Autophagy is a conserved survival pathway, which cells and tissues will activate during times of stress. It is characterized by the formation of double-membrane vesicles called autophagosomes inside the cytoplasm. The molecular mechanisms and the signalling components involved require specific control to ensure correct activation. The present chapter describes the formation of autophagosomes from within omegasomes, newly identified membrane compartments enriched in PI3P (phosphatidylinositol 3-phosphate) that serve as platforms for the formation of at least some autophagosomes. We discuss the signalling events required to nucleate the formation of omegasomes as well as the protein complexes involved.
- ULK complex
Autophagy is a catabolic process in which proteins and organelles are sequestered into double-membrane vesicles for degradation in lysosomes. It is conserved in all organisms, from simple eukaryotes such as yeast to higher eukaryotes such as humans, where it has developed multiple functions. All cells have a basal level of autophagy that has a housekeeping role and helps eliminate harmful material. However, the levels of autophagy can increase dramatically in response to changes of the cellular environment such as nutrient deprivation or invasion of a pathogen. Three forms of autophagy are described which are termed macroautophagy, microautophagy and chaperone-mediated autophagy. Macroautophagy, commonly referred to as autophagy, is the subject of the present chapter. It involves the formation of double-membrane vesicles known as autophagosomes, which fuse with lysosomes. Autophagy is a survival mechanism which cells activate during times of stress and it is antagonistic to apoptosis.
Much of what is known about the core machinery of autophagy was discovered in yeast, and genes that are involved in this pathway are termed Atg (autophagy-related) . The proteins coded by these genes are organized into distinct complexes that are involved at specific stages of the process. In broad terms, formation of autophagosomes involves an initiation stage, a nucleation stage, an elongation stage and finally closure of the double-membrane structure to form an autophagosome. The master regulator through which many signals converge for the initiation of autophagy is the mTOR (mechanistic target of rapamycin; also known as mammalian target of rapamycin) protein kinase [2–5]. Under nutrient-rich growth conditions, mTOR is active and autophagy is suppressed by the mTOR-induced phosphorylation of Atg1, the only kinase among the Atg proteins. mTOR can form two complexes (mTORC1 and mTORC2), which differ in sensitivity to rapamycin and in subunit composition. The subunits in each of these complexes that determine the sensitivity to rapamycin are known as raptor (regulatory-associated protein of mTOR) for mTORC1 and rictor (rapamycin-insensitive companion of mTOR) for mTORC2. mTORC1 is the complex that directly regulates the autophagic pathway. Upon starvation, or in response to other stimuli, mTORC1 is inactivated which rapidly results in dephosphorylation of Atg1 and the induction of autophagy. In mammalian cells, there are two Atg1 homologues known as ULK (uncoordinated-51-like kinase) 1 and ULK2. There is some redundancy between the two ULKs with ULK1 having the main role . ULK is one of the earliest proteins activated during the induction of autophagy, and is in complex with Atg13, Atg101 and FIP200 (focal adhesion kinase family-interacting protein 200 kDa) (the ULK complex). This ULK complex is essential for autophagosome formation and is thought to interact directly with mTOR under nutrient-rich conditions. During starvation, the ULK complex dissociates from mTOR and can be recruited to the site of autophagosome formation  (Figure 1). Concomitant to the physical disengagement from mTOR, a series of phosphorylation/dephosphorylation events involving ULK1, Atg13 and FIP200 ensures that the complex arriving at the autophagosome formation site is competent for the downstream steps. Recently, it has been shown that AMPK (AMP-activated protein kinase) can also play a role in the regulation of ULK during autophagy. The evidence implies that AMPK directly interacts with the ULK complex and phosphorylates several sites important for ULK activation. This is enhanced by mTOR inhibitors such as rapamycin, indicating that mTORC1 negatively regulates this association. Regulation of autophagy by both mTORC1 and AMPK allows the cells to respond to a wide range of stimuli [7,8].
Autophagosomes in yeast form at a site known as the PAS (phagophore assembly site), whereas in mammalian cells there are multiple such sites at any one time . Once the ULK complex is at the PAS, it helps recruit the next complex in the pathway, namely the Vps34 complex I. Figure 1 highlights the key components required for the formation of the autophagosomes, and involvement of the ULK complex and Vps34 complex I.
The Vps34 complex I consists of Atg14 L, Beclin1 (Atg6), Vps34 [class III PI3K (phosphoinositide 3-kinase)] and Vps15. The lipid kinase Vps34 is crucial in producing the lipid signalling molecule PI3P (phosphatidylinositol 3-phosphate) resulting in recruitment of other Atg proteins and allowing for expansion of the autophagosomal membrane [1,2,5]. Membrane expansion is mediated by the actions of the Atg5–Atg12–Atg16 complex, and by vesicles containing Atg9 and cycling between the PAS and organelles. The Atg5–Atg12–Atg16 complex mediates the conjugation of Atg8, known in mammalian cells as MAP1LC3 (microtubule-associated protein 1 light-chain 3) or simply LC3, with PE (phosphatidylethanolamine) as it becomes incorporated into the forming autophagosomes. LC3 is a very abundant protein of the autophagosome inner and outer membrane and remains associated with autophagosomes until their degradation in lysosomes [3,5]. As the autophagosomal membrane expands and begins to close on itself, it also sequesters within it cytoplasmic material (proteins or whole organelles) destined for delivery to lysosomes for degradation.
