Pacer Mediates the Function of Class III PI3K and HOPS Complexes in Autophagosome Maturation by Engaging Stx17

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1 Article Pacer Mediates the Function of Class III PI3K and HOPS Complexes in Autophagosome Maturation by Engaging Stx17 Graphical Abstract Authors Xiawei Cheng, Xiuling Ma, Xianming Ding,..., Wei Liu, Weihua Gong, Qiming Sun Correspondence (Q.S.), (W.G.) In Brief Cheng et al. demonstrate that Pacer antagonizes Rubicon to mediate the function of the UVRAG-PI3KC3 subcomplex in late steps of autophagy. Pacer recruits HOPS complex to regulate the fusion specificity of autophagosomes with late endosomes/lysosomes by anchoring to the autophagosomal Stx17. Highlights d Pacer antagonizes Rubicon to activate the UVRAG-PI3KC3 subcomplex d d Pacer recruits PI3KC3 and HOPS complex to promote autophagosome maturation Pacer s autophagosome association is mediated by Stx17 and phosphoinositides Cheng et al., 2017, Molecular Cell 65, March 16, 2017 ª 2017 Elsevier Inc.

2 Molecular Cell Article Pacer Mediates the Function of Class III PI3K and HOPS Complexes in Autophagosome Maturation by Engaging Stx17 Xiawei Cheng, 1 Xiuling Ma, 1 Xianming Ding, 1 Lin Li, 2 Xiao Jiang, 1 Zhirong Shen, 2 She Chen, 2 Wei Liu, 1,3 Weihua Gong, 4, * and Qiming Sun 1,5, * 1 Department of Biochemistry and Molecular Biology, School of Medicine, Zhejiang University, Hangzhou , China 2 National Institute of Biological Sciences, Beijing , China 3 Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou , China 4 Department of Surgery, Second Affiliated Hospital of School of Medicine, Zhejiang University, Hangzhou , China 5 Lead Contact *Correspondence: (Q.S.), (W.G.) SUMMARY Class III PI3-kinase (PI3KC3) is essential for autophagy initiation, but whether PI3KC3 participates in other steps of autophagy remains unknown. The HOPS complex mediates the fusion of intracellular vesicles to lysosome, but how HOPS specifically tethers autophagosome to lysosome remains elusive. Here, we report Pacer (protein associated with UVRAG as autophagy enhancer) as a regulator of autophagy. Pacer localizes to autophagic structures and positively regulates autophagosome maturation. Mechanistically, Pacer antagonizes Rubicon to stimulate Vps34 kinase activity. Next, Pacer recruits PI3KC3 and HOPS complexes to the autophagosome for their site-specific activation by anchoring to the autophagosomal SNARE Stx17. Furthermore, Pacer is crucial for the degradation of hepatic lipid droplets, the suppression of Salmonella infection, and the clearance of protein aggregates. These results not only identify Pacer as a crucial multifunctional enhancer in autophagy but also uncover both the involvement of PI3KC3 and the mediators of HOPS s specific tethering activity in autophagosome maturation. INTRODUCTION Macroautophagy (hereafter referred to as autophagy) is a lysosomal degradative pathway, which is central to development and homeostasis (He and Klionsky, 2009; Nakatogawa et al., 2009). The deregulation of autophagy is tightly associated with a variety of human diseases (Levine and Kroemer, 2008; Mizushima et al., 2008). One of the essential regulators of autophagy is class III PI3-kinase (PI3KC3), which produces phosphatidylinositol 3-phosphate (PtdIns(3)P), a key lipid required for autophagy (Noda et al., 2010; Simonsen and Tooze, 2009; Yu et al., 2015). Two distinct mammalian PI3KC3 subcomplexes, namely, the Atg14 and UVRAG subcomplexes, were identified and characterized independently by us and several other groups (Itakura et al., 2008; Liang et al., 2006; Matsunaga et al., 2009; Sun et al., 2008; Zhong et al., 2009). Despite a consensus in the field that UVRAG participates in endocytosis, the function of UVRAG in autophagy is still controversial (Farré et al., 2010; Itakura et al., 2008, 2012; Knævelsrud et al., 2010; Levine et al., 2015; Liang et al., 2006; L}orincz et al., 2014; Takahashi et al., 2007; Yu et al., 2015). More importantly, it is still unknown whether PI3KC3 complex functions in other steps of autophagy besides autophagy initiation. Rubicon was initially identified as an inhibitor of the UVRAG- PI3KC3 subcomplex (Matsunaga et al., 2009; Zhong et al., 2009). We and others have shown that Rubicon negatively regulates endosome maturation and autophagy by inhibiting HOPS recruitment, Rab7 GTPase activation, and PI3KC3 kinase activity (Kim et al., 2015; Sun et al., 2010, 2011; Tabata et al., 2010). Despite these findings, it remains unclear how Rubicon, an endosome-resident protein (Matsunaga et al., 2009; Sun et al., 2010; Zhong et al., 2009), inhibits autophagy. Moreover, Rubicon knockdown (KD) significantly induces autophagy without additional stimuli, indicating that other players may be involved in Rubicon-regulated autophagy (Matsunaga et al., 2009; Noda et al., 2010; Sun et al., 2011; Zhong et al., 2009). Fusion of vesicles to lysosome is driven by tethering and SNARE-mediated fusion. How autophagosome selectively fuses to lysosome is poorly characterized. The homotypic fusion and vacuole protein sorting (HOPS) complex is a tether involved in the fusion step during endocytosis and autophagy (Balderhaar and Ungermann, 2013). Before tethering, HOPS is recruited to different types of vesicles, including endosomes and autophagosomes, by various membrane proteins (Jiang et al., 2014; Liang et al., 2008; McEwan et al., 2015; Takáts et al., 2014). In mammalian cells, only Stx17 has previously been shown to recruit HOPS to autophagosomes for fusion to lysosomes (Jiang et al., 2014). It remains intriguing how Stx17 selectively targets HOPS to autophagosome, given that Stx17 resides also on mitochondria and endoplasmic reticulum (ER) (Arasaki et al., 2015; Hamasaki et al., 2013). Molecular Cell 65, , March 16, 2017 ª 2017 Elsevier Inc. 1029

