Membrane trafficking events should occur in a spatiotemporally regulated manner to deliver vesicles and their cargo proteins to the correct destination. The class III phosphatidylinositol 3-kinase (PI3-kinase; hereafter PI3-kinase indicates this class III kinase) participates in the regulation of membrane trafficking. PI3-kinase is a lipid kinase that specifically phosphorylates phosphatidylinositol at the D-3 position of the inositol ring to produce phosphoinositide 3-phosphate (PI3-P) (Schu et al., 1993; Stack and Emr, 1994). The generated PI3-P is recognized by downstream effectors; these effector molecules concentrate to a restricted area of the membrane where the subsequent membrane trafficking events occur (reviewed in Martin, 1998; Odorizzi et al., 2000). PI3-kinase is involved in membrane trafficking events, including endocytosis, the sorting of soluble vacuolar proteins, and autophagy (Schu et al., 1993; Munn and Riezman, 1994; Kihara et al., 2001). Currently, the mechanisms by which PI3-kinase mediates these diverse functions while maintaining the fidelity of each membrane-trafficking event remains poorly understood.

The involvement of PI3-kinase in membrane trafficking has been extensively studied in yeast. Vps34p, the only PI3-kinase encoded by yeast, is required for the sorting of nascent soluble vacuolar hydrolases, such as carboxypeptidase Y (CPY), via endosomes (Robinson et al., 1988; Schu et al., 1993). Yeast cells harboring point mutations in the Vps34p lipid kinase domain missort CPY into the secretory pathway. The function of Vps34p is dependent on the protein kinase activity of Vps15p, a serine/threonine kinase; therefore, Δvps15 cells display a similar phenotype to that seen for Δvps34 cells (Herman and Emr, 1990; Herman et al., 1991a, b; Stack et al., 1993, 1995). PI3-kinase is also involved in protein transport in mammalian cells. Proteins resident in lysosomes, an organelle functionally equivalent to the yeast vacuole, are erroneously secreted after treatment with PI3-kinase inhibitors (Brown et al., 1995; Davidson, 1995), suggesting a conserved function for PI3-kinases in protein sorting into the vacuole/lysosome in both yeast and mammalian cells. The human homologue of Vps15p associates with the Vps34p homologue in vivo (Volinia et al., 1995).

Autophagy is another major pathway delivering proteins into the vacuole/lysosome. Using this process, cells degrade large quantities of their cytoplasmic content to recycle the components in response to severe shortages of nutrients. This process is composed of multiple steps that are well conserved among eukaryotes. On induction of autophagy, the cytoplasmic contents are enclosed into a double membrane structure called an autophagosome. The autophagosome then fuses with a vacuole/lysosome; the inner membrane structures and packaged cytoplasmic macromolecules are degraded within this compartment (Baba et al., 1994; Mizushima et al., 2001; reviewed in Klionsky and Ohsumi, 1999). Microscopic analysis suggested that the majority of autophagy-related (Atg) proteins localize to a perivacuolar structure called pre-autophagosomal structure (PAS) at which autophagosomes are thought to originate (Suzuki et al., 2001). Because subcellular fractionation and electron microscopic studies have revealed that this poorly characterized structure does not contain vacuolar, endoplasmic reticulum (ER), Golgi, or late-endosomal markers, autophagosome formation likely involves a novel process that is distinct from the mechanisms governing the classical endomembrane system (reviewed in Noda et al., 2002). PI3-kinase also functions in the membrane dynamics of autophagy (Kiel et al., 1999). Δvps34 and Δvps15 mutant cells exhibited a complete loss of autophagic activity as seen for other autophagy-defective mutants (atg mutants) (Tsukada and Ohsumi, 1993; Thumm et al., 1994; Kihara et al., 2001). Two additional genes essential for autophagy, ATG14 and ATG6/VPS30, are subunits of a PI3-kinase complex (Kihara et al., 2001). In addition, autophagy is suppressed in mammalian cells treated with PI3-kinase inhibitors (Petiot et al., 2000), confirming the conserved role of PI3-kinase in autophagy from yeast to mammals.

