Beclin 1 Forms Two Distinct Phosphatidylinositol 3-Kinase Complexes with Mammalian Atg14 and UVRAG

Cell Culture and Transfection

HeLa and human embryonic kidney (HEK)293T cells were cultured in DMEM supplemented with 10% fatal bovine serum (FBS), 50 μg/ml penicillin, and streptomycin in a 5% CO₂ incubator. NIN3T3 cells were maintained in DMEM containing 10% bovine calf serum and the antibiotics. For starvation, cells were washed with phosphate-buffered saline (PBS) and incubated in amino acid-free DMEM without FBS. FuGENE 6 reagent (Roche Diagnostics, Tokyo, Japan) or Lipofectamine 2000 reagent (Invitrogen, Tokyo, Japan) were used for transfection. Stable cell lines were generated using a retroviral expression system (Hara et al., 2008). E64d and pepstatin were purchased from Peptide Institute (Osaka, Japan), and chloroquine and wortmannin were purchased from Sigma-Aldrich (St. Louis, MO).

Plasmids

cDNAs encoding human Atg14 (AK131251/FLJ 16178, obtained from the Department of Biotechnology, National Institute of Technology and Evaluation, Tokyo, Japan) and human UVRAG (IMAGE 618651) were amplified by polymerase chain reaction (PCR) and subcloned into p3xFLAG CMV10 (Sigma-Aldrich) or pCI-neo (Promega, Tokyo, Japan) together with hemagglutinin (HA) or Myc epitope tag. pCMV5 HA-Beclin 1, pcDNA3 FLAG-Vps34, and pcDNA3 MEF-p150 plasmids were kindly provided by Dr. Tamotsu Yoshimori (Osaka University, Osaka, Japan). FLAG-Rab5, FLAG-Rab7, and FLAG-Rab9 plasmids were kindly provided by Dr. Mitsunori Fukuda (Tohoku University, Sendai, Japan). Rab9 was subcloned into pCI-neo together with the HA epitope tag. For retroviral expression, cDNAs encoding human Atg14, UVRAG, and Vps34 were subcloned into pMXs-puro or pMXs-IP vector (provided by T. Kitamura, The University of Tokyo, Tokyo, Japan). A small interference RNA (siRNA)-resistant Atg14 silent mutant was created by substituting four nucleotides in the Atg14 siRNA-targeting region (A582T, A585T, C588A, and T591A).

Antibodies

To generate antibodies against human Atg14 and UVRAG, human Atg14 cDNA (amino acid residues 270–404) and human UVRAG cDNA (amino acid residues 137-419) were subcloned into pGEX-6p-1 (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). Resulting glutathione transferase (GST)-fused Atg14 and UVRAG were used to immunize rabbits. Antisera were affinity-purified. Mouse monoclonal anti-HA (16B12) and anti-Myc (9E10) antibodies, and rabbit polyclonal anti-HA antibody, were purchased from Covance Research Products (Princeton, NJ). Mouse monoclonal anti-FLAG antibody (M2), anti-β-actin, and anti-Atg5 antibody were purchased from Sigma-Aldrich. Rat monoclonal anti-green fluorescent protein (GFP) antibody (GF090R) was purchased from Nacalai Tesque (Kyoto, Japan) and used for immunocytochemistry. Rabbit polyclonal anti-GFP antibody was purchased from Invitrogen and used for immunoprecipitation and immunoblotting. Guinea pig polyclonal anti-p62 antibody was purchased from Progen (Heidelberg, Germany). The polyclonal antibodies against microtubule-associated protein light chain 3 (LC3) (SK2-6) (Kabeya et al., 2000), Vps34, Beclin 1 (Kihara et al., 2001a), and Atg16L1 (Mizushima et al., 2003) were described previously.

Immunoprecipitation and Immunoblotting

Cell lysates were prepared in a lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100, 1 mM phenylmethanesulfonyl fluoride, 1 mM Na₃VO₄, and protease inhibitor cocktail [Complete EDTA-free protease inhibitor; Roche Diagnostics]). The lysates were clarified by centrifugation at 14,000 rpm for 15 min and were subjected to immunoprecipitation by using specific antibodies in combination with protein G-Sepharose (GE Healthcare). Precipitated immunocomplexes were washed 6 times in a wash buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100) and boiled in sample buffer. Samples were subsequently separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to Immobilon-P polyvinylidene diflouride membranes (Millipore, Billerica, MA). Immunoblot analysis was performed with the indicated antibodies and visualized with SuperSignal West Pico Chemiluminescent substrate (Pierce Chemical, Rockford, IL).

