ABSTRACT

Autophagy is a pivotal cytoprotective process that secures cellular homeostasis, fulfills essential roles in development, immunity and defence against pathogens, and determines the lifespan of eukaryotic organisms. However, autophagy also crucially contributes to the development of age-related human pathologies, including cancer and neurodegeneration. Macroautophagy (hereafter referred to as autophagy) clears the cytoplasm by stochastic or specific cargo recognition and destruction, and is initiated and executed by autophagy related (ATG) proteins functioning in dynamical hierarchies to form autophagosomes. Autophagosomes sequester cytoplasmic cargo material, including proteins, lipids and organelles, and acquire acidic hydrolases from the lysosomal compartment for cargo degradation. Prerequisite and essential for autophagosome formation is the production of phosphatidylinositol 3-phosphate (PtdIns3P) by phosphatidylinositol 3-kinase class III (PI3KC3, also known as PIK3C3) in complex with beclin 1, p150 (also known as PIK3R4; Vps15 in yeast) and ATG14L. Members of the human WD-repeat protein interacting with phosphoinositides (WIPI) family play an important role in recognizing and decoding the PtdIns3P signal at the nascent autophagosome, and hence function as autophagy-specific PtdIns3P-binding effectors, similar to their ancestral yeast Atg18 homolog. The PtdIns3P effector function of human WIPI proteins appears to be compromised in cancer and neurodegeneration, and WIPI genes and proteins might present novel targets for rational therapies. Here, we summarize the current knowledge on the roles of the four human WIPI proteins, WIPI1–4, in autophagy.

This article is part of a Focus on Autophagosome biogenesis. For further reading, please see related articles: ‘ERES: sites for autophagosome biogenesis and maturation?’ by Jana Sanchez-Wandelmer et al. (J. Cell Sci.128, 185-192) and ‘Membrane dynamics in autophagosome biogenesis’ by Sven R. Carlsson and Anne Simonsen (J. Cell Sci.128, 193-205).

Introduction

Autophagy is a cytoprotective mechanism involving the degradation of proteins, lipids and organelles in the lysosomal compartment. By executing constitutive clearance of the cytoplasm and permitting the recycling of the degraded material, basal autophagy is important for cellular survival in all eukaryotes. The cytoplasmic material becomes sequestered in unique double-membraned vesicles called autophagosomes, either stochastically or by specific cargo recognition. Autophagosomes are formed by the elongation and closure of a membrane precursor, called the phagophore or isolation membrane (Fig. 1), and they acquire acidic hydrolases from lysosomes for cargo degradation. Upon starvation or a variety of cellular insults, autophagy is induced above the basal level to compensate for nutrient shortage and to provide monomeric constituents, such as amino acids, and energy for recycling processes (Choi et al., 2013; Feng et al., 2014; Jiang and Mizushima, 2014; Ohsumi, 2014; Shibutani and Yoshimori, 2014; Yang and Klionsky, 2010).

Fig. 1.

Human WIPI proteins as essential PtdIns3P effectors at the nascent autophagosome. (A) Upon the initiation of autophagy, localized PtdIns3P production at the ER recruits human WIPI proteins, as shown for WIPI1 and WIPI2. PtdIns3P is also bound by DFCP1 with unknown consequences. ER dynamics permit the formation of the phagophore from omegasome or cradle-like ER structures and ATG9-positive membranes, by an as-yet-unknown mechanism. WIPI proteins, as shown for WIPI1, WIPI2 and WIPI4, localize to the phagophore. Here, WIPI2 recruits the ATG12–ATG5–ATG16L complex (highlighted in green) by direct binding to ATG16L1. Subsequently, LC3 is conjugated to PtdEtn. WIPI1 is also essential for LC3 lipidation, but the functional relationship between WIPI1 and WIPI2 is unknown. At the autophagosome, LC3 decorates the inner and outer membrane as it stays conjugated to PtdEtn. Proteins highlighted in red (WIPI, DFCP1) specifically bind to PtdIns3P. Cytosolic LC3 is highlighted in blue. (B–D) There is evidence that WIPI1 (B, endogenous WIPI1), WIPI2B (C, GFP–WIPI2B) and WIPI2D (D, GFP–WIPI2D) also become membrane proteins of the inner (IM) and outer (OM) autophagosomal membrane. AP, autophagosome; E, E-face (of note, the E- or P-face terminology with regard to the monolayers of the inner or outer autophagosomal membrane might change once the membrane origin of autophagosomes is identified). Scale bars: 200 µm. The images in B–D were reproduced from Proikas-Cezanne and Robenek, 2011, under the terms of the Creative Commons Attribution License (© 2011 The Authors Journal compilation, © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd). The schematic drawing of the endoplasmic reticulum was obtained from Motifolio.

Fig. 1.

Human WIPI proteins as essential PtdIns3P effectors at the nascent autophagosome. (A) Upon the initiation of autophagy, localized PtdIns3P production at the ER recruits human WIPI proteins, as shown for WIPI1 and WIPI2. PtdIns3P is also bound by DFCP1 with unknown consequences. ER dynamics permit the formation of the phagophore from omegasome or cradle-like ER structures and ATG9-positive membranes, by an as-yet-unknown mechanism. WIPI proteins, as shown for WIPI1, WIPI2 and WIPI4, localize to the phagophore. Here, WIPI2 recruits the ATG12–ATG5–ATG16L complex (highlighted in green) by direct binding to ATG16L1. Subsequently, LC3 is conjugated to PtdEtn. WIPI1 is also essential for LC3 lipidation, but the functional relationship between WIPI1 and WIPI2 is unknown. At the autophagosome, LC3 decorates the inner and outer membrane as it stays conjugated to PtdEtn. Proteins highlighted in red (WIPI, DFCP1) specifically bind to PtdIns3P. Cytosolic LC3 is highlighted in blue. (B–D) There is evidence that WIPI1 (B, endogenous WIPI1), WIPI2B (C, GFP–WIPI2B) and WIPI2D (D, GFP–WIPI2D) also become membrane proteins of the inner (IM) and outer (OM) autophagosomal membrane. AP, autophagosome; E, E-face (of note, the E- or P-face terminology with regard to the monolayers of the inner or outer autophagosomal membrane might change once the membrane origin of autophagosomes is identified). Scale bars: 200 µm. The images in B–D were reproduced from Proikas-Cezanne and Robenek, 2011, under the terms of the Creative Commons Attribution License (© 2011 The Authors Journal compilation, © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd). The schematic drawing of the endoplasmic reticulum was obtained from Motifolio.

The formation of autophagosomes necessitates the concerted and sequential action of ATG proteins, originally identified in yeast (Itakura and Mizushima, 2010; Klionsky et al., 2003; Mizushima et al., 2011; Nakatogawa et al., 2009). ATG proteins are regulated by conserved nutrient and energy-dependent signaling cascades that crucially involve the mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) (Fig. 2C). Both mTOR and AMPK control the Unc-51-like kinases ULK1 and ULK2 (Meijer and Codogno, 2011; Mizushima, 2010; Russell et al., 2014). Nutrient availability activates protein synthesis and cell growth through the mTOR complex 1 (mTORC1), an essential switch towards anabolic pathways. When activated by growth factors through RHEB proteins, or by amino acids through RAG proteins (Dennis et al., 2011), mTORC1 inhibits autophagy by associating with and phosphorylating the autophagy-related protein ULK1, a serine/threonine-specific protein kinase that complexes with ATG13 and FIP200 (also known as RB1CC1) (Ganley et al., 2009; Hosokawa et al., 2009; Jung et al., 2009; Kim et al., 2011). By contrast, nutrient starvation permits autophagy initiation. AMPK, functioning as a switch towards catabolic pathways, activates autophagy in response to low cellular ATP levels either indirectly, through mTORC1 inactivation (Corradetti et al., 2004; Gwinn et al., 2008; Meijer and Dubbelhuis, 2004), or directly, through ULK1 phosphorylation (Egan et al., 2011; Kim et al., 2011).

Fig. 2.

WIPI proteins bind to PtdIns3P and PI(3,5)P2 through a conserved cluster of residues within the β-propeller. (A) Conserved amino acids specific for the PROPPIN family have been identified and are depicted in the homology model of human WIPI1. Red, homologous residues; magenta, invariant residues. (B) The conserved residues cluster at opposite sides of the WIPI1 β-propeller. Homologous residues mediate multiple regulatory protein–protein interactions, such as the interaction between WIPI2 and ATG16L1 (indicated by asterisks in C), and invariant residues confer specific binding to PtdIns3P or PtdIns(3,5)P2. (C) PtdIns3P-binding specificity is necessary for the functional contribution of WIPI proteins to autophagosome formation. AMPK activates ULK1 (in complex with ATG13 and FIP200) directly, by site-specific phosphorylation, and indirectly, by inhibiting mTORC1. Downstream of ULK1 activation, PI3KC3 (in complex with beclin 1, ATG14L and PIK3R4) produces PtdIns3P, which is subsequently bound by WIPI1 and WIPI2. WIPI1/2 binding to PtdIns3P is further controlled by members of the MTMR family, which hydrolyze PtdIns3P to PtdIns, and probably also by FIG4, which dephosphorylates PtdIns(3,5)P2. Of note, WIPI1 and WIPI2 also bind specifically to PtdIns(3,5)P2, the product of PIKFYVE activity, but the biological function of this binding is unknown. During autophagy initiation, WIPI1 and WIPI2 decode the PtdIns3P signal to confer LC3 lipidation (LC3–PtdEtn, LC3 conjugation to PtdEtn) through direct binding between WIPI2 and ATG16L (white asterisks) in complex with ATG12 conjugated to ATG5. WIPI4 bound to ATG2 is considered to function downstream of WIPI1/2 and LC3. The WIPI1 homology model (A,B) is reprinted by permission from Macmillan Publishers Ltd: Oncogene (Proikas-Cezanne et al., 2004), copyright 2004.

Fig. 2.

WIPI proteins bind to PtdIns3P and PI(3,5)P2 through a conserved cluster of residues within the β-propeller. (A) Conserved amino acids specific for the PROPPIN family have been identified and are depicted in the homology model of human WIPI1. Red, homologous residues; magenta, invariant residues. (B) The conserved residues cluster at opposite sides of the WIPI1 β-propeller. Homologous residues mediate multiple regulatory protein–protein interactions, such as the interaction between WIPI2 and ATG16L1 (indicated by asterisks in C), and invariant residues confer specific binding to PtdIns3P or PtdIns(3,5)P2. (C) PtdIns3P-binding specificity is necessary for the functional contribution of WIPI proteins to autophagosome formation. AMPK activates ULK1 (in complex with ATG13 and FIP200) directly, by site-specific phosphorylation, and indirectly, by inhibiting mTORC1. Downstream of ULK1 activation, PI3KC3 (in complex with beclin 1, ATG14L and PIK3R4) produces PtdIns3P, which is subsequently bound by WIPI1 and WIPI2. WIPI1/2 binding to PtdIns3P is further controlled by members of the MTMR family, which hydrolyze PtdIns3P to PtdIns, and probably also by FIG4, which dephosphorylates PtdIns(3,5)P2. Of note, WIPI1 and WIPI2 also bind specifically to PtdIns(3,5)P2, the product of PIKFYVE activity, but the biological function of this binding is unknown. During autophagy initiation, WIPI1 and WIPI2 decode the PtdIns3P signal to confer LC3 lipidation (LC3–PtdEtn, LC3 conjugation to PtdEtn) through direct binding between WIPI2 and ATG16L (white asterisks) in complex with ATG12 conjugated to ATG5. WIPI4 bound to ATG2 is considered to function downstream of WIPI1/2 and LC3. The WIPI1 homology model (A,B) is reprinted by permission from Macmillan Publishers Ltd: Oncogene (Proikas-Cezanne et al., 2004), copyright 2004.

Following ULK1 activation and subsequent ATG13 and FIP200 phosphorylation, ULK1 stimulates PtdIns3P production through beclin 1 phosphorylation, which leads to the activation of the phosphatidylinositol 3-kinase class III (PI3KC3, also known as PIK3C3) (Russell et al., 2013). Subsequently, PI3KC3 in complex with beclin 1, p150 (also known as PIK3R4; Vps15 in yeast) and ATG14L translocates to the initiation site for autophagosome formation, with ATG14L playing a crucial role in directing this PI3KC3 complex to the endoplasmic reticulum (ER) (Matsunaga et al., 2010). At the ER, the PI3KC3 complex stabilizes the ULK complex (Karanasios et al., 2013; Koyama-Honda et al., 2013). PI3KC3-mediated PtdIns3P production at the ER was identified by visualizing dynamic PtdIns3P-enriched ER structures called omegasomes (Axe et al., 2008) or cradles (Hayashi-Nishino et al., 2009). Omegasomes foster the formation of the phagophore, which is thought to form de novo by an as-yet-unknown mechanism (Roberts and Ktistakis, 2013; Simonsen and Stenmark, 2008). However, phagophore expansion is probably mediated by membrane uptake from endomembranes as well as from semiautonomous organelles (Lamb et al., 2013; Shibutani and Yoshimori, 2014).