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4 In this study, we report protein associated with UVRAG as autophagy enhancer (Pacer) as a regulator of PI3KC3 and HOPS in autophagosome maturation. We show that Pacer and Rubicon form a molecular switch for fine-tuning of autophagy by engaging the autophagosomal SNARE Stx17. Our data provide insight into autophagosome maturation, and they demonstrate a pivotal role for Pacer in bacterial infection, hepatic lipid homeostasis, and protein aggregate clearance. RESULTS Identification of Pacer as a Protein Associated with Autophagic Structures Bioinformatic analysis showed that Rubicon shares significant sequence similarity with PLEKHM1 and a potential tumor suppressor (Huisman et al., 2013), KIAA0226L or C13orf18, which is named Pacer in this study. Human Pacer contains 662 amino acids and it shares 23% and 14% sequence similarity with Rubicon and PLEKHM1, respectively (Figure 1A). Similar to Rubicon and PLEKHM1, Pacer possesses a conserved Rubicon homology (RH) domain at its carboxyl terminus. However, it lacks the RPIP8, UNC-14, and NESCA (Run) domain found in Rubicon and PLEKHM1 and the LC3 interaction region (LIR) motif in PLEKHM1 (Figure 1B). Pacer is a vertebrate-specific gene, as Pacer homologs only exist in zebrafish and other higher organisms (Figure S1). As Rubicon and PLEKHM1 play important roles in both autophagy and endocytosis (McEwan et al., 2015; Tabata et al., 2010), we investigated whether Pacer functions in these pathways as well. Interestingly, we found that Pacer is reticularly distributed in 85% of cells, while in other cells Pacer forms absolute cytosolic puncta, which co-localized with the autophagy marker LC3 and partially with the lysosome marker LAMP1 (Figure 1C). In contrast, no apparent overlap with the early endosome marker EEA1 and the Golgi marker GM130 was observed (Figures 1C, 1D, and S2A). Further analysis by co-staining for Atg16L, the early stage autophagy marker, and DFCP1, the Omegasome marker, indicated that Pacer associates with mature rather than early autophagic structures (Figures 1C, 1D, and S2A). Notably, Pacer puncta were augmented by the autophagy inducer Torin1 (a potent mtor kinase inhibitor) (Figures 1E and 1F). To study Pacer s role in autophagy, we prepared an antibody that recognizes both endogenous and exogenous Pacers (Figures S2B S2D), and we found that endogenous Pacer was predominantly co-fractionated with membrane proteins (P100), including LAMP1 and LC3-II, in a biochemical fractionation assay (Figures 1G and 1H, lanes 1 4). The membrane association of Pacer was increased in Torin1-treated cells (Figure 1H, lane 3), and it accumulated in Bafilomycin A-treated cells (Figure 1H, lane 4). As a control, the LC3-I species was present in cytosolic fractions (S100) (Figure 1H, lanes 5 8). These results demonstrate that Pacer is mainly associated with autophagic structures in vivo, and Pacer s membrane targeting is promoted by autophagic stimuli. Pacer Is Essential for Autophagosome Maturation We observed that Pacer KD caused significant p62 accumulation (Figure 2A, lanes 1 and 6), indicating that Pacer KD delayed p62 degradation in resting cells. When autophagy was stimulated by Torin1, p62 levels steadily dropped as expected (Figure 2A, lanes 2 5 and 7 10), and, notably, Pacer KD significantly decreased the degradation rate of p62 (Figure 2B). Pacer overexpression (OE) not only reduced p62 and LC3-II levels (Figure 2C) but also significantly increased the degradation rate of p62 and LC3-II (Figure 2D). Using transmission electron microscopy, we observed significant accumulation of autophagic vacuoles in Pacer knockout (KO) cells generated by CRISPR-Cas9 technology, under both Torin1-treated and untreated conditions (Figures 2E and 2F), confirming that Pacer mediates the degradation of autophagic vacuoles. As expected, p62 accumulation in Pacer KO cells was further elevated by blocking lysosome activity using Bafilomycin A (Figure S2E). Furthermore, in autophagosome maturation assays (Kimura et al., 2007) (Figures 2G 2I), Pacer KD largely blocked autophagosome maturation, as measured by the mcherry+gfp puncta in both starved and untreated U2OS cells, while Pacer OE enhanced autophagosome maturation in HeLa cells (Figure S2F). In addition, the overlap of LC3 and LAMP1, another indicator of autophagosome maturation, was reduced in Pacer-depleted cells (Figure S2G). Taken together, these results demonstrate that Pacer is required for autophagosome maturation. Pacer Is a Component of the UVRAG-PI3KC3 Subcomplex To understand how Pacer regulates autophagy, we searched for Pacer-interacting proteins by tandem affinity purification (TAP) using the protocol we developed previously (Sun et al., 2008). Mass spectrometric (MS) analysis of the proteins that co-immunoprecipitated (coip) with Pacer indentified PI3KC3 subunits, HOPS components, and other proteins or protein complexes (Figure 3A). Another proteomic study also indicated that Figure 1. Identification of Pacer as an