**Abstract**

In eukaryotes, lipids are not only an important constituent of the plasma membrane but also used to generate specialized membrane-bound organelles, including temporary compartments with critical functions. As such, lipids play a key role in intracellular homeostasis—the ability of a cell to maintain stable internal conditions upon changes in its extracellular environment. Autophagy, one of the cellular processes through which eukaryotic cells strive for survival under stress, is heavily dependent on lipid and membrane trafficking through the *de novo* formation of autophagosomes—temporary, large, and double-bilayered organelles in which materials are encapsulated for recycling. This chapter discusses what we know about lipid homeostasis and trafficking during autophagy and autophagosome formation and comments on future directions of the field.

**Keywords:** lipids, lipid homeostasis, lipid trafficking, autophagy, autophagosomes

#### **1. Introduction**

Careful control of cellular lipid pathways plays an important role in a cell's ability to maintain stable internal conditions in the face of an ever-changing extracellular environment. This is particularly true as it relates to cellular self-eating or autophagy, a process brought about by proteins collectively known as the Atg (in yeast) or ATG (in mammals, **A**u**T**opha**G**y) proteins [1]. Macroautophagy (herein referred to as autophagy) is the catabolic process by which unneeded or damaged cellular components are sequestered as cargo into unique double-membrane vesicles called autophagosomes [1]. Once made, autophagosomes deliver their contents for breakdown by docking and fusing with the cell's degradative organelle, the lysosome in animal cells or the vacuole in plant and yeast cells. Following the breakdown of these materials, the components can undergo efflux to enable recycling and reuse by cells. Autophagy can be activated in many ways, including by starvation and cellular damage. When induced by starvation, autophagy allows for the recycling of nutrients to sustain metabolism in the absence of extracellular nutrients. Under other conditions, such as when specific cellular components are damaged, autophagy can take more specific forms, targeting the damaged components for sequestration into autophagosomes and delivery to lysosomes/ vacuoles for destruction and recycling of the generated materials. The damaged components sequestered by more targeted forms of autophagy include mitochondria (mitophagy), ribosomes (ribophagy), and peroxisomes (pexophagy) [2–4].

Dysregulation of autophagy has been linked to a variety of human disease states, including cancer, neurodegenerative disease, and heart disease [5]. Mutations in ATG genes can result in autosomal recessive human genetic conditions including diseases like Niemann-Pick Type C1, a progressive lipid storage disorder associated with impaired autophagosome maturation and characterized by neurodegeneration [6], Gaucher disease, a disorder related to the inability to breakdown specific cargo once autophagosomes fuse with the lysosome and characterized by hematologic symptoms [7], and Pompe disease, commonly coupled with glycogen accumulation in autophagic compartments and lysosomes, often resulting in myopathies [8, 9]. These relationships between autophagy and human disease have generated much interest in attaining a better understanding of autophagic processes, especially its hallmark—autophagosome formation [1, 5].

Autophagosomes differ from smaller, more traditional types of transport vesicles in that they form *de novo* (anew) rather than budding off of a pre-existing donor organelle, range from 600 to 900 nm rather than 60–100 nm in diameter, and are delimited by two bilayer membranes rather than by a single bilayer [1]. Autophagosome formation takes place at distinct cellular locations called preautophagosomal structures (PAS). Anew, membrane materials from different intracellular lipid sources are brought to the PAS to ultimately nucleate, form, and complete autophagosome biogenesis—highlighting the important role of lipid homeostasis in autophagy. Much effort has been dedicated to understanding the lipid trafficking events leading to autophagosome formation, the unique hallmark of autophagy. In this review, we discuss the progress that has been made in understanding this relationship between autophagosome formation and lipid homeostasis.

We begin this discussion by surveying the diversity of lipids present inside cells, including evidence on the role of each lipid type in autophagy. We discuss how membrane composition helps establish organelle identity, in order to place autophagosome formation within a larger context of lipid homeostasis. After considering how these lipids relate to autophagy, we discuss the membrane trafficking events leading to autophagosome formation. We conclude by commenting on the questions that remain unanswered at the intersection of autophagy and lipid trafficking.

