Emerging roles of ATG proteins and membrane lipids in autophagosome formation

Autophagosome biogenesis is a dynamic membrane event, which is executed by the sequential function of autophagy-related (ATG) proteins. Upon autophagy induction, a cup-shaped membrane structure appears in the cytoplasm, then elongates sequestering cytoplasmic materials, and finally forms a closed double membrane autophagosome. However, how this complex vesicle formation event is strictly controlled and achieved is still enigmatic. Recently, there is accumulating evidence showing that some ATG proteins have the ability to directly interact with membranes, transfer lipids between membranes and regulate lipid metabolism. A novel role for various membrane lipids in autophagosome formation is also emerging. Here, we highlight past and recent key findings on the function of ATG proteins related to autophagosome biogenesis and consider how ATG proteins control this dynamic membrane formation event to organize the autophagosome by collaborating with membrane lipids.


Introduction
Under nutrient limited conditions cells survive by degrading their cellular contents to maintain homeostasis, energy levels, and building blocks. Macro-autophagy (hereafter, autophagy) is an intracellular degradation system that delivers cytoplasmic materials to the lysosome/vacuole. In response to autophagy induction, a cupshaped membrane structure termed the phagophore (also known as the isolation membrane) appears in the cytoplasm. Once the phagophore elongates enough to accommodate its substrates, it closes and seals to form a double membrane structure, called an autophagosome (Fig. 1a). Initially autophagy was mainly analyzed in rat liver hepatocytes by electron microscopy 1-3 and biochemical enzyme assays 4,5 . However, after the discovery of autophagy in the yeast Saccharomyces cerevisiae 6 , and the isolation of the yeast ATG (autophagy-related) genes [7][8][9] , research into autophagy has been transformed from a descriptive phenomenon into a biochemical and molecular field using model organisms, cell culture systems and in vitro reconstitution approaches. Core Atg/ATG proteins are functionally categorized into discrete units: the Atg1/ULK complex, the class III phosphatidylinositol 3-kinase (PI3K) complex, the Atg2-Atg18/WIPI4 complex, Atg9 vesicle, the Atg12 conjugation system, including ATG12-5-16L1 and WIPI2B, and the Atg8/LC3 conjugation system [10][11][12] . This molecular understanding of the autophagic machinery has led to the discovery of its importance in tumorigenesis 13 , mammalian development 14,15 , lipid metabolism 16 , degradation of intracellular pathogens 17 , and neurodegeneration 18,19 . In recent years, diverse physiological and pathological roles of autophagy have been uncovered 20 .
Autophagosome formation is driven by the ATG proteins, but during this dynamic membrane remodeling lipids are major constituents of autophagic membranes. Although the lipid composition of autophagosomes remains obscure, phosphatidylinositol 3-phosphate (PI3P) and phosphatidylethanolamine (PE) are crucial for autophagosome formation. The role of PI3P in autophagy was guided by a study showing the inhibitory activity of 3-methyladenine in rat hepatocytes 21 . Subsequently, it was shown that class III PI3K is a target of 3methyladenine 22,23 . In line with this, Vps34 was  32,[34][35][36]48,56,58,97,127,161,172 and yeast cells (c) 33,46,47,55,126,130,154,155 . Their localizations are categorized into five groups: -, not detectable; -/+, transient; +, weakly detectable; ++, easily detectable; +++, clearly detectable. Note that ATG/ Atg proteins show punctate structures on the ER-related membranes rather than a typical ER-like pattern. ERES ER exit sites, ERGIC ER-Golgi intermediate compartment.
identified as a PI3K in yeast 24 and mammals 25 , and a binding partner of Vps30/Atg6 26 . Finally, the Vps34-Vps15-Vps30/Atg6-Atg14 complex was characterized as an autophagy-specific class III PI3K complex 26 . PI3P is a minor lipid, but its formation is crucial for membrane recruitment of ATG proteins and the early stage of autophagosome formation (Fig. 1a). In contrast, PE is a major phospholipid in eukaryotic cells. Under starved conditions, Atg8/LC3/GABARAP proteins are bound to autophagic membranes through covalent conjugation to PE (Fig. 1a), which is a hallmark of autophagy 27 . Although Atg8/LC3/GABARAP proteins can be also conjugated to phosphatidylserine (PS) in vitro, PE is the major target of Atg8/LC3/GABARAP conjugation in vivo 28,29 .
