The simple phosphoinositide PtdIns3P has been shown to control cell growth downstream of amino acid signalling and autophagy downstream of amino acid withdrawal. These opposing effects depend in part on the existence of distinct complexes of Vps34 (vacuolar protein sorting 34), the kinase responsible for the majority of PtdIns3P synthesis in cells: one complex is activated after amino acid withdrawal to induce autophagy and another regulates mTORC1 (mammalian target of rapamycin complex 1) activation when amino acids are present. However, lipid-dependent signalling almost always exhibits a spatial dimension, related to the site of formation of the lipid signal. In the case of PtdIns3P-regulated autophagy induction, recent data suggest that PtdIns3P accumulates in a membrane compartment dynamically connected to the endoplasmic reticulum that constitutes a platform for the formation of some autophagosomes. For PtdIns3P-regulated mTORC1 activity, a spatial context is not yet known: several possibilities can be envisaged based on the known effects of PtdIns3P on the endocytic system and on recent data suggesting that activation of mTORC1 depends on its localization on lysosomes.
Lipid-dependent signalling involves the timely formation of lipid molecules at distinct cellular membranes. These lipid signals in turn can either modify the physical properties of the membrane on which they are formed, or they can attract specific protein effectors which translocate to the membranes (or become altered in activity if already present on these membranes) to mediate downstream actions. Given that all cellular membranes are sites of formation of lipid signals, pathways of lipid signalling can have significant effects on cellular physiology, from growth decisions to intracellular trafficking.
Nutrient sensing is critical for cellular homoeostasis and it is underpinned by complex signalling circuits. A central regulator is the protein kinase TOR (target of rapamycin) [1,2]. In the presence of plentiful nutrients, TOR is active and regulates positively a number of signalling pathways involved in protein and lipid synthesis, cytoskeletal organization and mitochondrial function (Figure 1). In the absence of nutrients, and most significantly of amino acids, TOR becomes inactive and this immediately initiates a pathway of nutrient generation from intracellular sources, termed autophagy [3,4] (Figure 1). The purpose of autophagy is to deliver proteins to the lysosomes for degradation and subsequent nutrient generation, thus allowing cells to survive under starvation. Many additional functions of autophagy as a quality-control mechanism against endogenous and exogenous insults have also been described [3,4].
Simplified diagram of TORC1 activation downstream of amino acids
The simple phosphoinositide signal PtdIns3P has been shown to be important for the initiation of autophagy in response to amino acid withdrawal and following TOR inactivation, because the lipid kinase responsible for most of its formation, Vps (vacuolar protein sorting) 34, is an essential activity for the autophagic response in all organisms studied  (Figure 1). Paradoxically, PtdIns3P and Vps34 are also shown to be positive regulators of TOR when amino acids are present, at least in mammalian cells [6,7] (Figure 1). For the remainder of the present paper, we review what is known about the role of PtdIns3P in the induction of autophagy, and as a positive signal upstream of TOR during normal growth.
PtdIns3P formation and functional Vps34 complexes
PtdIns3P is formed either by phosphorylation of phosphatidylinositol on the 3-position of the inositol ring, or by dephosphorylation at the 4- or 5-position of more complex phosphoinositides such as PtdIns(3,4)P2 or PtdIns(3,5)P2 (Figure 2). The phosphorylation reaction in yeast is mediated by a single protein, Vps34. In higher eukaryotes, PtdIns3P can be formed either by Vps34 or by another class of lipid kinases termed class II [or PI3K (phosphoinositide 3-kinase)-C2] of which three isoforms are known: PI3K-C2α, PI3K-C2β and PI3K-C2γ [8,9]. The type II enzymes are thought to be responsive to growth factor signals and of low activity basally. For several additional reasons, they are probably not involved in the PtdIns3P generation that we discuss in the present paper: the α isoform appears to be relatively insensitive to pharmacological inhibition, whereas most PtdIns3P responses linked to autophagy or TOR activity are sensitive to inhibitors such as wortmannin; the β isoform appears to be principally involved in cell migration and clathrin-mediated endocytosis; and the γ isoform has restricted expression [10–12]. However, these enzymes can, in principle, make PtdIns3P, and the possibility still exists that they could provide functional compensation for Vps34 under some circumstances.