Phagophore expansion requires two autophagosomal ubiquitin-like conjugation systems, the ATG12 and the LC3 (microtubule-associated protein 1A/1B-light chain 3) systems, both of which are necessary for the conjugation of LC3 to phosphatidylethanolamine (LC3–PtdEtn or LC3-II), a process that is referred to as LC3 lipidation (Hamasaki et al., 2013; Kabeya et al., 2000). Downstream of PtdIns3P production but prior to LC3 lipidation, the ATG16L1 complex, composed of ATG12 conjugated to ATG5 and associated with ATG16L (ATG12–ATG5–ATG16L) is recruited to the initiation site of autophagosome formation (Itakura and Mizushima, 2010), and functions as an E3-like ligase on the ATG3–LC3 conjugate to mediate the lipidation of LC3 (Fujita et al., 2008; Sakoh-Nakatogawa et al., 2013). LC3–PtdEtn, as a membrane protein of the nascent and mature autophagosome, fulfills at least three crucial functions: (1) it supports hemifusion events during phagophore expansion and closure, (2) it enables specific cargo recognition through binding to proteins harboring an LC3-interacting region (LIR) motif, and (3) it enables adaptor protein docking (Nakatogawa et al., 2008; Slobodkin and Elazar, 2013; Wild et al., 2014). The LC3-positive autophagosome sequesters cytoplasmic material and fuses with endosomes and lysosomes for cargo breakdown, and the degraded material is transported to the cytoplasm.

It is still not clear how autophagy is regulated during the succession of events from the production of ER-localized PtdIns3P to the de novo formation of PtdIns3P-enriched nascent autophagosomes (O'Farrell et al., 2013). Early studies made use of proteins harboring PtdIns3P-binding FYVE (conserved in Fab1, YOTB, Vac1 and EEA1) domains in order to visualize intracellular PtdIns3P on endosomes and nascent autophagosomes (Axe et al., 2008; Gillooly et al., 2000; Obara et al., 2008). By using the double FYVE domain-containing protein 1 (DFCP1, also known as ZFYVE1), the omegasome-like formation of the ER was identified in mammalian cells upon starvation, prior to phagophore formation (Axe et al., 2008). It has been suggested that, upon ER-localized PtdIns3P production by PI3KC3, DFCP1 binds to the ER through an interaction between its FYVE domain and PtdIns3P, rather than through an internal ER-targeting sequence in DFCP1 (Axe et al., 2008). Hence, DFCP1 can be considered as a PtdIns3P effector protein. However, the downstream consequences of this interaction remain unknown, because small interfering (si)RNA-mediated downregulation of DFCP1 does not appear to negatively affect autophagy (Axe et al., 2008; O'Farrell et al., 2013). Another FYVE-domain-containing protein, the autophagy-linked FYVE-protein (Alfy, also known as WDFY3), functions during the degradation of large cytoplasmic aggregates, which are characteristic of neurodegenerative diseases; however, Alfy does not appear to play a role during starvation-induced autophagy (Clausen et al., 2010; Filimonenko et al., 2010; Finley et al., 2003; Simonsen et al., 2004).

PROPPINs (β-propellers that bind phosphoinositides) are currently the only known PtdIns3P-binding proteins with conserved and essential PtdIns3P effector function in autophagy from yeast to human (Thumm et al., 2013). The human PROPPIN family members are called WD-repeat protein interacting with phosphoinositides (WIPI), and consist of the four members (WIPI1 to WIPI4) along with their splice variants (Proikas-Cezanne et al., 2004). Human WIPI proteins are considered to bridge PtdIns3P production and LC3 lipidation (Lamb et al., 2013). In this Commentary, we summarize the current status of knowledge on the four individual human WIPI members.

The human WIPI gene and protein family

The identification of the human WIPI genes was based on human liver cDNA library screening for novel p53 inhibitory factors (Waddell et al., 2001), leading to the identification of a partial 5′-truncated cDNA encoding human WIPI1 (WIPI1α) that localized to a region on human chromosome 17q known to be frequently imbalanced in human cancer (Box 1). On this basis, the four human WIPI members, WIPI1 to WIPI4, were identified through BLAST searching the human genome, and were then cloned and found to be ubiquitously expressed in normal human tissue, with high expression in skeletal muscle and heart (Proikas-Cezanne et al., 2004). Moreover, all WIPI genes were found to be aberrantly expressed in different human tumors (Box 1) (Proikas-Cezanne et al., 2004). WIPI1 gene expression is positively regulated by TFEB in liver (Settembre et al., 2013) and by PU.1 during neutrophil differentiation (Brigger et al., 2014), and is epigenetically repressed by the histone methyltransferase G9a (also known as EHMT2) (Artal-Martinez de Narvajas et al., 2013). Interestingly, upon starvation or activation of naive T cells, G9a dissociates from the WIPI1 promoter, leading to increased WIPI1 expression upon induction of autophagy (Artal-Martinez de Narvajas et al., 2013). WIPI2 gene expression has been found to be negatively regulated by ZKSCAN3, a master transcriptional repressor of autophagy and lysosome biogenesis (Chauhan et al., 2013).

As endogenous WIPI1 localizes in an autophagy-specific manner to cytoplasmic membranes that are positive for LC3, human WIPI1 was functionally described as a novel autophagy factor (Proikas-Cezanne et al., 2004), with a function related to that of ancestral yeast Atg18 (Barth and Thumm, 2001; Dove et al., 2004; Guan et al., 2001) in autophagy (Box 2). Furthermore, it was proposed that WIPI homologs in all eukaryotes share an evolutionarily conserved function in autophagy, which is compromised in human cancer (Proikas-Cezanne et al., 2004). Subsequent siRNA-mediated knockdown studies on human WIPI members have demonstrated an essential function in mammalian autophagy (Dooley et al., 2014; Gaugel et al., 2012; Liu and Ryan, 2012; Mauthe et al., 2011; Polson et al., 2010), as observed for WIPI homologs in lower eukaryotes, including Drosophila melanogaster (Scott et al., 2004), Arabidopsis thaliana (Xiong et al., 2005), Caenorhabditis elegans (Liu and Ryan, 2012) and Dictyostelium discoideum (Calvo-Garrido et al., 2014). In addition, further data point towards a crucial role of human WIPI members in cancer (Box 1) and other human pathologies, including neurodegeneration (Haack et al., 2012; Hayflick et al., 2013; Saitsu et al., 2013), phospholipidosis (Sawada et al., 2005) and cardiovascular diseases (Chasman et al., 2009). Moreover, WIPI members might contribute to human aging and longevity as ATG-18, the WIPI1/2 homolog in C. elegans, has been found to be essential for lifespan determination (Box 3).

The four human WIPI members were also described independently based on their sequence homology to yeast Atg18, and WIPI49 (WIPI1β) was shown to localize to the trans-Golgi and endosomal membranes in monkey cells (Jeffries et al., 2004). Importantly, GST-purified WIPI49 was shown to bind to PtdIns3P, phosphatidylinositol 5-phosphate (PtdIns5P) and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2], with a preference for PtdIns3P (Jeffries et al., 2004). Specific binding of human WIPI1 and also WIPI2 to these phosphoinositides with a preference towards PtdIns3P was confirmed in subsequent studies (Dooley et al., 2014; Gaugel et al., 2012; Polson et al., 2010; Proikas-Cezanne et al., 2007).

WIPI members fold into seven-bladed β-propeller proteins

Human WIPI proteins contain seven WD repeats that fold into an open ‘Velcro’-arranged seven-bladed β-propeller, as predicted by homology modeling of human WIPI1 and as proposed for all WIPI homologs (Proikas-Cezanne et al., 2004). Indeed, the seven-bladed β-propeller structure was demonstrated by structural analyses of the WIPI homolog Hsv2 in Kluyveromyces lactis (Baskaran et al., 2012a; Baskaran et al., 2012b; Krick et al., 2012) and in Kluyveromyces marxianus (Watanabe et al., 2012). Conserved and invariant residues from yeast to human were identified throughout the seven WD repeats that form two clusters at opposite sides of the WIPI β-propeller (depicted in WIPI1; Fig. 2A,B) (Proikas-Cezanne et al., 2004). One of these clusters, harboring invariant residues within blades 5 and 6, the most conserved blades (Fig. 2A,B, magenta), was predicted to function as a phosphoinositide-binding site (Proikas-Cezanne et al., 2004). Within this cluster, two crucial arginine residues (R226 and R227 in human WIPI1) within an FRRG motif were identified as being essential for phosphoinositide binding in yeast homologs (Dove et al., 2004; Krick et al., 2006) and human WIPI1 (Gaugel et al., 2012; Jeffries et al., 2004; Proikas-Cezanne et al., 2007). The opposite site of the β-propeller, harboring a cluster of homologous residues (Fig. 2A,B, red), was proposed to associate with regulatory proteins (Proikas-Cezanne et al., 2004).

Indeed, based on the structural analysis of Hsv2, invariant residues were identified in the protein that confer specific binding to two individual phosphoinositides (Baskaran et al., 2012a; Baskaran et al., 2012b; Krick et al., 2012; Watanabe et al., 2012). Furthermore, alanine-scanning mutagenesis demonstrated that the cluster of invariant residues in human WIPI1 confers specific binding to PtdIns3P at phagophore membranes (Gaugel et al., 2012). It has been shown that identical amino acids in WIPI1 confer the ability to bind to either PtdIns3P or PtdIns(3,5)P2 (Gaugel et al., 2012), indicating that one pool of WIPI members might bind to PtdIns3P and another pool to PtdIns(3,5)P2. Of note, it was suggested that PtdIns3P-bound yeast Atg18 functions in autophagy, whereas PtdIns(3,5)P2-bound Atg18 fulfills additional functions in alternative vesicle pathways (Box 2) (Efe et al., 2007).

A crucial arginine, R110, within the cluster of conserved residues in WIPI1 (Fig. 2A,B, red) was found to bind to an as-yet-unidentified regulator that should inhibit the binding of WIPI1 to PtdIns3P at phagophore membranes (Gaugel et al., 2012). Recently, autophagy regulation through arginine residues (R108, R125) within the cluster of conserved residues in human WIPI2B was reported, with these residues being found to confer specific binding to ATG16L1, an event preceding LC3 lipidation (Dooley et al., 2014). Interestingly, although these crucial arginines are conserved in all WIPI members, the binding of ATG16L1 is specific to WIPI2B (Dooley et al., 2014), supporting the idea of non-redundant and distinct functional contributions of WIPI members and splice variants to the process of autophagy.

WIPI proteins belong to an ancient seven-bladed β-propeller protein family referred to as PROPPINs

Phylogenetic analysis has demonstrated that human WIPI proteins belong to an ancient seven-bladed β-propeller protein family, with two paralogous groups, one containing human WIPI1 and WIPI2 and the ancestral yeast Atg18, and the other containing human WIPI3 and WIPI4 (Behrends et al., 2010; Polson et al., 2010; Proikas-Cezanne et al., 2004). This ancient β-propeller protein family was proposed to be named PROPPIN, for ‘β-propellers that bind phosphoinositides’ (Michell et al., 2006). Both paralogous groups of the PROPPIN family include representatives from plants, fungi and animals, and the WIPI3 and WIPI4 group is also present in some protozoans (Behrends et al., 2010; Polson et al., 2010; Proikas-Cezanne et al., 2004). Phylogenetic analysis further demonstrated that vertebrates have undergone an additional duplication in each of the two paralogous groups, indicating that the four human WIPI proteins should fulfill distinct functional roles in autophagy (Behrends et al., 2010; Polson et al., 2010; Proikas-Cezanne et al., 2004). Because the prototypical β-propeller was differentiated into its seven blades at the time of evolutionary divergence, phosphoinositide-binding likely represents an ancestral function of the PROPPIN family (Gaugel et al., 2012; Proikas-Cezanne et al., 2004).

