Golgi, endosome and vacuole to form the pre-autophagosomal structure (PAS) or isolation membrane (IM). (B) PI in the PAS is converted to PI3P via Vps34 and Atg8-PE is enriched, allowing for IM expansion and closure. (C) Additional lipid membrane from Atg9-Atg27 vesicle trafficking allows for further expansion of the IM. (D) Autophagosome (AV) maturation is completed once PI3P is converted into PI by myotubularin phosphatases such as Ymr1. The mature AV can fuse to the vacuole which has converted PI3P into PI(3,5)P2 via Fab1 kinase. PI(3,5)P2 permits fusion of AV and endosomes. Vacuole lipid homeostasis is further aided by Snx4 trafficking of PS and Atg27* 

Initially, activated or triggered by upstream inhibition of mTOR, core autophagy proteins collect at the PAS structure. These core factors include the ULK1 complex, ATG13, FIP200, ATG101, (yeast Atg1, Atg13, Atg17, Atg29 and Atg31) [13, 14]. The mobilization of these factors ultimately leads to the PAS recruitment and activation of the PI3K/Vps34 kinase complex which allows for the generation of PI3P at this site (from PI) (**Figure 2A**) [13, 14, 30, 31]. This enzyme activity drives forward phagophore initiation/nucleation, allowing additional recruitment of other proteins like WIPI proteins (Atg18 in yeast). WIPI proteins are key PI3P effectors. PI3K regulator proteins like ATG14 (Atg14 in yeast) are also recruited to the PAS, allowing for the modulation of initiation/nucleation of phagophore formation [13, 14]. ATG14 is also able to mediate homotypic fusion of single-membrane vesicles at the PAS, allowing more traditional membrane vesicles delivered to the PAS to fuse and contribute to the nucleation and growth of the phagophore [13, 14]. In this way, the phagophore has been found to accept lipid inputs from the ER, ER exit sites (ERES), the Golgi, the plasma membrane and recycling endosomes for its growth

**92**

tures at any given time.

*from the endosome and vacuole.*

and expansion (**Figure 2A**) [13, 14].

The lipidation of Atg8 (LC3) onto PE lipid molecules in the growing phagophore membrane is key for autophagosome formation. Other Atg proteins like ATG16L1 (yeast Atg16) as well as ATG12-ATG5 are required for Atg8 lipidation with PE. Interestingly, some of these required Atg factors like ATG16L1 partly reside in the recycling endosome and have been found to localize to the PAS in a PX-BAR/ SNX protein-dependent manner (for example, SNX18) [13, 14]. While not considered core Atg proteins, PX-BAR/SNX proteins are membrane remodeling proteins that have also been found to be required for autophagosome formation.

While the exact lipid composition of the phagophore/autophagosome is still under debate, because of the findings described above, PI3P is thought to be one of the most abundant lipid species present in these structures. In fact, recent findings identify the ATG protein ATG2A as a lipid shuttle factor that facilitates PI3P-dependent autophagosome growth [42–44]. Other phosphoinositides such as PI4P, PI(4,5)P2 and PI(3,5)P2 have also been suggested to play a role in the expansion of the phagophore [13, 14]. Apart from these, as mentioned above, sphingosine-1-phosphate (S1P) and ceramide, also contribute to autophagy. And while, Atg8/LC3 lipidation resulting in LC3-PE is important for the expansion and closure of the autophagosome, the intracellular membranes that are the source of this PE still remain to be identified. PE is produced from phosphatidylserine (PS) in mitochondria, which might be one of the sources of PE (**Figure 2A**). PE might also be shuttled from the ER, plasma membrane and recycling endosomes. It has been recently shown by Ma et al. [39], that PS trafficking by sorting nexin Snx4 in yeast is required for maintaining the correct lipid composition of the vacuole for autophagosome fusion in yeast (**Figure 2C**). Thus, maintenance of organelle lipid identity through proper trafficking and lipid homeostasis are crucial for autophagy as well.

## *4.1.3 ATG9 trafficking*

ATG9 (Atg9 in yeast) is a six-transmembrane protein that is required for autophagosome formation [45–52]. ATG9 trafficking is one of the most studied topics in the autophagy field, highlighting the important role it plays in autophagy—the autophagic phenotypes of null mutants are very penetrant. While this is the case, we still lack detailed information about the function of this protein during autophagy. Yeast studies have shown that Atg9 is localized to PAS as well as cytoplasmic vesicles of 30–60 nm diameter that bud-off of the late Golgi. It is believed that Atg9 shuttles between the PAS and its cytoplasmic vesicle pool upon autophagy induction (**Figure 2C**). At the PAS, Atg9 associates with Atg1, Atg2 as well as Atg18. Since Atg9 is a transmembrane protein, its trafficking directly affects the funneling or channeling of membrane to the PAS and the growing autophagosome. The Atg9 vesicles are thought to originate at the Golgi and contain fusion factors such as subunits of the TRAPIII (Trs85) complex that are responsible for fusion with the growing autophagosome [45, 46, 52]. Once Atg9 reaches the lysosome/vacuole as autophagosomes fuse, some studies suggest that the protein can be recycled out of the vacuolar membrane for reuse in new rounds of autophagosome formation. These studies are synergistic with findings that Atg9 traffics through endosomal compartments, as this might be an intermediary step important for recycling.

This recycling model is supported by the observation that mutations in the retromer complex, when combined with mutations in tethering factor Trs85, can abrogate trafficking of Atg9 to the PAS. Similarly the combination mutations with GARP subunits that are responsible for tethering vesicles to the Golgi from the endosome, with Trs85 result in defective autophagy. Atg9 trafficking is influenced by other autophagy proteins such as Atg27 and Atg23 [53–56]. Atg23 is a peripheral membrane protein that facilitates the anterograde trafficking of Atg9 from the Golgi to the PAS. Atg27, another transmembrane protein that facilitates formation of Atg9 vesicles at the late Golgi, is itself observed to be present in early/late endosomes, PAS, Golgi and vacuolar membrane. Atg27 can be retrieved from the vacuole in a process that is Snx4-dependent. Furthermore, earlier studies identified a C terminal tyrosine (YSAV) motif in Atg27 that is important for the proper delivery of Atg27 to the vacuole as well as to maintain Atg9 pools at the endosome that can be mobilized to different compartments during autophagy (**Figure 2C**). Taken together, the complexity of Atg9 trafficking synergizes with the hypothesis that a collection of different membranes such as endosome and Golgi all contribute to the lipid identity of the autophagosome.