Considering ATG protein recruitment to autophagic membranes is both spatially and temporally controlled, changes in the amount of PI3P and PE on autophagic membranes are unlikely to fully explain how ATG proteins execute their functions and accomplish autophagosome biogenesis. Here, we review key studies and recent findings on the molecular machinery underlying autophagosome formation, and focus on the recent literature investigating the co-operation of ATG proteins with membrane lipids during autophagy, and the emerging roles of membrane lipids in autophagosome formation.
The ULK/Atg1 complex: an initiator of autophagosome formation The ULK/Atg1 complex is one of the earliest factors to target to membrane structures at the phagophore formation site (Fig. 1b, c). This complex composed of four components (ULK1/2, ATG13, FIP200/RB1CC1, and ATG101) in mammals (Fig. 2a), and five components (Atg1, Atg13, Atg17, Atg29, and Atg31) in yeast (Fig. 2b), is a pivotal signal transducer of autophagy. So far, the identification of ULK/Atg1 substrates has been largely confined to ones involved in autophagy 30,31 . There is accumulating evidence showing that membrane recruitment of ULK/Atg1 complex at the initiation stage does not require downstream ATG proteins. In genetic hierarchical analyses, the puncta formation of ULK/Atg1 complex is independent of other Atg complexes in both yeast and mammalian cells 32,33 . A comprehensive biochemical analysis has shown that ULK complex components are bound to membranes even in the absence of downstream ATG proteins 34 . Importantly, under starved conditions the formation of the ULK complex puncta can occur during inhibition of PI3P synthesis although the size and lifespan of puncta are reduced [34][35][36] . Thus, the ULK/Atg1 complex is first recruited to membrane structures independently of PI3P and downstream ATG proteins, and then its membrane localization is stabilized by PI3P 36 . Yet, which components of the ULK/Atg1 complex act as determinants for membrane recruitment is unclear.
To date, there have been several reports on the direct membrane association of the ULK/Atg1 complex components. A putative lipid-binding domain has been identified in ULK/Atg1 proteins. The C-terminus of ULK1 associates with cell membranes (Fig. 2a, red line) 37 , and in line with this, the Atg1 EAT domain (Fig. 2b, red line), a region that corresponds to the ULK1 C-terminus, binds to liposomes with a preference for small highly curved vesicles 38,39 , suggesting that the ULK/Atg1 C-terminus serves as a membrane curvature sensor. However, the Atg1 EAT domain needs to interact with Atg13 to induce autophagy 40,41 , which hinders the membrane association of Atg1 EAT domain 38 . Therefore, it is unclear if the Atg1 EAT domain in fully assembled Atg1 complex can associate with membranes. A recent paper has proposed that the Atg13-free Atg1 EAT domain is involved in later stages of autophagosome biogenesis 42 .
ATG13/Atg13 also has a putative lipid-binding region. Membrane binding of human ATG13 is regulated by a cluster of arginine/lysine residues at the N-terminus (Fig. 2a, blue line) via an electrostatic interaction with negatively charged phospholipids, such as phosphatidic acid and phosphatidylinositol phosphates (PIPs) 36 . Similarly, the C-terminus of yeast Atg13 has positively charged residues (Fig. 2b, blue line) and directly binds to liposomes containing negatively charged lipids in vitro. Thus, the lipid binding ability of Atg13 seems to be conserved from yeast to human, although the domain carrying the cluster of arginine/lysine residues is not conserved 39,43 .
Secretory pathway proteins can participate in membrane targeting of the ULK/Atg1 complex components. In yeast, a Rab GTPase Ypt1, which is essential for ER-Golgi and Golgi traffic, recruits Atg1 to the preautophagosomal structures (PAS) via an interaction with the N-terminus of Atg1 (Fig. 2b), although other Atg1 complex components, Atg13 and Atg17, are localized to the PAS independently of Ypt1 44 . The initiation step has also been spatially linked to COPII vesicles, which mediate ER-to-Golgi transport [45][46][47][48] . In mammalian cells, SEC12, the activator of COPII assembly, is associated with the FIP200 C-terminus, although this interaction is mainly required for FIP200 function in the remodeling of ER exit sites (ERES), specialized ER regions for COPII vesicle formation 48 . A recent study has revealed that ULK1 and FIP200 interact with integral ER proteins, VAP proteins (VAPA and VAPB), which can tether the ER to other cell membranes at membrane contact sites 49 . VAP proteins directly bind FFAT (two phenylalanines in an acidic tract) motifcontaining proteins to the ER 50 . Consistent with this, ULK1 and FIP200 have functional FFAT motifs, suggesting that membrane association of ULK1 and FIP200 is regulated by VAP proteins. Therefore, Ypt1, SEC12, and VAPs facilitate the initiation of autophagy by recruitment of the ULK complex components. However, given that autophagic phenotypes caused by the inhibition of these interactions are partial 44,48,49 , we suggest the leading players for the recruitment of the Atg1/ULK complex are still missing (Fig. 2c).