Autophagosome-Associated Protein (A) Amino acid sequence alignment of full-length Pacer, Rubicon, and PLEKHM1. Amino acids in similarity were highlighted in black and gray. (B) Schematic representation of full-length Pacer, Rubicon, and PLEKHM1 with the boundaries of RH, RUN, and LIR indicated. RH, Rubicon homology domain; RUN, domain for RPIP8, UNC-14, and NESCA; LIR, LC3 interaction region. (C) The co-localization of Pacer with different organelle markers. Scale bars, 10 mm. (D) Quantification of the co-localization described in (C) by percentage. (E and F) U2OS cells transfected with Pacer-GFP (E) were treated with Torin1, then fixed and analyzed by confocal microscopy, and are quantified in (F). Data are shown as mean ± SD (**p < 0.01). (G) Workflow of organelle membrane isolation by ultra-centrifugation. (H) 293T cells were treated with Torin1, Bafilomycin A, or Torin1 plus Bafilomycin A. Then cell lysates were prepared and separated into P100 and S100 by ultracentrifugation, followed by western blot analysis. C, control; T, Torin1; B, Bafilomycin A; TB, Torin1 plus Bafilomycin A. See also Figures S1 and S2. Molecular Cell 65, , March 16,

5 1032 Molecular Cell 65, , March 16, 2017 (legend on next page)

6 C13orf18 (Pacer in this study) is a potential PI3KC3-binding protein (Behrends et al., 2010). Additional immunoprecipitation assays showed that endogenous Pacer is indeed associated with UVRAG, Beclin1, Beclin2 (He et al., 2013), Vps34, and p150, but not with Atg14, hnrbf2, or Rubicon (Figures 3B and S3A). Therefore, Pacer is a component of the UVRAG-Beclin1, 2-PI3KC3 subcomplex. Further immunoprecipitation assays showed that the coiledcoil domain (CCD) of Beclin1 was sufficient to co-immunoprecipitate with Pacer (Figures S3B and S3C), while the regions flanking UVRAG s CCD, but not the CCD of UVRAG, interacted with Pacer (Figures S3D and S3E). As the CCDs of UVRAG and Beclin1 are indispensable and sufficient for Beclin1-UVRAG interactions (Itakura et al., 2008; Matsunaga et al., 2009; Sun et al., 2008; Zhong et al., 2009), we concluded that Pacer may interact with UVRAG in order to be associated with Beclin1, and UVRAG is, therefore, likely the direct binding partner of Pacer. Pacer Stimulates PI3KC3 Kinase Activity by Antagonizing Rubicon We further identified that Pacer s mutants containing amino acids interacted with endogenous UVRAG and Beclin1 (Figure 3C, lanes 2 8). Sequence alignment revealed that a region corresponding to amino acids of human Pacer is highly conserved among all Pacer homologs identified in different species (Figure 3D), and Pacer likely forms a coiled-coil a helix (Figure S3F). Furthermore, Pacer was able to pull down endogenous Beclin1 and UVRAG as efficiently as wild-type (WT) Pacer (Figures 3E and 3F, lanes 2 and 3), indicating that Pacer binds to UVRAG through its residues , a motif exhibiting high similarity in sequence and structure with Rubicon (Figure 3D). The observation that UVRAG is the direct binding partner for both Pacer and Rubicon (Sun et al., 2011), together with the sequence alignment data, led us to hypothesize that Pacer and Rubicon might compete for binding to UVRAG. To test this hypothesis, we conducted an in vitro competition assay using purified recombinant proteins (Figure S3G). Indeed, full-length Rubicon and Pacer, but not PacerD40, interacted with UVRAG in vitro, confirming their direct interaction (Figure S3H). More importantly, WT Pacer, but not PacerD40, which is unable to interact with UVRAG, was able to dissociate Rubicon from UVRAG in a dose-dependent manner (Figures S3I and S3J). We further examined their competition by immunoprecipitating endogenous Beclin1 complexes from Pacer WT, KO, or OE cells. As shown in Figure 3G, Pacer KO did not change the association of Rubicon with PI3KC3, but it did decrease the amount of UVRAG in the complex (lane 7 compared to lane 5). Pacer OE reduced endogenous Rubicon levels by 50% (lane 3 compared to lane 1 or 2), and it totally abolished its association with PI3KC3 (lane 9 compared to lane 5). More importantly, Pacer OE enhanced the association among Beclin1, UVRAG, and Vps34 without affecting Atg14 (lane 9 compared to lane 5), indicating that Pacer is specifically required for the stability and integrity of the UVRAG-PI3KC3 subcomplex. To further prove the direct competition between Rubicon and Pacer in vitro, we generated another mutant, Pacer-5A, by mutating the amino acids VEKEN (underlined amino acid in Figures 3D and S3F) into AAAAA. Pacer-5A largely lost its binding to UVRAG (Figure 3H), and, thus, it was incapable of preventing the Rubicon-UVRAG interaction (Figure 3I). The instability of Rubicon in Pacer OE cells was likely due to the dissociation of Rubicon from the PI3KC3 complex by Pacer (Figure S3K). Moreover, stimulating autophagy by starvation enhanced the association between the PI3KC3 complex and Pacer, leading to Rubicon dissociation (Figure 3J). These data demonstrate that Pacer competes with Rubicon for direct binding to the UVRAG complex, which is further promoted by autophagic stress in vivo. To test whether Pacer affects PI3KC3 kinase activity, Vps34 complexes were IP from Pacer OE, WT, and KO cells, respectively, and tested for PtdIns(3)P biogenesis in vitro. Vps34 isolated from Pacer OE cells yielded the highest PtdIns(3)P levels, while Pacer KO led to a significant reduction of Vps34 activity (Figure 3K). Consistently, WT Pacer, but