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Beclin-1-interacting autophagy protein Atg14L targetsthe SNARE-associated protein Snapin to coordinateendocytic trafficking

Hee Jin Kim1, Qing Zhong2, Zu-Hang Sheng3, Tamotsu Yoshimori4, Chengyu Liang1 and Jae U. Jung1,*1Department of Molecular Microbiology and Immunology, University of Southern California, Keck School of Medicine, Los Angeles, CA 90033, USA2Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA3Synaptic Function Section, National Institute of Neurobiological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA4Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan

*Author for correspondence (jaeujung@usc.edu)

Accepted 11 June 2012Journal of Cell Science 125, 4740–4750� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.100339

SummaryAutophagy is a highly regulated membrane remodeling process that allows the lysosome-mediated degradation of cytoplasmic entities

by sequestrating them in double-membrane autophagosomes. Autophagy is hence highly intertwined with the endocytic traffickingpathway, sharing similar molecular machinery. Atg14L, also known as Beclin 1-associated autophagy-related key regulator (Barkor),directly interacts with Beclin 1 through its coiled-coil domain and enhances phosphatidylinositol 3-phosphate kinase class III (PI3KC3)

activity to induce autophagosome membrane nucleation, highlighting its essential role in the early stage of mammalian autophagy. Here,we report a novel function of Atg14L in the endocytic trafficking pathway wherein Atg14L binds to and colocalizes with the fusogenicSNARE effector protein Snapin to facilitate endosome maturation. Atg14L specifically binds to Snapin and this interaction effectively

facilitates endosomal maturation without affecting autophagic cargo degradation. Consequently, atg14l knockdown significantlydelayed the late stage of endocytic trafficking, as evidenced by the retarded kinetics of internalized surface receptor degradation. Thisphenotype was effectively complemented by wild-type Atg14L or Beclin 1-binding mutant, but not by its Snapin-binding mutant. Takentogether, our study demonstrates that Atg14L functions as a multivalent trafficking effector that regulates endosome maturation as well

as autophagosome formation, reflecting the complexity of the crosstalk between autophagic and endocytic vesicle trafficking in highereukaryotes.

Key words: Atg14L, Snapin, Autophagosome maturation, Endosome maturation, Autophagy

IntroductionMacroautophagy (hereafter called autophagy) is a membrane

trafficking process dedicated to the maintenance of homeostasis

by allowing the self-cannibalization of intracellular constituents

through the lysosomal degradation pathway (Klionsky, 2005).

Unique de novo double-membrane structures termed isolation

membranes (IMs) enwrap cytoplasmic contents to generate

autophagosomes, which subsequently undergo stepwise

maturation into hybrid-like organelles with degradative

capabilities termed autolysosomes. Specifically, autophagosomes

utilize the microtubule network to migrate and sequentially fuse

with components of the endocytic pathway, such as early and late

endosomes and multi-vesicular bodies (MVBs), producing

amphisomes. Autophagosomes and/or amphisomes eventually fuse

with lysosomes to generate autolysosomes, in which the sequestered

cargo is degraded into building blocks for macromolecules and

recycled to the cytoplasm (Chen and Klionsky, 2011; Simonsen and

Tooze, 2009).

Despite profound differences in structure, autophagosomes

topologically and mechanistically resemble endosomes during

maturation as they share a number of vesicle-trafficking

components (Orsi et al., 2010). For instance, both autophagic

and endocytic trafficking processes require the class C vacuolar

protein sorting (Vps-C)/homotypic fusion and protein sorting

(HOPS) complex, a central regulator governing multiple

trafficking events at the vacuolar/lysosomal compartments

(Nickerson et al., 2009). In yeast, the Vps-C/HOPS complex

functions as a tethering apparatus by promoting nucleotide

exchange of Ypt7/Rab7 and drives membrane fusion by

catalyzing the assembly of soluble N-ethylmaleimide sensitive

factor (NSF) attachment protein receptor (trans-SNARE)

complexes (Sato et al., 2000; Stroupe et al., 2006; Wurmser

et al., 2000). Our previous studies have shown that UV radiation

resistance-associated gene (UVRAG), initially characterized as a

component of the class III posphatidylinositol-3-kinase (PI3KC3)

complex during autophagosome formation, interacts with the

Vps-C/HOPS complex and promotes autophagosome/endosome

maturation, thus serving as a convergence point of the autophagic

and endocytic pathways (Liang et al., 2006; Liang et al., 2008).

Consisting of three core components – hVps34, hVps15 and

Beclin 1 – the activity and specificity of the PI3KC3 complex is

tightly modulated by numerous regulators during autophagosome

biogenesis and endocytic trafficking (Lindmo and Stenmark,

2006). It has been increasingly recognized that, as in yeast, the

mammalian PI3KC3 complex containing UVRAG predominantly

functions in autophagosome/endosome maturation, whereas

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the yeast Atg14-like (Atg14L)-harboring complex inducesautophagosome membrane nucleation. Atg14L, also known as

Beclin 1-associated autophagy-related key regulator (Barkor), wasidentified to directly interact with Beclin 1 through its coiled-coildomain and to be excluded from the PI3KC3 complex carrying

UVRAG (Itakura et al., 2008; Matsunaga et al., 2009; Sun et al.,2008; Zhong et al., 2009). The N-terminus of Atg14L contains theconserved cysteine repeats necessary for endoplasmic reticulum(ER) localization and thereby recruits the PI3KC3 complex to the

initiating IMs originated from the ER, a potential origin ofautophagosomal membranes (Matsunaga et al., 2010). Moreover,the C-terminal amphipathic alpha helix in the Barkor/Atg14L

autophagosome targeting sequence (BATS) domain wassuggested to be essential for autophagosome targeting bystabilizing the highly curved early autophagic membrane in a

phosphatidylinositol 3-phosphate (PI3P)-dependent manner (Fanet al., 2011; Lu et al., 2011). These support the notion that Atg14Lis crucial for the early phase of autophagosome formation.

Originally identified as a neuronal SNARE protein, Snapin wassuggested to modulate priming of synaptic vesicles for theirsynchronized fusion during neurotransmission (Pan et al., 2009).

Further studies have extended the fusogenic function of Snapin in awide array of membrane fusion events in non-neuronal cells such asendosome-mediated cytokinesis, phagosome maturation, and insulinexocytosis (Ilardi et al., 1999; Buxton et al., 2003; Gromley et al.,

2005; Song et al., 2011; Tiwari et al., 2009). Snapin executes thefusogenic function in part by associating with proteins in vesicleSNARE (v-SNARE) complexes on donor membranes and drives the

fusion with trans SNARE (t-SNARE) complexes on targetmembranes such as lysosomal membranes (Buxton et al., 2003;Lu et al., 2009; Pan et al., 2009). Moreover, Snapin has been

recently implicated to coordinate the retrograde transport of lateendosomes and lysosomal maturation by interacting with the dyneinmotor complex, thereby contributing to efficient autophagic-

lysosomal function (Cai et al., 2010). These suggest that Snapin isa candidate molecular target for autophagy and lysosome regulation.

As part of our endeavor to delineate the biological functions of

mammalian Atg14L, we screened novel interacting proteins ofAtg14L using a yeast two-hybrid system. Our results showed thatAtg14L directly binds to Snapin and that this interaction

accelerates endosomal maturation without affecting autophagiccargo degradation. Our findings reveal that Atg14L, previouslyconsidered to be solely a Beclin 1-binding autophagy protein,plays a novel role in the late stage of endocytic trafficking in

conjunction with Snapin.