Many EM (electron microscopy) studies looking at autophagosome formation concluded that the membranes must be related to the ER (endoplasmic reticulum) [9,10], but clear ER-derived markers that become part of the autophagosomal membrane are lacking. Previous work from our group proposed the idea that specialized membrane compartments linked to the ER and enriched in PI3P (termed omegasomes due to their Ω shape) may be sites of autophagosome formation . A novel PI3P-binding protein named DFCP1 (double FYVE domain-containing protein 1) was used to illustrate the relationship between the ER, omegasomes and autophagosome formation. DFCP1 has an ER targeting domain and two FYVE domains, localizing to the ER and Golgi, instead of endosomes  (Figure 2A). During starvation, DFCP1 translocates from the cytosol to distinct puncta throughout the cytoplasm (Figure 2B) which partially colocalize with LC3 and Atg5 and are identified as sites of autophagosome formation. The translocation of DFCP1 could be blocked by PI3K inhibitors such as wortmannin or 3-methyladenine, or by using siRNA to knock down levels of Vps34 and Beclin1, indicating that it requires formation of PI3P. It is also apparent that omegasome expansion is seen in close apposition to Vps34-containing vesicles, providing a potential way for the delivery of the PI3P-synthesizing machinery to these sites (Figure 2C). These DFCP1-containing puncta are clearly connected to the ER and their dynamic behaviour during autophagy is reflected in changes to the underlying ER structure. Figure 2(D) illustrates that as omegasomes form and expand (green colour) the underlying ER also expands (blue colour) leading to an almost complete colocalization. In terms of the omegasome and LC3 dynamics, it is apparent that LC3 puncta (red colour) form within omegasomes and at the point of maximal expansion the LC3 membranes exit omegasomes accompanied by gradual diminishment of the omegasome signal. The LC3 particle exiting omegasomes requires 3–5 min to mature and become acidic, supporting the idea that autophagosomes are not mature when leaving the omegasome. The connection between expanding omegasomes and the underlying ER is perhaps more clearly seen by TIRF (total internal reflection fluorescence) microscopy (Figure 2E).
The idea that the ER is one site of autophagosome formation is supported by previous electron tomography studies showing many examples of the ER ribbon weaving through the autophagosome membrane [13,14]. Other data in the literature imply mitochondria as a source for autophagosome membrane, suggesting this organelle contributes as much as the ER membrane to autophagosome formation . Recent work by Hamasaki et al.  indicates that the ER–mitochondria contact sites appear to label with early autophagy proteins during starvation and disruption of these contact sites results in a decrease in autophagy. Thus it is possible that autophagosomes are also induced at the junction of the ER and mitochondria . Similarly, inhibition of endocytosis has a detrimental effect on autophagy, reducing the number of autophagosomes, suggesting the plasma membrane may provide membrane for autophagosomes . The contribution of membranes from several organelles to the phagophore need not be mutually exclusive; it is possible that, depending on the conditions and the stimulus, many cellular membranes may contribute to the formation of autophagosomes .
Regulation of omegasome formation
The generation of the lipid PI3P is an important factor for the initiation of autophagy and for localizing effector proteins to the site of autophagosome formation. The most prominent PI3P-binding protein is Atg18. In mammals, Atg18 homologues are called WIPI (WD-repeat protein interacting with phosphoinositides) family members including WIPI1, WIPI2 and WIPI4, all of which have been shown to be involved in autophagy [18–20]. Similar to DFCP1, WIPI2 is recruited to the omegasomes during PI3P production, and is required for LC3 lipidation and closure of the double-membrane structure. In cells down-regulated for WIPI2, omegasomes marked with DFCP1 are enhanced even under basal conditions, indicating that WIPI2 is downstream of DFCP1 and is involved in the maturation of the autophagosome as it leaves the omegasome . A similar result holds for WIPI4 .
The Vps34 complex can be regulated in multiple ways and this is illustrated in Figure 3 [21,22]. One of the key proteins of the complex is Beclin1 with an essential role in autophagy; as much as 50% of cellular Beclin1 is in complex with Vps34 and when overexpressed can lead to certain forms of cancer . Under nutrient-rich conditions, autophagy is inhibited due to Beclin1 binding to Bcl-2 family members . However, during starvation, the Bcl-2 proteins can become phosphorylated and no longer bind Beclin1, allowing autophagy to occur. Once Beclin1 is free from Bcl-2 interactions, it can bind to other proteins such as UVRAG (UV radiation resistance-associated gene), Ambra1, Atg14 L or Rubicon to regulate autophagy . UVRAG and Atg14 L binding to the PI3K complex are mutually exclusive and evidence suggests that both are involved in the early stages of autophagosome formation. Atg14 can localize to the omegasomes without binding to the PI3K complex, and it is thought that its role is to recruit the complex to the omegasome and stimulate production of PI3P . UVRAG increases the interaction between Beclin1 and Vps34, thus promoting autophagy. Similarly, downstream of these early events, Bif-1 binds to UVRAG rather than Beclin1, and can also be a positive inducer of autophagy [26,27]. On the other hand, Rubicon has been shown to inhibit autophagy. Rubicon has been shown to bind to the Beclin1 complexes containing UVRAG and inhibits autophagosome maturation by inhibiting the kinase activity of Vps34, and inhibiting degradation in lysosomes .