Late stage recruitment of ULK/Atg1 complex during autophagosome formation requires LC3/GABARAP/Atg8 proteins. ULK/Atg1 complex components have a LC3/ GABARAP/ATG8-interacting (LIR/AIM) motif and bind to ATG8 family proteins (Fig. 2a, b) [51][52][53] . These interactions have been proposed to be involved in autophagosome maturation and/or a negative feedback by degrading the ULK/Atg1 complex via autophagy. Atg9 vesicles: a membrane source for autophagosome formation ATG9A/Atg9 is a multi-spanning membrane protein essential for the initiation of autophagosome formation (Fig. 1a). ATG9A/Atg9 cycles between different organelle compartments via vesicular transport pathways and delivered to the autophagosome formation site in response to induction of autophagy. In yeast Atg9 localizes to vesicular and tubular structures at the PAS under starved condition 54 . Atg9-containing vesicles, the diameter of which are 30-60 nm, are highly mobile within the cytoplasm 55 . As a small part of yeast Atg9 localizes to the autophagosomal outer membrane, it has been proposed that Atg9 vesicles become a seed membrane for phagophore formation in yeast 55 . In contrast, mammalian ATG9A is not obviously incorporated into autophagosomal membranes. Rather, ATG9A is found on clusters of vesicles and/or tubules in the vicinity of phagophores and transiently associated with the autophagosomal membranes 32,56-58 . Accordingly, it is thought that ATG9A supplies key components, such as proteins and lipids, to the autophagosomal membranes by transient association.
Despite the apparent differences in the localization of ATG9A during autophagy initiation, the two models proposed in yeast and mammalian cells are not mutually exclusive.
Recently, the machinery sorting ATG9A/Atg9 to different locations in the cell has become increasingly clear. Atg9 distribution is partly regulated by the Atg1 complex via its physical interactions with other Atg proteins (Fig. 3a). Cytoplasmic regions of Atg9 interact with Atg17 39,59 . The N-terminus of Atg9 also binds to Atg13 HORMA domain, which facilitates recruitment of Atg9 vesicles to the PAS 60 . Atg9 is phosphorylated by Atg1 kinase, and this phosphorylation is required for recruitment of downstream factors such as Atg18 and Atg2, while it does not affect the PAS recruitment of Atg9 61 . As in yeast, the trafficking of ATG9A is dependent on the ULK complex 37,62,63 although further analysis is needed to confirm whether ATG9A directly interacts with the ULK complex.
The essential role of ATG9A/Atg9 in autophagy remains elusive, but a recent study has revealed a close relationship between ATG9A vesicle and PI4P metabolism 81 . The BAR-domain-containing protein Arfaptin-2 and two PI4Ks (PI4KIIα and PI4KIIIβ) have been identified as components of ATG9A vesicles. ATG9A vesicles transport PI4KIIIβ to the ER membrane promoting PI4P production at the initiation site, which facilitates recruitment of the ULK complex and initiation of autophagy. In this model, Arfaptin-2 has been proposed to serve as a regulator of ATG9A vesicles by modulating ATG9A exit from the Golgi complex 81 . PI4KIIα may also be delivered by ATG9A vesicles to provide PI4P for later stages of autophagosome maturation 82 . Thus, a key role of ATG9A in autophagy may be to supply PI4P to autophagosomal membranes. Very recently, using single-particle cryoelectron microscopy the structure of Arabidopsis thaliana ATG9 was reported at sub-nanometer resolution 83 . Future structural studies at even higher resolution are needed to reveal some of the unresolved roles of ATG9A/ Atg9.