ResultsAtg14L binds to and colocalizes with Snapin

To further explore the biological roles of Atg14L during autophagy

and/or other cellular processes, we performed a yeast two-hybridscreen using full-length Atg14L as bait. Along with Beclin 1,which was previously shown to directly bind to Atg14L (Itakura

et al., 2008; Matsunaga et al., 2009; Sun et al., 2008; Zhong et al.,2009), Snapin was identified as a putative interaction partner(supplementary material Fig. S1A). Co-immunoprecipitation

verified the efficient and specific binding between endogenousAtg14L and Snapin as well as between epitope-tagged Atg14L andSnapin (Fig. 1A; supplementary material Fig. S1B). In search of

an Atg14L mutant that specifically loses Snapin-binding abilitywhile preserving Beclin 1-interacting activity, extensive bindingmapping studies were conducted. Firstly, a series of Atg14L

truncation mutants fused with mammalian glutathione S-transferase (GST) was constructed to define the region

responsible for Snapin interaction (Fig. 1B). GST pull-downshowed that Atg14L possesses three independent sites sufficientfor Snapin binding: residues 71–106 (A), 270–320 (B) and 419–492 (C) (Fig. 1C). Accordingly, these three regions were

individually deleted or in combination to identify the specificSnapin-binding site in the context of full-length Atg14L (Fig. 1D).Region 419–492 was modified to D419–480 since the last 10

amino acids of the Atg14L BATS domain are requiredfor autophagosome targeting (Fan et al., 2011). Co-immunoprecipitation showed that any Atg14L mutant devoid of

residues 419–480 lost Snapin-interacting activity (Fig. 1E). Wefurther confirmed that the Atg14L D419–480 mutant, also denotedas DC or mSnapin, failed to interact with Snapin, while it stillefficiently bound to the core components of class III PI3K

complex, Beclin 1 and hVps34 (supplementary material Fig. S1C–F). Consistent with previous studies (Matsunaga et al., 2009; Sunet al., 2008), the Atg14L D71–180 (mBeclin 1) mutant carrying a

deletion of its coiled-coil domain evidently lost Beclin-1-interacting activity, while retaining Snapin-binding ability(supplementary material Fig. S1C–F). Furthermore, confocal

analysis revealed the substantial colocalizations of mCherry-fused Snapin with EGFP-fused Atg14L wild type (WT) [Pearson’scorrelation coefficient (PCC)50.778 Manders overlap coefficient

(MOC)50.802] and EFGP–Atg14L (419–492) truncation mutant(PCC50.892, MOC50.899), while EFGP–Atg14L mSnapinmutant exhibited a minimal level of colocalization (PCC50.602,MOC50.645; Fig. 1F). Hence, these mutational analyses

identified the Atg14L mutants defective in Snapin- or Beclin-1-binding ability, mSnapin and mBeclin 1, respectively, which wereselected for the following functional studies.

Snapin enhances autophagosome maturation

We firstly examined whether Snapin plays an important role in

the autophagy pathway. Upon autophagy induction, solublemicrotubule-associated protein light chain 3 (LC3-I) is convertedto a lipidated form (LC3-II) that preferentially associates with thegrowing autophagosome membrane in puncta formations (Kabeya

et al., 2000). Thus, we monitored the effect of Snapin expressionon autophagosome biogenesis with fluorescent microscopy aftertransiently expressing green fluorescent protein (GFP)-tagged LC3

(GFP–LC3) in snapin+/+ and snapin2/2 mouse embryonicfibroblasts (MEFs). Compared with WT MEFs, snapin-deficientMEFs displayed significant increases in both the percentage of

GFP–LC3-puncta-positive cells and the number of GFP–LC3puncta per cell with or without rapamycin treatment (Fig. 2A).Furthermore, LC3-II and an autophagic substrate p62/SQSTMprotein levels were substantially increased in snapin-deficient

MEFs compared with WT MEFs, whereas reintroduction of snapin

transgene into snapin-deficient MEFs reduced LC3-II and p62/SQSTM to levels comparable to those in WT MEFs (Fig. 2B).

These results collectively indicate that autophagosomes aresubstantially accumulated upon the ablation of Snapinexpression. Because autophagosomes can be accumulated due to

either upregulation of autophagosome formation or blockade ofautophagosome maturation, we sought to distinguish between thetwo possibilities by treating the cells with vacuolar H+ ATPase

inhibitor bafilomycin A1 that abrogates autophagosomematuration (Fig. 2C). Bafilomycin A1 treatment increased LC3-II and p62/SQSTM protein levels in snapin+/+ MEFs, while

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Fig. 1. Atg14L binds to and colocalizes with Snapin. (A) Atg14L interacts with Snapin. HEK293T whole-cell extracts (WCEs) were subjected to co-

immunoprecipitation (co-IP) with anti-Atg14L antibody, followed by immunoblotting (IB) using anti-Snapin, anti-Beclin 1 or anti-Atg14L antibody. 5% of the

WCE was used as input. (B) Schematic representation of Atg14L and its truncation mutants constructed in pEBG-GST. (C) Atg14L residues 106–138, 270–320 or

419–492 (indicated as red bars in B) individually interact with Snapin. At 48 h post-transfection with Atg14L-GST wild type (WT) or truncation mutants and

Snapin-AU1, HEK293T WCEs were subjected to GST pulldown (GST-PD), followed by IB using anti-AU1 or anti-GST antibody. (D) Schematic representation

of Atg14L and its deletion mutants constructed in pEF-IRES-puro-FLAG. A, B and C, indicate residues 106–138, 270–320 and 419–480 of Atg14L, respectively.

(E) Atg14L aa419–480 (C) deletion abolishes Snapin binding. At 48 h post-transfection with Atg14L-FLAG WT or mutants and Snapin-AU1, HEK293T WCEs

were subjected to co-IP with anti-FLAG antibody, followed by IB using anti-AU1 or anti-FLAG antibody. Atg14L-DC was used as a Snapin binding mutant

(mSnapin in D). (F) Atg14L WT but not mSnapin mutant colocalizes with Snapin. At 48 h post-transfection with Atg14L-FLAG WT (416–492), or mSnapin

mutant and Snapin-AU1, HeLa cells were stained with anti-FLAG (green) or anti-AU1 (red) antibody and Hoechst 33342 (blue; to stain the nuclei), followed by

confocal microscopy analysis. Scale bars: 5 mm.

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snapin2/2 MEFs exhibited no detectable change (Fig. 2C),

suggesting that the aberrant accumulation of autophagosomes

after Snapin deletion results from the impairment of

autophagosome maturation rather than hyper-induction of

autophagosome formation. Collectively, these results indicate

that Snapin is pivotal for autophagy, especially during

autophagosome maturation.