### **2. Structure/function diversity of lipids and membranes in eukaryotes**

Lipids are the only biologically relevant macromolecules that cannot be categorized as a polymer—their diversity is not dependent on monomer sequence like that of complex carbohydrates, proteins, or nucleic acids. The diversity of lipids is instead dependent on their chemical structure and the unique properties that are attained by combining different lipid molecules in the context of biological membrane bilayers [10–12]. The structural diversity of lipids in biological membranes can be divided into three different groups: glycerophospholipids (also often referred to as phospholipids), sphingolipids, and sterols (**Figure 1**). These three types of amphipathic lipids, when combined in different ratios and leaflet asymmetries, give rise to a variety of biological membrane bilayer properties such as fluidity and curvature. Lipids in biological membranes can be covalently modified through the attachment of carbohydrate or phosphate moieties through glycosylation or phosphorylation, respectively. These modified lipids also contribute to the functional diversity of biological membranes. The properties of a membrane bilayer are also influenced by the proteins embedded within it. Some transmembrane proteins can be post-translationally modified to contain chemical groups like carbohydrates

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*Intracellular Lipid Homeostasis and Trafficking in Autophagy*

fied by covalent attachment to lipid groups as well.

**2.1 Glycerophospholipids**

**Figure 1.**

uting to its amphipathic nature [10–12].

(glycoproteins) and phosphate groups (phosphoproteins) that also inform membrane function. Some membrane and peripheral membrane proteins can be modi-

*be found in cell membranes, namely (A) phospholipids, (B) sphingolipids, and (C) sterols.*

*Chemical structures of selected cell membrane lipids. Examples of some of the different types of lipids that can* 

Glycerophospholipids or phospholipids are the major component of biological membranes and their name evokes the building blocks used for their synthesis, including a three-carbon glycerol backbone (**Figure 1A**). This glycerol molecule is modified so that two of its hydroxyl groups (the ones attached to carbons 1 and 2) are esterified to covalently attach long-chain fatty acids to its backbone [10–12]. The fatty acid esterified to carbon 1 is often saturated (no carbon to carbon doublebonds) and the fatty acid esterified to carbon 2 is often unsaturated (with carbon to carbon double bonds present) [10–12]. Carbon to carbon double bonds in fatty acids have bends associated with them, which modify interactions with adjacent molecules and contribute to the fluidity of the bilayer [10–12]. The hydroxyl group on the third carbon of the glycerol backbone is covalently attached or esterified to phosphoric acid, adding a highly polar head group to the phospholipid and contrib-

Glycerophospholipid diversity is determined by the specific fatty acids and potentially alcohol-modified phosphoric acid groups attached to the glycerol backbone. A cell can generate a large variety of more than 100 glycerophospholipids, each with a different combination of fatty acids on carbons 1 and 2 and a head group on carbon 3 [10–12]. Glycerophospholipids are named depending on the head group attached to carbon 3. Common glycerophospholipids in cells include: phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol (PI) [10–12]. PE is the simplest phospholipid, with just a phosphoric acid moiety as a head group, while the other common types have alcohol-modified phosphoric acid head groups [10–12]. PI in particular is the kind of lipid that plays a major role in determination of organelle identity, as described later in this review [10–12].

Certain phospholipids and lipid-modifying enzymes are key for autophagy. For example, the PI3Kinase complex (Class III and potentially Class II) and its substrate PI are essential for autophagosome formation [13, 14]. Moreover, soluble autophagyrelated proteins known to be required for the induction of autophagosome formation like Atg1 are known to be recruited to membrane enriched with PI and PI3P [13, 14].

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

**Figure 1.**

*Cell Growth*

homeostasis.

Dysregulation of autophagy has been linked to a variety of human disease states, including cancer, neurodegenerative disease, and heart disease [5]. Mutations in ATG genes can result in autosomal recessive human genetic conditions including diseases like Niemann-Pick Type C1, a progressive lipid storage disorder associated with impaired autophagosome maturation and characterized by neurodegeneration [6], Gaucher disease, a disorder related to the inability to breakdown specific cargo once autophagosomes fuse with the lysosome and characterized by hematologic symptoms [7], and Pompe disease, commonly coupled with glycogen accumulation in autophagic compartments and lysosomes, often resulting in myopathies [8, 9]. These relationships between autophagy and human disease have generated much interest in attaining a better understanding of autophagic processes, especially its

Autophagosomes differ from smaller, more traditional types of transport vesicles in that they form *de novo* (anew) rather than budding off of a pre-existing donor organelle, range from 600 to 900 nm rather than 60–100 nm in diameter, and are delimited by two bilayer membranes rather than by a single bilayer [1]. Autophagosome formation takes place at distinct cellular locations called preautophagosomal structures (PAS). Anew, membrane materials from different intracellular lipid sources are brought to the PAS to ultimately nucleate, form, and complete autophagosome biogenesis—highlighting the important role of lipid homeostasis in autophagy. Much effort has been dedicated to understanding the lipid trafficking events leading to autophagosome formation, the unique hallmark of autophagy. In this review, we discuss the progress that has been made in understanding this relationship between autophagosome formation and lipid

We begin this discussion by surveying the diversity of lipids present inside cells,

including evidence on the role of each lipid type in autophagy. We discuss how membrane composition helps establish organelle identity, in order to place autophagosome formation within a larger context of lipid homeostasis. After considering how these lipids relate to autophagy, we discuss the membrane trafficking events leading to autophagosome formation. We conclude by commenting on the questions that remain unanswered at the intersection of autophagy and lipid trafficking.