Recent studies by single-particle electron microscopy and crystal structural analysis have provided many insights into how lipid-binding domains individually contribute to the membrane association of the PI3KC3-C1 complex 95,98,99 . The PI3KC3-C1 complex demonstrates a two-armed V-shaped architecture (Fig. 4c). One arm contains the helical and lipid kinase domains of Vps34/VPS34 and the Vps15/VPS15 N-terminal myristoylation site. The other arm includes the C-terminal domains of Vps30/BECN1 and Atg14/ATG14L, corresponding to BARA and BATS domains, respectively 95,98 .
The crystal structure of Drosophila melanogaster Vps34 revealed a C-terminal loop region can interact with membranes and allow Vps34 activation 93 . The VPS34 Cterminus is regulated by acetylation at K771, which hinders the affinity of VPS34 for its substrate PI 100 . Aromatic residues in a surface loop of the BARA domain of Vps30/ BECLIN1 serve as a hydrophobic finger to mediate direct association with membranes 94,95,101,102 . VPS34 catalytic site geometry is strongly influenced by the presence of VPS15, indicating that VPS15 has a central role in scaffolding complex assembly 103 . Accordingly, it is thought that the PI3KC3-C1 complex is associated with membranes via the tips of the two arms of the PI3KC3-C1 complex carrying the Vps34/VPS34 C-terminus, the myristoylated Vps15/VPS15 N-terminus, the aromatic finger in Vps30/BECN1 and the ATG14L C-terminal  BATS domain (Fig. 4c). The BATS domain binds small liposomes containing PI3P or PI(4,5)P 2 and senses membrane curvature via an amphipathic helix loop (ALPS motif, Fig. 4a) 96 . Thus, the VPS34 C-terminus determines the orientation of the PI3KC3-C1 complex, while ATG14L1 BATS domain is critical to sense membrane curvature and mediate the lipid specificity to target membranes 95,99 .
The PI3KC3-C1 complex components also contain functional LIR motifs and interact preferentially with GABARAP and GABARAPL1 (Fig. 4a). As the LIR motif in ATG14L is in close apposition to its BATS domain, these two motifs might work as a coincidence detector for specific targeting of autophagic membranes 104 .

Atg18/WIPI proteins: a transmitter of PI3P signals
Atg18/Atg21/WIPI proteins belong to the PROPPINs (β-propellers that bind polyphosphoinositides) family and work as PI3P effectors in autophagy. Atg21 is likely to be restricted to yeast and function mainly in selective autophagy 105,106 . A major role of Atg18/WIPI proteins is to transmit PI3P signals to downstream ATG proteins (Fig. 5a). In yeast, Atg18 interacts with Atg2 and regulates PAS recruitment of Atg2 33,107 . In mammals, the Atg18 homolog WIPI4 interacts with ATG2 proteins [108][109][110] . WIPI2B most prominently functions in LC3 lipidation in mammals 111 , via its unique interaction with ATG16L1 10 . Thus, individual WIPI proteins have some preferences for their binding partners. This is the case also for C.elegans: the WIPI3/4 ortholog EPG-6 binds to ATG-2, while the WIPI1/2 ortholog ATG-18 does not 112 . As C. elegans atg-18 and epg-6 mutants show different autophagic defects, it is thought that they act at distinct steps 112 . These functional differences of Atg18/WIPI proteins in autophagy are presumably caused by differences in their binding partners 10,109,112 .
Atg18/WIPI proteins target to autophagic membranes by directly interacting with PI3P via a conserved FRRG motif (Fig. 5a, FRRG or LRRG) 113,114 . The PI3P binding of Atg18 proteins is required for full autophagic activity 107,115 . Structural analyses have revealed that Atg18 proteins fold into a seven-bladed β-propeller and contain two PI3P binding pockets at blades 5 and 6 which are composed of two arginine residues located in the conserved FRRG motif 116,117 . As Atg2/ATG2-binding and Atg16L1-binding sites are located at the opposite surface from the FRRG motif 10,108,110,118,119 , this architecture enables Atg18/WIPIs to interact with PI3P and downstream ATG proteins simultaneously.