not PacerD40, which does not bind UVRAG, stimulated Vps34 kinase activity similarly to UVRAG. Furthermore, a GFP-FVYE2 construct was transfected at a low level to monitor the PtdIns(3)P biogenesis in vivo (Gillooly et al., 2000). In line with in vitro data, Pacer OE increased cellular PtdIns(3)P levels, as indicated by GFP-FYVE2-labeled puncta (Figure S4A). In contrast, Pacer KD significantly reduced endogenous PtdIns(3)P levels, similar to treatment with 3-MA, a PI3KC3 kinase inhibitor. Thus, Pacer positively regulates PI3KC3 kinase activity both in vitro and in vivo, and Pacer is required for endogenous PtdIns(3)P biogenesis. Figure 2. Pacer Is Essential to Autophagosome Maturation (A) 293T cells were transfected with scrambled small hairpin RNA (shrna) or Pacer shrna, cells were treated with Torin1 for different times, and then analyzed by western blot. (B) The p62 levels in (A) were quantified by a Phosphorimager and normalized with that of GAPDH, and the relative p62 levels are shown as the percentage of the initial p62 content. (C) 293T stable cell lines harboring either empty or Pacer-FLAG vector were treated with Torin1 and collected at different time points and analyzed by western blot (*non-specific band). (D) The p62 levels in (C) were quantified as described above. (E and F) Control and Pacer KO U2OS cells were treated with Torin1 or DMSO for 2 hr and analyzed by transmission electron microscopy (E). Arrows indicate autophagic vacuole. The autophagic vacuole per cross-sectioned cell under EM was calculated and is summarized in (F). Scale bars, 0.5 mm. Data are shown as mean ± SD (**p < 0.01). (G I) mcherry-gfp-lc3 was expressed in Pacer KD and WT U2OS cells, respectively. Cells were starved using starvation medium. LC3 was monitored by fluorescence microscope (G). GFP-negative mcherry-positive (GFP-mCherry+) puncta, which indicate autolysosomes, were quantified and are summarized in (H) and (I). Scale bars, 10 mm. Data are shown as mean ± SD (**p < 0.01). See also Figure S2. Molecular Cell 65, , March 16,

7 Figure 3. Pacer Positively Regulates PI3KC3 Kinase Activity by Antagonizing Rubicon (A) Silver staining of the tandem affinity-purified Pacer complexes from 293T cells. The marked bands were identified by mass spectrometry. (B) Endogenous Pacer was IP using homemade Pacer antibody, and then it was analyzed by western blot for PI3KC3 components. (C) FLAG-tagged full-length Pacer or mutants were expressed individually in 293T cells, and anti-flag immunoprecipitation was performed and analyzed by western blot. (D) Sequence homology analysis of Pacer and Rubicon from different species. The blue highlights amino acids with high similarity among all of the listed sequences. (E and F) GFP-tagged full-length Pacer, aa or aa was IP and analyzed by western blot (E). The dissection of interaction is summarized in (F). (G) Endogenous PI3KC3 complexes were IP from cells with Pacer WT, OE, or KO using anti-beclin1 antibody, and then they were analyzed by western blot. (H) Pacer-WT or Pacer-5A was IP and analyzed by western blot. (I) Pacer-WT or Pacer-5A was expressed individually in 293T cells. Immunoprecipitation was performed using anti-uvrag antibody and analyzed by western blot. (J) Endogenous PI3KC3 complexes were IP from control cells and cells treated with starvation medium or Torin1 using anti-beclin1 antibody, and they were analyzed by western blot. (K) Measure Pacer activity using in vitro PI3KC3 kinase assay. In-vitro-synthesized P 32 -labeled PtdIns(3)P was separated in thin-layer chromatography and analyzed by autoradiography. See also Figures S3 and S4. Pacer Regulates UVRAG s Autophagic Activity by Targeting UVRAG to Autophagosomes through Its PATS Motif To investigate how Pacer localizes to autophagosomes, we analyzed by deletion mutagenesis which region of Pacer is essential for autophagosome targeting. In this way, we mapped the autophagosome membrane-targeting sequence (PATS) of Pacer to aa (Pacer aa )(Figure 4A). We found that Pacer 1 200aa was sufficient for autophagosome targeting, but not as efficient as Pacer 1 300aa or Pacer aa (Figures 4B 1034 Molecular Cell 65, , March 16, 2017

8 Figure 4. Pacer Modulates UVRAG s Activity in Autophagy by Targeting UVRAG to Autophagosomes (A C) GFP-tagged full-length Pacer and mutants were expressed in U2OS cells, and they were analyzed for punctum formation (A). The capability of forming LC3-overlapped puncta is summarized in (B) and (C). Scale bars, 10 mm. Data are shown as mean ± SD (**p < 0.01). (D and E) UVRAG and LC3 co-localization was analyzed in Pacer OE, KD, and rescued cells (D), and it is quantified in (E). Scale bars, 10 mm. Data are shown as mean ± SD (**p < 0.01). (F and G) UVRAG depends on Pacer to function in autophagy. UVRAG was overexpressed in either Pacer KO or WT cells, which were then treated with Torin1 (F). Autophagy activity was measured by analyzing p62 level (G). See also Figure S4. and 4C). This indicates that Pacer associates with autophagosomes independently of its interaction with UVRAG, and it implies that its UVRAG interaction may induce Pacer aa to adopt an optimal conformation for maximal autophagosome targeting. Consequently, mutants missing the PATS failed entirely to form cytosolic puncta, demonstrating an essential role for PATS in Pacer s autophagosome association. We subsequently investigated whether Pacer is able to direct UVRAG to autophagosomes. First, we