Pathways of PtdIns3P formation and consumption
Vps34 was originally identified as a protein necessary for vacuolar protein sorting in yeast, and it is in the context of endosomal protein trafficking that this protein is best understood [13–15]. Subsequent work in yeast suggested that Vps34 exists in two distinct complexes: complex I is composed of Vps34, Vps15 (a putative kinase myristoylated at the N-terminus that enhances Vps34 activity), Vps30 and Vps38, whereas in complex II, Vps38 is replaced by Atg14 [16,17] (Figure 1). A useful simplification is that complex I is involved in endosomal activities, whereas the replacement of Vps38 with Atg14 (an essential autophagy-specific protein) makes complex II important during autophagy (Figure 1). The composition of the two Vps34 complexes is conserved in higher eukaryotes including mammals, with some additional elaborations [5,18]. Beclin 1 is the mammalian homologue of Vps30, whereas UVRAG (UV radiation resistance-associated gene) is functionally related to Vps38 [19,20]. Additional mammalian proteins involved in the ‘endocytic’ Vps34 complex include Rubicon (Run domain and cysteine-rich domain-containing Beclin 1-interacting protein), a negative regulator that may indirectly also affect autophagy, and Bif-1 (Bax-interacting factor 1), a curvature-sensing positive regulator that may also affect autophagy [21–23]. The Vps34 complex involved in autophagy contains additional interacting partners, most of them affecting the interaction of Vps34 with Beclin 1. The anti-apoptotic protein Bcl-2 was identified as a binding partner of Beclin 1 that could sequester the protein away from Vps34, resulting in lower activity . Another Beclin 1 partner is Ambra1 (activating molecule in Beclin 1-regulated autophagy 1), a protein that shows antagonistic activity to that of Bcl-2 .
The first crystal structure of a Vps34 catalytic core was recently described for the Drosophila enzyme by Williams and colleagues . An important element in the structure is a C-terminal helix that appears to associate with the substrate-binding loop and the catalytic loop, rendering the enzyme potentially inactive in the cytosol. Upon membrane binding, the helix would shift by binding to the membrane and allow higher catalytic activity. The properties of this C-terminal loop provide an interesting clue for identifying potential regulators of Vps34 in vivo. The crystal structure of Vps34 also addressed a problem in understanding Vps34 function, namely the absence of a specific pharmacological inhibitor . Uniquely for Vps34 among other PI3Ks, the ATP-binding pocket is more constricted. This explains why other specific inhibitors do not work very well, and it offers opportunities for the design of better compounds. A specific Vps34 inhibitor working at low concentrations would be extremely useful, since, at present, the two compounds in wide use have several problems: wortmannin also inhibits most other PI3K enzymes, whereas 3-methyladenine is used at 10 mM concentration in vivo and is likely to have additional non-specific effects.
Despite the absence of a specific Vps34 inhibitor, we know a great deal about the function of PtdIns3P in many cellular processes . Critical to this has been the identification of two protein modules that bind PtdIns3P with high specificity: the FYVE (conserved in Fab1, YOTB, Vac1 and EEA1) domain and the PX (Phox homology) domain [15,27–29] (Figure 2). Proteins containing these domains have been implicated in endosomal fusion, endosomal sorting, phagocytosis, autophagy, and, more recently, cytokinesis and tumour suppression (reviewed in ). Using constructs derived from these PtdIns3P modules, it has also been possible to survey cellular membranes for PtdIns3P accumulation, and the results suggest that PtdIns3P is found primarily on membranes of endocytic/phagosomal origin, in agreement with the distribution of most effectors [14,15]. A relatively unique occurrence of PtdIns3P is on membranes of autophagosomes and their precursors, which is discussed further below. The function of PtdIns3P in the various cellular pathways appears to be two-fold. On membranes where PtdIns3P is constitutively present, such as on early endosomes, it appears to provide important localization clues for fusion or sorting. On membranes where PtdIns3P accumulates following a signal (such as on phagosomes or on autophagosomes), PtdIns3P appears to serve as a nucleation and expansion site for downstream effectors. In the latter examples, PtdIns3P may constitute a mechanism to provide identity to a membrane compartment formed de novo.