In addition, a hydrophobic loop in blade 6 serves as a membrane anchor by inserting deeply into the lipid bilayer (Fig. 5a, 6CD loop) 117,120 . In Komagataella phaffii (previously known as Pichia pastoris), PIPs binding of PpAtg18 is modulated by phosphorylation of the loop region 121 . Of note, this loop region of yeast Atg18 can fold into an amphipathic α-helix, and is likely to be well conserved among Atg18/WIPI homologs 122 . The property of the loop might be one of main reasons why Atg18/ WIPI proteins preferentially bind to smaller liposomes in vitro 120 and localize to the edge of the phagophore in vivo 46 . Thus, direct membrane association of Atg18/ WIPI proteins is controlled by two factors, membrane anchoring via the hydrophobic loop in blade 6 and PI3Pbinding via the FRRG motif 117,120 . Furthermore, Atg2-Atg18 complex formation 107,119,123 and Atg18/WIPI oligomerization 109,124 can further stabilize the membrane association of Atg18/WIPI proteins.

Atg2/ATG2: a membrane tether and lipid transfer protein
The precise role of Atg2/ATG2 in autophagy has recently been established. Single particle EM analyses have resolved the architecture of Atg2-Atg18 and ATG2A/B-WIPI4 complexes 108,110 . Atg2/ATG2 and Atg18/WIPI4 demonstrate a rod-shaped and a globular structure, respectively. Atg2 has membrane-binding regions at both ends of the rod structure (Fig. 5b, red lines), the length of which is about~200 Å (Fig.  5c) 110,125,126 , and can tether small liposomes in vitro 110,126 . WIPI4 is stably attached to one end of the ATG2 rod. In a complex with WIPI4, ATG2A can tether a PI3P-containing vesicle to another PI3P-free vesicle 110 . In line with this, high-resolution microscopy analyses have shown that Atg2/ATG2 localizes to the edge of the phagophore in close apposition to the ERES in yeast 46,47 and contact sites between ER and autophagosomal membranes in mammalian cells 127 . The N-terminal 46 residues of Atg2 localizes to the ER, and the membrane-binding region in the C-terminal region is required for the targeting of the Atg2-Atg18 complex to the PAS 126 . Furthermore, both N-terminal and C-terminal regions of Atg2 are required to restore autophagy deficiency in atg2△ cells. Thus, Atg2/ATG2 serves as a tether for early autophagic structures to the ER membranes by collaborating with Atg18 in yeast and WIPI4 in mammals 110,126 . Yet, the contribution of WIPI4 is limited in mammals 128,129 . Instead, a functional LIR/GIM (LC3/ GABARAP-interacting motif) has been found in ATG2, which is indispensable for autophagy 129 . Atg9 also facilitates Atg2-dependent contact site formation in yeast 130 . Thus, multiple factors contribute to Atg2/ATG2-dependent tethering activity.
In addition to the tethering function of Atg2/ATG2, it possesses lipid transfer activity. Recently, the crystal structure of the N-terminus of Atg2 has been solved revealing a tubular architecture with a hydrophobic cavity that can harbor tens of glycerophospholipids at once (Fig. 5b, green lines) 131 , and can transfer phospholipids with little head group specificity using in vitro liposome assays 127,131 . The Atg2/ATG2-dependent lipid transfer depends on packing defects and negatively charged membranes 123,131 . In line with these in vitro data, the Nterminal region of Atg2/ATG2 is essential for autophagic flux in both yeast and mammalian cells [125][126][127]131 . Surprisingly, overexpression of the Atg2 N-terminus can restore autophagy deficiency in ATG2A/B DKO cells 127 , suggesting that the tethering function of Atg2/ATG2 can be rescued by overexpression of the lipid transfer domain. Given that the Vps13 N-terminus, a homologous lipid transfer domain 132 , can be substituted for the corresponding region of Atg2 during autophagy 131 , the major function of Atg2/ATG2 in autophagy is lipid transport. How Atg2/ATG2 proteins accomplish unidirectional lipid transport and grow the phagophore membranes are key issues to be solved (Fig. 5c).