**2. Structure/function diversity of lipids and membranes in eukaryotes**

Lipids are the only biologically relevant macromolecules that cannot be categorized as a polymer—their diversity is not dependent on monomer sequence like that of complex carbohydrates, proteins, or nucleic acids. The diversity of lipids is instead dependent on their chemical structure and the unique properties that are attained by combining different lipid molecules in the context of biological membrane bilayers [10–12]. The structural diversity of lipids in biological membranes can be divided into three different groups: glycerophospholipids (also often referred to as phospholipids), sphingolipids, and sterols (**Figure 1**). These three types of amphipathic lipids, when combined in different ratios and leaflet asymmetries, give rise to a variety of biological membrane bilayer properties such as fluidity and curvature. Lipids in biological membranes can be covalently modified through the attachment of carbohydrate or phosphate moieties through glycosylation or phosphorylation, respectively. These modified lipids also contribute to the functional diversity of biological membranes. The properties of a membrane bilayer are also influenced by the proteins embedded within it. Some transmembrane proteins can be post-translationally modified to contain chemical groups like carbohydrates

hallmark—autophagosome formation [1, 5].

**86**

*Chemical structures of selected cell membrane lipids. Examples of some of the different types of lipids that can be found in cell membranes, namely (A) phospholipids, (B) sphingolipids, and (C) sterols.*

(glycoproteins) and phosphate groups (phosphoproteins) that also inform membrane function. Some membrane and peripheral membrane proteins can be modified by covalent attachment to lipid groups as well.

## **2.1 Glycerophospholipids**

Glycerophospholipids or phospholipids are the major component of biological membranes and their name evokes the building blocks used for their synthesis, including a three-carbon glycerol backbone (**Figure 1A**). This glycerol molecule is modified so that two of its hydroxyl groups (the ones attached to carbons 1 and 2) are esterified to covalently attach long-chain fatty acids to its backbone [10–12]. The fatty acid esterified to carbon 1 is often saturated (no carbon to carbon doublebonds) and the fatty acid esterified to carbon 2 is often unsaturated (with carbon to carbon double bonds present) [10–12]. Carbon to carbon double bonds in fatty acids have bends associated with them, which modify interactions with adjacent molecules and contribute to the fluidity of the bilayer [10–12]. The hydroxyl group on the third carbon of the glycerol backbone is covalently attached or esterified to phosphoric acid, adding a highly polar head group to the phospholipid and contributing to its amphipathic nature [10–12].

Glycerophospholipid diversity is determined by the specific fatty acids and potentially alcohol-modified phosphoric acid groups attached to the glycerol backbone. A cell can generate a large variety of more than 100 glycerophospholipids, each with a different combination of fatty acids on carbons 1 and 2 and a head group on carbon 3 [10–12]. Glycerophospholipids are named depending on the head group attached to carbon 3. Common glycerophospholipids in cells include: phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol (PI) [10–12]. PE is the simplest phospholipid, with just a phosphoric acid moiety as a head group, while the other common types have alcohol-modified phosphoric acid head groups [10–12]. PI in particular is the kind of lipid that plays a major role in determination of organelle identity, as described later in this review [10–12].

Certain phospholipids and lipid-modifying enzymes are key for autophagy. For example, the PI3Kinase complex (Class III and potentially Class II) and its substrate PI are essential for autophagosome formation [13, 14]. Moreover, soluble autophagyrelated proteins known to be required for the induction of autophagosome formation like Atg1 are known to be recruited to membrane enriched with PI and PI3P [13, 14].

Another lipid component essential for autophagosome formation is PE [13, 14]. PE lipids present in the autophagosome are important for the ability of this structure to associate with Atg8, another essential autophagy protein. This is because successful recruitment of Atg8 to autophagosomes involves Atg8 lipidation to the head group of PE, ultimately yielding a mature autophagosome that is decorated by lipidated Atg8 [13, 14]. In fact, PE is the lipid that contributes the most to autophagosome expansion [13, 14].