Snapin promotes endocytic trafficking

Since Snapin appears to serve as an important modulator of the late

endocytic fusion machinery besides its established role in

regulating synaptic vesicle fusion, we sought to explore the role

of Snapin in endocytic trafficking. We firstly examined the

degradation kinetics of epidermal growth factor receptor (EGFR)

as the receptor-ligand complex is internalized from the cell surface

into endosomes, which ultimately fuse with lysosomes for

degradation. The results showed that EGFR degradation was

severely delayed in snapin-depleted MEFs compared with WT

MEFs (Fig. 3A). Subsequently, HeLa cells transfected with an

empty vector or Snapin were exposed to Alexa-Fluor-488-

conjugated EGF to visualize the effect of Snapin expression on

the internalization, endocytic transport, and lysosomal degradation

of EGFR associated with the exogenous fluorescent ligand

(Fig. 3B). At 15 minutes post-stimulation, EGF was detected in

small puncta structures in vector-expressing cells (PCC50.460,

MOC50.388), whereas in Snapin-expressing cells it was present in

cytoplasmic aggregates that were considerably colocalized with

lysosomal-associated membrane protein 1 (LAMP1)-positive

compartments (PCC50.496, MOC50.521). This difference

became more pronounced at 30 minutes post-incubation (vector

control, PCC50.605, MOC50.632; Snapin overexpression,

PCC50.694, MOC50.644). Yet, there were no noticeable

differences in the amounts of internalized EGF between snapin+/+

and snapin2/2 MEFs at 15 minutes post-stimulation

(supplementary material Fig. S2A,B). To further confirm the role

of Snapin in endolysosomal trafficking, we then assessed the

lysosomal proteolysis kinetics of a self-quenched red BODIPY dye-

conjugated bovine albumin serum (DQ-Red BSA) after small

hairpin RNA (shRNAmir)-mediated gene silencing of snapin

in HeLa cells (Fig. 3C; supplementary material Fig. S2C).

Consistently, DQ-Red BSA degradation rate was attenuated in

HeLa cells expressing Snapin-specific shRNAmir compared with

those carrying scrambled shRNAmir, indicating that ablation of

the Snapin expression compromises endolysosomal trafficking.

Altogether, these findings suggest that Snapin facilitates the

trafficking of endocytic cargo to late endosomes and lysosomes

for subsequent decomposition.

Atg14L–Snapin interaction is not required for

autophagosome maturation

Given that Snapin positively regulates autophagosome maturation

(Fig. 2), we next investigated whether the Atg14L–Snapin

Fig. 2. Snapin enhances autophagosome maturation. (A) Depleting Snapin increases the percentage of cells with GFP–LC3-labeled puncta. At 16 h post-

transfection with GFP-LC3, snapin+/+ (WT) and snapin2/2 (KO) MEFs were treated with DMSO or rapamycin (2 mM) for 3 h. Both the percentage of GFP–LC3-

positive cells with more than three puncta and the number of GFP–LC3 puncta per cell were quantified as the means 6 s.d. of 200 cells (from three independent

experiments); *P#0.01. (B) Depleting Snapin inhibits LC3 conversion and p62 degradation, which is recovered upon the reintroduction of Snapin–AU1. Snapin

WT and KO MEFs stably expressing Snapin–AU1 or control vector were treated with DMSO or rapamycin (2 mM) for 3 h and WCEs were subjected to IB

using anti-LC3, anti-p62, anti-AU1, anti-Snapin, or anti-b-Actin antibody. (C) Depleting Snapin leads to autophagosome accumulation. Snapin WT and KO MEFs

were treated with DMSO or bafilomycin A1 (0.1 mM) for the indicated time periods and WCEs were subjected to IB using anti-LC3, anti-p62, anti-Snapin or

anti-b-Actin antibody.

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interaction functions in the late stage of autophagy. In an attempt to

specifically monitor the role of Atg14L–Snapin binding during

autophagosome maturation independent of the Atg14L–Beclin 1

interaction, we complemented atg14l knockout mouse embryonic

stem cells (ESCs) with Atg14L Snapin- or Beclin 1-binding-

deficient mutant (mSnapin or mBeclin 1, respectively;

supplementary material Fig. S1) and monitored the steady-state

levels of LC3-II and p62/SQSTM in atg14l2/2 ESCs stably

expressing vector, Atg14L WT, mSnapin, or mBeclin 1. Compared

with atg14l+/+ ESCs, LC3-II was reduced and p62/SQSTM was

markedly accumulated in atg142/2 ESCs in the absence and

presence of rapamycin; this phenotype was readily rescued by

introducing the atg14l WT transgene into atg14l-deficient ESCs

(Fig. 4A). Interestingly, ectopic expression of the Atg14L

mSnapin mutant effectively rescued autophagy defects, whereas

the mBeclin 1 mutant expression failed to do so. These suggest that

the Atg14L–Snapin interaction has little or no effect on autophagic

cargo degradation. This phenotype was further confirmed by

transiently introducing monomeric red fluorescent protein

(mRFP)–GFP tandem fluorescently tagged LC3 (tfLC3) into

atg14l ESCs (Fig. 4B). tfLC3 can be used as a probe to distinguish

autophagosome formation from maturation due to the distinct pKa

values of the two fluorescent proteins. In short, tfLC3 is stable in

newly formed autophagosomes, giving rise to red and green

fluorescence, whereas green fluorescence is lost in acidic

autolysosomal compartments due to the high pKa value of GFP,

thus showing only red fluorescence (Kimura et al., 2007). No

detectable difference was observed between atg14l2/2-WT ESCs

and atg14l2/2-mSnapin ESCs; the proportion of vesicles that

solely exhibited red fluorescence was similar over the time course

Fig. 3. Snapin promotes endocytic trafficking. (A) Depleting

Snapin attenuates EGFR degradation. Snapin WT and KO MEFs

were treated with EGF (200 ng/ml) for the indicated time periods and

WCEs were subjected to IB with anti-EGFR, anti-Snapin or anti-b-

Actin antibody. (B) Overexpressing Snapin promotes EGF-

stimulated endocytic trafficking. HeLa cells stably expressing

Snapin–AU1 or control vector were exposed to Alexa-Fluor-488–

EGF (green) for the indicated time periods and stained with anti-

LAMP1 antibody (red) and Hoechst 33342 (blue, to stain the nuclei)

for confocal microscopy analysis. Scale bars: 20 mm. (C) Snapin

gene silencing suppresses proteolysis of DQ-Red BSA in lysosomes.

HeLa cells stably expressing Snapin or non-silencing control

shRNAmir were loaded with 20 mg/ml DQ-Red BSA for the

indicated time periods and subjected to Flow cytometry analysis.

Background (gray peak) represents samples without the BSA loading.

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of rapamycin treatment. These results collectively indicate that theAtg14L–Snapin interaction is not required for autophagosome

maturation.

Atg14L is required for endosome maturation

Provided that the autophagic and endocytic pathways converge atthe endosome prior to lysosome-mediated degradation, we nextasked whether Atg14L was functionally linked to endolysosomal

transport through its interaction with Snapin. Firstly, we observedthat Atg14L and Snapin were colocalized with the late endosomalproteins LAMP1 or CD63, but not with the early endosomal

proteins EEA1 or Rab5 (supplementary material Fig. S3). We then

tested the role of Atg14L expression in endocytic vesicle trafficking

by silencing atg14l expression and examining the EGF-stimulation-

mediated endocytic transport of EGFR to lysosomes. This showed

that silencing atg14l expression in HeLa cells resulted in marked

retardation of EGFR degradation, while it induced no gross

alterations of organelle structures and distributions (Fig. 5A;

supplementary material Fig. S4A). By striking contrast, silencing

atg5 expression, another autophagy essential gene, had little or no

effect on EGFR degradation kinetics (supplementary material Fig.