More recently, ATG16L1 was found to have the ability to bind lipids. ATG16L1 has three membrane-binding domains, in the N-terminal region, the coiled-coil domain (CCD) and the β-isoform-specific region. The N-terminal region, which contains an amphipathic helix (Fig. 6b, red lines), is universally required for the lipidation of LC3 family proteins, while the β-isoform-specific region (Fig. 6b, purple lines) is essential for VPS34independent LC3 lipidation at perturbed endosomes 149 , while the CCD has an intrinsic ability to bind lipids that is also required for LC3 lipidation (Fig. 6b, green lines) 150 . In summary, the Atg16/ATG16L1 complex is recruited to the target membranes through multiple-interacting partners, including both proteins and lipids. These dependencies can be changed in response to a physiological context 149-151 . Atg3/ATG3: an executor of Atg8/LC3 lipidation Atg3/ATG3 acts as the E2-like enzyme that catalyzes lipidation of Atg8/LC3 family proteins by localizing to the PAS and the phagophore (Fig. 6a) [152][153][154][155] . An amphipathic helix found in the N-terminus of Atg3 is critical for its association with membranes and Atg8/LC3 lipidation (Fig. 6c, red lines) [156][157][158] . This region is involved in the preferential association of Atg3/ATG3 with membranes containing high curvatures and/or conical lipids, such as PE 156 . On the other hand, lipidation of Atg8/LC3 family proteins is stimulated by acidic phospholipids in vitro 29,158 . This is probably due to the fact that membrane association of ATG3 relies on membrane charge in addition to lipid packing defects, which lead to cavities for the interaction of peripheral proteins 158 . Atg3 localization is also regulated by its interaction with Atg8 as a positive feedback to facilitate Atg8 lipidation 155 . In addition, acetylation at K19 and K48 of Atg3 significantly enhances the membrane association of Atg3 in the presence of physiological level of PE 159,160 . Overall, these multiple factors mediate Atg3 membrane targeting in vivo.

The ER membrane: a platform for autophagosome formation
The membrane origins and sources for the autophagosome remain under debate, as several membrane sources have been proposed in mammals, including the ER 161-163 , mitochondria 164 , the PM 165 , the ERGIC 166 , the recycling endosome 167,168 , and lipid droplets (LD) 169 . Among these membrane sources it is becoming clear that the ERrelated membranes are the primary membrane source and serve as a platform for autophagosome formation. In support of evidence showing the contribution of the ER membranes, omegasomes have been well characterized as an autophagy-related ER structure (ER subdomain): it is a PI3P-enriched membrane labeled by DFCP1, formed in response to starvation and dynamically connected to the ER and the phagophores 161 . Membrane contacts between the ER and phagophore have been observed by electron microscopy. Rough ER membranes are attached to both the outer and inner surfaces of cup-shaped phagophores 162,163 . Correlative light and electron microscopy (CLEM) and correlative cryo-fluorescence and cryo-soft X-ray microscopy (cryo-CLXM) have shown that omegasomes are composed of clusters of tubular/vesicular elements, part of which are continuous with the phagophores and/or the ER membranes 170,171 . These ER subdomains may also form organelle contact sites between the ER and other compartments, such as the mitochondria 172 , LDs 173 , and the PM 174 . It has been also shown that the edge regions of phagophore membranes are attached to the ERES [46][47][48]175 and that COPII coat subunits are required for autophagosome formation 45,176,177 and a COPII cargo protein is incorporated into autophagic membranes 178 , suggesting ER-derived COPII vesicles could contribute to autophagic membranes.
ATG proteins are sequentially recruited to the ER and autophagic membranes in response to autophagy induction (Fig. 1b, c). Their localization is changed depending on different stages of autophagosome formation. In mammalian cells, the ULK complex is first recruited to the ER membrane to initiate autophagy. At middle and late stages of autophagosome formation, the ULK complex also localizes to the omegasome, the phagophore and autophagosome 32,34 . ATG14L, a component of mammalian PI3KC3-C1, targets to both the ER-related membranes and phagophore to generate PI3P 96,97,172 , while the lipid transfer protein ATG2 is specifically localized to the ER and phagophore contact sites 127 . The unique localization of ATG2 presumably reflects its dual membrane targeting and tethering function. WIPI2B and ATG16L1 are mainly localized to the phagophore dependently on PI3P under starvation condition 10 . Given that it has been proposed that the ERGIC is a key membrane source for LC3 lipidation 166 , ATG12-5-16L1 and ATG3 are thought to be recruited to the ERGIC in addition to the phagophore membrane to execute LC3 lipidation. Similarly, yeast Atg proteins are also distributed to distinct compartments during autophagosome formation. Atg1 complex, Atg14, Atg12-5-16 complex, and Atg3 localize to the phagophore 46,47,154,155 , while Atg2, Atg18, and Atg9 are enriched at the edge regions of phagophore membranes, which is close apposition to the ERES 46,47,130 . On the other hand, there are some differences between yeast and mammalian cells. There are no counterparts to ERGIC and the omegasomes in yeast, but instead the phagophore is formed in the vicinity of the ERES and the vacuole 46,47,130 . Some Atg proteins such as Atg13, Atg17, and Atg14 are distributed to the vacuole and phagophore contact sites 46 . Further analyses are needed to reveal how the complex membrane targeting of Atg proteins is achieved in yeast and mammalian cells.