WIPI1

WIPI1 was the first WIPI family member to be identified as having a role in autophagy. Prototypical cell treatments that induce autophagy above the basal level, such as starvation and treatment with rapamycin, promote a striking relocalization of cytoplasmic WIPI1 to ATG12-, ATG16L- and LC3-positive autophagosomal membranes (Gaugel et al., 2012; Itakura and Mizushima, 2010; Proikas-Cezanne et al., 2007; Proikas-Cezanne et al., 2004; Vergne et al., 2009). Furthermore, prototypical cell treatments that inhibit autophagy, such as blocking the generation of PtdIns3P, prevent the formation of autophagosomal membranes, causing WIPI1 to remain in the cytoplasm (Gaugel et al., 2012; Proikas-Cezanne et al., 2004; Vergne et al., 2009). In the cytoplasm, WIPI1 moves bidirectionally on microtubules, and the relocalization of cytoplasmic WIPI1 to autophagosomal membranes upon autophagy induction was found to be assisted by labile microtubules (Geeraert et al., 2010). Autophagosomal membranes that become decorated with WIPI1 upon autophagy induction have been shown to resemble both phagophores and autophagosomes (Proikas-Cezanne and Robenek, 2011; Proikas-Cezanne et al., 2007) (Fig. 1B). Freeze-fracture replica immunolabelling identified endogenous WIPI1, as well as overexpressed GFP–WIPI1, in both the inner and the outer autophagosomal membrane (Proikas-Cezanne and Robenek, 2011). Moreover, upon autophagy induction, WIPI1 localizes prominently to the plasma membrane and the ER, particularly the nuclear envelope (Proikas-Cezanne and Robenek, 2011), which suggests that these structures act as sources of membrane for autophagosomes. The specific localization of WIPI1 at autophagosomal membranes can be observed as punctate structures by using fluorescence microscopy. Consequently, quantifying the number of WIPI1-puncta-positive cells and the number of WIPI1 puncta per cell was introduced as a new method to assess autophagy (Klionsky et al., 2012; Proikas-Cezanne et al., 2007). Subsequently, WIPI1 puncta formation assessments have been used as the basis for automated high-throughput high-content image acquisition and analysis (Mauthe et al., 2011; Pfisterer et al., 2011). Apart from reflecting autophagy induction, an increase in the number of WIPI1 puncta can also be due to an imposed block in autophagic flux, e.g. by treatment with Ca2+-mobilizing compounds (Engedal and Mills, 2014), according to an increase of LC3 punctae upon autophagic flux inhibition (Ganley et al., 2011). Of note, not only does the monitoring of WIPI1 protein localization represent a robust tool to assess autophagy, but the abundance of WIPI1 mRNA also reliably reflects the level of autophagosome formation (Tsuyuki et al., 2014).

Evidence for an essential PtdIns3P effector function of WIPI1 has been provided by several approaches. The induction of autophagy by starvation or rapamycin treatment results in specific and rapid relocalization of cytoplasmic WIPI1 to phagophores and autophagosomes; this can be counteracted by co-treatment with compounds that inhibit PtdIns3P production, such as wortmannin or LY294002 (Proikas-Cezanne et al., 2007; Proikas-Cezanne et al., 2004). The localization of WIPI1 to the phagophore is also counteracted upon functional inhibition of PtdIns3P production, either by siRNA-mediated downregulation of PI3KC3 (Itakura and Mizushima, 2010) or in genetically deficient PI3KC3 (Devereaux et al., 2013) or Vps15 (Nemazanyy et al., 2013) backgrounds. In line with this, mutant WIPI1 variants that are unable to bind to PtdIns3P are also unable to localize to autophagosomal membranes (Gaugel et al., 2012; Proikas-Cezanne et al., 2007). Moreover, treatment with YM201636, a selective PIKFYVE inhibitor (Jefferies et al., 2008) that prevents the phosphorylation of PtdIns3P to PtdIns(3,5)P2 – thereby leading to PtdIns3P accumulation – results in an enhanced localization of WIPI1 to the phagophore and autophagosome (Gaugel et al., 2012). It is not only the pool of PtdIns3P and PtdIns(3,5)P2 that regulates autophagy, but also the balance between PtdIns3P production and hydrolysis, with the latter being controlled by myotubularin phosphatases (MTMR) that remove D3-positioned phosphate from PtdIns3P or PtdIns(3,5)P2 (Fig. 2C). Downregulation of Jumpy (also known as MTMR14) or MTMR3 was found to significantly increase the amount of WIPI1-decorated autophagosomal membranes and, thus, autophagosome formation (Taguchi-Atarashi et al., 2010; Vergne et al., 2009).

The positive regulation of the PtdIns3P effector activity of human WIPI1 follows the canonical route to autophagosome formation, i.e. the inhibition of mTORC1, activation of ULK1 and PI3KC3, and production of PtdIns3P (Codogno et al., 2012). It has been demonstrated that mTORC1 inhibition, either by rapamycin administration (Proikas-Cezanne et al., 2007), siRNA-mediated mTOR downregulation (Gaugel et al., 2012) or FOXO-mediated glutamine production (van der Vos et al., 2012), promotes the localization of WIPI1 at the nascent autophagosome. This specific localization of WIPI1 is counteracted by the downregulation of ULK1 or PI3KC3 (Itakura and Mizushima, 2010; McAlpine et al., 2013). Hence, the activation of ULK1 and PI3KC3, followed by localized PtdIns3P production, rapidly attracts WIPI1 through its PtdIns3P-binding specificity. Subsequently, PtdIns3P-bound WIPI1 at the nascent autophagosome is required for the conjugation of LC3 to PtdEtn (Fig. 1), as siRNA-mediated downregulation of WIPI1 counteracts LC3 lipidation (Mauthe et al., 2011). The role of WIPI1 as a crucial PtdIns3P effector in autophagy has been further underlined by the finding that WIPI1 also functions in selective autophagy, including mitophagy (Itakura et al., 2012) and xenophagy (Kageyama et al., 2011; Mauthe et al., 2012). Accordingly, WIPI1 prominently colocalizes with the autophagy adaptor p62 (also known as SQSTM1) (van der Vos et al., 2012).

The PtdIns3P-effector function of WIPI1 not only depends on ULK-mediated activation of PI3K3C and PtdIns3P production during the initiation step of autophagy, but also on Ca2+ signaling, as Ca2+ chelation counteracts starvation-induced accumulation of WIPI1 at autophagosomal membranes (Engedal and Mills, 2014; Pfisterer et al., 2011). AMPK, one of the conserved regulators of autophagy, can be activated by Ca2+ signaling through calmodulin-dependent kinase kinase α/β (CAMKKα and -β; also known as CAMKK1 and -2) (Means, 2008). Accordingly, cytoplasmic Ca2+ levels have been found to positively regulate autophagy through CaMKKα/β-mediated activation of AMPK followed by the inhibition of mTORC1 (Høyer-Hansen et al., 2007). However, Ca2+-mediated induction of autophagy has been suggested to also occur in a non-canonical way, bypassing AMPK (Grotemeier et al., 2010). Accordingly, although WIPI1 localization at autophagosomal membranes is ablated upon Ca2+ chelation, WIPI1-positive autophagosomes are formed in AMPK-deficient mouse embryonic fibroblasts, probably involving an AMPK-independent route through CAMKI (Pfisterer et al., 2011). It is noteworthy that the PtdIns3P effector function of WIPI1 does not respond to glucose starvation (McAlpine et al., 2013; Pfisterer et al., 2011), which generally activates AMPK. Glucose starvation has been suggested to signal independently of PtdIns3P to trigger autophagy (McAlpine et al., 2013).

WIPI2

Initial expression analysis showed that WIPI2 is ubiquitously expressed in normal human tissues, with high levels in heart and skeletal muscle, and it appears to be aberrantly expressed in matched human pancreatic, kidney and uterine tumor samples (Proikas-Cezanne et al., 2004) (Box 1). Interestingly, non-silent mutations in WIPI2 have been identified in large-scale analyses (Box 1, Table 1).

Table 1.
WIPI mutations identified in human tumors
graphic
graphic
*

nonsense mutations; fs, frame shift; SCC, squamous cell carcinoma; ALL, acute lymphoblastic leukemia; RCC, renal cell carcinoma.

In the course of the cloning of the human WIPI gene and protein family, WIPI2 splice variants have been identified that also encode seven-bladed β-propeller WIPI2 proteins (Proikas-Cezanne et al., 2004). So far, only the splice variants WIPI2B and WIPI2D (Proikas-Cezanne et al., 2004) follow the pattern of WIPI1, whereby punctate structures prominently increase upon autophagy induction. By contrast, full-length WIPI2A and WIPI2C show no increase in punctate staining upon autophagy induction (Mauthe et al., 2011). This indicates that different WIPI2 splice variants fulfill distinct functional roles, one of which is essential in autophagy (Lamb et al., 2013). Like WIPI1 (Itakura and Mizushima, 2010), WIPI2 functions downstream of ULK1 (McAlpine et al., 2013) and specifically binds to PtdIns3P at early autophagosomal membranes that are decorated with ULK1, DFCP1, ATG16L1 and LC3 during starvation-induced autophagy (Polson et al., 2010). Therefore, WIPI2 localizes to both ER-associated omegasomes and phagophore membranes (Polson et al., 2010). Immunofreeze fracture electron microscopy provided evidence that WIPI2B (Fig. 1C) and WIPI2D (Fig. 1D) further localize at the inner and outer membrane of autophagosomes (Proikas-Cezanne and Robenek, 2011). As is the case for the distribution of WIPI1, WIPI2 also appears to be enriched in the inner membrane of the autophagosome (Fig. 1B–D). This asymmetry seems to be distinct from the localization of PtdIns3P in yeast, which is enriched in the luminal monolayers of the autophagosome, thereby suggesting different initiating processes of autophagosome formation in yeast and mammals (Cheng et al., 2014). Immunofreeze fracture electron microscopy provides further evidence that WIPI2B and WIPI2D also localize at the plasma membrane and to the Golgi and ER area (Proikas-Cezanne and Robenek, 2011).

Functionally, siRNA-mediated downregulation of WIPI2 showed that WIPI2 is essential for LC3 lipidation (Mauthe et al., 2011; Polson et al., 2010). Interestingly, WIPI2 downregulation results in an accumulation of ATG9 (also known as ATG9A) at DFCP1-positive omegasomes (Orsi et al., 2012), suggesting that WIPI2 might have a role in the retrieval of ATG9 from early autophagosomal membranes, a functional role that has been assigned to the yeast Atg18 (Reggiori et al., 2004). However, WIPI2 was not found to be necessary for the recruitment of ATG9 to the omegasomes (Orsi et al., 2012).

Importantly, a recent study demonstrates that WIPI2 connects PtdIns3P production with LC3 lipidation at the onset of autophagy, because WIPI2 has been found to specifically bind to ATG16L1, thereby recruiting the ATG12–ATG5–ATG16L1 complex that is required for LC3 lipidation (Dooley et al., 2014). This indicates a scaffold function for WIPI2, in that it bridges PI3KC3-mediated PtdIns3P production and LC3 lipidation during phagophore formation and expansion. WIPI2 is also though to play a functional role in bridging the PtdIns3P signal with LC3 lipidation in xenophagy, as shown for Salmonella clearance (Dooley et al., 2014). In this context it has been reported previously that during xenophagy of Shigella, WIPI2 interacts with TECPR1, which also interacts with ATG5 (Ogawa et al., 2011). Moreover, a functional requirement for TECPR1 in regulating xenophagy of Shigella was extended to a general role of TECPR1 in selective autophagy, as TECPR1-deficient mouse embryonic fibroblasts were found to be defective in degrading depolarized mitochondria and protein aggregates (Ogawa et al., 2011).

The PtdIns3P-effector function of WIPI2 has also been shown to be crucial in non-canonical autophagy that is triggered by LRRK2 protein kinase activity (Manzoni et al., 2013) or rVP1, a recombinant capsid protein of the foot and mouth disease virus (Liao et al., 2014). By contrast, autophagy triggered by glucose starvation appears to be independent of WIPI2, as is the case for WIPI1 (McAlpine et al., 2013).

WIPI3

WIPI3 (also known as WDR45L) was found to be ubiquitously expressed in normal human tissue and aberrantly expressed in ovarian, uterus and kidney cancers in an initial expression analysis (Proikas-Cezanne et al., 2004). WIPI3 should also fold into a seven-bladed β-propeller that specifically binds to phosphoinositides (Proikas-Cezanne et al., 2004); however, this has not been demonstrated as yet, and the functional role of WIPI3 is unknown.