Evidence that PtdIns3P regulates autophagosome formation
Autophagosomes are double membrane vesicles that are formed during autophagy. Their function is to engulf cytosolic proteins and/or organelles for delivery to the lysosomes . Formation of autophagosomes requires PtdIns3P at an early stage and downstream of TOR inactivation [5,17]. In yeast, elimination of Vps34 or its replacement by an inactive allele results in complete suppression of autophagosomes. In Drosophila, elimination of Vps34 has a strong inhibitory effect on autophagosome formation , whereas, in mammalian cells, down-regulation of Vps34 in several different cell lines affects significantly the autophagic response (reviewed in ). Although mice carrying a Vps34 knockout die as embryos , animals with conditional deletion of Vps34 in neurons have been generated . Surprisingly, small-diameter Vps34 mutant neurons appear to mount an autophagic response that requires canonical downstream autophagy genes. It will be interesting to determine whether this is a neuron-specific phenomenon, or whether additional mechanisms for generating autophagosomes, independent of Vps34, exist in animal tissues. These PtdIns3P-independent mechanisms of autophagy induction are currently well recognized as requiring further investigation .
Indirect evidence that PtdIns3P is involved in autophagosome formation has also been provided by the identification of two 3-phosphatases of the myotubularin-related family, MTMR14 (Jumpy) and MTMR3, that negatively regulate autophagy [35,36]. Functional inactivation of Jumpy , and in later work of MTMR3 , results in enhancement of basal and stimulated autophagy, indicating that formation and consumption of PtdIns3P are tightly coupled under normal autophagic induction. More recently, the dual-domain PTPσ (protein tyrosine phosphatase σ) was also shown to negatively regulate autophagy via consumption of the PtdIns3P signal .
An additional piece of evidence suggesting the involvement of PtdIns3P in autophagy is the identification of several autophagy-specific proteins capable of binding PtdIns3P [5,17]. In yeast, Atg20 and Atg24 contain a PX domain, and are involved in selective autophagy. In both yeast and mammalian cells, the Atg18 protein (WIPI in mammals) is essential for an early step during autophagy and has been shown to bind to PtdIns3P [38–40].
Omegasomes are PtdIns3P-enriched autophagosome platforms
To explore how PtdIns3P may regulate autophagy, Ohsumi and colleagues used a FYVE domain-derived probe and showed that yeast autophagosomes are enriched in PtdIns3P, and that autophagy induction transports a significant amount of PtdIns3P to the vacuole . Interestingly, the distribution of PtdIns3P was asymmetrical on the autophagosomal membrane, with the inner surface containing much stronger label. In many other recent publications, the FYVE-derived probe has been shown to label nascent autophagosomes. Thus PtdIns3P appears to be a component of the autophagosomal membrane, and a reasonable hypothesis is that it enables some trafficking or fusion step during autophagosome maturation and fusion with lysosomes. These data, however, do not explain how PtdIns3P is involved at the very early steps of autophagosome formation.
Work from our group has suggested a possible mechanism addressing this . We found that a novel protein termed DFCP1 (double FYVE domain-containing protein 1), which normally localizes on ER (endoplasmic reticulum) and Golgi membranes, translocates upon amino acid starvation to omegasomes, a novel punctate compartment closely connected to the ER (Figure 3). Formation of omegasomes was sensitive to Vps34 and Beclin 1 function and was inhibited by wortmannin and 2-methyladenine. Live imaging experiments suggested that at least some autophagosomes are formed within omegasomes. Subsequent work suggested that several pathogens known to induce autophagy also induce omegasomes, whereas depletion of WIPI2 (a mammalian equivalent of Atg18) increases omegasome numbers [40,43,44]. Finally, DFCP1 and WIPI2 co-localize on omegasomes and neither becomes part of mature autophagosomes . Taken together, these data suggest that omegasomes are ER-derived transient precursors of autophagosomes that are enriched in PtdIns3P. The geometry of these precursors and their connection to the ER have been described using three-dimensional electron microscopic tomography [45,46].