observed that UVRAG depletion caused no difference in Pacer-LC3 co-localization (Figure S4B), while Pacer KD or OE significantly altered the percentage of UVRAG-LC3 overlap (Figures 4D and 4E). This indicates that Pacer is the targeting molecule. Second, using a similar strategy reported before (Fan et al., 2011), we fused the PATS to the N terminus of UVRAG, and we showed that the chimera protein PATS-UVRAG, but not UVRAG, exhibited robust co-localization with LC3 puncta (Figures S4C and S4D). Consistently, co-expression of either WT Pacer or PATS with UVRAG redistributed UVRAG to autophagosomes (Figures S4E and S4F), and UVRAG co-expression enhanced the punctum formation of WT Pacer and PATS 1-fold (Figure S4G). This demonstrates that Pacer depends on its PATS motif to relocate UVRAG from endosomes or the cytosol to autophagosomes. Indeed, UVRAG OE enhanced the degradation of p62 in WT cells, but not in Pacer KO cells (Figures 4F and 4G), indicating that Pacer mediates UVRAG s function in autophagy. Overall, these data demonstrate that Pacer targets UVRAG to autophagosomes to modulate UVRAG s autophagic activity. Molecular Cell 65, , March 16,

9 Figure 5. Pacer Activates PI3KC3 and HOPS on Autophagosomes (A and B) Pacer recruits Beclin1 and Vps34 to autophagosomes. U2OS cells co-expressed with Pacer-GFP and Beclin1-hemagglutinin (HA) or Vps34-HA (A) were fixed and stained with anti-ha and LC3 antibodies, analyzed by confocal microscopy, and are quantified in (B). Scale bars, 10 mm. Data are shown as mean ± SD (**p < 0. 01). (C) Pacer co-localizes with UVRAG, Beclin1, and Vps34. Scale bars, 10 mm. (D) U2OS stable cell lines for Pacer-FLAG or empty vector were transfected with GFP-FYVE2 at a low level. Cells were then fixed, stained for LC3, analyzed by confocal microscopy, and quantified for the co-localization of LC3 and GFP-FYVE2. Scale bars, 10 mm. Data are shown as mean ± SD (**p < 0.01). (E) Endogenous Pacer coip with HOPS. (legend continued on next page) 1036 Molecular Cell 65, , March 16, 2017

10 Pacer Directs PI3KC3 and HOPS Complexes to Autophagosomes We next investigated whether Pacer recruits Beclin1-PI3KC3 to autophagosomes. Beclin1 normally does not form cytosolic foci, but it resides on trans-golgi networks or remains in soluble form in the cytosol (Kihara et al., 2001). Remarkably, Pacer co-expression induced Beclin1 to form puncta that co-localized with Pacer and LC3 (Figures 5A and 5B). Likewise, Pacer co-expression increased the number of Vps34 puncta, which mostly co-localized with LC3. As predicted, we also detected the co-localization of Pacer and UVRAG with either Vps34 or Beclin1 (Figure 5C). In contrast, the UVRAG interaction-defective mutants, Pacer-5A and PacerD40, failed to recruit PI3KC3 to autophagic structures as efficiently as Pacer WT (Figures S5A and 5B). These data demonstrate that Pacer targets the UVRAG-Beclin1-Vps34 subcomplex to autophagosomes. We next examined PtdIns(3)P biogenesis on autophagosomes by expressing the PtdIns(3)P probe GFP-FYVE2 at a low level. Normally GFP-FYVE2 largely co-localized with EEA1 and partially with LC3, indicating PtdIns(3)P is more abundant on endosomes than autophagosomes (Gillooly et al., 2000). We found that Pacer OE or KD altered the co-localization of GFP-FYVE2 and LC3 in both U2OS and HEK293 cells (Figures 5D and S5B). In addition, Pacer WT, but not PacerD( ), rescued PtdIns(3)P biogenesis in Pacer-deficient cells (Figure S5C). Not surprisingly, PacerD( ) failed to complement the defect of LC3-II degradation in Pacer KO cells (Figure S5D). Furthermore, Pacer-5A exhibited impaired activity in promoting PtdIns(3)P biogenesis (Figure S5E), and, accordingly, Pacer-5A was ineffective in boosting autophagosome maturation (Figure S5F). These data demonstrate that PtdIns(3)P biogenesis on autophagosomes is enhanced by Pacer-directed redistribution of PI3KC3 complexes to autophagosomes, which is important for autophagosome maturation. In addition to function as a subunit of PI3KC3 complex, UVRAG also recruits the HOPS complex to facilitate endocytic and autophagic fusion with lysosomes, which is independent of Beclin1 and PI3KC3 (Liang et al., 2008). During Pacer-TAP- MS analysis, we found that Pacer coip with parts of the HOPS complex, namely Vps16, Vps18, and Vps33 (Figure 3A). Their interactions were confirmed by immunoprecipitation assays (Figures 5E, 5F, S6A, and S6B). Interestingly, Pacer interacted with HOPS independently of UVRAG, as the mutant PacerD , which lacks the UVRAG-binding motif, and Pacer-5A interacted with HOPS in a manner similar to WT Pacer (Figures 5F, S6A, and S6B). We further studied whether Pacer targets the HOPS complex to autophagosomes. Each subunit of HOPS rarely co-localized with LC3 when expressed alone in Pacer WT or KD cells (Figure S6C), and they either formed cytosolic puncta that overlapped with endosomal markers (Liang et al., 2008) or exhibited a diffuse form in the cytosol. However, Pacer co-expression changed their subcellular distribution (Figure 5G), and most of their puncta co-localized with LC3, indicating Pacer also recruits the HOPS complex to autophagosomes. Notably, Pacer-5A was equally effective in targeting the HOPS complex (Figures S6D and S6E), although this mutant exhibited impaired activity in redistributing the PI3KC3 complex (Figures S5A and 5B), indicating that Pacer-HOPS and Pacer-PI3KC3 are functionally separable. As the