WIPI4

WIPI4 (also known as WDR45) encodes a seven-bladed β-propeller protein that is expected to specifically bind to phosphoinositides; it is ubiquitously expressed at high levels in skeletal muscle and heart, and appears to be overexpressed in pancreatic and kidney cancer samples in an initial expression analysis (Proikas-Cezanne et al., 2004). The localization of WIPI4 has been analyzed in normal rat kidney (NRK) cells, and the protein shows a diffuse pattern in dividing cells (Lu et al., 2011). Specifically upon starvation, WIPI4 relocates to autophagosomal membranes and colocalizes with LC3 (Lu et al., 2011), but unlike WIPI1 and WIPI2, WIPI4 was not found on autophagosomes as analyzed by immunofreeze fracture electron microscopy (T.P.-C. and Horst Robenek, unpublished observations). Functionally, WIPI4 was found to be essential for proper omegasome maturation and autophagosome formation, as siRNA-mediated downregulation of WIPI4 impaired the formation of autophagosomes and increased the number of ATG5-positive phagophores (Lu et al., 2011). Thus WIPI4 was shown to be essential for autophagy in mammalian cells. The WIPI4 homolog EPG-6 in C. elegans is thought to function downstream of ATG-18, in complex with ATG-2 (Lu et al., 2011). From this, it is possible that mammalian WIPI4 binds to ATG2 and acts downstream of LC3 in autophagosome formation (Fig. 2C). However, the role of the WIPI members in recruiting ATG2 in human cells warrants further detailed studies, as it was also found that ATG2 functions hierarchically at the same position as WIPI1 (Itakura and Mizushima, 2010; Velikkakath et al., 2012), and if WIPI1 is not bound to PtdIns3P at omegasomes, ATG2 is not recruited to the initiation site of autophagosome formation (Pfisterer et al., 2014).

Strikingly, WIPI4 was found to be mutated de novo and causative for SENDA (static encephalopathy of childhood with neurodegeneration in adulthood), a sporadic NBIA (neurodegeneration with brain iron accumulation) subtype (Haack et al., 2012; Hayflick et al., 2013; Saitsu et al., 2013). Interestingly, although the WIPI4 gene is localized on the X chromosome, the phenotype in female and male patients has been found to be indistinguishable (Hayflick et al., 2013; Saitsu et al., 2013). Functionally, it has been shown that the de novo mutations in the WIPI4 gene, which probably result in the production of unstable WIPI4 protein variants, indeed confer a drastic reduction in autophagic function in lymphoblastoid cell lines derived from SENDA patients (Saitsu et al., 2013). Furthermore, this study showed that mutant WIPI4 proteins in SENDA patients were less abundant when compared with wild-type WIPI4 in unaffected individuals and that lipidated LC3 and LC3-positive autophagosomal membranes accumulated in these patients without being completely utilized in the process of autophagy (Saitsu et al., 2013). Furthermore, accumulated LC3-positive autophagosomal membranes were also positive for ATG9 and were classified as abnormal autophagic structures, and autophagy occurred at only very low levels (Saitsu et al., 2013). Thus, WIPI4 mutations in SENDA patients lead to the accumulation of early autophagosomal membranes and improper autophagic degradation. These findings are of utmost importance for both future diagnosis and classification of SENDA. Moreover, identifying WIPI4 mutations in SENDA patients provides the first direct evidence that autophagy malfunction is causative of neurodegeneration in humans (Saitsu et al., 2013).

Conclusions

Evidence suggests that WIPI1, WIPI2 and WIPI4 function as essential and non-redundant PtdIns3P effectors downstream of PI3KC3-mediated PtdIns3P production at the onset of autophagy. By this means, WIPI2 recruits the ATG12–ATG5–ATG16L1 complex, which mediates LC3 lipidation, for which WIPI1 is also essential. In addition, WIPI4 interacts with ATG2 and is anticipated to function downstream of LC3. It is of urgent interest to decipher the individual contributions of the WIPI members at the nascent autophagosome, as their concerted action and regulation should hold clues for the further understanding of autophagy initiation and for the understanding of autophagosomal membrane formation. On the basis of their important role in autophagy, the function of WIPI family members is likely to be compromised in a great variety of human pathologies that exhibit defective or misregulated autophagy, as indicated by the identification of WIPI mutations in cancer and neurodegeneration. Hence, a better understanding of WIPI proteins could help to elucidate the principles of autophagosome formation in health and disease.

Box 1. WIPIs and cancer

Based on the initial observation that WIPI1 is encoded within a segment on the long arm of chromosome 17 [see the box figure, fluorescent in situ hybridization analysis localizing human WIPI1 to 17q24.3 (indicated by the arrows)], which is allelically imbalanced in a variety of tumors, subsequent WIPI gene expression analysis in matched human tumor samples suggested that there is aberrant expression of all human WIPI genes in different tumors (Proikas-Cezanne et al., 2004). Interestingly, aberrant expression of WIPI1 in 50% of human skin tumor samples and melanoma cell lines (Proikas-Cezanne et al., 2004) correlates with the high abundance of WIPI1 protein (http://www.proteinatlas.org/ENSG00000070540/cancer) and the identification of WIPI1 mutations in melanoma (Table 1). WIPI1 expression was also upregulated in 40% of cervical carcinoma patients. Moreover, non-silent mutations have been identified in all human WIPI members in large-scale genome analyses. In particular, three mutant WIPI1 and WIPI4 variants were found in analyses of lung cancer (Table 1).

In addition to the original report that beclin 1 induces autophagy and inhibits tumorigenesis (Liang et al., 1999), subsequent evidence showed that autophagy contributes to tumor development and therapy resistance (Liu and Ryan, 2012; White, 2012). Whether and how WIPI members contribute to tumorigenesis and malignancy, however, needs to be addressed in large-scale tumor patient analyses with regard to WIPI mutations, expression status and functional consequences for autophagy. Moreover, it needs to be determined whether WIPI mutations found in different tumors represent driver or passenger mutations. Nevertheless, in primary AML patient samples, a significantly repressed expression of WIPI1 was recently reported, in line with the functional dependence on WIPI-mediated autophagy during neutrophil differentiation (Brigger et al., 2014).

graphic
Box 2. Atg18 function in yeast autophagy

In yeast, three WIPI homologs have been identified: Atg18, Atg21 and Hsv2 (Barth et al., 2002; Barth and Thumm, 2001; Georgakopoulos et al., 2001; Guan et al., 2001). Atg18 has been reported to function in both autophagy and the cytoplasm-to-vacuole (Cvt) pathway (Barth and Thumm, 2001; Guan et al., 2001), and is predicted to fold into a seven-bladed β-propeller with six loops interconnecting the individual propeller blades (Dove et al., 2004). Atg18 specifically binds to both PtdIns3P and PtdIns(3,5)P2 (Dove et al., 2004; Strømhaug et al., 2004) through a FRRG motif (Krick et al., 2006), with higher affinity for PtdIns(3,5)P2in vitro (Dove et al., 2004; Rieter et al., 2013). Functionally, the PtdIns3P effector activity of Atg18 was proposed to be essential for autophagy, whereas the PtdIns(3,5)P2 effector activity was proposed to be required for vacuole morphology and inheritance, and retrograde transport (Efe et al., 2007). The essential PtdIns3P-effector function of Atg18 in Saccharomyces cerevisiae is interconnected with the function of Atg2 (Barth and Thumm, 2001; Shintani et al., 2001; Strømhaug et al., 2001; Wang et al., 2001), as Atg18 binds to Atg2, and the Atg18–Atg2 complex localizes to the autophagosomal membrane through the binding of Atg18 to PtdIns3P (Obara et al., 2008; Rieter et al., 2013; Suzuki et al., 2013; Suzuki et al., 2007). Thus, recruitment of Atg18–Atg2 depends on PtdIns3P, and as recently found, is facilitated by Atg1-mediated phosphorylation of Atg9 (Papinski et al., 2014). Vice versa, Atg9 cycling through the yeast pre-autophagosomal structure depends on Atg18 and Atg2 (Reggiori et al., 2004).

Yeast Atg21, which also binds to PtdIns3P and PtdIns(3,5)P2 (Dove et al., 2004; Strømhaug et al., 2004), strictly functions in the Cvt pathway (Barth et al., 2002; Krick et al., 2008; Strømhaug et al., 2004). However, both Atg18 and Atg21 enable the recruitment of Atg8 (LC3) to the nascent autophagosome (Meiling-Wesse et al., 2004; Nair et al., 2010).

Hsv2 has been shown to function in micronucleophagy in S. cerevisiae (Krick et al., 2008). The protein structure of K. lactis Hsv2 was recently identified (Baskaran et al., 2012a; Krick et al., 2012), and the data confirmed the predicted seven-bladed β-propeller, as for yeast Atg18 (Dove et al., 2004) and human WIPI1 (Jeffries et al., 2004; Proikas-Cezanne et al., 2004). Furthermore, the two crucial arginines within the FRRG motif that have been identified in Atg18 (Dove et al., 2004; Krick et al., 2006) are situated in two different pockets of K. lactis Hsv2 and have been suggested to form two phospholipid-binding sites (Baskaran et al., 2012a; Krick et al., 2012). Mutation of the corresponding two phospholipid-binding sites in S. cerevisiae Atg18 revealed that the two sites are not redundant, but work cooperatively to confer the function of Atg18 in autophagy (Baskaran et al., 2012a). Moreover, the loop in blade 6 of K. lactis Hsv2, which is proposed to be responsible for membrane docking, was also found to be important for the autophagic function of Atg18 (Baskaran et al., 2012a).

In summary, Atg18, Atg21 and Hsv2 display different specificities in autophagic pathways and, furthermore, have been found to localize to the endosomal compartment in yeast (Krick et al., 2008). These data clearly indicate the need to search for further, non-autophagic functions of the human WIPI members as well as to dissect their different autophagic specificities.

Box 3. Atg-18 controls lifespan in C. elegans

Beside maintaining cellular homeostasis and securing cellular survival under conditions of stress, autophagy plays an important role in the regulation of lifespan and in aging and age-related diseases in multiple model organisms (Cuervo, 2008; Rubinsztein et al., 2011). Most of the signals regulating lifespan are connected to nutrient availability and metabolic activity, such as dietary restriction, mitochondrial respiration and the nutrient-sensing insulin–IGF-1 and TOR pathway (Gelino and Hansen, 2012). C. elegans carrying a mutation in the DAF-2 insulin-like receptor, live more than twice as long as wild-type nematodes (Kenyon et al., 1993) and exhibit increased autophagic activity, which is dependent on the ATG gene bec-1 (beclin 1) (Meléndez et al., 2003). Subsequent studies using C. elegans have reinforced the idea that ATG genes are essential for long-lived phenotypes and that ATG mutants age faster and have shorter lives than wild-type nematodes (Hars et al., 2007; Meléndez et al., 2003; Tóth et al., 2008). However, autophagy might not always be beneficial for longevity, as RNAi-mediated downregulation of unc-51 (ULK1 homolog) or atg-9 (ATG9 homolog) actually increased the lifespan of C. elegans S6 kinase rsks-1 mutants (Hashimoto et al., 2009).

C. elegans possesses two homologs of the human WIPI family: ATG-18 (a WIPI1/2 homolog) and EPG-6 (a WIPI3/4 homolog) (Proikas-Cezanne et al., 2004), both of which are necessary for proper autophagosome formation (Lu et al., 2011). Regarding lifespan regulation only the function of ATG-18 has been investigated so far, and the data demonstrate that the depletion of ATG-18 function decreased the lifespan of both wild-type and long-lived daf-2 mutant nematodes (Hashimoto et al., 2009; Meléndez et al., 2003; Tóth et al., 2008). Moreover, atg-18 mutant nematodes showed accelerated aging, represented by an earlier decline in locomotory activity and accumulation of lipofuscin granules when compared to that of wild-type nematodes (Hashimoto et al., 2009; Tóth et al., 2008). Furthermore, ATG-18 is also necessary for the increased lifespan observed upon the downregulation of LET-363, the C. elegans ortholog of TOR, the major inhibitor of autophagy (Tóth et al., 2008; Vellai et al., 2003). For future studies it will be interesting to investigate the role of EPG-6 in general lifespan regulation in C. elegans, as it has been shown that EPG-6-mutant L1 larvae have reduced lifespans in the absence of food (Lu et al., 2011).

Acknowledgements

We apologize to researchers whose work we were unable to cite due to length constraints.