S4B). Using Alexa-Flour-488-conjugated EGF, we also ascertained

Fig. 4. Atg14L–Snapin interaction is not required for autophagosome maturation. (A) atg14l knockout cells complemented with Atg14L mSnapin are not

autophagy defective. atg14l+/+ (WT) and atg14l2/2 (KO) mouse embryonic stem cells (mESCs) stably expressing Atg14L–FLAG WT, mSnapin, or mBeclin 1 or

control vector were treated with DMSO or rapamycin (2 mM) for 3 h. The WCEs were subjected to IB using anti-LC3, anti-p62, anti-Atg14L or anti-b-Actin antibody.

The asterisk indicates a non-specific band above endogenous Atg14L. (B) Autophagosome maturation is competent in atg14l knockout cells complemented with

Atg14L mSnapin mutant. atg14l KO mESCs stably expressing Atg14L–FLAG WT or mSnapin mutant were electroporated with ptfLC3. At 24 h post-electroporation,

cells were treated with DMSO or rapamycin (2 mM) for the indicated time periods and stained with Hoechst 33342 (blue, to stain the nuclei), followed by confocal

microscopy analysis. GFP–LC3 (green) represents autophagosomes, and mRFP–LC3 represents autophagosomes and autolysosomes. Since green fluorescence is lost

in acidic autolysosomal compartments, the yellow punctae of the merged view indicate autolysosomes. Scale bars: 5 mm.

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whether silencing atg14l expression affected the internalization,

endocytic trafficking, and lysosome-mediated degradation of

EGFR. Small interfering RNA (siRNA)-mediated knockdown

of atg14l expression in HeLa cells substantially reduced

the colocalization of Alexa-Fluor-488-conjugated EGF with

the LAMP1-positive lysosomal compartments compared with

scrambled siRNA treatment (Fig. 5B). However, there was no

detectable difference in the amounts of internalized EGF between

the scrambled siRNA-treated cells and the Atg14L-specific siRNA-

treated cells at 15 minutes post-incubation (supplementary material

Fig. S4C,D), indicating that the levels of EGF endocytosis are not

affected by atg14l gene knockdown. However, the lysosomal

proteolysis kinetics of DQ-Red BSA was considerably lower in cells

treated with atg14l-specific siRNA compared with scrambled

siRNA (Fig. 5C; supplementary material Fig. S4E). Altogether,

these results suggest that Atg14L is a potential positive regulator of

endocytic trafficking.

Atg14L facilitates endolysosomal trafficking through itsinteraction with Snapin

To determine whether the Atg14L–Snapin interaction functions

in the endolysosomal pathway, we continued testing the effects

of Snapin expression on EGF-stimulated endosomal transport

under atg14l-silenced conditions. Ectopic expression of Snapin

profoundly facilitated EGFR degradation in cells treated with

scrambled shRNAmir, whereas no obvious effect was observed in

cells treated with atg14l-specific shRNAmir (Fig. 6A), indicating

that Snapin-mediated endocytic trafficking depends on Atg14L

expression. To further support that Atg14L–Snapin interaction is

required for endosomal transport, HeLa cells stably expressing

Fig. 5. Atg14L is required for endocytic trafficking. (A) atg14l

gene silencing attenuates EGFR degradation. HeLa cells stably

expressing atg14l-specific or non-silencing control shRNAmir were

treated with EGF (200 ng/ml) for the indicated time periods and the

WCEs were subjected to IB with anti-EGFR, anti-Atg14L or anti-b-

Actin antibody. (B) atg14l gene silencing represses EGF-stimulated

endocytic trafficking. At 48 h post-transfection with atg14l-specific

or non-specific control siRNA, HeLa cells were exposed to Alexa-

Fluor-488–EGF (green) for the indicated time periods and stained

with anti-LAMP1 antibody (red) and Hoechst 33342 (blue, to stain

the nuclei) for confocal microscopy analysis. Scale bars: 20 mm.

(C) atg14l gene silencing suppresses proteolysis of DQ-Red BSA in

lysosomes. At 48 h post-transfection with atg14l-specific or non-

specific control siRNA, HeLa cells were loaded with 20 mg/ml DQ-

Red BSA for the indicated time periods and subjected to flow

cytometry analysis. The fold change in fluorescence of DQ-Red BSA

was quantified as the means 6 s.d. (from three independent

experiments). Asterisks indicate statistical significance determined

by Student’s paired t-test with a two-tailed distribution; **P#0.05;

**P#0.005.

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the atg14l-specific shRNAmir were reconstituted with GST-fused

Atg14L WT, mSnapin or mBeclin 1 transgene, each carrying silent

mutations at the target sequence to confer resistance to the atg14l-

specific shRNAmir. Introduction of atg14l-specific shRNAmir-

resistant GST–Atg14L WT or GST–Atg14L mBeclin 1 mutant, but

not the GST–Atg14 mSnapin mutant, restored efficient EGFR

degradation kinetics (Fig. 6B), highlighting the importance of

Atg14L–Snapin binding in the endocytic trafficking of EGFR.

Given that three regions of Atg14L (aa71–106, aa270–320 and

aa419–492) were individually sufficient for Snapin binding, we

next examined whether the expression of any of these regions

disrupted the Atg14L interaction with Snapin and thus exerted a

dominant-negative effect on EGFR endocytic trafficking. Indeed,

expression of the GST-fused Atg14L (419–492) truncation mutant

suppressed the Atg14L–Snapin interaction, thereby causing a

significant delay in EGFR degradation without affecting

autophagic flux (supplementary material Fig. S5A,B; Fig. 6C).

These results provide compelling evidence that Atg14L

participates in endolysosomal trafficking through its interaction

with Snapin.

DiscussionIn this report, we demonstrate that Beclin 1-binding autophagy

protein Atg14L binds to SNARE-associated protein Snapin to

promote endosomal maturation in mammalian cells. This finding

is surprising given that Atg14(L) in both yeast and mammals has

been primarily regarded as a specificity factor that enables the

PI3KC3 complex to function in autophagy induction. There are

indeed two distinct subsets of PI3KC3 complexes in yeast: the

Vps38-containing PI3KC3 complex acts in vacuolar protein

sorting, a phenomenon equivalent to late endosomal fusion with

lysosomes in mammals, whereas the Atg14-containing PI3KC3

complex targets the complex to the pre-autophagosomal structure

(Klionsky et al., 2008; Kihara et al., 2001). Likewise, recent

studies have established mammalian Atg14L as a predominantly

positive regulator of the PI3KC3 complex via its coiled-coil

domain-mediated interaction with Beclin 1, highlighting the

importance of Atg14L in the early stage of mammalian

autophagy (Itakura et al., 2008; Matsunaga et al., 2009; Sun

et al., 2008; Zhong et al., 2009). Further studies have elaborated

the underlying mechanisms of Atg14L-dependent autophagosome

formation: the N-terminal cysteine repeats of Atg14L direct the

PI3KC3 complex to produce PI3P at local ER membranes and

presumably cradle the precursor membranes of autophagosomes

(Matsunaga et al., 2010). Its C-terminal BATS domain then binds

to PI3P at the sprouting membranes to sense and/or stabilize the

membrane curvature (Fan et al., 2011; Lu et al., 2011). These

studies comprehensively demonstrate the vital roles of Atg14L in

the early stage of autophagosome biogenesis. Our results have

revealed an additional function of Atg14L in endosomal

Fig. 6. Atg14L facilitates endolysosomal trafficking

through its interaction with Snapin. (A) Overexpressing

Snapin in atg14l knockdown cells fails to promote EGFR

degradation. HeLa cells stably expressing atg14l-specific

or non-silencing control shRNAmir were transiently

transfected with Snapin–AU1 or control vector. At 48 h

post-transfection, cells were treated with EGF (200 ng/ml)