HOPS complex subunit Vps39 is a guanosine diphosphate (GDP) exchange factor for Rab7 (Wurmser et al., 2000), we then measured Rab7 and LC3 co-localization to see whether HOPS recruitment by Pacer activates lysosomal Rab7 to promote tethering. Indeed, Pacer OE enhanced Rab7- LC3 co-localization (Figure 5H), even though Pacer only exhibited extremely low affinity to Rab7 compared to Rubicon and PLEKHM1 (Figure S6F). Collectively, Pacer targets both PI3KC3 and HOPS complexes to autophagosomes for their site-specific activation. Pacer Directly Interacts with Phosphoinositides and Autophagosomal Stx17 To further understand how Pacer targets autophagic structures, we studied whether Pacer directly interacts with vesicular lipids. Indeed, recombinant Pacer and Rubicon purified from insect cells selectively bound to PtdIns(3)P, PtdIns(4)P, and PtdIns(5)P in vitro (Figures 6A 6C). When performing Pacer s subcellular localization analysis, we observed that the puncta of Pacer and UVRAG were almost perfectly overlapped, and the signal intensity of their individual puncta was largely identical (Figures S7A, S7C, and S7D). In contrast, 65% of the Atg14 co-localized with Pacer, and, for each individual overlapped puncta, the intensity of Pacer and Atg14 signal was complementary to each other (Figures S7B, S7C, and S7E), implying that Pacer and Atg14 may occupy autophagosomes in a sequential manner. This led us to postulate that Pacer may interact or co-localize with Stx17, the only known Atg14-interacting molecule on autophagic structures (Diao et al., 2015). As predicted, Pacer and UVRAG co-localized with Stx17 almost perfectly, while Pacer and Atg14 overlapped with Stx17 in a mixed manner (Figure 6D). Indeed, Pacer directly interacted with Stx17 in an affinity similar to Atg14 (Figures 6E 6H), and their interaction was mediated by aa, the minimum region of Pacer for autophagosome targeting (Figures 6I and 6J). Notably, these Pacer mutants displayed autophagosome association as long as they bound to Stx17 (Figure 6J). Accordingly, Stx17 KD significantly reduced the relative number of Pacer puncta (Figure 6K). Consistently, both WT Pacer and PATS, but not PacerD , co-localized with Stx17 on autophagosomes (Figures 6L and S7F). Furthermore, Pacer and Stx17 (F) FLAG-tagged Pacer WT, Pacer , or PacerD was co-expressed with Vps33-HA and analyzed by anti-flag immunoprecipitation, followed by anti-ha western blot. (G) Pacer recruits the HOPS complex to autophagosomes. Pacer-GFP was co-expressed with HA-tagged HOPS subunits, which were then fixed, stained for LC3 and HA, analyzed by confocal microscopy, and quantified for the co-localization of LC3 and HOPS subunits. Scale bars, 10 mm. Data are shown as mean ± SD (***p < 0.001). (H) The co-localization of Rab7 and LC3 was analyzed in Pacer OE and control cells. Scale bars, 10 mm. Data are shown as mean ± SD (**p < 0.01). See also Figures S5 and S6. Molecular Cell 65, , March 16,

11 1038 Molecular Cell 65, , March 16, 2017 (legend on next page)

12 co-localized with Vps34, Beclin 1, or Beclin 2 (Figure S7G), indicating that Pacer targets the UVRAG-PI3KC3 subcomplex to autophagosomes by anchoring to the autophagosomal Stx17. In addition, Pacer unlikely affects the formation of the Stx17 SNARE complex (Figure S7H), and Pacer may interact with Stx17 independently of UVRAG (Figure S7I). These data reveal that Stx17 mediates Pacer s autophagosome targeting, which might be assisted by autophagosomal phosphoinositides. Pacer Is Required for Autophagy-Mediated Suppression of Bacterial Replication, Lipid Degradation, and Protein Aggregate Clearance In Vivo To further investigate the physiological function of Pacer, we performed a bacterial infection assay, a hepatic lipid accumulation assay, and a protein clearance assay. Autophagy plays an essential role to suppress the infections of some bacteria, including Salmonella typhimurium (Birmingham et al., 2006). Pacer WT, KO, and OE U2OS cells were infected with Salmonella marked with red fluorescence protein (RFP), and the uptake of Salmonella was monitored by microscopy. Pacer KO cells were more permissive for intracellular replication of Salmonella than WT cells, visualized by the high number of red fluorescent bacteria in the cytosol, while the Pacer OE cells were more resistant to bacterial replication than WT cells, enabling significantly reduced number of red fluorescent bacteria to survive (Figure 7A). In a quantitative assay to measure the in vivo bacterial growth, we detected an 10-fold increase in bacterial replication in Pacer KO over Pacer WT cells (Figure 7B). In contrast, Pacer OE resulted in a 10-fold decrease in bacterial replication compared to WT cells. In addition, Pacer OE significantly reduced bacterial replication in HEK293 cells (Figure 7C), while this effect was largely compromised in Atg7-KO HEK293 cells (Figure 7C). These observations were confirmed in quantitative assays (Figure 7D). These data indicate that Pacer is critical for autophagy-mediated suppression of bacterial replication in vivo. In addition, Pacer depletion led to significant lipid accumulation in hepatocytes (Figures 7E and 7F), phenocopying Atg5 or Atg7 deletion in hepatocytes (Karsli-Uzunbas et al., 2014; Martinez-Lopez and Singh, 2015; Singh et al., 2009). Therefore, Pacer maintains lipid homeostasis in hepatocytes as an essential autophagy regulator. Furthermore, we measured the dynamics of