*4.1.1 Initiation*

*Cell Growth*

The two main molecular determinants of organelle membrane identity in cells are lipid content, specifically PIs, and association with activated small GTPases (such as the Rab family of proteins) [19, 20]. These membrane elements can easily be remodeled and changed, in contrast to transmembrane proteins and other integral membrane elements, allowing for the dynamic regulation and sculpting of membranes. Small GTPases exhibit diversity that correlates with specific subcellular locations, such that Rab 1 (ER and Golgi), Rab 2 (cis Golgi), Rab 4/11 (Recycling endosomes), and Rab 5 (Early endosomes, plasma membrane, clathrin coated vesicles) each localize to the membranes of different organelles [19, 20].

Small GTPases like Rabs exist in two forms: a GTP-bound active and membrane

Organelle identity is also determined by specific forms of PI, with phosphate groups covalently linked to positions 3, 4, or 5 of their inositol ring [11, 12, 15, 19–22]. The plasma membrane contains predominantly PI4P and PI(4,5)P2, with the latter synthesized from the former [19–22]. These two can be phosphorylated by PI3-kinases (PI3K) to generate signaling lipids [19–22]. PI(4,5)P2 can also be cleaved to generate diacylglycerol (DAG; which can activate autophagic response) and I(1,4,5)P3 second messengers [19–22]. PI4P can also be found in the Golgi [19, 20]. Early endosomes are characterized by the presence of PI(3)P through the function of PI3-OH kinases like Vps34, which require small GTPases like Rab5 to function at the proper location [13, 14, 19–22]. This requirement for a Rab GTPase to produce the corresponding PI species characteristic of the compartment highlights the close functional relationship between the two key determinants of organelle identity [19–22]. The presence of PI(3,5)P (synthesized from PI3P) is characteristic

The lipid composition of organelles is influenced by exchange of materials between

them. There are different ways in which such exchanges can take place, including vesicle-mediated membrane trafficking between organelles, lipid droplet function, and direct exchange of lipid species via membrane contact sites between organelles such as the ER and Golgi. While the ER is the central site for intracellular lipid synthesis, lipid trafficking is essential for lipids to move from the ER to different organelles in order to maintain homeostatic membrane composition required for morphology, signaling and cellular processes. Moreover, it is common for organelles like the Golgi and endosome to change their lipid and membrane compositions as they mature. Much of what we know about the lipid content of intracellular organelles or compartments is due to lipidomics studies that have served as a tool to identify lipid species in these membranes [23–27]. Biochemical fractionation and/or extraction techniques are used to isolate organellar membranes of interest and subject them to mass spectrometry for lipid identification and lipidome determination, similar to proteomics studies that have been used to catalog the protein content of many of these organelles [27]. Different efficiencies and membrane targets can be obtained depending on how the samples are prepared and treated during extraction leading up to ionization and mass spectrometry analysis [27]. Different types of data processing allow for a range of lipidomics applications, from species identification and quantification to pathway and network analysis [27]. These techniques have been thoroughly reviewed elsewhere and outside of the scope of this review article [27]. While proteomic studies have shed light on the proteins that reside in autophagosomes [28–31], an autophagosome lipidome has yet to be experimentally determined. Through microscopy and biochemical methods, we know that the lipid identity of autophagosomes is defined largely by the presence of PE, PI and PI3P.

associated form, and a GDP-bound inactive cytosolic form. While Rabs can be lipidated (prenylated) at their C-terminus, facilitating their anchoring to the membrane, what is thought to determine Rab-membrane interaction specificity are

the proteins that facilitate their GDP/GTP exchange [19, 20].

of late endosomes [19–22].

**90**

During the initial stages of autophagy, essential Atg proteins are recruited to PAS structures to nucleate the gathering of membrane *de novo* and to generate


#### **Table 2.**

*Organellar lipid contributions to autophagic processes and autophagosome formation.*

#### **Figure 2.**

*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 from the endosome and vacuole.*

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 structures at any given time.

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

**93**

*Intracellular Lipid Homeostasis and Trafficking in Autophagy*

Generation of PI3P at PAS sites functions as a scaffold for ATG/Atg proteins to associate with the PAS, helping in the expansion, elongation and curvature genera-

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

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.

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.

tion needed for autophagosome formation to proceed successfully [13, 14].

that have also been found to be required for autophagosome formation.

*DOI: http://dx.doi.org/10.5772/intechopen.89683*

*4.1.2 Expansion*

*4.1.3 ATG9 trafficking*

Generation of PI3P at PAS sites functions as a scaffold for ATG/Atg proteins to associate with the PAS, helping in the expansion, elongation and curvature generation needed for autophagosome formation to proceed successfully [13, 14].
