**3. Organelle lipid identity**

Each membrane-bound organelle in the cell is characterized by a membrane bilayer with a particular lipid composition, leading to unique physical properties and the ability to recruit a specific set of interacting partners (**Table 1**). For this reason, organelle identity is not only defined by the collection of lipids displayed by the membrane of a particular organelle, but also by the set of proteins and molecules that are able to associate and interact with it. It is also important to recognize that these membranes can be remodeled and change their properties over time as cells strive to respond to stimuli.


**Table 1.** *Lipid content of different organelle membranes.*

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 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].

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 of late endosomes [19–22].

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.

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**Table 2.**

*4.1.1 Initiation*

*Intracellular Lipid Homeostasis and Trafficking in Autophagy*

In the following section of this review, we discuss in detail the molecular processes that bring about lipid homeostasis and trafficking during autophagy, particularly in

**4. Understanding autophagosomes: a non-traditional membrane vesicle**

Current efforts to better understand autophagy are focused on the molecular details leading to the formation of autophagosomes, a double-membraned vesicle unlike any other in size and structure, that serves as the hallmark of autophagy. These approaches have mainly focused on understanding the intracellular trafficking of autophagy-related transmembrane proteins that are thought to help deliver lipids to the growing autophagosome via vesicle transport. Insights have also been obtained from assessing the influence of lipid-dependent enzymes as well as lipidsynthesis and transport pathways on autophagy. For example, mutation of PI3K enzymes severely impairs autophagy [32, 33], pointing at the importance PI and some of its phosphorylated forms in autophagic processes. In these ways, we have identified that PE and PI lipids play an important role in autophagy (**Table 2**) [14,

The process of autophagy is often thought about to happen in different stages—initiation or nucleation of phagophores (cup-shaped, double-membraned autophagosome precursors), expansion of phagophores, completion or closure of autophagosomes, fusion of autophagosomes with the lysosome/vacuole, and the efflux of materials from the lysosome/vacuole for reuse (**Figure 2**). Because our review aims to focus on the lipid homeostasis and trafficking during autophagy, we focus our discussion below on the first four stages of autophagy (initiation through fusion).

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

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

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

34–41]. We discuss these findings below.

**4.1 Membrane dynamics during autophagy**

autophagosome formation.

In the following section of this review, we discuss in detail the molecular processes that bring about lipid homeostasis and trafficking during autophagy, particularly in autophagosome formation.
