**5. Conclusion**

*Cell Growth*

of the autophagosome.

off the lysosomal membrane.

*4.1.4 Autophagosomal fusion with the lysosome/vacuole*

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

After the autophagosome is completed, the double membrane vesicle is ready

Another aspect that is key for late autophagy stages to proceed is intact lysosomal/vacuolar lipid homeostasis and lipid identity. For example, yeast vacuoles have unique lipid composition that is different from other membrane organelles within the cell. Vacuoles are enriched with lipids such as, myo-inositols, PI3P, and typically PI3,5P2, among other lipids such as ergosterol, diacylglycerol and some sphingolipids (**Figure 2D**). The specific lipid identity of vacuoles is important for the recruitment of fusion factors such as SNARES Ypt7 and HOPS that allow fusion of autophagosomes. Thus, the specific lipid identity of vacuole is important for its physiological function and its fusion with the late endosome and autophagosome.

for fusion with the lysosome/vacuole. The fusion of the autophagosome not only requires components of the fusion machinery as well as PI3P turnover, but it also requires the disassembly or retrieval of several ATG proteins from the autophagosome. In yeast, phosphoinositide phosphatases, including those from the myotubularin protein family, like Ymr1, along with Sjl2 and Sjl3 are important for removal of PI3P from completed autophagosomes, making autophagosomes fusion-competent (**Figure 2D**). In mammalian cells, PI3P phosphatase MYM-3 acts similarly to Ymr1 in promoting autophagosome maturation and fusion. Fusion of autophagosomes with the lysosome is mediated by RAB7-like protein Ypt7 along with the HOPS (Homotypic fusion and vacuole protein sorting) tethering complex and SNARE (Soluble Ethylmaleimide-Sensitive Factor Attachment Protein Receptor) proteins. Three Q–SNAREs: Vam3, Vti1 and Vam7 have been identified in yeast along with R-SNARE Ykt6 as key for this fusion step. Vamp7 is a sorting nexin family protein containing a PX domain that interacts with PI3P. Studies suggest that Vam7 interacts with Atg17-Atg29-Atg31 trimer complex via Atg17 interaction. In mammalian cells, SNAREs such as SYN12, SNAP29 and VAMP7/8 are responsible for autophagosome fusion with the lysosome. Interestingly, the PI3K VPS34 has also been linked with later stages of autophagy, including lysosomes returning to normal or regenerating once fusion with autophagosomes has taken place. Other phosphorylated lipid species like PI4P and PI(4,5)P2 can also facilitate lysosomal regeneration, allowing for new rounds of autophagy to occur. This might also trigger the formation of recycling vesicles packaging ATG protein cargo for recycling

**94**

While progress has been made in understanding the molecular underpinnings of autophagosome formation, our understanding has been primarily advanced by understanding the functions of proteins that lead and are required for this autophagic vesicle to form [28–31]. Because the extensive level of membrane remodeling that takes place during autophagy formation, much will be gained by investigating the process using methods that focus on the membrane and lipid biology of the process [23–27]. This new perspective has the potential of changing the way we have conventionally understood the remodeling of membranes for vesicle formation.

**Table 2** describes the lipid contributions of different intracellular organelles or donor membranes to the progression of autophagy and/or autophagosome formation. The autophagy processes or steps benefiting from these contributions are also indicated.