Immunocytochemistry

Cells grown on coverslips were washed with PBS and fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. Fixed cells were permeabilized with 50 μg/ml digitonin in PBS for 15 min, blocked with 3% bovine serum albumin in PBS and incubated with primary antibody for 1 h. After washing, cells were incubated with AlexaFluor 488-conjugated goat anti-rat immunoglobulin G (IgG) or AlexaFluor 564-conjugated goat anti-rabbit or anti-mouse IgG secondary antibody (Invitrogen) for 1 h and examined under a fluorescence microscope (IX81; Olympus, Tokyo, Japan) equipped with a charge-coupled device camera (ORCA ER; Hamamatsu Photonics, Hamamatsu, Japan). Quantification of GFP-LC3 dots was performed using the Top Hat algorithm of MetaMorph version 7.0 (Molecular Devices, Tokyo, Japan) as described previously (Kuma et al., 2004).

RNAi

Stealth RNAi oligonucleotides were used for siRNA experiments (Invitrogen). The sequences used were as follows: human Atg14-1-siRNA sense, 5′-GGCAAAUCUUCGACGAUCCCAUAUA-3′ and antisense, 5′-UAUAUGGGAUCGUCGAAGAUUUGCC-3′; human Atg14-2-siRNA sense, 5′-GAUCACAACGGAGACACCAGCAUUA-3′ and antisense, 5′-UAAUGCUGGUGUCUCCGUUGUGAUC-3′; human UVRAG-siRNA sense, 5′-CAUCAGCUCCUUGAUACCUACUUUA-3′ and antisense, 5′-UAAAGUAGGUAUCAAGGAGCUGAUG-3′; uman Beclin 1-siRNA sense, 5′-CCACUCUGUGAGGAAUGCACAGAUA-3′ and antisense, 5′-UAUCUGUGCAUUCCUCACAGAGUGG-3′; and human Vps34-siRNA sense, 5′-CCCAUGAGAUGUACUUGAACGUAAU-3′ and antisense, 5′-AUUACGUUCAAGUACAUCUCAUGGG-3′.

For a negative control, Medium GC duplex of stealth RNAi Negative Control Duplexes (Invitrogen) was used. The Stealth RNAi oligonucleotides were transfected into HeLa cells by using Lipofectamine RNAi MAX according to the manufacturer's protocols. After 2 d, the cells were again transfected with the same siRNA and cultured for additional 3 d before analysis, unless otherwise specified.

Reverse Transcriptase (RT)-PCR

Total RNA was extracted from mouse tissues using Isogen (Nippon Gene, Toyama, Japan) according to the manufacturer's instruction. Trace contamination of DNA was removed by DNase digestion (Promega). cDNA was synthesized from 2 μg of total RNA by using ReverTra Ace (Toyobo Engineering, Osaka, Japan). PCR amplification was performed using the following primers: mouse Atg14 forward, 5′-CATAACAACCCCGCCTACAC-3′ and reverse, 5′-TGCGTTCAGTTTCCTCACTG-3′; mouse UVRAG forward, 5′-GCAGAAGGACTCCCTGAGTG-3′ and reverse, 5′-GGAAGTCCTCGGAATTAGGC-3′; and mouse β-actin forward, 5′-CTGGGTATGGAATCCTGTGG-3′; reverse, 5′-GTACTTGCGCTCAGGAGGAG-3′.

Real-Time PCR

Real-time PCR was performed on a Thermal Cycler Dice (Takara, Kyoto, Japan) by using SYBR premix EX Taq (Takara). The primer sets used were as follows: human Vps34 forward, 5′-AAGCAGTGCCTGTAGGAGGA-3′ and reverse, 5′-TGTCGATGAGCTTTGGTGAG-3′; human Beclin 1 forward, 5′-TGTCACCATCCAGGAACTCA-3′ and reverse, 5′-CTGTTGGCACTTTCTGTGGA-3′; human Atg14 forward, 5′-ATGAGCGTCTGGCAAATCTT-3′ and reverse, 5′-CCCATCGTCCTGAGAGGTAA-3′; human UVRAG forward, 5′-CTGTTTGGATGGGCTGAAAT-3′ and reverse, 5′-TGCGAACACAGTTCTGATCC-3′; and human β-actin forward, 5′-ATTGCCGACAGGATGCAGAA-3′ and reverse, 5′-ACATCTGCTGGAAGGTGGACAG-3′. Amplicon expression in each sample was normalized to its β-actin mRNA content.