Spatial considerations in the regulation of autophagy and growth by TORC1 and Vps34
In live imaging experiments, omegasome formation appears to require a continuous interaction of Vps34-containing lysosomes with the ER, suggesting that ER–lysosome interaction may deliver Vps34 to omegasomes  (Figure 3). A possible mechanism for delivering Vps34 to the ER precursor membrane is suggested by recent work showing that Atg14, the protein that confers autophagy specificity on the Vps34 complex, contains an ER-targeting epitope that is necessary for its function during autophagy . Atg14 (or Barkor) also contains a region capable of recognizing PtdIns3P in the context of a sharply curved membrane . These observations can be fitted into a model whereby initial delivery of Vps34 to omegasomes would initiate a positive-feedback loop that would result in large amounts of PtdIns3P being formed there . The signal(s) activating Atg14 under these conditions remains elusive.
It was mentioned above that, in general, PtdIns3P appears to function in some cases as a means to provide identity to a membrane (for example, during phagocytosis), whereas in other cases, it appears to enable membrane maturation (fusion and sorting) events. The quick accumulation of PtdIns3P on autophagosomal precursors and its occurrence on mature autophagosomes suggests that during autophagy PtdIns3P is used as both an identity and a maturation signal.
It should be emphasized that other pathways of autophagosome formation depending on mitochondria and the plasma membrane have been described recently [50,51]. It is not clear whether analogous mechanisms for PtdIns3P formation and consumption are also at play in those cases. In addition, Vps34 functional complexes can be formed during starvation in response to Ralb in a pathway dependent on exocyst subassemblies . These data suggest additional pathways for autophagosome biogenesis in mammalian cells.
Activation of TORC1 downstream of amino acids and PtdIns3P involvement
All eukaryotes contain functional homologues of TOR that are responsible for integrating nutrient sensing with growth control [1,2]. In mammalian cells, levels of amino acids are very important signals to a specific complex of TOR termed TORC1 (TOR complex 1) composed of the TOR catalytic subunit, the small GTPase Rheb and several other associated proteins  (Figure 1). Overexpression of Rheb is sufficient to activate TORC1, such that it becomes insensitive to amino acid withdrawal and autophagy is not induced . Conversely, upon down-regulation of Rheb, amino acids cannot activate TORC1. The Rag GTPases are also involved in amino-acid-dependent TORC1 activation [55,56]. These proteins exist as a heterotetrameric assembly, and TORC1 activation is strongest when RagA/B are in the GTP-bound form and RagC/D are in the GDP-bound form. Interestingly, the Rag GTPases localize to the lysosomes, and recent work has suggested that localization of TORC1 on lysosomes, via the Rag GTPases and a second complex termed Ragulator, is essential for TORC1 activation  (Figure 3). Conversely, amino acid withdrawal induces a very quick redistribution of TORC1 from lysosomes to the cytosol and inactivation . An attractive hypothesis is that amino acids activate a particular configuration of the Rag proteins (residing on lysosomes via interaction with the Ragulator complex), and this causes TORC1 to translocate to the lysosomal surface where it could be activated by interacting with Rheb (Figure 3). Upon starvation, TORC1 translocation from the lysosomes could result in derepression of downstream targets and the induction of autophagy. One such target is ULK1, an essential autophagy kinase homologous with yeast Atg1, whose inactivation downstream of TORC1 has been described previously [58–60].