Funding

We acknowledge financial support from the International Max Planck Research School ‘From Molecules to Organsims’, Tuebingen; and the Ministry of Science, Research and the Arts Baden Wuerttemberg (MWK Stuttgart, Landesforschungsschwerpunkt), Germany.

References

Artal-Martinez de Narvajas
A.
,
Gomez
T. S.
,
Zhang
J. S.
,
Mann
A. O.
,
Taoda
Y.
,
Gorman
J. A.
,
Herreros-Villanueva
M.
,
Gress
T. M.
,
Ellenrieder
V.
,
Bujanda
L.
<et al. (
2013
).
Epigenetic regulation of autophagy by the methyltransferase G9a.
Mol. Cell. Biol.
33
,
3983
3993
.
Axe
E. L.
,
Walker
S. A.
,
Manifava
M.
,
Chandra
P.
,
Roderick
H. L.
,
Habermann
A.
,
Griffiths
G.
,
Ktistakis
N. T.
(
2008
).
Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum.
J. Cell Biol.
182
,
685
701
.
Barth
H.
,
Thumm
M.
(
2001
).
A genomic screen identifies AUT8 as a novel gene essential for autophagy in the yeast Saccharomyces cerevisiae.
Gene
274
,
151
156
.
Barth
H.
,
Meiling-Wesse
K.
,
Epple
U. D.
,
Thumm
M.
(
2002
).
Mai1p is essential for maturation of proaminopeptidase I but not for autophagy.
FEBS Lett.
512
,
173
179
.
Baskaran
S.
,
Ragusa
M. J.
,
Boura
E.
,
Hurley
J. H.
(
2012a
).
Two-site recognition of phosphatidylinositol 3-phosphate by PROPPINs in autophagy.
Mol. Cell
47
,
339
348
.
Baskaran
S.
,
Ragusa
M. J.
,
Hurley
J. H.
(
2012b
).
How Atg18 and the WIPIs sense phosphatidylinositol 3-phosphate.
Autophagy
8
,
1851
1852
.
Behrends
C.
,
Sowa
M. E.
,
Gygi
S. P.
,
Harper
J. W.
(
2010
).
Network organization of the human autophagy system.
Nature
466
,
68
76
.
Berger
M. F.
,
Hodis
E.
,
Heffernan
T. P.
,
Deribe
Y. L.
,
Lawrence
M. S.
,
Protopopov
A.
,
Ivanova
E.
,
Watson
I. R.
,
Nickerson
E.
,
Ghosh
P.
<et al. (
2012
).
Melanoma genome sequencing reveals frequent PREX2 mutations.
Nature
485
,
502
506
.
Bettegowda
C.
,
Agrawal
N.
,
Jiao
Y.
,
Sausen
M.
,
Wood
L. D.
,
Hruban
R. H.
,
Rodriguez
F. J.
,
Cahill
D. P.
,
McLendon
R.
,
Riggins
G.
<et al. (
2011
).
Mutations in CIC and FUBP1 contribute to human oligodendroglioma.
Science
333
,
1453
1455
.
Biankin
A. V.
,
Waddell
N.
,
Kassahn
K. S.
,
Gingras
M. C.
,
Muthuswamy
L. B.
,
Johns
A. L.
,
Miller
D. K.
,
Wilson
P. J.
,
Patch
A. M.
,
Wu
J.
<et al. 
Australian Pancreatic Cancer Genome Initiative
(
2012
).
Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes.
Nature
491
,
399
405
.
Brigger
D.
,
Proikas-Cezanne
T.
,
Tschan
M. P.
(
2014
).
WIPI-dependent autophagy during neutrophil differentiation of NB4 acute promyelocytic leukemia cells.
Cell Death Dis.
5
,
e1315
.
Calvo-Garrido
J.
,
King
J. S.
,
Muñoz-Braceras
S.
,
Escalante
R.
(
2014
).
Vmp1 regulates PtdIns3P signaling during autophagosome formation in Dictyostelium discoideum.
Traffic
15
,
1235
1246
.
Carlsson
S. R.
,
Simonsen
A.
(
2015
).
Membrane dynamics in autophagosome biogenesis.
J. Cell Sci.
128
,
193
205
.
Chasman
D. I.
,
Paré
G.
,
Mora
S.
,
Hopewell
J. C.
,
Peloso
G.
,
Clarke
R.
,
Cupples
L. A.
,
Hamsten
A.
,
Kathiresan
S.
,
Mälarstig
A.
<et al. (
2009
).
Forty-three loci associated with plasma lipoprotein size, concentration, and cholesterol content in genome-wide analysis.
PLoS Genet.
5
,
e1000730
.
Chauhan
S.
,
Goodwin
J. G.
,
Chauhan
S.
,
Manyam
G.
,
Wang
J.
,
Kamat
A. M.
,
Boyd
D. D.
(
2013
).
ZKSCAN3 is a master transcriptional repressor of autophagy.
Mol. Cell
50
,
16
28
.
Cheng
J.
,
Fujita
A.
,
Yamamoto
H.
,
Tatematsu
T.
,
Kakuta
S.
,
Obara
K.
,
Ohsumi
Y.
,
Fujimoto
T.
(
2014
).
Yeast and mammalian autophagosomes exhibit distinct phosphatidylinositol 3-phosphate asymmetries.
Nat. Commun.
5
,
3207
.
Choi
A. M.
,
Ryter
S. W.
,
Levine
B.
(
2013
).
Autophagy in human health and disease.
N. Engl. J. Med.
368
,
1845
1846
.
Clausen
T. H.
,
Lamark
T.
,
Isakson
P.
,
Finley
K.
,
Larsen
K. B.
,
Brech
A.
,
Øvervatn
A.
,
Stenmark
H.
,
Bjørkøy
G.
,
Simonsen
A.
<et al. (
2010
).
p62/SQSTM1 and ALFY interact to facilitate the formation of p62 bodies/ALIS and their degradation by autophagy.
Autophagy
6
,
330
344
.
Codogno
P.
,
Mehrpour
M.
,
Proikas-Cezanne
T.
(
2012
).
Canonical and non-canonical autophagy: variations on a common theme of self-eating?
Nat. Rev. Mol. Cell Biol.
13
,
7
12
.
Corradetti
M. N.
,
Inoki
K.
,
Bardeesy
N.
,
DePinho
R. A.
,
Guan
K. L.
(
2004
).
Regulation of the TSC pathway by LKB1: evidence of a molecular link between tuberous sclerosis complex and Peutz-Jeghers syndrome.
Genes Dev.
18
,
1533
1538
.
Cuervo
A. M.
(
2008
).
Autophagy and aging: keeping that old broom working.
Trends Genet.
24
,
604
612
.
Dennis
M. D.
,
Baum
J. I.
,
Kimball
S. R.
,
Jefferson
L. S.
(
2011
).
Mechanisms involved in the coordinate regulation of mTORC1 by insulin and amino acids.
J. Biol. Chem.
286
,
8287
8296
.
Devereaux
K.
,
Dall'Armi
C.
,
Alcazar-Roman
A.
,
Ogasawara
Y.
,
Zhou
X.
,
Wang
F.
,
Yamamoto
A.
,
De Camilli
P.
,
Di Paolo
G.
(
2013
).
Regulation of mammalian autophagy by class II and III PI 3-kinases through PI3P synthesis.
PLoS ONE
8
,
e76405
.
Dooley
H. C.
,
Razi
M.
,
Polson
H. E.
,
Girardin
S. E.
,
Wilson
M. I.
,
Tooze
S. A.
(
2014
).
WIPI2 links LC3 conjugation with PI3P, autophagosome formation, and pathogen clearance by recruiting Atg12-5-16L1.
Mol. Cell
55
,
238
252
.
Dove
S. K.
,
Piper
R. C.
,
McEwen
R. K.
,
Yu
J. W.
,
King
M. C.
,
Hughes
D. C.
,
Thuring
J.
,
Holmes
A. B.
,
Cooke
F. T.
,
Michell
R. H.
<et al. (
2004
).
Svp1p defines a family of phosphatidylinositol 3,5-bisphosphate effectors.
EMBO J.
23
,
1922
1933
.
Dulak
A. M.
,
Stojanov
P.
,
Peng
S.
,
Lawrence
M. S.
,
Fox
C.
,
Stewart
C.
,
Bandla
S.
,
Imamura
Y.
,
Schumacher
S. E.
,
Shefler
E.
<et al. (
2013
).
Exome and whole-genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity.
Nat. Genet.
45
,
478
486
.
Efe
J. A.
,
Botelho
R. J.
,
Emr
S. D.
(
2007
).
Atg18 regulates organelle morphology and Fab1 kinase activity independent of its membrane recruitment by phosphatidylinositol 3,5-bisphosphate.
Mol. Biol. Cell
18
,
4232
4244
.
Egan
D.
,
Kim
J.
,
Shaw
R. J.
,
Guan
K. L.
(
2011
).
The autophagy initiating kinase ULK1 is regulated via opposing phosphorylation by AMPK and mTOR.
Autophagy
7
,
643
644
.
Engedal
N.
,
Mills
I. G.
(
2014
).
Endosomal signaling and oncogenesis.
Methods Enzymol.
535
,
179
200
.
Feng
Y.
,
He
D.
,
Yao
Z.
,
Klionsky
D. J.
(
2014
).
The machinery of macroautophagy.
Cell Res.
24
,
24
41
.
Filimonenko
M.
,
Isakson
P.
,
Finley
K. D.
,
Anderson
M.
,
Jeong
H.
,
Melia
T. J.
,
Bartlett
B. J.
,
Myers
K. M.
,
Birkeland
H. C.
,
Lamark
T.
<et al. (
2010
).
The selective macroautophagic degradation of aggregated proteins requires the PI3P-binding protein Alfy.
Mol. Cell
38
,
265
279
.
Finley
K. D.
,
Edeen
P. T.
,
Cumming
R. C.
,
Mardahl-Dumesnil
M. D.
,
Taylor
B. J.
,
Rodriguez
M. H.
,
Hwang
C. E.
,
Benedetti
M.
,
McKeown
M.
(
2003
).
blue cheese mutations define a novel, conserved gene involved in progressive neural degeneration.
J. Neurosci.
23
,
1254
1264
.
Fujita
N.
,
Itoh
T.
,
Omori
H.
,
Fukuda
M.
,
Noda
T.
,
Yoshimori
T.
(
2008
).
The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy.
Mol. Biol. Cell
19
,
2092
2100
.
Ganley
I. G.
,
Lam
H.
,
Wang
J.
,
Ding
X.
,
Chen
S.
,
Jiang
X.
(
2009
).
ULK1.ATG13.FIP200 complex mediates mTOR signaling and is essential for autophagy.
J. Biol. Chem.
284
,
12297
12305
.
Ganley
I. G.
,
Wong
P. M.
,
Gammoh
N.
,
Jiang
X.
(
2011
).
Distinct autophagosomal-lysosomal fusion mechanism revealed by thapsigargin-induced autophagy arrest.
Mol. Cell
42
,
731
743
.
Gaugel
A.
,
Bakula
D.
,
Hoffmann
A.
,
Proikas-Cezanne
T.
(
2012
).
Defining regulatory and phosphoinositide-binding sites in the human WIPI-1 β-propeller responsible for autophagosomal membrane localization downstream of mTORC1 inhibition.
J. Mol. Signal.
7
,
16
.
Geeraert
C.
,
Ratier
A.
,
Pfisterer
S. G.
,
Perdiz
D.
,
Cantaloube
I.
,
Rouault
A.
,
Pattingre
S.
,
Proikas-Cezanne
T.
,
Codogno
P.
,
Poüs
C.
(
2010
).
Starvation-induced hyperacetylation of tubulin is required for the stimulation of autophagy by nutrient deprivation.
J. Biol. Chem.
285
,
24184
24194
.
Gelino
S.
,
Hansen
M.
(
2012
).
Autophagy – an emerging anti-aging mechanism.
J. Clin Exp. Pathol.
Suppl. 4
,
006
.
Georgakopoulos
T.
,
Koutroubas
G.
,
Vakonakis
I.
,
Tzermia
M.
,
Prokova
V.
,
Voutsina
A.
,
Alexandraki
D.
(
2001
).
Functional analysis of the Saccharomyces cerevisiae YFR021w/YGR223c/YPL100w ORF family suggests relations to mitochondrial/peroxisomal functions and amino acid signalling pathways.