Electron Microscopy

Conventional electron microscopy was performed as described previously (Hara et al., 2008). For morphometric analysis of autophagosomes, at least 10 cells of each sample were analyzed using MetaMorph image analysis software, version 6.2 (MDS Analytical Technologies, Sunnyvale, CA).

Yeast Studies

Total cell lysates were prepared and subjected to immunoblot analysis as described previously (Mizushima et al., 1999). Separation of extracellular and intracellular forms of carboxypeptidase Y (CPY) was performed essentially as described previously (Gabriely et al., 2007). Five OD₆₀₀ U of mid-log phase yeast cells were incubated in 500 μl of YPD medium for 1 h at 30°C. Then, 5 μl of 1 M NaN₃ was added, and the cell cultures were cooled on ice for 10 min. Culture samples were centrifuged to separate the cells (containing the intracellular fraction) from the medium (containing the extracellular fraction). The culture medium was precipitated with 10% trichloroacetic acid and 10% Triton X-100 for 10 min at 4°C and centrifuged at 15,000 rpm for 30 min. Samples were washed with chilled acetone and resuspended in 200 μl of SDS sample buffer. The yeast Atg14 and Vps38 expression plasmids, Ape1 and CPY antibodies, and Δapg14 and Δvps38 strains were generous gifts from Drs. Yoshinori Ohsumi and Keisuke Obara (National Institute for Basic Biology, Okazaki, Japan).

Identification of Mammalian Homologues of Atg14 and Vps38 (UVRAG)

Mammalian homologues of Atg14 and Vps38 have not been successfully identified by simple BLAST database search. We therefore performed a position specific iterative (PSI)-BLAST search of the National Center for Biotechnology Information (NCBI) database by using Saccharomyces cerevisiae Atg14 (NP_009686) and Candida glabrata Atg14 (XP_445209) (Altschul et al., 1997). PSI-BLAST is more sensitive to weak but biologically relevant sequence similarities than conventional BLAST, because we can create a position-specific score matrix for each query protein sequence (Jones and Swindells, 2002). After two or three iterated profiling, a putative mammalian homologue, KIAA0831, was identified in both human and mouse databases. Human KIAA0831 (NP_055739) is a 492-amino acid protein with no obvious sequence motifs except the coiled-coil domain, and it has weak homology to S. cerevisiae Atg14 (13.1% identity and 37.2% similarity) (Figure 1A). Likewise, we performed a similar PSI-BLAST search by using S. cerevisiae Vps38 (NP_013464) and C. glabrata Vps38 (XP_445092), and identified UVRAG as a putative Vps38 homologue. UVRAG shows 10.2% identity and 31.9% similarity to yeast Vps38 (Figure 1B). Both KIAA0831 and UVRAG transcripts were widely expressed in mouse tissues (Figure 1C).

KIAA0831 homologues were widely found in species where the Atg14 homologue has not been identified. Similarly, UVRAG homologues were found in many species in which Vps38 has been unknown. We then constructed an unrooted neighbor-joining (NJ) tree of these proteins (Figure 1D). According to the tree topology, the aligned sequences were classified into four groups: the Atg14, Vps38, KIAA0831, and UVRAG homologues. The Atg14 homologues were closely related to the KIAA0831 homologues, but not to the UVRAG homologues. The UVRAG homologues were located between the Atg14 and Vps38 homologues, but they seemed to be closer to the Vps38 homologues. Both human KIAA0831 and yeast Atg14 have a coiled-coil domain in their N-terminal region, whereas UVRAG and yeast Vps38 possess the coiled-coil domain in their center region (Figure 1E). Based on these findings together with the results described below, we named KIAA0831 as human Atg14. Our sequence analysis also suggests that UVRAG could be the functional counterpart of Vps38.