for the indicated time periods and WCEs were subjected to

IB with anti-EGFR, anti-AU1, anti-Atg14L or anti-b-Actin

antibody. (B) Complementing atg14l knockdown cells

with Atg14L WT but not mSnapin rescues retardation in

EGFR degradation. HeLa cells stably expressing atg14l-

specific or non-silencing control shRNAmir were

transiently transfected with Atg14L–GST full-length,

D419–480 (mSnapin), and D71–480 (mBeclin 1) resistant

to pGIPZ lentiviral shRNAmir together with control

vector. At 48 h post-transfection, cells were treated with

EGF (200 ng/ml) for the indicated time periods and WCEs

were subjected to IB with anti-EGFR, anti-Atg14L, or anti-

b-Actin antibody. Asterisk indicates a non-specific band

above the endogenous Atg14L. (C) Expression of Atg14L

(419–492) truncation mutant induces EGFR degradation.

At 48 h post-transfection with GST-fused Atg14L (419–

492) truncation mutant or control vector, HeLa cells were

treated with EGF (200 ng/ml) for the indicated time

periods and WCEs were subjected to IB with anti-EGFR,

anti-GST or anti-b-Actin antibody.

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maturation in concert with Snapin in mammalian cells. We foundthat although the Atg14L–Snapin interaction is not required for the

internalization of endocytic cargos, it promotes their endomembranetrafficking and lysosome-dependent degradation. Abrogation ofAtg14L–Snapin binding led to significant retardation of stimulation-induced EGFR degradation, underscoring the importance of this

interaction in the late stage of the endolysosomal pathway. Incontrast, EGFR degradation kinetics were not altered whensilencing other autophagy essential gene atg5, indicating that

endolysosomal defects observed in atg14l knockdown cells is aspecific phenotype of Atg14L rather than a general effect arisingfrom global autophagic failure. These results indicate that Atg14L

is a multivalent trafficking effector that regulates not onlyautophagosomal formation, but also late endosomal maturation,which may concurrently facilitate the transport of autophagic andendocytic cargos to digestive compartments.

Our findings provide compelling evidence that the functionof Atg14L is not exclusively confined to the regulation ofautophagosome formation but extends to the modulation

of dynamic membrane trafficking at the late stage of theendolysosomal pathway. Indeed, the multivalent activity ofmammalian Vps34 regulators in membrane trafficking has been

well exemplified by UVRAG, a putative mammalian homolog ofyeast Vps38 (Itakura et al., 2008). Our previous studies havedemonstrated that UVRAG not only interacts with Beclin 1 topromote PI3KC3 lipid kinase activity for autophagosome formation,

but also targets the HOPS complex and activates Rab7 GTPaseactivity to trigger autophagosomal or endosomal fusion withlysosomes (Liang et al., 2006; Liang et al., 2008). These findings

collectively imply that, unlike their yeast counterparts, mammalianVps34 regulators, Atg14L and UVRAG, are bestowed withpleiotropic functions owing to the complexity of endolysosomal

systems in higher eukaryotes. Exploring specific spatiotemporalcues for Atg14L- versus UVRAG-mediated membrane traffickingin the endocytic and autophagic pathways would provide a better

understanding of their seemingly parallel, yet specific actions.

Intriguingly, unlike the UVRAG–HOPS interaction, theAtg14L–Snapin binding had no pronounced impact onautophagy (Fig. 4). This appears quiet counterintuitive,

considering the primary role of Atg14L per se in autophagy aswell as the observed effect of Snapin on autophagosomematuration (Fig. 2). It is possible that Snapin is involved in

autophagosome formation as a SNARE component based on therecent findings delineating the role of SNAREs in autophagosomebiogenesis (Moreau et al., 2011; Nair et al., 2011). However, we

found that disrupting Atg14L–Snapin interaction by the Atg14L(419–492) truncation mutant led to a dominant-negative effecton EGFR degradation without affecting autophagy (Fig. 6C;supplementary material Fig. S5B). Moreover, a recent study (Cai

et al., 2010) has shown the roles of Snapin in the regulation of lateendosomal trafficking and subsequent lysosome maturation viainteraction with Dynein intermediate chain (DIC). Specifically,

Snapin contributes to lysosome maturation by recruiting lysosomalhydrolases-containing late endosomes to the dynein motorcomplex for their trafficking along microtubules, thereby

bringing late endosomes and immature lysosomes into sufficientproximity to allow highly efficient fusion events. Interestingly, thisstudy also shows that autolysosome formation is intact under

Snapin-depleted conditions while cargo clearance is severelydiminished, indicating that Snapin is involved in autophagypathway by ensuring lysosome maturation for the final degradation

of autophagic cargo. Importantly, the study shows that Snapin

interaction with the DIC affects the ‘migration’ of late endosomes

along microtubules rather than the ‘fusion’ of late endosomes

or autophagosomes with lysosomes. However, since atg14l

expression showed no effect on the Snapin–DIC interaction

(supplementary material Fig. S6A). On the other hand, the role of

Snapin in SNARE-mediated membrane fusion suggests that the

Atg14L–Snapin interaction may regulate endosomal fusions

by targeting the SNARE machinery. Indeed, Snapin has been

shown to associate with the LAMP1-positive late endosomal

compartments and interact with the late endosomal SNARE

proteins Syntaxin 8, Vti1b and VAMP8 (Lu et al., 2009). Likewise,

we found that Atg14L was colocalized with Syntaxin 8 and Vti1b

(supplementary material Fig. S6B). Hence, it is tempting to

speculate that Atg14L regulates endolysosomal trafficking, such as

homotypic fusion between late endosomes or heterotypic fusion

with lysosomes, by targeting Snapin together with late endosomal

SNARE complex. Finally, given the roles of PI3P in several

endosomal functions (Lindmo and Stenmark, 2006), Atg14L–

Snapin binding may rely on coincident recognition of PI3P. Our

mapping study showed that Snapin interaction requires the C-

terminal residues 419–480 of Atg14L (Fig. 1), largely overlapping

with the BATS domain (residues 413–492) that preferentially

binds to PI3P on highly curved membranes (Fan et al., 2011).

Moreover, Snapin has also been reported to directly interact with

PI3P (Tiwari et al., 2009). Therefore, it is possible that Atg14L and

Snapin bind to PI3P and utilize these lipid moieties to trigger

endosomal maturation. Yet, further studies are necessary to

elaborate the molecular mechanisms of Atg14L-mediated

endolysosomal trafficking.