Several independent reports have also shown that Vps34 is a positive regulator of TORC1, downstream of amino acids [6,7] (Figure 1). The evidence is based on functional inactivation of Vps34 by pharmacological or siRNA (small interfering RNA) treatments and on sequestration of endogenous PtdIns3P by overexpression of FYVE domain-derived probes [61,62]. In all of these settings, phosphorylation of downstream targets of TORC1 was very suppressed. In knockout mice for the Pik3c3 gene, which codes for Vps34, death occurs between E (embryonic day) 7.5 and E8.5 of embryogenesis, and there is substantial evidence for severely reduced cell proliferation . In addition, levels of phosphorylation of the downstream target of TORC1 ribosomal protein S6 are strongly reduced . All of these data suggest that mammalian Vps34 regulates nutrient sensing upstream of TORC1. The situation is different in Drosophila, where genetic ablation of Vps34 affected endocytosis and autophagy, but not TOR activity, so there appears to be species variation for this signalling circuit .
A study from Thomas and colleagues has provided some molecular details on the regulation of TORC1 by Vps34 and its lipid product . These authors reported that amino acids induce a rise in cytosolic calcium which drives binding of Ca2+/calmodulin to Vps34 and activation of the lipid kinase. The subsequent elevation of PtdIns3P induces the translocation of TORC1 to a PtdIns3P ‘signalosome’, resulting in the activation of TORC1. At the time of publication of that study , the data showing that Rag GTPases direct TOR to a Rab7 compartment were not available, so it is not clear whether the PtdIns3P signalosome is related to a Rab7-positive lysosome. Although the connection between calcium and Vps34 appears to be variable , other recent work has also suggested that activation of TORC1 downstream of Vps34 may depend on trafficking events that are known to be regulated by PtdIns3P [65,66]. Backer and colleagues showed that the maturation of early to late endosomes that depends on Rab5/Rab7 exchange is critical for TOR activity , whereas work in Drosophila and mammalian cells also implicated Rab5 in TOR activation . Significantly, Rab5 and PtdIns3P are frequently used as two coincident signals for the translocation of proteins to endosomal membranes [67,68], suggesting a connection between the Rab5 requirement and Vps34 upstream of TORC1 activation. In addition, Vps34 and its binding partner Vps15 have been shown to interact with Rab7 .
On the basis of the available data several possibilities can be envisaged for the regulation of TORC1 by Vps34 (Figure 3). One possibility is that Vps34 activity creates a PtdIns3P-enriched region on the lysosomal membrane which enhances the translocation of active TORC1 in response to the Rag GTPases and to Rheb. Another possibility is that this PtdIns3P region recruits an unknown PtdIns3P effector with a critical function in TORC1 activation. Finally, it is possible that PtdIns3P regulates a trafficking step leading from endosomes to lysosomes which is required either for the trafficking of TORC1 components or for amino acid sensing by the upstream regulators of TORC1.
In addition to the many unknowns about the Vps34–TORC1 interaction, an important set of future questions concerns the role of the lysosomes as co-ordinators of both growth and autophagy. On the one hand, it is surprising that an organelle that was considered an end point of trafficking pathways is beginning to take an important role in the biology of TORC1. On the other hand, given that lysosomes can generate nutrients under both normal growth and during autophagy, it is perhaps logical to expect that regulators of this process would reside on their surface. In support of this notion, recent work has shown that lysosomal mobility is dynamic depending on nutrient status, and that lysosome homoeostasis after a round of starvation and autophagy is an important TOR activity in multiple experimental systems [70,71]. Future work will probably identify important proteins that regulate cell fate decisions from the lysosomal surface.
Signalling 2011: a Biochemical Society Centenary Celebration: A Biochemical Society Focused Meeting held at the University of Edinburgh, U.K., 8–10 June 2011. Organized and Edited by Nicholas Brindle (Leicester, U.K.), Simon Cook (The Babraham Institute, U.K.), Jeff McIlhinney (Oxford, U.K.), Simon Morley (University of Sussex, U.K.), Sandip Patel (University College London, U.K.), Susan Pyne (University of Strathclyde, U.K.), Colin Taylor (Cambridge, U.K.), Alan Wallace (AstraZeneca, U.K.) and Stephen Yarwood (Glasgow, U.K.).
Our work is supported by the Biotechnology and Biological Sciences Research Council.