Human Atg14 Interacts with Beclin 1 and Vps34 but Not with UVRAG

We first analyzed complex formation of human Atg14, Beclin 1, Vps34, and UVRAG. Atg14, Beclin 1, and UVRAG were N-terminally fused to the Myc-, HA-, and FLAG-tags, respectively, and transiently coexpressed in HEK293T cells. When the cell lysate was precipitated with HA-Beclin 1, both Atg14 and UVRAG were detected in the precipitates (Figure 2A). However, if we precipitated the lysate with Myc-Atg14, only Beclin 1, but not UVRAG, was coprecipitated. Likewise, Atg14 was not coprecipitated with UVRAG, whereas Beclin 1 was precipitated. These findings suggest that Atg14 and UVRAG form distinct Beclin 1 complexes. These interactions were also observed in HeLa cells and NIH3T3 cells (Supplemental Figure 1).

Vps34 contains three distinct domains: the N-terminal C2 domain, central accessory domain and C-terminal catalytic domain. A previous study showed that the C2 domain of Vps34 is sufficient to bind UVRAG (Liang et al., 2006); thus, we determined which region of Vps34 Atg14 interacts with. Full-length FLAG-Vps34, FLAG-Vps34N (1-282 amino acids, containing the C2 domain), or FLAG-Vps34C (283-887) was coexpressed with HA-UVRAG and HA-Atg14 (Figure 2B). Immunoprecipitation analysis revealed that Atg14 as well as UVRAG interacts with the N-terminal C2 domain of Vps34, suggesting that one Vps34 molecule might interact with either Atg14 or UVRAG through the C2 domain. Beclin 1 was also specifically coprecipitated with Vps34N, as reported previously (Liang et al., 2006).

To analyze the endogenous Atg14 complex, we generated antibodies against human Atg14 and UVRAG. Anti-Atg14 antibody was able to precipitate endogenous Atg14, Vps34, and Beclin 1, but not UVRAG (Figure 2C). Likewise, endogenous Vps34 and Beclin 1, but not Atg14, were coprecipitated with anti-UVRAG antibody. These data confirm the interactions of endogenous proteins, which were shown in the transfection experiments.

We confirmed that the Atg14 protein complex also contained p150, which is a regulatory subunit of Vps34 (Figure 2D). All these data suggest that human Atg14 and UVRAG form two distinct PI3-kinase complexes with Beclin 1, Vps34, and p150.

Beclin 1 and Vps34 Are Important for Expression Levels of Atg14 and UVRAG

We next performed siRNA-mediated knockdown experiments using HeLa cells, because knockdown efficiency is very high in these cells. HeLa cells were transfected with specific siRNAs to knockdown endogenous Vps34, Beclin 1, Atg14, and UVRAG expressions. At 72 h after the transfection, respective siRNA, but not a control siRNA, significantly reduced endogenous expression of each protein (Figure 3A). In addition to these direct effects, treatment with Vps34 siRNA also decreased Beclin 1, Atg14, and UVRAG expression levels (Figure 3A, lane 2). Atg14 and UVRAG protein levels also decreased in cells treated with Beclin 1 siRNA (Figure 3A, lane 3). Furthermore, siRNA against either Atg14 or UVRAG also modestly decreased Beclin 1 protein levels (Figure 3A, lanes 4 and 5), and the knockdown of both Atg14 and UVRAG reduced the Beclin 1 expression level more markedly (Figure 3A, lane 6). Changes in the mRNA levels were only found in each specific siRNA target (Figure 3B). These results suggest that the presence of Beclin 1 and Vps34 is important for the stability of both Atg14 and UVRAG, and Beclin 1 stability depends on Vps34, Atg14, and UVRAG.

Atg14 Is Present on Isolation Membranes

In yeast, Atg14 is present on PAS and is thought to function at the early stage of autophagosome formation. We therefore determined the intracellular localization of human Atg14. We used NIH3T3 cells stably expressing Atg14 N-terminally fused to GFP, because the numbers of isolation membrane and autophagosome are larger in NIH3T3 cells than in HEK293T and HeLa cells, which allowed us to perform morphological analysis more precisely. In these cells, although the GFP-Atg14 signal was primarily observed as a diffuse or reticular pattern in the cytoplasm, it was also detected as punctate dots, whose number increased during starvation (Figure 4A). These dots partially overlapped with LC3-positive dots, which represented autophagic vacuoles (Figure 4A, arrowheads, ∼25% of the total LC3 puncta). However, GFP-Atg14 was not present on most of the large LC3 dots (Figure 4A, arrows), raising the possibility that GFP-Atg14 might be present on only intermediate structures, but not on complete autophagosomes. Because the Atg12-Atg5 conjugate and Atg16L1 specifically localize on elongating isolation membranes (phagophore) (Mizushima et al., 2001), we tested their colocalization with Atg14. We observed better colocalization between GFP-Atg14 and endogenous Atg5, and between GFP-Atg14 and endogenous Atg16L1 after autophagy induction by starvation (Figure 4, B and C). Approximately 85% of Atg16L1 dots were positive for Atg14, and ∼75% of Atg14 dots were positive for Atg16L1. These data suggested that Atg14 primarily localizes on isolation membranes, but not on established autophagosomes.