In summary, our data indicate that Atg14L targets two

independent molecular machineries, the PI3KC3–Beclin 1

complex and the SNARE-associated Snapin, to accomplish its

dual roles in autophagosome formation and endosome maturation,

respectively. Our study suggests that Atg14L functions as a

multifaceted trafficking effector that orchestrates the autophagic

and endocytic pathways depending on its binding partners,

which allows proper coordination of these two distinct, yet

interconnected trafficking processes.

Materials and MethodsCell culture

HEK293T, HeLa and mouse embryonic fibroblast (MEF) cells were cultured inDulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetalbovine serum and 1% penicillin–streptomycin (Invitrogen). Snapin WT and KOMEFs were generously provided by Dr Zu-Hang Sheng (National Institutes ofHealth, MD) and immortalized with LXSN-E6/E7 retroviral vector containinghuman papilloma virus 16 E6 and E7 oncogenes using a standard protocol ofselection using 200 mg/ml of neomycin (Sigma). Transient transfections wereperformed with Lipofectamine 2000TM (Invitrogen) or calcium phosphate(Clontech) following the manufacturer’s instructions. HeLa and MEF stable celllines were established by transfection followed by selection using 2 mg/ml ofpuromycin (Invitrogen). Mouse embryonic stem cells were graciously provided byDr Tamotsu Yoshimori (Osaka University, Japan) and cultured in DMEMsupplemented with 20% fetal bovine serum (Invitrogen), 1% penicillin–streptomycin (Invitrogen), 2 mM L-glutamine (Invitrogen), 0.1 mM non-essential amino acids (Invitrogen), 1 mM 2-mercapthoethanol (Invitrogen), and1000 units/ml of Leukemia Inhibitory Factor (Sarkar et al., 2007) (Millipore) onplastic plates coated with 0.1% gelatin (Millipore). Mouse ES stable cell lines wereestablished by electroporation followed by selection using 2 mg/ml of puromycin.

Plasmid construction

Full-length cDNA corresponding to the coding sequence of human atg14l genewas purchased from Open Biosystems and subcloned into the pGBKT7 vectorbetween NdeI and SalI restriction sites to serve as bait in yeast two-hybridscreening. All plasmids for transient and stable expression in mammalian cells

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were derived from the pEF-IRES-puro vector encoding C-terminal FLAG or AU1epitope tag or the pEBG GST fusion vector. Full-length and truncation mutant cDNAfragments corresponding to the coding sequence of atg14l gene were amplified fromthe template cDNA by polymerase chain reaction (PCR) and subcloned into the pEF-IRES-puro-FLAG vector between EcoRI and XbaI restriction sites or the pEBG vectorbetween BamHI and NotI restriction sites. Because the atg14l gene contains a BamHIrestriction site in its open reading frame, the PCR amplicon was digested with BglIIthat generates different but compatible overhangs with BamHI. Atg14L deletionmutants were generated by two-step PCR-directed mutagenesis. Full-length cDNAfragment corresponding to the coding sequence of the snapin gene was purchasedfrom Open Biosystems and subcloned into the pEF-IRES-puro vector containingFLAG or AU1 epitope tag between AflII and XbaI restriction sites. pEGFP-LC3plasmid was kindly provided by Dr Tamotsu Yoshimori (Osaka University, Japan),and ptfLC3 plasmid was purchased from Addgene. pEGFP-Atg14L plasmid wasgenerously provided by Qing Zhong (University of California, Berkeley, CA), andAtg14 mutants (D419–480) and (419–492) were subcloned into the pEGFP-C2 vectorbetween restriction sites BglII and EcoRI. Snapin was subcloned into pmCherry-C1vector between restriction sites BglII and EcoRI.

Gene silencing

For siRNA-mediated gene silencing, HeLa cells were transfected with stealthRNAiTM siRNA duplex specific for human Atg14L (59-CCACUGCAUACCCU-CAGGAAUCUAA-39) and non-specific scrambled siRNA (Invitrogen) usingLipofectamine RNAiMAXTM (Invitrogen) according to the protocol provided bythe manufacturer. At 48–72 h post-transfection, gene silencing was confirmed byimmunoblotting and the cells were analyzed for EGF endocytosis and DQ-RedBSA dequenching assays. For shRNAmir-mediated gene silencing, HeLa cellswere transduced with pGIPZ lentiviral shRNAmir specific for human Atg14L (59-CAGCATGTAAATTTAGATCAA-39), Snapin (59-CCACAGAACTGTGCCG-CATAA-39), Atg5 (59-CTTGGAACATCACAGTACA-39) or non-silencingshRNAmir control (Open Biosystems) for 72 h following the manufacturer’sinstruction. The stable transfectants were selected with 2 mg/ml of puromycin.Atg14L–GST full-length, D419–480 (mSnapin), and D71–480 (mBeclin 1)resistant to pGIPZ lentiviral shRNAmir-mediated gene silencing were generatedby two-step PCR-directed mutagenesis using the following mutagenic primer (theshRNAmir targeting sequence is capitalized): 59-gtttttctcagCACGTGAACCTGG-ACCAGttacaaccac-39.

Yeast two-hybrid screen

The human leukocyte MatchmakerTM cDNA library (Clontech) was screened usingfull-length human Atg14L as bait. Yeast transformation was performed usingMatchmaker GAL4 Two-Hybrid System 3 (Clontech) according to the protocolprovided by the manufacturer.

Immunoprecipitation and in vivo GST pulldown

HEK293T cells were harvested and lysed in NP40 buffer [50 mM Tris-HCl(pH 8.0), 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40] supplemented with acomplete protease inhibitor cocktail (Roche). The whole-cell extracts were pre-clearing with Sepharose beads for 2 h at 4 C. For immunoprecipitation, the whole-cell extracts were incubated with 1–2 mg of the indicated antibodies at 4 C for 8–12 h. After adding protein A/G agarose beads, incubation was continued for anadditional 2 h. For in vivo GST pulldown, the whole-cell extracts were incubatedwith 50% slurry of glutathione-conjugated Sepharose beads (GE Healthcare) for2 h at 4 C. The precipitates were then extensively washed with lysis buffer andeluted by boiling with Laemmli sample buffer (Sigma) for 5 min.

Immunoblotting

Polypeptides were resolved by SDS-PAGE (SDS-PAGE) and transferred onto aPVDF membrane (Bio-Rad). Immunodetection was achieved with anti-FLAG(Sigma), anti-GST (Santa Cruz Biotech), anti-AU1 (Covance), anti-V5 (Invitrogen),anti-b-Actin (Santa Cruz Biotech), anti-Atg14L (Sigma and homemade), anti-Snapin (Synaptic Systems), anti-Beclin 1 (BD Biosciences), anti-LC3 (Cosmo Bio),anti-p62 (MBL), anti-EGFR (Millipore), anti-Atg5 (MBL), or anti-DIC (Millipore)antibody for 8–12 h at 4 C. To produce Atg14L polyclonal antibody, the peptide(APGCGPRPLARDLVDSVDDAEGLYVAVERCP) was immunized to rabbits andthe antiserum from rabbits was subjected to affinity purification. The membrane waswashed three times with PBS-T and incubated with the appropriate secondaryantibody for 1 hr at room temperature. The proteins were visualized bychemiluminescence reagent (Denville) and detected by Fuji Phosphor Imager.