Membrane recruitment of the ULK complex precedes omegasome formation in mammalian cells. In highresolution analysis using super-resolution microscopy, the initiation of autophagosome formation occurs in regions of the ER, where the ULK complex and the ATG9A vesicles are associated 58 . Interestingly, ERlocalized phospholipid synthesizing enzymes, such as PI synthase and PS synthase, are enriched in close proximity to the autophagosome initiation site 34 , implying a close relationship between phospholipid synthesis and autophagosome formation. The enrichment of these enzymes might facilitate PI3P generation and ATG2-dependent lipid transfer. VMP1 and TMEM41B: a key regulator of ion homeostasis during autophagosome formation? VMP1 (also known as TMEM49) is a multi-spanning membrane protein localized at the ER and required for autophagosome formation. The VMP1 gene is absent in yeast, but conserved in most higher eukaryotes. Given its ER localization, VMP1 has been thought to be a key player in an ER-related event essential for autophagosome formation. Although VMP1-GFP puncta have been observed after overexpression, this puncta formation is not essential for the autophagy function of VMP1 179,180 . Impaired VMP1 function causes not only accumulation of abnormal autophagic structures 181,182 , but also pleiotropic effects, such as protein secretion defects 183,184 , impaired lipoprotein secretion 185 , abnormal distribution of PI4P and phospholipid metabolizing enzymes 186 , accumulation of lipid droplets 179,187 , and enhanced ER-organelle contact sites 179,187 . Accordingly, VMP1 function is not limited to autophagy, and the molecular mechanism underlying the abnormalities of VMP1-deficient cells remains unclear.
A recent study has revealed a new functional link between VMP1 and a calcium pump SERCA (sarcoendoplasmic reticulum calcium transport ATPase). VMP1 interacts with SERCAs that transport cytosolic Ca 2+ into the ER lumen and positively regulates their Ca 2 + -ATPase activity, suggesting that VMP1 coordinates multiple organelle contact sites by maintaining local Ca 2 + levels via SERCA activity regulation 179 . More detail about VMP1 function has been provided by studies on TMEM41B, a newly identified autophagy-related gene in genome-wide CRISPR screens 180,188,189 . Interestingly, TMEM41B localizes to the ER and shares a similar protein structure with VMP1. As TMEM41B and VMP1 form a complex, and overexpression of VMP1 restores impaired autophagic flux in TMEM41B KO cells, it has been proposed that they may be half-transporters and function together in autophagy as a full transporter by forming a complex. More work is needed to support this exciting hypothesis and the role of VMP1 and TMEM41B in ion homeostasis during autophagosome formation.