#### **2.2 Sphingolipids**

The backbone of sphingolipids is not glycerol but sphingosine, an amino alcohol synthesized from palmitoyl-CoA and serine [10–12]. The covalent attachment of the sphingosine backbone to a fatty acid through an amide linkage yields an amphipathic sphingolipid (**Figure 1B**) [10–12]. Common examples of this type of lipid in cells include ceramides (the simplest form of sphingolipid), phosphosphingolipids (also known as sphingomyelins, the only phospholipids without a glycerol backbone), and glycosphingolipids [10–12]. The differences and diversity between these sphingolipids is dependent on the type of fatty acid and additional head groups attached to the sphingosine backbone. Sphingolipids were discovered and can be predominantly found in nervous tissue. This type of lipid can also be found in lower eukaryotes such as budding yeast [10–12].

Sphingolipids such as sphingosine-1-phosphate (S1P) and ceramides have been found to have an effect on autophagy [13, 14]. S1P, as part of its well-known role in fostering proliferation and cell survival, can induce autophagy in a way that engages elements usually associated with apoptosis [13, 14]. Ceramides, in their well-known role in promoting cell cycle arrest, promote cell death while engaging elements associated with autophagy [13, 14].

#### **2.3 Sterols**

Sterols are four-ringed steroid molecules that contain a hydroxyl functional group at position 3 and as well as a variety of potential side chains (**Figure 1C**) [10–12]. These lipids can be present in plant, animal, and microbial cells such as budding yeast. The predominant form of sterol lipid in animal cells is cholesterol. In other organisms, the lipid forms most similar to cholesterol in function are called by different names (**Figure 1C**). For example, the functional yeast equivalent of cholesterol is referred to as ergosterol (**Figure 1C**).

Sterols in eukaryotes have been shown to influence specific forms of autophagy including chaperone-mediated autophagy (CMA), pexophagy (autophagy of peroxisomes) and lipophagy (autophagy of lipid droplets) [13, 14]. These types of specific autophagy are beyond the scope of this review and are therefore not discussed.

#### **2.4 Lipid contributions to membrane structure/function**

#### *2.4.1 Membrane curvature*

The concept of membrane curvature refers to the lipid composition asymmetry between the two leaflets of a membrane bilayer [11, 15, 16]. The membrane curvature of an area can be changed, not only by altering its lipid composition, but also through the function of specialized proteins that can bind and remodel membranes. Early autophagosomes, often referred to as phagophores, have a high degree of curvature and are enriched for PI and PI3P [13, 14]. This combination of curvature

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**Table 1.**

*Intracellular Lipid Homeostasis and Trafficking in Autophagy*

molecules and rendering it easier to bend and deform [11, 17].

and lipid composition is thought to help recruit a collective of autophagy-related proteins such as Atg1 and Atg3 to the site of autophagosome formation [13, 14].

Membrane fluidity refers to the ability of a membrane to sustain diffusiondriven movement of molecules within it [11, 17]. In membranes, diffusion takes place laterally [11, 15, 17]. The presence of unsaturated and sterol lipid structures enhances membrane fluidity, ultimately allowing for more lateral diffusion of

This relationship between membrane fluidity and bending is highly relevant to autophagosome membranes [13, 14]. Early autophagosomal structures are characterized by high levels of curvature, suggesting that fluidity is key for autophagosome formation [13, 14]. In fact, autophagosomes are enriched for unsaturated lipids, and abrogation of enzyme functions that mediate desaturation can have

Membranes are not even in composition throughout. Instead, they can have areas of differential lipid composition such as lipid rafts. Lipid rafts are generally more tightly packed (less fluid) than neighboring membrane material and their edges are usually rich in sphingolipids and cholesterol [11, 15, 17, 18]. Lipid rafts, because of their differential lipid composition, can act as scaffolds for non-clathrin-

While no specific lipid microdomains have been identified as characteristic of autophagosome membranes, it is known that lipid rafts associated with the endoplasmic reticulum and mitochondria can contribute to autophagosome formation

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

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

inhibitory effects on autophagy [13, 14].

mediated internalization dynamics [11, 15, 17, 18].

*2.4.2 Membrane fluidity*

*2.4.3 Lipid microdomains*

from these lipid sources [13, 14].

**3. Organelle lipid identity**

cells strive to respond to stimuli.

*Lipid content of different organelle membranes.*

and lipid composition is thought to help recruit a collective of autophagy-related proteins such as Atg1 and Atg3 to the site of autophagosome formation [13, 14].