Yeast
18
,
1155
1171
.
Gillooly
D. J.
,
Morrow
I. C.
,
Lindsay
M.
,
Gould
R.
,
Bryant
N. J.
,
Gaullier
J. M.
,
Parton
R. G.
,
Stenmark
H.
(
2000
).
Localization of phosphatidylinositol 3-phosphate in yeast and mammalian cells.
EMBO J.
19
,
4577
4588
.
Grotemeier
A.
,
Alers
S.
,
Pfisterer
S. G.
,
Paasch
F.
,
Daubrawa
M.
,
Dieterle
A.
,
Viollet
B.
,
Wesselborg
S.
,
Proikas-Cezanne
T.
,
Stork
B.
(
2010
).
AMPK-independent induction of autophagy by cytosolic Ca2+ increase.
Cell. Signal.
22
,
914
925
.
Guan
J.
,
Stromhaug
P. E.
,
George
M. D.
,
Habibzadegah-Tari
P.
,
Bevan
A.
,
Dunn
W. A.
 Jr
,
Klionsky
D. J.
(
2001
).
Cvt18/Gsa12 is required for cytoplasm-to-vacuole transport, pexophagy, and autophagy in Saccharomyces cerevisiae and Pichia pastoris.
Mol. Biol. Cell
12
,
3821
3838
.
Gwinn
D. M.
,
Shackelford
D. B.
,
Egan
D. F.
,
Mihaylova
M. M.
,
Mery
A.
,
Vasquez
D. S.
,
Turk
B. E.
,
Shaw
R. J.
(
2008
).
AMPK phosphorylation of raptor mediates a metabolic checkpoint.
Mol. Cell
30
,
214
226
.
Haack
T. B.
,
Hogarth
P.
,
Kruer
M. C.
,
Gregory
A.
,
Wieland
T.
,
Schwarzmayr
T.
,
Graf
E.
,
Sanford
L.
,
Meyer
E.
,
Kara
E.
<et al. (
2012
).
Exome sequencing reveals de novo WDR45 mutations causing a phenotypically distinct, X-linked dominant form of NBIA.
Am. J. Hum. Genet.
91
,
1144
1149
.
Hamasaki
M.
,
Shibutani
S. T.
,
Yoshimori
T.
(
2013
).
Up-to-date membrane biogenesis in the autophagosome formation.
Curr. Opin. Cell Biol.
25
,
455
460
.
Hars
E. S.
,
Qi
H.
,
Ryazanov
A. G.
,
Jin
S.
,
Cai
L.
,
Hu
C.
,
Liu
L. F.
(
2007
).
Autophagy regulates ageing in C. elegans.
Autophagy
3
,
93
95
.
Hashimoto
Y.
,
Ookuma
S.
,
Nishida
E.
(
2009
).
Lifespan extension by suppression of autophagy genes in Caenorhabditis elegans.
Genes Cells
14
,
717
726
.
Hayashi-Nishino
M.
,
Fujita
N.
,
Noda
T.
,
Yamaguchi
A.
,
Yoshimori
T.
,
Yamamoto
A.
(
2009
).
A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation.
Nat. Cell Biol.
11
,
1433
1437
.
Hayflick
S. J.
,
Kruer
M. C.
,
Gregory
A.
,
Haack
T. B.
,
Kurian
M. A.
,
Houlden
H. H.
,
Anderson
J.
,
Boddaert
N.
,
Sanford
L.
,
Harik
S. I.
<et al. (
2013
).
β-propeller protein-associated neurodegeneration: a new X-linked dominant disorder with brain iron accumulation.
Brain
136
,
1708
1717
.
Hosokawa
N.
,
Hara
T.
,
Kaizuka
T.
,
Kishi
C.
,
Takamura
A.
,
Miura
Y.
,
Iemura
S.
,
Natsume
T.
,
Takehana
K.
,
Yamada
N.
<et al. (
2009
).
Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy.
Mol. Biol. Cell
20
,
1981
1991
.
Høyer-Hansen
M.
,
Bastholm
L.
,
Szyniarowski
P.
,
Campanella
M.
,
Szabadkai
G.
,
Farkas
T.
,
Bianchi
K.
,
Fehrenbacher
N.
,
Elling
F.
,
Rizzuto
R.
<et al. (
2007
).
Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2.
Mol. Cell
25
,
193
205
.
Imielinski
M.
,
Berger
A. H.
,
Hammerman
P. S.
,
Hernandez
B.
,
Pugh
T. J.
,
Hodis
E.
,
Cho
J.
,
Suh
J.
,
Capelletti
M.
,
Sivachenko
A.
<et al. (
2012
).
Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing.
Cell
150
,
1107
1120
.
Itakura
E.
,
Mizushima
N.
(
2010
).
Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins.
Autophagy
6
,
764
776
.
Itakura
E.
,
Kishi-Itakura
C.
,
Koyama-Honda
I.
,
Mizushima
N.
(
2012
).
Structures containing Atg9A and the ULK1 complex independently target depolarized mitochondria at initial stages of Parkin-mediated mitophagy.
J. Cell Sci.
125
,
1488
1499
.
Jefferies
H. B.
,
Cooke
F. T.
,
Jat
P.
,
Boucheron
C.
,
Koizumi
T.
,
Hayakawa
M.
,
Kaizawa
H.
,
Ohishi
T.
,
Workman
P.
,
Waterfield
M. D.
<et al. (
2008
).
A selective PIKfyve inhibitor blocks PtdIns(3,5)P(2) production and disrupts endomembrane transport and retroviral budding.
EMBO Rep.
9
,
164
170
.
Jeffries
T. R.
,
Dove
S. K.
,
Michell
R. H.
,
Parker
P. J.
(
2004
).
PtdIns-specific MPR pathway association of a novel WD40 repeat protein, WIPI49.
Mol. Biol. Cell
15
,
2652
2663
.
Jiang
P.
,
Mizushima
N.
(
2014
).
Autophagy and human diseases.
Cell Res.
24
,
69
79
.
Jiao
Y.
,
Yonescu
R.
,
Offerhaus
G. J.
,
Klimstra
D. S.
,
Maitra
A.
,
Eshleman
J. R.
,
Herman
J. G.
,
Poh
W.
,
Pelosof
L.
,
Wolfgang
C. L.
<et al. (
2014
).
Whole-exome sequencing of pancreatic neoplasms with acinar differentiation.
J. Pathol.
232
,
428
435
.
Joseph
C. G.
,
Hwang
H.
,
Jiao
Y.
,
Wood
L. D.
,
Kinde
I.
,
Wu
J.
,
Mandahl
N.
,
Luo
J.
,
Hruban
R. H.
,
Diaz
L. A.
 Jr
<et al. (
2014
).
Exomic analysis of myxoid liposarcomas, synovial sarcomas, and osteosarcomas.
Genes Chromosomes Cancer
53
,
15
24
.
Jung
C. H.
,
Jun
C. B.
,
Ro
S. H.
,
Kim
Y. M.
,
Otto
N. M.
,
Cao
J.
,
Kundu
M.
,
Kim
D. H.
(
2009
).
ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery.
Mol. Biol. Cell
20
,
1992
2003
.
Kabeya
Y.
,
Mizushima
N.
,
Ueno
T.
,
Yamamoto
A.
,
Kirisako
T.
,
Noda
T.
,
Kominami
E.
,
Ohsumi
Y.
,
Yoshimori
T.
(
2000
).
LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.
EMBO J.
19
,
5720
5728
.
Kageyama
S.
,
Omori
H.
,
Saitoh
T.
,
Sone
T.
,
Guan
J. L.
,
Akira
S.
,
Imamoto
F.
,
Noda
T.
,
Yoshimori
T.
(
2011
).
The LC3 recruitment mechanism is separate from Atg9L1-dependent membrane formation in the autophagic response against Salmonella.
Mol. Biol. Cell
22
,
2290
2300
.
Karanasios
E.
,
Stapleton
E.
,
Manifava
M.
,
Kaizuka
T.
,
Mizushima
N.
,
Walker
S. A.
,
Ktistakis
N. T.
(
2013
).
Dynamic association of the ULK1 complex with omegasomes during autophagy induction.
J. Cell Sci.
126
,
5224
5238
.
Kenyon
C.
,
Chang
J.
,
Gensch
E.
,
Rudner
A.
,
Tabtiang
R.
(
1993
).
A C. elegans mutant that lives twice as long as wild type.
Nature
366
,
461
464
.
Kim
J.
,
Kundu
M.
,
Viollet
B.
,
Guan
K. L.
(
2011
).
AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1.
Nat. Cell Biol.
13
,
132
141
.
Klionsky
D. J.
,
Cregg
J. M.
,
Dunn
W. A.
 Jr
,
Emr
S. D.
,
Sakai
Y.
,
Sandoval
I. V.
,
Sibirny
A.
,
Subramani
S.
,
Thumm
M.
,
Veenhuis
M.
<et al. (
2003
).
A unified nomenclature for yeast autophagy-related genes.
Dev. Cell
5
,
539
545
.
Klionsky
D. J.
,
Abdalla
F. C.
,
Abeliovich
H.
,
Abraham
R. T.
,
Acevedo-Arozena
A.
,
Adeli
K.
,
Agholme
L.
,
Agnello
M.
,
Agostinis
P.
,
Aguirre-Ghiso
J. A.
<et al. (
2012
).
Guidelines for the use and interpretation of assays for monitoring autophagy.
Autophagy
8
,
445
544
.
Koyama-Honda
I.
,
Itakura
E.
,
Fujiwara
T. K.
,
Mizushima
N.
(
2013
).
Temporal analysis of recruitment of mammalian ATG proteins to the autophagosome formation site.
Autophagy
9
,
1491
1499
.
Krauthammer
M.
,
Kong
Y.
,
Ha
B. H.
,
Evans
P.
,
Bacchiocchi
A.
,
McCusker
J. P.
,
Cheng
E.
,
Davis
M. J.
,
Goh
G.
,
Choi
M.
<et al. (
2012
).
Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma.
Nat. Genet.
44
,
1006
1014
.
Krick
R.
,
Tolstrup
J.
,
Appelles
A.
,
Henke
S.
,
Thumm
M.
(
2006
).
The relevance of the phosphatidylinositolphosphat-binding motif FRRGT of Atg18 and Atg21 for the Cvt pathway and autophagy.
FEBS Lett.
580
,
4632
4638
.
Krick
R.
,
Henke
S.
,
Tolstrup
J.
,
Thumm
M.
(
2008
).
Dissecting the localization and function of Atg18, Atg21 and Ygr223c.
Autophagy
4
,
896
910
.
Krick
R.
,
Busse
R. A.
,
Scacioc
A.
,
Stephan
M.
,
Janshoff
A.
,
Thumm
M.
,
Kühnel
K.
(
2012
).
Structural and functional characterization of the two phosphoinositide binding sites of PROPPINs, a β-propeller protein family.
Proc. Natl. Acad. Sci. USA
109
,
E2042
E2049
.
Lamb
C. A.
,
Yoshimori
T.
,
Tooze
S. A.
(
2013
).
The autophagosome: origins unknown, biogenesis complex.
Nat. Rev. Mol. Cell Biol.
14
,
759
774
.
Le Gallo
M.
,
O'Hara
A. J.
,
Rudd
M. L.
,
Urick
M. E.
,
Hansen
N. F.
,
O'Neil
N. J.
,
Price
J. C.
,
Zhang
S.
,
England
B. M.
,
Godwin
A. K.
<et al. 
NIH Intramural Sequencing Center (NISC) Comparative Sequencing Program
(
2012
).
Exome sequencing of serous endometrial tumors identifies recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes.
Nat. Genet.
44
,
1310
1315
.
Liang
X. H.
,
Jackson
S.
,
Seaman
M.
,
Brown
K.
,
Kempkes
B.
,
Hibshoosh
H.
,
Levine
B.
(
1999
).
Induction of autophagy and inhibition of tumorigenesis by beclin 1.
Nature
402
,
672
676
.
Liao
J.
,
Wu
C. X.
,
Burlak
C.
,
Zhang
S.
,
Sahm
H.
,
Wang
M.
,
Zhang
Z. Y.
,
Vogel
K. W.
,
Federici
M.
,
Riddle
S. M.
<et al. (
2014
).
Parkinson disease-associated mutation R1441H in LRRK2 prolongs the “active state” of its GTPase domain.
Proc. Natl. Acad. Sci. USA
111
,
4055
4060
.
Liu
E. Y.
,
Ryan
K. M.
(
2012
).
Autophagy and cancer—issues we need to digest.
J. Cell Sci.
125
,
2349
2358
.
Lu
Q.
,