puromycin-induced ubiquitin (Ub) and p62/sqstm1-positive protein aggregates in U2OS cells. Silencing Pacer expression caused the appearance of Ub-positive (Ub+) puncta in untreated cells that were rarely present in control cells (Figure 7G, NT). Moreover, while control cells efficiently cleared puromycininduced p62+/ub+ protein aggregates, recovery of Pacerdepleted cells was not apparent even after 3 hr (Figures 7G and 7H), indicating that Pacer also functions in clearing protein aggregates. DISCUSSION Elucidating how PI3KC3 complexes are targeted to different membranes is the key for understanding the spatiotemporal control of PtdIns(3)P biogenesis and related vesicular trafficking. PtdIns(3)P participates in nearly all aspects of endosomal function (Di Paolo and De Camilli, 2006). In contrast, little is known about the requirement of PtdIns(3)P for other aspects of autophagy, although the significance of PtdIns(3)P in autophagy initiation has been well characterized (Itakura et al., 2008; Matsunaga et al., 2009, 2010; Obara et al., 2006; Sun et al., 2008; Takahashi et al., 2007; Zhong et al., 2009). Studies have shown that autophagosomes indeed require a steady level of PtdIns(3)P to recruit proteins, including TECRP1, FYCO1, and Mon1-Ccz1, for late-stage progress in mammalian cells (Cabrera et al., 2014; Chen et al., 2012; Pankiv et al., 2010; Yu et al., 2015). Moreover, PtdIns(3)P has a high turnover rate in vivo (Gillooly et al., 2000), indicating a necessity of continuous PtdIns(3)P biogenesis. In this study, we provide the evidence that Pacer localizes PI3KC3 to autophagosomes for site-specific PtdIns(3)P production to support late-stage autophagy processes. Pacer regulates PI3KC3 activity through at least three different mechanisms. First, Pacer displaces Rubicon to unleash PI3KC3 catalytic activity; second, Pacer enhances the integrity of PI3KC3 complexes to directly stimulate their activity; and, third, Pacer targets the PI3KC3 complex to autophagosome membranes for robust in situ PtdIns(3)P synthesis. Recently, Stx17 was shown to play crucial roles in both early and late stages of autophagy (Diao et al., 2015; Hamasaki et al., 2013; Itakura et al., 2012; Jiang et al., 2014; Takáts et al., 2013, 2014). We propose here that Stx17 may act as a platform to coordinate Atg14- and Pacer-PI3KC3 complexes in a sequential manner to ensure proper autophagy progression. However, one puzzle that remains to be solved is why Pacer preferentially binds to Stx17 on autophagosomes and not to the Stx17 on ER or on early autophagic membrane Figure 6. Pacer Directly Binds to Phosphoinositides and Stx17 for Autophagosome Targeting (A) Schematic of a PIP Strip spotted with eight phosphoinositides and other bioactive lipids. (B and C) PIP Strips were incubated with recombinant proteins for full-length Pacer or Rubicon (0.5 mg/ml) as indicated overnight at 4 C and probed with anti- FLAG antibody (B); the binding affinity as indicated by dot intensity is quantified in (C). Data are shown as mean ± SD (**p < 0.01). (D) Co-localization analysis of Pacer and Stx17 with UVRAG or Atg14. Scale bars, 10 mm. (E) Endogenous Pacer coip with Stx17. (F) Analysis of the interaction between Pacer and Stx17 using Atg14 as the positive control. (G) Analysis of the interaction between Pacer and Stx17, SNAP29, or VAMP8. (H) Recombinant GST, GST-Stx17, GST-VAMP8, and GST-SNAP29 were purified as indicated. Recombinant GST-tagged proteins were allowed to bind to beads first, incubated with recombinant protein for FLAG-Pacer (full length), washed three times, and analyzed by anti-flag western blot. (I and J) Dissection of the Stx17-interacting region of Pacer using co-immunoprecipitation (I). The interaction is summarized in (J). (K) Pacer-autophagosome targeting was measured in control or Stx17 KD U2OS cells. Scale bars, 5 mm. Data are shown as mean ± SD (**p < 0.01). (L) Analysis of Stx17, Pacer, and LC3 co-localization. Scale bars, 10 mm. See also Figure S7. Molecular Cell 65, , March 16,

13 Figure 7. Pacer Regulates Bacterial Infection, Lipid Hemostatsis, and Protein Aggregate Clearance (A) Pacer is indispensable for autophagy-mediated suppression of bacterial replication in vivo. Cells were infected with WT RFP-marked S. typhimurium (red) for 4 hr and analyzed by immunostaining for intracellular replicated bacteria. Cells were counterstained with anti-tubulin antibody (green). Scale bars, 10 mm. (B) Quantification of the intracellular bacterial growth. Cells were infected with S. typhimurium for the indicated times. The infected cells were treated with gentamicin sulfate to block extracellular bacterial amplification and then lysed, and internalized bacteria were plated on Petri dishes. The replication of bacteria was quantified by numeration of the colony-forming units on the Petri plates. Data are shown as mean ± SD (***p < 0.001). (C) Pacer WT, OE, Atg7 KO, and Pacer OE/Atg7 KO HEK293 cells were used for infection assay as described in (A). (D) The replication of bacteria was quantified by numeration of the colony-forming units on the Petri plates. Data are shown as mean ± SD (***p < 0.001). (E) HepG2 cells stably expressing Pacer shrna or Pacer were subjected to starvation and then fixed and analyzed by Nile red staining. (F) Analysis of lipid drops by transmission electron microscopy and quantification in Pacer WT and KD HepG2 cells. Data are shown as mean ± SD (*p < 0.05). (G and