PI3-Kinase Activity Is Not Required for Starvation-induced Atg14 Puncta Formation

To test whether Atg14 puncta formation depends on PI3-kinase activity, we used a PI3-kinase inhibitor wortmannin, which is known to inhibit autophagy (Blommaart et al., 1997). Wortmannin completely inhibited starvation-induced Atg16L1 puncta formation as described previously for the Atg5 puncta (Mizushima et al., 2001). However, starvation-induced Atg14 puncta remained in wortmannin-treated cells, suggesting that Vps34 activity is not required for Atg14 puncta formation (Figure 5, A and B).

We next examined localization of an Atg14 mutant unable to bind Vps34 and Beclin 1. Yeast Atg14 interacts with Vps34 and Vps30/Atg6 via the coiled-coil domain. We therefore designed a human Atg14 mutant (Atg14ΔCC), which lacks the coiled-coil domain (71-184 amino acid residues). In NIH3T3 and HeLa cells, both Vps34 and Beclin 1 were coprecipitated with Atg14, but not with Atg14ΔCC (Figure 5C), confirming that the coiled-coil region is important for the complex formation in mammals as well. Nonetheless, both wild-type Atg14 and Atg14ΔCC accumulated to punctate structures during starvation, which were colocalized with Atg16L1 and partially with LC3 (Figure 5D) These results suggest that Atg14 can localize to isolation membranes or its precursor structures independently of Vps34 and Beclin 1.

UVRAG Is Present on Rab9-positive Endocytic Compartments

We next examined the intracellular localization of UVRAG. In NIH3T3 cells stably expressing UVRAG N-terminally fused to GFP (GFP-UVRAG), numerous dots were observed even under nutrient-rich conditions. Most of the GFP-UVRAG dots were distinct from LC3 (Figure 6A). Endogenous UVRAG was also not colocalized with GFP-LC3 even under starvation conditions (Figure 6B, starvation). We tested other autophagy factors such as Atg16L1 and Atg14, but failed to detect colocalization with UVRAG (Figure 6, C and D). These data suggest that these UVRAG dots do not represent autophagy-related structures. Considering the possibility that UVRAG might be a homolog of Vps38, we then analyzed the endocytic pathway. Among several marker proteins tested, UVRAG showed clear colocalization with Rab9 (∼80%), one of the late endosome markers (Figure 6, E and F). UVRAG also colocalized but only partially with other endosome markers such as Rab5 and Rab7 (Supplemental Figure 2). It was shown that Rab9 and Rab7 localize distinct type of late endosomes. Rab9-positive compartments contain mannose 6-phosphate receptors and the tail-interacting protein of 47-kDa (TIP47) and are involved in retrograde transport from late endosomes to TGN (Lombardi et al., 1993; Barbero et al., 2002). In contrast, Atg14 did not colocalize with Rab9 (Figure 6G). Therefore, UVRAG primarily resides in these endocytic compartments, not in the autophagy pathway.

Vps34 Is Present on Rab9-positive Endocytic Compartments and Starvation-induced Atg14-positive Structure

Because Vps34 interacts with both Atg14 and UVRAG, it is expected to localize to autophagic and endocytic Rab9-positive compartments. We generated Vps34 constructs tagged with GFP at its N terminus (GFP-Vps34) or C terminus (Vps34-GFP). Although GFP-Vps34 showed no punctate structures (data not shown), Vps34-GFP showed numerous dot structures under nonstarvation conditions (Figure 7A). These Vps34-GFP dots were colocalized with coexpressed HA-UVRAG almost completely. Under starvation conditions, ∼30% of Vps34-GFP dots were colocalized with coexpressed HA-Atg14 (Figure 7B). These results suggest that Atg14 and UVRAG function together with Vps34 in these compartments.