Yang
P.
,
Huang
X.
,
Hu
W.
,
Guo
B.
,
Wu
F.
,
Lin
L.
,
Kovács
A. L.
,
Yu
L.
,
Zhang
H.
(
2011
).
The WD40 repeat PtdIns(3)P-binding protein EPG-6 regulates progression of omegasomes to autophagosomes.
Dev. Cell
21
,
343
357
.
Manzoni
C.
,
Mamais
A.
,
Dihanich
S.
,
Abeti
R.
,
Soutar
M. P.
,
Plun-Favreau
H.
,
Giunti
P.
,
Tooze
S. A.
,
Bandopadhyay
R.
,
Lewis
P. A.
(
2013
).
Inhibition of LRRK2 kinase activity stimulates macroautophagy.
Biochim. Biophys. Acta
1833
,
2900
2910
.
Matsunaga
K.
,
Morita
E.
,
Saitoh
T.
,
Akira
S.
,
Ktistakis
N. T.
,
Izumi
T.
,
Noda
T.
,
Yoshimori
T.
(
2010
).
Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L.
J. Cell Biol.
190
,
511
521
.
Mauthe
M.
,
Jacob
A.
,
Freiberger
S.
,
Hentschel
K.
,
Stierhof
Y. D.
,
Codogno
P.
,
Proikas-Cezanne
T.
(
2011
).
Resveratrol-mediated autophagy requires WIPI-1-regulated LC3 lipidation in the absence of induced phagophore formation.
Autophagy
7
,
1448
1461
.
Mauthe
M.
,
Yu
W.
,
Krut
O.
,
Krönke
M.
,
Götz
F.
,
Robenek
H.
,
Proikas-Cezanne
T.
(
2012
).
WIPI-1 positive autophagosome-like vesicles entrap pathogenic staphylococcus aureus for lysosomal degradation.
Int. J. Cell Biol.
2012
,
179207
.
McAlpine
F.
,
Williamson
L. E.
,
Tooze
S. A.
,
Chan
E. Y.
(
2013
).
Regulation of nutrient-sensitive autophagy by uncoordinated 51-like kinases 1 and 2.
Autophagy
9
,
361
373
.
Means
A. R.
(
2008
).
The year in basic science: calmodulin kinase cascades.
Mol. Endocrinol.
22
,
2759
2765
.
Meijer
A. J.
,
Codogno
P.
(
2011
).
Autophagy: regulation by energy sensing.
Curr. Biol.
21
,
R227
R229
.
Meijer
A. J.
,
Dubbelhuis
P. F.
(
2004
).
Amino acid signalling and the integration of metabolism.
Biochem. Biophys. Res. Commun.
313
,
397
403
.
Meiling-Wesse
K.
,
Barth
H.
,
Voss
C.
,
Eskelinen
E. L.
,
Epple
U. D.
,
Thumm
M.
(
2004
).
Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure during the Cvt pathway.
J. Biol. Chem.
279
,
37741
37750
.
Meléndez
A.
,
Tallóczy
Z.
,
Seaman
M.
,
Eskelinen
E. L.
,
Hall
D. H.
,
Levine
B.
(
2003
).
Autophagy genes are essential for dauer development and life-span extension in C. elegans.
Science
301
,
1387
1391
.
Michell
R. H.
,
Heath
V. L.
,
Lemmon
M. A.
,
Dove
S. K.
(
2006
).
Phosphatidylinositol 3,5-bisphosphate: metabolism and cellular functions.
Trends Biochem. Sci.
31
,
52
63
.
Mizushima
N.
(
2010
).
The role of the Atg1/ULK1 complex in autophagy regulation.
Curr. Opin. Cell Biol.
22
,
132
139
.
Mizushima
N.
,
Yoshimori
T.
,
Ohsumi
Y.
(
2011
).
The role of Atg proteins in autophagosome formation.
Annu. Rev. Cell Dev. Biol.
27
,
107
132
.
Muzny
D. M.
,
Bainbridge
M. N.
,
Chang
K.
,
Dinh
H. H.
,
Drummond
J. A.
,
Fowler
G.
,
Kovar
C. L.
,
Lewis
L. R.
,
Morgan
M. B.
,
Newsham
I. F.
<et al. (
2012
).
Comprehensive molecular characterization of human colon and rectal cancer.
Nature
487
,
330
337
.
Nair
U.
,
Cao
Y.
,
Xie
Z.
,
Klionsky
D. J.
(
2010
).
Roles of the lipid-binding motifs of Atg18 and Atg21 in the cytoplasm to vacuole targeting pathway and autophagy.
J. Biol. Chem.
285
,
11476
11488
.
Nakatogawa
H.
,
Oh-oka
K.
,
Ohsumi
Y.
(
2008
).
Lipidation of Atg8: how is substrate specificity determined without a canonical E3 enzyme?
Autophagy
4
,
911
913
.
Nakatogawa
H.
,
Suzuki
K.
,
Kamada
Y.
,
Ohsumi
Y.
(
2009
).
Dynamics and diversity in autophagy mechanisms: lessons from yeast.
Nat. Rev. Mol. Cell Biol.
10
,
458
467
.
Nemazanyy
I.
,
Blaauw
B.
,
Paolini
C.
,
Caillaud
C.
,
Protasi
F.
,
Mueller
A.
,
Proikas-Cezanne
T.
,
Russell
R. C.
,
Guan
K. L.
,
Nishino
I.
<et al. (
2013
).
Defects of Vps15 in skeletal muscles lead to autophagic vacuolar myopathy and lysosomal disease.
EMBO Mol. Med.
5
,
870
890
.
Nikolaev
S. I.
,
Rimoldi
D.
,
Iseli
C.
,
Valsesia
A.
,
Robyr
D.
,
Gehrig
C.
,
Harshman
K.
,
Guipponi
M.
,
Bukach
O.
,
Zoete
V.
<et al. (
2012a
).
Exome sequencing identifies recurrent somatic MAP2K1 and MAP2K2 mutations in melanoma.
Nat. Genet.
44
,
133
139
.
Nikolaev
S. I.
,
Sotiriou
S. K.
,
Pateras
I. S.
,
Santoni
F.
,
Sougioultzis
S.
,
Edgren
H.
,
Almusa
H.
,
Robyr
D.
,
Guipponi
M.
,
Saarela
J.
<et al. (
2012b
).
A single-nucleotide substitution mutator phenotype revealed by exome sequencing of human colon adenomas.
Cancer Res.
72
,
6279
6289
.
O'Farrell
F.
,
Wang
S.
,
Katheder
N.
,
Rusten
T. E.
,
Samakovlis
C.
(
2013
).
Two-tiered control of epithelial growth and autophagy by the insulin receptor and the ret-like receptor, stitcher.
PLoS Biol.
11
,
e1001612
.
Obara
K.
,
Sekito
T.
,
Niimi
K.
,
Ohsumi
Y.
(
2008
).
The Atg18-Atg2 complex is recruited to autophagic membranes via phosphatidylinositol 3-phosphate and exerts an essential function.
J. Biol. Chem.
283
,
23972
23980
.
Ogawa
M.
,
Yoshikawa
Y.
,
Kobayashi
T.
,
Mimuro
H.
,
Fukumatsu
M.
,
Kiga
K.
,
Piao
Z.
,
Ashida
H.
,
Yoshida
M.
,
Kakuta
S.
<et al. (
2011
).
A Tecpr1-dependent selective autophagy pathway targets bacterial pathogens.
Cell Host Microbe
9
,
376
389
.
Ohsumi
Y.
(
2014
).
Historical landmarks of autophagy research.
Cell Res.
24
,
9
23
.
Orsi
A.
,
Razi
M.
,
Dooley
H. C.
,
Robinson
D.
,
Weston
A. E.
,
Collinson
L. M.
,
Tooze
S. A.
(
2012
).
Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy.
Mol. Biol. Cell
23
,
1860
1873
.
Papinski
D.
,
Schuschnig
M.
,
Reiter
W.
,
Wilhelm
L.
,
Barnes
C. A.
,
Maiolica
A.
,
Hansmann
I.
,
Pfaffenwimmer
T.
,
Kijanska
M.
,
Stoffel
I.
<et al. (
2014
).
Early steps in autophagy depend on direct phosphorylation of Atg9 by the Atg1 kinase.
Mol. Cell
53
,
471
483
.
Peifer
M.
,
Fernández-Cuesta
L.
,
Sos
M. L.
,
George
J.
,
Seidel
D.
,
Kasper
L. H.
,
Plenker
D.
,
Leenders
F.
,
Sun
R.
,
Zander
T.
<et al. (
2012
).
Integrative genome analyses identify key somatic driver mutations of small-cell lung cancer.
Nat. Genet.
44
,
1104
1110
.
Pfisterer
S. G.
,
Mauthe
M.
,
Codogno
P.
,
Proikas-Cezanne
T.
(
2011
).
Ca2+/calmodulin-dependent kinase (CaMK) signaling via CaMKI and AMP-activated protein kinase contributes to the regulation of WIPI-1 at the onset of autophagy.
Mol. Pharmacol.
80
,
1066
1075
.
Pfisterer
S. G.
,
Bakula
D.
,
Frickey
T.
,
Cezanne
A.
,
Brigger
D.
,
Tschan
M. P.
,
Robenek
H.
,
Proikas-Cezanne
T.
(
2014
).
Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells.
J. Lipid Res.
55
,
1267
1278
.
Polson
H. E.
,
de Lartigue
J.
,
Rigden
D. J.
,
Reedijk
M.
,
Urbé
S.
,
Clague
M. J.
,
Tooze
S. A.
(
2010
).
Mammalian Atg18 (WIPI2) localizes to omegasome-anchored phagophores and positively regulates LC3 lipidation.
Autophagy
6
,
506
522
.
Proikas-Cezanne
T.
,
Robenek
H.
(
2011
).
Freeze-fracture replica immunolabelling reveals human WIPI-1 and WIPI-2 as membrane proteins of autophagosomes.
J. Cell. Mol. Med.
15
,
2007
2010
.
Proikas-Cezanne
T.
,
Waddell
S.
,
Gaugel
A.
,
Frickey
T.
,
Lupas
A.
,
Nordheim
A.
(
2004
).
WIPI-1alpha (WIPI49), a member of the novel 7-bladed WIPI protein family, is aberrantly expressed in human cancer and is linked to starvation-induced autophagy.
Oncogene
23
,
9314
9325
.
Proikas-Cezanne
T.
,
Ruckerbauer
S.
,
Stierhof
Y. D.
,
Berg
C.
,
Nordheim
A.
(
2007
).
Human WIPI-1 puncta-formation: a novel assay to assess mammalian autophagy.
FEBS Lett.
581
,
3396
3404
.
Pugh
T. J.
,
Weeraratne
S. D.
,
Archer
T. C.
,
Pomeranz Krummel
D. A.
,
Auclair
D.
,
Bochicchio
J.
,
Carneiro
M. O.
,
Carter
S. L.
,
Cibulskis
K.
,
Erlich
R. L.
<et al. (
2012
).
Medulloblastoma exome sequencing uncovers subtype-specific somatic mutations.
Nature
488
,
106
110
.
Reggiori
F.
,
Tucker
K. A.
,
Stromhaug
P. E.
,
Klionsky
D. J.
(
2004
).
The Atg1-Atg13 complex regulates Atg9 and Atg23 retrieval transport from the pre-autophagosomal structure.
Dev. Cell
6
,
79
90
.
Rieter
E.
,
Vinke
F.
,
Bakula
D.
,
Cebollero
E.
,
Ungermann
C.
,
Proikas-Cezanne
T.
,
Reggiori
F.
(
2013
).
Atg18 function in autophagy is regulated by specific sites within its β-propeller.
J. Cell Sci.
126
,
593
604
.
Roberts
R.
,
Ktistakis
N. T.
(
2013
).
Omegasomes: PI3P platforms that manufacture autophagosomes.
Essays Biochem.
55
,
17
27
.
Roberts
K. G.
,
Morin
R. D.
,
Zhang
J.
,
Hirst
M.
,
Zhao
Y.
,
Su
X.
,
Chen
S. C.
,
Payne-Turner
D.
,
Churchman
M. L.
,
Harvey
R. C.
<et al. (
2012
).
Genetic alterations activating kinase and cytokine receptor signaling in high-risk acute lymphoblastic leukemia.
Cancer Cell
22
,
153
166
.
Rubinsztein
D. C.
,
Mariño
G.
,
Kroemer
G.
(
2011
).
Autophagy and aging.
Cell
146
,
682
695
.
Rudin
C. M.
,
Durinck
S.
,