In addition to indirect effects via Beclin1, the formation of PI3P during omegasome nucleation and expansion could be regulated by direct effects on Vps34 itself. Previous studies indicate that at least three kinases phosphorylate Vps34 including cyclin-dependent kinase 1, cyclin-dependent kinase 5 and protein kinase D. Phosphorylation by the first two results in inhibition, whereas protein kinase D phosphorylation has been shown to increase Vps34 activity [29,30]. Undoubtedly more proteins will be identified which can regulate Vps34 directly. The challenge will be to differentiate effects on the endocytic function of Vps34 (mediated by complex II) versus the autophagic function (mediated by complex I).
Omegasomes during pathogen-induced autophagy
Autophagy has a complicated relationship with pathogen infection. In some cases pathogens usurp the autophagic machinery for replication and expansion, whereas in other cases pathogens activate autophagy during infection and this can be part of an innate immune response . Beclin1 is a key player in autophagy and during viral infection appears to be the common target protein in several instances. For example, the herpes simplex virus protein ICP34.5 binds to Beclin1 to prevent activation of autophagy and clearance of infection . A screen of non-structural proteins from infectious bronchitis virus revealed that one of them (nsp6) was capable of inducing omegasomes that led to autophagosome formation. The equivalent proteins from mammalian coronaviruses and porcine reproductive and respiratory syndrome viruses, in all cases multi-spanning ER transmembrane proteins, were all capable of inducing omegasomes leading to autophagosomes . Similarly, Salmonella infection has been shown to trigger formation of omegasomes before their maturation into autophagosomes in a pathway also requiring the small GTPase Rab1, a known regulator of ER-to-Golgi traffic . Thus in the case of both virally and bacterially induced autophagy it appears that an omegasome intermediate is used.
Termination of the PI3P signal: role for several 3-phosphatases
Lipid signalling depends not only on the generation of the signal (which is usually rapid and localized), but also on its consumption. In the case of PI3P during autophagy, several 3-phosphatases have been shown to be involved, with none so far being essential on its own. These phosphatases all belong to the MTM (myotubularin) family, they are ubiquitously expressed and are known to play a role in the endocytic pathway. A recent siRNA screen has revealed that three such phosphatases play a role in the regulation of autophagy, Jumpy (MTMR14), MTMR6 and MTMR7, with Jumpy having a stronger role [35,36], whereas another group implicated MTMR3 in the termination of the signal during autophagosome formation . Knockdown of the expression of both MTMR3 and Jumpy increased autophagosome number and both proteins localized to the phagophore membrane [36,37]. One possibility to keep in mind is that multiple pools of PI3P may be involved in autophagosome formation, one during the induction step related to omegasomes and one at later steps that could mediate maturation and/or fusion with the lysosomes. It will be important in future studies to differentiate between these steps in order to provide additional insights into the specificity of these 3-phosphatases during autophagosome formation.
Part of the requirement for PI3P formation during the induction of autophagy can be understood in the context of omegasome action. These omegasomes are transient precursors from which autophagosomes form. The advantage of such a transient precursor appears to be two-fold  (Figure 4). It allows the cells to be able to respond to a wide range of nutrient states, and it also makes for a smooth and economical approach to a new steady state of autophagosome formation in response to a starvation signal. However, it should be noted that autophagy appears to be regulated in many ways and at multiple points, and the pathway involving omegasome intermediates is one of several leading to autophagosome formation. Among the many unknowns in the field, perhaps the plasticity of the autophagic response and the underlying rationale for such plasticity will be important questions for future studies.
• Autophagy is a conserved catabolic process in which proteins and organelles are sequestered into double-membrane vesicles (termed autophagosomes) for degradation in lysosomes.
• Formation of autophagosomes is the decisive step during autophagy, and many proteins (generally termed Atg from work in yeast) are involved in the process.
• Induction of autophagosomes is regulated by the protein kinase ULK and its associated proteins, and by the lipid kinase Vps34 and its associated proteins.
• One function of the Vps34 complex during autophagy is the generation of PI3P-enriched and ER-connected membrane platforms, termed omegasomes, within which autophagosomes are formed.
• Omegasome intermediates are also evident during viral or bacterial-induced autophagy.
• The current view in the field is that autophagosome formation may involve multiple membranes sources (mitochondria, Golgi and plasma membrane) depending on cellular state.
Our work is supported by the Biotechnology and Biological Sciences Research Council.
- © The Authors Journal compilation © 2013 Biochemical Society