Confocal immunofluorescence microscopy

HeLa cells were fixed with 4% (w/v) paraformaldehyde (PFA) in phosphatebuffered saline (PBS) for 30 min, permeabilized with 0.2% (v/v) Triton X-100 inPBS for 10 min, and blocked with 10% goat serum (Invitrogen) in PBS for 1 h.Primary antibody staining was performed using anti-FLAG (Sigma), anti-GFP(Santa Cruz Biotech), anti-LAMP1 (Abcam), anti-CD63 (Santa Cruz Biotech),anti-EEA1 (BD Biosciences), anti-Rab5 (Santa Cruz Biotech), Syntaxin 8 (BD

Biosciences) or Vti1b (BD Biosciences) antibody diluted in PBS containing 1% goatserum for 1–2 h at room temperature. The cells were washed three times with PBS andincubated with the appropriate secondary antibody diluted in PBS containing 1% goatserum for 1 h at room temperature. The cells were then washed three times with PBSand nucleus was stained using Hoechst 33342 (Invitrogen). After slide mounting usingfluorescent mounting medium (Dako), the confocal images were acquired using aNikon Eclipse C1 laser-scanning microscope (Nikon Instruments Inc.) fitted with a1006Nikon objective and Nikon Element image software.

Electron microscopy

For transmission electron microscopy (TEM) microscopy, the standard procedurewas applied. Briefly, the cells were fixed by KK and osmium tetroxide solutions,stained uranium salt, dehydrated, and infiltrated with Epon plastic (TED PELLA,Redding, CA). The samples were then embedded in Epon blocks and polymerizedovernight. After ultra thin sections were prepared (MT6000, Servall Instrument),samples were picked up on grids, followed by image analysis using JEOL JEM 2100electron microscope.

Autophagy analyses

For autophagy induction, cells were washed two times with PBS and incubated withrapamycin (Sigma) at a final concentration of 2 mM in serum-free DMEM (and 1000units/ml of LIF in the case of mouse ES cells) for 2–8 h (3 h in general). Forinhibition of autophagosome maturation, cells were incubated with bafilomycin A1(Sigma) at a final concentration of 0.1 mM in complete medium (containing 1000units/ml of LIF in the case of mouse ES cells) for 1 h. Autophagy was assessed bymonitoring redistribution of GFP–LC3 or tfLC3, LC3 conversion, and p62degradation. For GFP–LC3 redistribution, MEFs were transfected with GFP–LC3plasmid. At 16 h post–transfection, GFP–LC3 was detected under normal conditionsand in the presence of rapamycin using an inverted fluorescence microscope. Thepercentage of GFP–LC3-positive cells with puncta staining was determined in threeindependent experiments. To quantify GFP–LC3-positive autophagosomes, sixrandom fields representing 200 cells were counted. For tfLC3 redistribution, mouseES cells were electroporated with ptfLC3 plasmid. After treatment with rapamycinfor the indicated time points at 24 h post-electroporation, the cells were fixed with4% PFA and processed for confocal microscopy analysis. To monitor LC3conversion or p62 degradation, cells were treated on ice for 30 min, lysed with 1%Triton X-100 buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 1%Triton X-100) supplemented with a complete protease inhibitor cocktail (Roche),and subjected to immunoblot analysis with anti-LC or anti-p62 antibody.

EGF endocytosis and EGFR degradation assays

For EGF uptake, cells were washed twice with PBS and starved in serum-freeDMEM for 2 h at 37 C. The cells were then washed with ice-cold PBS and incubatedon ice in uptake medium (20 mM HEPES and 2% BSA in DMEM) containing 5 mg/ml of EGF complexed to Alexa-Fluor-488–streptavidin (Invitrogen). Afterincubation on ice for 1 h, the cells were washed three times with ice-cold PBS toremove unbound ligand. The cells from one well were fixed with 4% PFA to give thetotal amount of bound ligand and the remaining wells were transferred to a 37 Cincubator. At each indicated time point, the cells were fixed with 4% PFA andprocessed for confocal microscopy analysis.

For EGFR degradation assay, cells were cultured to ,80% confluency, washedtwice with PBS and starved in serum-free DMEM for 2 h at 37 C. EGFRendocytosis was stimulated by adding 200 ng/ml of EGF (Sigma) in DMEMcontaining 20 mM HEPES and 0.2% BSA. At each time point after EGF stimulation,the cells were harvested and the amount of protein in each sample was measuredusing the BCA protein assay kit (Pierce) following the manufacturer’s protocol.Equal amount of the extracts was subjected to immunoblot analysis to quantify thetotal EGFR protein level.

BSA dequenching assay

HeLa cells were incubated with 20 mg/ml of DQTM-Red BSA (Invitrogen) incomplete DMEM at 37 C for the indicated time periods (Vazquez and Colombo,2009). After washing twice with ice-cold PBS to remove excess probe, the cells wereharvested and resuspended in 4% PFA. Red-fluorescent DQ-Red BSA was thenanalyzed by flow cytometry using FACSCanto II (BD Biosciences) and FlowJosoftware (Treestar, Inc.).

Statistics

Statistical analyses were performed using Student’s paired t-test with a two-taileddistribution. Values are expressed as mean 6 s.d. of at least three independentexperiments, unless otherwise noted. A P-value of #0.05 was consideredstatistically significant.

AcknowledgementsWe thank Stacy Lee for the manuscript preparation and all of themembers of the J.U.J. laboratory for their support and discussion.

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FundingThis work was partly supported by the Hastings Foundation (grantnumber 22-2107-1000 to J.U.J.), the Fletcher Jones Foundation[grant number 22-2101-1003 to J.U.J.], the Global ReaserchLaboratory Program from National Research Foundation of Korea(grant number K20815000001 to JUJ), and National Institute Health[grant numbers CA82057, CA31363, DE019085, AI073099, andAI083025 to J.U.J; CA140964 and AI083841 to C.L.], and AmericanCancer Society (grant number RGS-11-121-01-CCG to C.L.).Deposited in PMC for release after 12 month.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.100339/-/DC1

ReferencesBuxton, P., Zhang, X. M., Walsh, B., Sriratana, A., Schenberg, I., Manickam,

E. and Rowe, T. (2003). Identification and characterization of Snapin as aubiquitously expressed SNARE-binding protein that interacts with SNAP23 in non-neuronal cells. Biochem. J. 375, 433-440.

Cai, Q., Lu, L., Tian, J. H., Zhu, Y. B., Qiao, H. and Sheng, Z. H. (2010). Snapin-regulated late endosomal transport is critical for efficient autophagy-lysosomalfunction in neurons. Neuron 68, 73-86.

Chen, Y. and Klionsky, D. J. (2011). The regulation of autophagy – unansweredquestions. J. Cell Sci. 124, 161-170.

Fan, W., Nassiri, A. and Zhong, Q. (2011). Autophagosome targeting and membranecurvature sensing by Barkor/Atg14(L). Proc. Natl. Acad. Sci. USA 108, 7769-7774.

Gromley, A., Yeaman, C., Rosa, J., Redick, S., Chen, C. T., Mirabelle, S., Guha, M.,Sillibourne, J. and Doxsey, S. J. (2005). Centriolin anchoring of exocyst andSNARE complexes at the midbody is required for secretory-vesicle-mediatedabscission. Cell 123, 75-87.

Ilardi, J. M., Mochida, S. and Sheng, Z. H. (1999). Snapin: a SNARE-associatedprotein implicated in synaptic transmission. Nat. Neurosci. 2, 119-124.