Stawiski
E. W.
,
Poirier
J. T.
,
Modrusan
Z.
,
Shames
D. S.
,
Bergbower
E. A.
,
Guan
Y.
,
Shin
J.
,
Guillory
J.
<et al. (
2012
).
Comprehensive genomic analysis identifies SOX2 as a frequently amplified gene in small-cell lung cancer.
Nat. Genet.
44
,
1111
1116
.
Russell
R. C.
,
Tian
Y.
,
Yuan
H.
,
Park
H. W.
,
Chang
Y. Y.
,
Kim
J.
,
Kim
H.
,
Neufeld
T. P.
,
Dillin
A.
,
Guan
K. L.
(
2013
).
ULK1 induces autophagy by phosphorylating Beclin-1 and activating VPS34 lipid kinase.
Nat. Cell Biol.
15
,
741
750
.
Russell
R. C.
,
Yuan
H. X.
,
Guan
K. L.
(
2014
).
Autophagy regulation by nutrient signaling.
Cell Res.
24
,
42
57
.
Saitsu
H.
,
Nishimura
T.
,
Muramatsu
K.
,
Kodera
H.
,
Kumada
S.
,
Sugai
K.
,
Kasai-Yoshida
E.
,
Sawaura
N.
,
Nishida
H.
,
Hoshino
A.
<et al. (
2013
).
De novo mutations in the autophagy gene WDR45 cause static encephalopathy of childhood with neurodegeneration in adulthood.
Nat. Genet.
45
,
445
449, 449e441
.
Sakoh-Nakatogawa
M.
,
Matoba
K.
,
Asai
E.
,
Kirisako
H.
,
Ishii
J.
,
Noda
N. N.
,
Inagaki
F.
,
Nakatogawa
H.
,
Ohsumi
Y.
(
2013
).
Atg12-Atg5 conjugate enhances E2 activity of Atg3 by rearranging its catalytic site.
Nat. Struct. Mol. Biol.
20
,
433
439
.
Sanchez-Wandelmer
J.
,
Ktistakis
N. T.
,
Reggiori
F.
(
2015
).
ERES: sites for autophagosome biogenesis and maturation?
J. Cell Sci.
128
,
185
192
.
Sato
Y.
,
Yoshizato
T.
,
Shiraishi
Y.
,
Maekawa
S.
,
Okuno
Y.
,
Kamura
T.
,
Shimamura
T.
,
Sato-Otsubo
A.
,
Nagae
G.
,
Suzuki
H.
<et al. (
2013
).
Integrated molecular analysis of clear-cell renal cell carcinoma.
Nat. Genet.
45
,
860
867
.
Sawada
H.
,
Takami
K.
,
Asahi
S.
(
2005
).
A toxicogenomic approach to drug-induced phospholipidosis: analysis of its induction mechanism and establishment of a novel in vitro screening system.
Toxicol. Sci.
83
,
282
292
.
Scott
R. C.
,
Schuldiner
O.
,
Neufeld
T. P.
(
2004
).
Role and regulation of starvation-induced autophagy in the Drosophila fat body.
Dev. Cell
7
,
167
178
.
Seo
J. S.
,
Ju
Y. S.
,
Lee
W. C.
,
Shin
J. Y.
,
Lee
J. K.
,
Bleazard
T.
,
Lee
J.
,
Jung
Y. J.
,
Kim
J. O.
,
Shin
J. Y.
<et al. (
2012
).
The transcriptional landscape and mutational profile of lung adenocarcinoma.
Genome Res.
22
,
2109
2119
.
Seshagiri
S.
,
Stawiski
E. W.
,
Durinck
S.
,
Modrusan
Z.
,
Storm
E. E.
,
Conboy
C. B.
,
Chaudhuri
S.
,
Guan
Y.
,
Janakiraman
V.
,
Jaiswal
B. S.
<et al. (
2012
).
Recurrent R-spondin fusions in colon cancer.
Nature
488
,
660
664
.
Settembre
C.
,
De Cegli
R.
,
Mansueto
G.
,
Saha
P. K.
,
Vetrini
F.
,
Visvikis
O.
,
Huynh
T.
,
Carissimo
A.
,
Palmer
D.
,
Klisch
T. J.
<et al. (
2013
).
TFEB controls cellular lipid metabolism through a starvation-induced autoregulatory loop.
Nat. Cell Biol.
15
,
647
658
.
Shibutani
S. T.
,
Yoshimori
T.
(
2014
).
A current perspective of autophagosome biogenesis.
Cell Res.
24
,
58
68
.
Shintani
T.
,
Suzuki
K.
,
Kamada
Y.
,
Noda
T.
,
Ohsumi
Y.
(
2001
).
Apg2p functions in autophagosome formation on the perivacuolar structure.
J. Biol. Chem.
276
,
30452
30460
.
Simonsen
A.
,
Stenmark
H.
(
2008
).
Self-eating from an ER-associated cup.
J. Cell Biol.
182
,
621
622
.
Simonsen
A.
,
Birkeland
H. C.
,
Gillooly
D. J.
,
Mizushima
N.
,
Kuma
A.
,
Yoshimori
T.
,
Slagsvold
T.
,
Brech
A.
,
Stenmark
H.
(
2004
).
Alfy, a novel FYVE-domain-containing protein associated with protein granules and autophagic membranes.
J. Cell Sci.
117
,
4239
4251
.
Slobodkin
M. R.
,
Elazar
Z.
(
2013
).
The Atg8 family: multifunctional ubiquitin-like key regulators of autophagy.
Essays Biochem.
55
,
51
64
.
Stephens
P. J.
,
Tarpey
P. S.
,
Davies
H.
,
Van Loo
P.
,
Greenman
C.
,
Wedge
D. C.
,
Nik-Zainal
S.
,
Martin
S.
,
Varela
I.
,
Bignell
G. R.
<et al. 
Oslo Breast Cancer Consortium (OSBREAC)
(
2012
).
The landscape of cancer genes and mutational processes in breast cancer.
Nature
486
,
400
404
.
Stransky
N.
,
Egloff
A. M.
,
Tward
A. D.
,
Kostic
A. D.
,
Cibulskis
K.
,
Sivachenko
A.
,
Kryukov
G. V.
,
Lawrence
M. S.
,
Sougnez
C.
,
McKenna
A.
<et al. (
2011
).
The mutational landscape of head and neck squamous cell carcinoma.
Science
333
,
1157
1160
.
Strømhaug
P. E.
,
Bevan
A.
,
Dunn
W. A.
 Jr
(
2001
).
GSA11 encodes a unique 208-kDa protein required for pexophagy and autophagy in Pichia pastoris.
J. Biol. Chem.
276
,
42422
42435
.
Strømhaug
P. E.
,
Reggiori
F.
,
Guan
J.
,
Wang
C. W.
,
Klionsky
D. J.
(
2004
).
Atg21 is a phosphoinositide binding protein required for efficient lipidation and localization of Atg8 during uptake of aminopeptidase I by selective autophagy.
Mol. Biol. Cell
15
,
3553
3566
.
Suzuki
K.
,
Kubota
Y.
,
Sekito
T.
,
Ohsumi
Y.
(
2007
).
Hierarchy of Atg proteins in pre-autophagosomal structure organization.
Genes Cells
12
,
209
218
.
Suzuki
K.
,
Akioka
M.
,
Kondo-Kakuta
C.
,
Yamamoto
H.
,
Ohsumi
Y.
(
2013
).
Fine mapping of autophagy-related proteins during autophagosome formation in Saccharomyces cerevisiae.
J. Cell Sci.
126
,
2534
2544
.
Taguchi-Atarashi
N.
,
Hamasaki
M.
,
Matsunaga
K.
,
Omori
H.
,
Ktistakis
N. T.
,
Yoshimori
T.
,
Noda
T.
(
2010
).
Modulation of local PtdIns3P levels by the PI phosphatase MTMR3 regulates constitutive autophagy.
Traffic
11
,
468
478
.
Thumm
M.
,
Busse
R. A.
,
Scacioc
A.
,
Stephan
M.
,
Janshoff
A.
,
Kühnel
K.
,
Krick
R.
(
2013
).
It takes two to tango: PROPPINs use two phosphoinositide-binding sites.
Autophagy
9
,
106
107
.
Tóth
M. L.
,
Sigmond
T.
,
Borsos
E.
,
Barna
J.
,
Erdélyi
P.
,
Takács-Vellai
K.
,
Orosz
L.
,
Kovács
A. L.
,
Csikós
G.
,
Sass
M.
<et al. (
2008
).
Longevity pathways converge on autophagy genes to regulate life span in Caenorhabditis elegans.
Autophagy
4
,
330
338
.
Tsuyuki
S.
,
Takabayashi
M.
,
Kawazu
M.
,
Kudo
K.
,
Watanabe
A.
,
Nagata
Y.
,
Kusama
Y.
,
Yoshida
K.
(
2014
).
Detection of WIPI1 mRNA as an indicator of autophagosome formation.
Autophagy
10
,
497
513
.
van der Vos
K. E.
,
Eliasson
P.
,
Proikas-Cezanne
T.
,
Vervoort
S. J.
,
van Boxtel
R.
,
Putker
M.
,
van Zutphen
I. J.
,
Mauthe
M.
,
Zellmer
S.
,
Pals
C.
<et al. (
2012
).
Modulation of glutamine metabolism by the PI(3)K-PKB-FOXO network regulates autophagy.
Nat. Cell Biol.
14
,
829
837
.
Velikkakath
A. K.
,
Nishimura
T.
,
Oita
E.
,
Ishihara
N.
,
Mizushima
N.
(
2012
).
Mammalian Atg2 proteins are essential for autophagosome formation and important for regulation of size and distribution of lipid droplets.
Mol. Biol. Cell
23
,
896
909
.
Vellai
T.
,
Takacs-Vellai
K.
,
Zhang
Y.
,
Kovacs
A. L.
,
Orosz
L.
,
Müller
F.
(
2003
).
Genetics: influence of TOR kinase on lifespan in C. elegans.
Nature
426
,
620
.
Vergne
I.
,
Roberts
E.
,
Elmaoued
R. A.
,
Tosch
V.
,
Delgado
M. A.
,
Proikas-Cezanne
T.
,
Laporte
J.
,
Deretic
V.
(
2009
).
Control of autophagy initiation by phosphoinositide 3-phosphatase Jumpy.
EMBO J.
28
,
2244
2258
.
Waddell
S.
,
Jenkins
J. R.
,
Proikas-Cezanne
T.
(
2001
).
A “no-hybrids” screen for functional antagonizers of human p53 transactivator function: dominant negativity in fission yeast.
Oncogene
20
,
6001
6008
.
Walker
B. A.
,
Wardell
C. P.
,
Melchor
L.
,
Hulkki
S.
,
Potter
N. E.
,
Johnson
D. C.
,
Fenwick
K.
,
Kozarewa
I.
,
Gonzalez
D.
,
Lord
C. J.
<et al. (
2012
).
Intraclonal heterogeneity and distinct molecular mechanisms characterize the development of t(4;14) and t(11;14) myeloma.
Blood
120
,
1077
1086
.
Wang
C. W.
,
Kim
J.
,
Huang
W. P.
,
Abeliovich
H.
,
Stromhaug
P. E.
,
Dunn
W. A.
 Jr
,
Klionsky
D. J.
(
2001
).
Apg2 is a novel protein required for the cytoplasm to vacuole targeting, autophagy, and pexophagy pathways.
J. Biol. Chem.
276
,
30442
30451
.
Watanabe
Y.
,
Kobayashi
T.
,
Yamamoto
H.
,
Hoshida
H.
,
Akada
R.
,
Inagaki
F.
,
Ohsumi
Y.
,
Noda
N. N.
(
2012
).
Structure-based analyses reveal distinct binding sites for Atg2 and phosphoinositides in Atg18.
J. Biol. Chem.
287
,
31681
31690
.
White
E.
(
2012
).
Deconvoluting the context-dependent role for autophagy in cancer.
Nat. Rev. Cancer
12
,
401
410
.
Wild
P.
,
McEwan
D. G.
,
Dikic
I.
(
2014
).
The LC3 interactome at a glance.
J. Cell Sci.
127
,
3
9
.
Xiong
Y.
,
Contento
A. L.
,
Bassham
D. C.
(
2005
).
AtATG18a is required for the formation of autophagosomes during nutrient stress and senescence in Arabidopsis thaliana.
Plant J.
42
,
535
546
.
Yang
Z.
,
Klionsky
D. J.
(
2010
).
Eaten alive: a history of macroautophagy.
Nat. Cell Biol.
12
,
814
822
.

Competing interests

The authors declare no competing or financial interests.