Human Atg14 Is Required for Autophagosome Formation

Although human Atg14 does not complement the yeast atg14 deletion mutant (Supplemental Figure 3A), subcellular localization analyses of Atg14 suggested that Atg14 functions in the autophagy pathway. To prove this hypothesis, we determined the effect of Atg14 silencing on autophagy in HeLa cells by using two sets of siRNA against different regions of Atg14 (Atg14-1 and Atg14-2). Atg14 protein levels decreased by nearly 100% and ∼90% after Atg14-1 and Atg14-2 siRNA treatments, respectively (Figure 8A). We then performed autophagy flux assay (Tanida et al., 2005; Mizushima and Yoshimori, 2007). In control cells, the amount of LC3-II increased after chloroquine treatment, which increased lysosome pH, under both nutrient-rich and starvation condition, suggesting the high autophagy flux in HeLa cells. In Atg14 siRNA-treated cells, LC3-II was still observed in rather higher levels. However, the LC3-II amount did not change after chloroquine treatment in Atg14-1 siRNA-treated cells and only slightly increased in Atg14-2 siRNA-treated cells (Figure 8A), suggesting that autophagic flux is blocked in these cells. We also monitored the expression level of autophagy substrate p62, another indicator of autophagic degradation (Bjørkøy et al., 2005; Mizushima and Yoshimori, 2007). The amount of p62 decreased after autophagy induction by starvation in control cells, which was inhibited in Atg14 siRNA-treated cells (Figure 8A). We obtained similar results when we used lysosome protease inhibitors E64d and pepstatin A, instead of chloroquine (Figure 8B). We also examined the effect of Vps34 silencing on autophagy. Vps34 siRNA caused accumulation of LC3-II under both nonstarvation and starvation conditions as did Atg14 siRNA treatment (Figure 8C). Again, E64d and pepstatin treatment did not lead to further accumulation of LC3-II in Vps34 siRNA-treated cells. Starvation-dependent p62 reduction was not also observed in these cells. All these data indicate that autophagy flux is suppressed in cells treated with siRNA against Atg14 and Vps34, although LC3 conversion is not inhibited.

Finally, we determined at which step autophagy is perturbed. In HeLa cells stably expressing GFP-LC3, GFP-LC3 was mostly present in the nucleus under normal conditions, although its physiological significance is unknown (Kuma et al., 2007). GFP-LC3 was translocated to cytoplasmic dots when autophagy was normally induced (Figure 8D). However, the starvation induced GFP-LC3 puncta formation was markedly impaired in cells treated with siRNA against Atg14 and Vps34 (Figure 8, D and E). Instead, most GFP-LC3 remained in the nucleus in these cells. Electron microscopic analysis demonstrated that autophagosomes are virtually absent in Atg14 knockdown cells (Figure 8F). These fluorescence and electron microscopic analyses suggest that Atg14 is required for autophagosome formation rather than autophagosome maturation.

We also tested the effect of UVRAG knockdown on autophagy. In contrast to a previous report (Liang et al., 2006), we could not detect any inhibitory effect of UVRAG siRNA on autophagy flux and GFP-LC3 dot formation, although UVRAG expression was efficiently suppressed in the treated cells (Figure 8, H–J).

Coiled-Coil Domain of Atg14 Is Required for Autophagy

To investigate the role of Atg14 complex formation in autophagy, we analyzed the function of Atg14ΔCC, which is unable to interact with Beclin 1 and Vps34 (Figure 5C), by an Atg14 complementation assay. siRNA-resistant Atg14 and Atg14ΔCC were generated by introducing silent mutations in the siRNA target region, and they were expressed in Atg14-knockdown HeLa cells. In these cells, endogenous Atg14 was replaced with HA-Atg14 or HA-Atg14ΔCC (Figure 9A). Accumulation of p62 due to Atg14 depletion was restored by wild-type Atg14 but not by Atg14ΔCC (Figure 9A). LC3 flux was also restored by wild-type Atg14 but not by Atg14ΔCC (Figure 9B). Therefore, these data suggest that the coiled-coil domain of Atg14 is required for its involvement in autophagy probably through interaction with Vps34 and Beclin 1.