Itakura, E., Kishi, C., Inoue, K. and Mizushima, N. (2008). Beclin 1 forms twodistinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 andUVRAG. Mol. Biol. Cell 19, 5360-5372.

Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T.,

Kominami, E., Ohsumi, Y. and Yoshimori, T. (2000). LC3, a mammalianhomologue of yeast Apg8p, is localized in autophagosome membranes afterprocessing. EMBO J. 19, 5720-5728.

Kihara, A., Noda, T., Ishihara, N. and Ohsumi, Y. (2001). Two distinct Vps34phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidaseY sorting in Saccharomyces cerevisiae. J. Cell Biol. 152, 519-530.

Kimura, S., Noda, T. and Yoshimori, T. (2007). Dissection of the autophagosomematuration process by a novel reporter protein, tandem fluorescent-tagged LC3.Autophagy 3, 452-460.

Klionsky, D. J. (2005). The molecular machinery of autophagy: unanswered questions.J. Cell Sci. 118, 7-18.

Klionsky, D. J., Abeliovich, H., Agostinis, P., Agrawal, D. K., Aliev, G., Askew,

D. S., Baba, M., Baehrecke, E. H., Bahr, B. A., Ballabio, A. et al. (2008).Guidelines for the use and interpretation of assays for monitoring autophagy in highereukaryotes. Autophagy 4, 151-175.

Liang, C., Feng, P., Ku, B., Dotan, I., Canaani, D., Oh, B. H. and Jung, J. U. (2006).Autophagic and tumour suppressor activity of a novel Beclin1-binding proteinUVRAG. Nat. Cell Biol. 8, 688-698.

Liang, C., Lee, J. S., Inn, K. S., Gack, M. U., Li, Q., Roberts, E. A., Vergne, I.,

Deretic, V., Feng, P., Akazawa, C. et al. (2008). Beclin1-binding UVRAG targets

the class C Vps complex to coordinate autophagosome maturation and endocytictrafficking. Nat. Cell Biol. 10, 776-787.

Lindmo, K. and Stenmark, H. (2006). Regulation of membrane traffic byphosphoinositide 3-kinases. J. Cell Sci. 119, 605-614.

Lu, L., Cai, Q., Tian, J. H. and Sheng, Z. H. (2009). Snapin associates with lateendocytic compartments and interacts with late endosomal SNAREs. Biosci. Rep. 29,261-269.

Lu, Q., Yang, P., Huang, X., Hu, W., Guo, B., Wu, F., Lin, L., Kovacs, A. L., Yu,

L. and Zhang, H. (2011). The WD40 repeat PtdIns(3)P-binding protein EPG-6regulates progression of omegasomes to autophagosomes. Dev. Cell 21, 343-357.

Matsunaga, K., Saitoh, T., Tabata, K., Omori, H., Satoh, T., Kurotori, N., Maejima,

I., Shirahama-Noda, K., Ichimura, T., Isobe, T. et al. (2009). Two Beclin 1-bindingproteins, Atg14L and Rubicon, reciprocally regulate autophagy at different stages.Nat. Cell Biol. 11, 385-396.

Matsunaga, K., Morita, E., Saitoh, T., Akira, S., Ktistakis, N. T., Izumi, T., Noda,

T. and Yoshimori, T. (2010). Autophagy requires endoplasmic reticulum targeting ofthe PI3-kinase complex via Atg14L. J. Cell Biol. 190, 511-521.

Moreau, K., Ravikumar, B., Renna, M., Puri, C. and Rubinsztein, D. C. (2011).Autophagosome precursor maturation requires homotypic fusion. Cell 146, 303-317.

Nair, U., Jotwani, A., Geng, J., Gammoh, N., Richerson, D., Yen, W. L., Griffith, J.,

Nag, S., Wang, K., Moss, T. et al. (2011). SNARE proteins are required formacroautophagy. Cell 146, 290-302.

Nickerson, D. P., Brett, C. L. and Merz, A. J. (2009). Vps-C complexes: gatekeepersof endolysosomal traffic. Curr. Opin. Cell Biol. 21, 543-551.

Orsi, A., Polson, H. E. and Tooze, S. A. (2010). Membrane trafficking events thatpartake in autophagy. Curr. Opin. Cell Biol. 22, 150-156.

Pan, P. Y., Tian, J. H. and Sheng, Z. H. (2009). Snapin facilitates the synchronizationof synaptic vesicle fusion. Neuron 61, 412-424.

Sarkar, S., Davies, J. E., Huang, Z., Tunnacliffe, A. and Rubinsztein, D. C. (2007).Trehalose, a novel mTOR-independent autophagy enhancer, accelerates the clearanceof mutant huntingtin and alpha-synuclein. J. Biol. Chem. 282, 5641-5652.

Sato, T. K., Rehling, P., Peterson, M. R. and Emr, S. D. (2000). Class C Vps proteincomplex regulates vacuolar SNARE pairing and is required for vesicle docking/fusion. Mol. Cell 6, 661-671.

Simonsen, A. and Tooze, S. A. (2009). Coordination of membrane events duringautophagy by multiple class III PI3-kinase complexes. J. Cell Biol. 186, 773-782.

Song, W. J., Seshadri, M., Ashraf, U., Mdluli, T., Mondal, P., Keil, M., Azevedo, M.,

Kirschner, L. S., Stratakis, C. A. and Hussain, M. A. (2011). Snapin mediatesincretin action and augments glucose-dependent insulin secretion. Cell Metab. 13,308-319.

Stroupe, C., Collins, K. M., Fratti, R. A. and Wickner, W. (2006). Purification ofactive HOPS complex reveals its affinities for phosphoinositides and the SNAREVam7p. EMBO J. 25, 1579-1589.

Sun, Q., Fan, W., Chen, K., Ding, X., Chen, S. and Zhong, Q. (2008). Identification ofBarkor as a mammalian autophagy-specific factor for Beclin 1 and class IIIphosphatidylinositol 3-kinase. Proc. Natl. Acad. Sci. USA 105, 19211-19216.

Tiwari, S., Choi, H. P., Matsuzawa, T., Pypaert, M. and MacMicking, J. D. (2009).Targeting of the GTPase Irgm1 to the phagosomal membrane via PtdIns(3,4)P(2) andPtdIns(3,4,5)P(3) promotes immunity to mycobacteria. Nat. Immunol. 10, 907-917.

Vazquez, C. L. and Colombo, M. I. (2009). Assays to assess autophagy induction andfusion of autophagic vacuoles with a degradative compartment, using monodansyl-cadaverine (MDC) and DQ-BSA. Methods Enzymol. 452, 85-95.

Wurmser, A. E., Sato, T. K. and Emr, S. D. (2000). New component of the vacuolarclass C-Vps complex couples nucleotide exchange on the Ypt7 GTPase to SNARE-dependent docking and fusion. J. Cell Biol. 151, 551-562.

Zhong, Y., Wang, Q. J., Li, X., Yan, Y., Backer, J. M., Chait, B. T., Heintz, N. and

Yue, Z. (2009). Distinct regulation of autophagic activity by Atg14L and Rubiconassociated with Beclin 1-phosphatidylinositol-3-kinase complex. Nat. Cell Biol. 11,468-476.

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