Although only one functional lipid kinase is encoded by yeast, we have previously identified two distinct PI3-kinase complexes, complexes I and II, in this organisms (Kihara et al., 2001). Complex I functions in autophagy, whereas complex II functions in vacuolar protein sorting. Both of these complexes contain three common subunits, Vps15p, Vps34p, and Vps30p. Vps15p tethers the complex to the membrane; although this protein is myristylated at its N terminus, membrane association of the complex is not dependent solely on myristoylation (Herman et al., 1991b). Vps34p is recruited to the membrane and activated by Vps15p (Stack et al., 1995). The protein kinase activity of Vps15p is essential for complex formation; the lipid kinase activity of Vps34p is not. Whereas the function of Vps30p within these PI3-kinase complexes is not well understood, the homologous protein Beclin 1 has also been implicate in autophagy in both animals and plants (Liang et al., 1999; Liu et al., 2005). In addition to the common subunits, each complex also contains a unique factor. Atg14p is integrated into complex I, whereas Vps38p is specific for complex II. These additional subunits may act as connecter molecules, bridging Vps30p and Vps34p to allow complex formation (Kihara et al., 2001). These two complexes function in distinct biological processes; disruption of ATG14 does not affect vacuolar protein sorting, whereas deletion of VPS38 does not affect autophagy (Kametaka et al., 1998; Kihara et al., 2001). The mechanisms determining the specificity of these PI3-kinase complex functions have not been examined. Because both complexes exhibit PI3-kinase activity (Kihara et al., 2001), we assume that the primary functions of the two complexes are the same, namely, the production of PI3-P. Additional factors are likely necessary to direct each PI3-kinase complex to function in distinct biological processes, despite generating a common primary product. Atg14p and Vps38p may be these key factors, both conferring upon each complex a specificity of function and connecting Vps30p and Vps34p.

In this study, we investigated the function of Atg14p by deletion analysis. We also monitored the intracellular localization of the components of individual PI3-kinase complexes. Based on these analyses, we demonstrated the function of Atg14p in autophagy. These results led to our proposal of a model detailing the mechanisms by which PI3-kinase complexes I and II acquire distinct functions.

In this study, we focused on Atg14p, a specific component of PI3-kinase complex I that is essential for autophagy. Analyses using a deletion series of Atg14p indicated that the region containing coiled-coils I and II within the N-terminal half were essential and sufficient for the function in autophagy. This region was minimum essential for the localization of Atg14p to the PAS. The two coiled-coil structures in this region were responsible for interactions with Vps34p and Vps30p to form PI3-kinase complex I. These results indicate that the primary function of Atg14p is mediated by protein–protein interactions of the coiled-coils.

PI3-kinase complex I is essential for autophagy but dispensable for vacuolar protein sorting. Conversely, complex II functions in the sorting of vacuolar proteins but is dispensable for autophagy. How this specificity of the function is determined has not been elucidated. In this study, we investigated the intracellular localization of the two complexes by microscopy, which provided several important discoveries. All the components of complex I were present at the PAS. The localization of Vps34p and Vps30p to the PAS was dependent on the expression of Atg14p, indicating an essential role for Atg14p in the accumulation of PI3-kinase complex I at the PAS. Atg14p, however, was not essential for the localization of Vps34p and Vps30p to vacuolar membranes and endosomes. Vps38p, a specific component of complex II, did not localize to the PAS, but localized to endosomes and vacuolar membranes. Vps38p was not required for the PAS localization of Vps34p, Vps30p, and Vps15p but was essential for their localization to endosomes. Therefore, Vps38p plays a crucial role in the association of complex II with endosomes. From these results, we concluded that only complex I can accumulate at the PAS, whereas only complex II is found at endosomes. These unique localization patterns are mediated by the specific components of each complex, Atg14p and Vps38p. Here, we propose a model that the distinct functions of PI3-kinase complexes I and II are acquired by the specific association of each complex to a distinct compartment that is mediated by the specific components.

Unlike Vps34p and Vps30p, Vps15p localized to the PAS in the absence of Atg14p (Figure 9). The mechanism by which Vps15p localizes to the PAS is currently unknown. This Atg14p-independent localization of Vps15p suggests a potential mechanism by which Atg14p can promote the accumulation of complex I at the PAS. Atg14p may recruit Vps34p and Vps30p to the PAS, forming a complex with preexisting Vps15p on the membrane. In this way, Atg14p would not have to bring a preformed complex containing Vps15p to the PAS. Alternatively, Atg14p could prevent the dissociation of complex I, stabilizing it at the PAS. In any case, the interaction between certain PAS-localized proteins and the complex I subunits may be critical.