H) U2OS cells with Pacer KD were treated with 5 mg/ml puromycin for 2 hr, cells were either fixed in 4% paraformaldehyde (PFA) (0 h) or washed three times in DMEM (10% fetal bovine serum [FBS]) without puromycin, and they were incubated for a further 3 hr (3 h) to fixation and immunofluorescence analysis (G). NT represents U2OS cells without puromycin treatment. Data are quantified in (H) and shown as mean ± SD, *p < structures. One possibility is that the ER-associated or preautophagosomal Stx17 is fully occupied by Atg14-PI3KC3 and other molecules (Arasaki et al., 2015; Hamasaki et al., 2013). However, it remains to be determined how Stx17 switches to interact with Pacer. The other possibility is that Pacer s autophagosome targeting might be co-determined by Stx17 and the PIPs, 1040 Molecular Cell 65, , March 16, 2017 as Pacer directly binds to PtdIns(3)P, PtdIns(4)P, and PtdIns(5)P in vitro (Figures 6A 6C). Indeed, besides PtdIns(3)P, both PtdIns(4)P and PtdIns(5)P are components of autophagic membrane structures (Vicinanza et al., 2015; Wang et al., 2015). However, it will be necessary to investigate which PIP plays the major role in determining Pacer s autophagosome targeting, and it will also be interesting to see whether Pacer functions in non-canonical autophagosome formation and pathogen infections, both of which are regulated by PtdIns(5)P (Pendaries et al., 2006; Vicinanza et al., 2015). Identification of Pacer provides an opportunity for better understanding how the HOPS complex selectively tethers autophagosomes to lysosomes. As Pacer interacts with both Stx17 and HOPS, it is possible that Pacer mediates the interaction

14 between HOPS and Stx17; alternatively, Stx17, Pacer, and HOPS form a trimeric complex. The latter possibility is supported by some observations: first, Pacer binds to the HOPS complex independently of UVRAG (Figures 5F, S6A, and S6B), because the Pacer mutants lacking UVRAG-binding activity interact with the HOPS complex as efficiently as WT Pacer; second, Stx17 interacts with the HOPS complex independently of UVRAG (Jiang et al., 2014); and, third, Pacer utilizes different motifs to directly interact with Stx17 and UVRAG (Figures 6I and 6J). In this study, we propose a molecular switch model in autophagy regulation (Figure S7J), which consists of two molecules with opposite biological functions. Rubicon acts as a brake by sequestering UVRAG-PI3KC3 on endosomes or in the cytosol during autophagy progression. In contrast, Pacer displaces this brake after receiving upstream signaling, and it allows autophagosomes to mature. This model is in line with the observations that Pacer OE or Rubicon KD overrides upstream signaling to elevate autophagy flux and that mtor inhibition dissociates Rubicon from UVRAG-PI3KC3 to facilitate autophagosome maturation (Figure 3J) (Kim et al., 2015). In future study, it will be necessary to address whether the Pacer-Rubicon molecular switch is controlled by mtor and other upstream regulators, including ULK1 and AMPK. In conclusion, The work presented here not only demonstrates that PI3KC3 engages in late stages of autophagy, defining a broader range of PI3KC3 functions in the membrane-associated processes, but it also reveals the mechanism that HOPS requires both Pacer and Stx17 to function specifically in autophagosome-lysosome fusion. Moreover, the identification of Pacer in the regulation of bacterial infection, hepatic lipid storage, and protein aggregate clearance provides further insights into how autophagy is connected with human diseases. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d KEY RESOURCES TABLE d CONTACT FOR REAGENT AND RESOURCE SHARING d EXPERIMENTAL MODEL AND SUBJECT DETAILS B RFP labeled S.typhimurium construction and culture B Cell culture B Stable cell lines construction d METHOD DETAILS B Immunoprecipitation and western blot B Immunofluorescence B Fractionation by differential centrifugation B Insect cell recombinant protein purification B Tandem affinity purification and mass spectrometry B Autophagy analysis B PI3KC3 (hvps34) kinase assay B Bacterial infection assay B Nile red staining B Protein Aggregate Clearance Assay d QUANTIFICATION AND STATISTICAL ANALYSIS SUPPLEMENTAL INFORMATION Supplemental Information includes seven figures and can be found with this article online at AUTHOR CONTRIBUTIONS Q.S., W.G., and X.C. designed the experiments. X.C., X.M., X.D., and X.J. performed the experiments. L.L. and S.C. performed the mass spectrometry. Z.S. and W.L. contributed reagents. Q.S. and X.C. wrote the manuscript. All authors discussed the results and commented on the manuscript. ACKNOWLEDGMENTS We are grateful to Harald Stenmark, Beth Levine, Jae U. Jung, Chenyu Liang, and Honggang Wang for reagents. We are grateful to Christopher Proud for critical reading of the manuscript. We thank LiangyanWang and Tao Li for technical support in the kinase assay. We thank the Imaging Center of Zhejiang University School of Medicine for assistance with confocal microscopy and electron microscopy. This study was supported by the Zhejiang Provincial Funds for Distinguished Young Scientists (LR15C070001), the National Science Foundation for Young Scholars of China ( ), the National Science Foundation for Outstanding Young Scholars of China ( ), and the National Science Foundation for Post-doctoral Scientists of China (2015M581923). 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