Emerging roles of lipid metabolism in autophagosome biogenesis
There is accumulating evidence that autophagy can be regulated by sphingolipids [190][191][192] . Treatment with a shortchain ceramide (C2-ceramide) is able to induce autophagy in various different cell lines, such as human cervical cancer cells, colon cancer cells, and malignant glioma cells [193][194][195][196][197] . Consistent with these reports, de novo synthesis of ceramide is required for the induction of autophagy in stimulated RAW264.7 macrophage cells 198 and the mouse liver 199 , as well as in yeast 200 . The proautophagic effects by ceramide were proposed to be caused by upregulation of BECN1 activity and/or interfering with class I PI3K/Akt signaling pathway 194,197 , yet the molecular details remain to be elucidated. Sphingosine 1-phosphate (S1P) is a simple lysophospholipid known to promote cell survival. Recently, the diverse roles of S1P-metabolizing enzymes in autophagy are being discovered. Overexpression of sphingosine kinase 1 (SK1), which generates S1P from sphingosine, stimulates autophagy in MCF-7 cells and primary neurons 201,202 . Depletion of S1P phosphohydrolase 1, which mediates degradation of S1P by dephosphorylation, results in the induction of autophagy, suggesting that accumulation of S1P can promote autophagy 203 . S1P not only binds to S1P receptors at the cell surface 204,205 , but also acts on intracellular membranes 202,203 , but how intracellular S1P works on autophagy remains unclear. Notably, S1P is cleaved by SGPL1 (sphingosine phosphate lyase 1) into hexadecenal and ethanolamine phosphate, which can be consumed for the synthesis of PE. Given that autophagosome formation is compromised in SGPL1-deficient brains 206 , PE generated from the S1P degradation products might play a key role in autophagy in neurons. Sphingomyelin is also involved in autophagy. Overloading of sphingomyelin affects ATG9A transport and induces accumulation of immature autophagosomes 207 . The increase of ceramide 1-phosphate (C1P) at the Golgi induced by CPTP (C1P transfer protein) knockdown also affects ATG9A distribution 208 . Neutral sphingomyelinase 2 induces autophagy by increasing ceramide levels in the Golgi 209 .
The effect of fatty acids on autophagy has drawn some attention in recent years. Saturated fatty acid palmitate induces autophagy in several cell lines [210][211][212] , although it has an inhibitory effect in different experimental conditions 213 . Mono-unsaturated (oleate) and ω-6 polyunsaturated fatty acids (arachidonic acid) also activate autophagy [214][215][216] . Yet, it is reported that the underlying mechanisms of saturated fatty acids-and monounsaturated fatty acids-induced autophagy are not the same 216 . On the other hand, stearoyl-CoA desaturase, an enzyme generating mono-unsaturated fatty acids, is indispensable for efficient autophagosome formation [217][218][219] . Therefore, the synthetic pathway of mono-unsaturated fatty acids also contributes to autophagy. Finally, a recent study has shown that trans-unsaturated fatty acids inhibit autophagy induced by saturated fatty acids 220 , implying complicated connections between autophagic effects caused by different types of fatty acids. Further analyses are needed to elucidate how individual fatty acids act on autophagy.
Autophagosome formation can be affected by changes in phospholipid metabolism. PLD1 hydrolyzes phosphatidylcholine (PC) to generate phosphatidic acid (PA). In mammalian cells, PLD1 localizes to autophagosomerelated structures and facilitates autophagy [221][222][223] . It has been proposed that its product PA plays a key role in autophagosome formation 221,224 and/or lysosomal functions 223 . However, contrary to these findings, another study has shown that the inhibition of PLD1 results in an enhancement of autophagic flux 225 . Therefore, PLD1 might function as both positive and negative regulator of autophagy in a context-dependent manner. On the other hand, a role for lipid droplets (LDs) in autophagosome biogenesis has been reported. LDs are made of a hydrophobic core of neutral lipids and a surrounding phospholipid monolayer. In mammalian cells, a neutral lipase PNPLA5 positively regulates autophagy 169 . In line with this study, autophagy is impaired upon nitrogen starvation in LD-deficient yeast mutant 173,226 . Interestingly, a recent study has found that the autophagy deficiency in the LDdeficient yeast mutant can be improved by suppressing fatty acid synthesis or by restoring the altered phospholipid composition, indicating that LDs are not necessarily essential for autophagosome biogenesis, and that LDs play a key role in autophagy regulation by relieving metabolic stress caused by excess fatty acid synthesis and altered phospholipid composition 227 .

Concluding remarks
Thanks to the tremendous development of novel techniques, our knowledge of the molecular machinery underlying autophagosome formation has been expanded. Of note, accumulating evidence suggest that ATG proteins are directly involved in membrane lipid dynamics and organization during autophagy. While these findings have pushed the field forward, our current knowledge of lipid composition and distribution in autophagic membranes is very limited 228 . Therefore, advanced techniques to detect and evaluate lipid distribution and changes in lipid composition in vivo need to be further developed 229,230 . In vitro reconstitution 231 , in silico simulation 232 and theoretical analysis 233 are also required to obtain deeper insights into the relationship between ATG proteins and membrane lipids. In summary, it will be important to reveal how ATG proteins organize membrane lipids for understanding the detailed mechanisms of autophagosome biogenesis.