All of the components of PI3-kinase complexes I and II localized to the vacuolar membrane. At present, it is not clear whether the PI3-kinase complexes on the vacuolar membrane are functional. Vesicles undergoing anterograde transport finally fuse with vacuoles. Similarly, autophagosomes finally fuse with vacuoles (Baba et al., 1994; Kirisako et al., 1999). Therefore, it remains possible that the presence of complexes I and II at vacuolar membranes simply represents the consequence of membrane-trafficking events. Fab1p, a phosphoinositide kinase that phospholylates PI3-P to generate phosphoinositide-3,5-diphosphate, is involved in the regulation of vacuolar morphology (Yamamoto et al., 1995; Bonangelino et al., 1997; Gary et al., 1998; Dove et al., 2002, 2004). Alternatively, PI3-kinase complexes at vacuolar membranes may function in the maintenance of vacuolar homeostasis in combination with Fab1p and its possible effectors. It will be interesting to examine the vacuolar membrane localization of PI3-kinase complex subunits in various mutants defective in anterograde or retrograde transport, retrieval of the vacuolar membrane components, and autophagy (Tsukada and Ohsumi, 1993; Yamamoto et al., 1995; Bryant et al., 1998; Gary et al., 1998; Dove et al., 2002, 2004).

Vps34p and Vps15p remain localized to dot structures and vacuolar membranes in Δatg14Δvps38 cells (Figures ​(Figures77 and ​and9).9). These GFP-positive signals may represent Vps34p–Vps15p complex and/or a novel PI3-kinase complex containing Vps34p, Vps15p, and potentially novel undefined components. The existence of PI3-kinase complex(es) unique from both complexes I and II has been suggested (Kihara et al., 2001). Given that ~80% of WT PI3-kinase activity is retained in Δvps30 cells and that Δvps34 and Δvps15 cells have additional phenotypes beyond that of Δvps30 cells (Robinson et al., 1988; Raymond et al., 1992; Kihara et al., 2001), the putative novel complex likely does not contain Vps30p. This result would be in good agreement with our microscopy and cell fractionation data demonstrating that Vps30p was dispersed throughout the cytosol and did not localize to a specific compartment in Δatg14Δvps38 cells (Figure 8).

To date, genes with significant homology with ATG14 and VPS38 have not been found in animals and plants. Given the conserved roles of class III PI3-kinase in autophagy and protein sorting to the vacuole/lysosome from yeast to mammals (Petiot et al., 2000; Brown et al., 1995; Davidson, 1995), it would be interesting to search the functional homologues of Atg14p and Vps38p that potentially function to connect Vps34p and Vps30p in mammals.

We demonstrated that PI3-kinase complex I accumulates at the PAS in a manner dependent on Atg14p. What is the function of PAS-localized complex I in autophagy? In mammals, wortmannin and LY294002, two inhibitors of PI3-kinase, inhibit autophagy (Petiot et al., 2000), suggesting that the lipid kinase activity of a Vps34p homologue is required for this process. We have confirmed that the lipid kinase activity of Vps34p is also essential for autophagy in yeast (our unpublished data). In a variety of biological processes, PI3-P marks specific membrane sites to which downstream molecules need to be recruited (Martin, 1998; Odorizzi et al., 2000). One possible role of PI3-kinase complex I in autophagy may be to produce PI3-P at the PAS to recruit PI3-P-binding molecules, which in turn recruit additional downstream molecules to the PAS. Atg14p is required for the localization of Atg2p, Atg5p, and Atg8p to the PAS (Shintani et al., 2001; Suzuki et al., 2001). Therefore, complex I may act primarily at a relatively early point in the autophagosome formation, potentially during formation and/or maintenance of the PAS through the production of PI3-P.

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05-09-0841) on January 18, 2006.

Abbreviations used: ALP, alkaline phosphatase; API, aminopeptidase I; CPY, carboxypeptidase; PAS, preautophagosomal structure; PI3-kinase, phosphatidylinositol 3-kinase; PI3-P, phosphoinositide 3-phosphate; WT, wild-type.