hsatcei ato ou nAtpaooeboeei.For biogenesis. Autophagosome on Focus a of part is article This asl ria-ean ta.( al. et Proikas-Cezanne Tassula PtdIns3 essential [email protected]) ([email protected]; correspondence for *Authors Norway. Oslo, NO-0317 vnR Carlsson R. Sven biogenesis autophagosome in dynamics Membrane COMMENTARY ß uohgsm ihlssmslast erdto fthe products of degradation 1 of degradation recycling the to the and leads of material lysosomes sequestered Fusion with a a (autophagosome). into of autophagosome cytoplasm the vesicle nucleation engulf called to the double-membraned expands also with which phagophore, membrane), initiates (Stolz isolation (the dysfunctional pathway pathogens structure The and or double-membraned aggregates 2014). damaged protein al., of as et well removal of is as selective organelles, control it sequestration quality by essential but an random plays function autophagosomes, also autophagy forming that involving evident now into non-selective a process, material be to cytoplasmic survival considered time a conditions. long cell in pathophysiological a implicated for and was been physiological Autophagy has of best and set the autophagy diverse is of autophagy, form as characterized to referred hereafter Macroautophagy, Introduction PtdIns3 Atg, WORDS: KEY for sites Sanchez- ‘ERES: ( Jana al. articles: by et maturation?’ related Wandelmer and see biogenesis please autophagosome reading, further pathway. autophagy the of steps different at events required remodeling are and that shaping membrane the in of progress understanding recent the our review we Commentary, this In size. and shape autophagosome specific of membrane regulation the for responsible the are about that and events known membranes modeling autophagic is the little constitute but that autophagy, lipids for found essential been have be proteins of to number large A material. captured the of fusion degradation to for leading compartments, lysosomal primed bilayered and compartment, endosomal then with separate double is autophagosome a The growing autophagosome. creates the closure a upon in by which differ membrane, enclosed cytoplasmic can macroautophagy, become that During localities components operation. yielding functional cell, and eukaryotic complex composition the the This of for encapsulated. prerequisite design be a to spaces is compartmentalization defined membranes polarity, allowing of borders principles fundamental create on Based cell. the of living organization the to vital are membranes phospholipid Bilayered ABSTRACT 0 7Umea 87 901 eateto eia iceityadBohsc,Uiest fUmea of University Biophysics, and Biochemistry Medical of Department 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,1325doi:10.1242/jcs.141036 193–205 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. ˚ Sweden. , P .Cl Sci. Cell J. 2 fetr ttensetatpaooe by autophagosome’ nascent the at effectors nttt fBscMdclSine,Uiest fOslo, of University Sciences, Medical Basic of Institute 1, n neSimonsen Anne and * P uohgsm,Phagophore Autophagosome, , 128 .Cl Sci. Cell J. 8-9)ad‘IIproteins: ‘WIPI and 185-192) , 128 207-217). , 2, * ˚ SE- , idn fetrpoen n rti iae n phosphatases. modifying and these kinases lipid- lipid protein and the proteins, by and remodeling 2) proteins on regulated effector and Box tightly binding principles sculpting be see membrane general to enzymes, curvature, (for appear membrane processes pathway of steps autophagy several generation at the required are of remodeling and proteins. shaping RAB and (NSF) Membrane proteins (SNARE) factor receptor N-ethylmaleimide-sensitive protein attachment as soluble such act proteins, components, proteins Atg trafficking coat that membrane clear 2011). general it al., made with et has together (Klionsky field the 1) in Box and progress (see non-selective Recent autophagy the both of for types essential selective be to found and identified Lamb 1). 2013; (Fig. Codogno, 2013) al., and et (Klionsky macromolecules new into e abe l,21;Mzsiae l,21 n o 1. Box selective and 2011 al., autophagy, in et of of Mizushima 2013; hierarchy induction al., occurring upon complex et regulation Lamb the their see membrane events of and proteins reviews the Atg detailed modeling discuss For autophagy. briefly and we dynamics functional Finally, the of curvature autophagosome. and to composition reactions membrane fusion the and obtain tethering our including in closure, and in expansion events 3-phosphate (PtdIns) nucleation progress phosphatidylinositol modeling the of (PtdIns3 role in membrane recent the involved and those process early as on such biogenesis, the focus phagophore of will understanding Commentary This ulaea nitiaemmrnu tutr,tre the omega originally labeled spots letter were (ER)-associated termed reticulum Greek Omegasomes endoplasmic the as pictures). structure, identified to microscopy membranous resemblance electron its in intricate to (due phagophores an is starvation, omegasome it upon at but least cell, at nucleate the that, in phagophore accepted (PAS) locations eukaryotes, largely different now higher structure at occur In might pre-autophagosomal 2010). nucleation near Ohsumi, the origin and single called (Suzuki a from al., vacuole, originate phagophore et to the the Lamb found yeast, been 2013b; In has al., 2014). membrane see Yoshimori, et reviews, and Hamasaki Shibutani recent 2014; 2013; (for Elazar, membrane and preexisting Abada a on nucleation unknown. mostly lipid still are constitutive size and the have determining shape in involved but cells phagophore proteins modeling formation, mammalian membrane and and phagophore species mechanisms yeast in the of in involved understanding Recent our 2013). proteins to (Tooze, substantially Atg contributed decades for identity precursor of debate and of membrane origin studies matter its cup-shaped a and been autophagy, small have of induction a upon is formed phagophore The membrane phagophore The phagophore the response: autophagy of early formation the in events modeling Membrane ag ubro uohg-eae Ag rtishv been have proteins (Atg) autophagy-related of number large A ti eeal gedta hgpoe r formed are phagophores that agreed generally is It P n te iis swl sltrseso phagophore of steps later as well as lipids, other and ) enovo de 193 by

Journal of Cell Science COMMENTARY Bdmn ta. 01 ege l,21;vndrVate al., et Vaart der van 194 2010; al., et Golgi Geng the 2011; 2013), ER–Golgi al., al., the et et 2010), (Bodemann sites al., (Ge et outer exit (ERGIC) Zoppino ER compartment the 2013; intermediate The al., sources. that et membrane (Graef also phagophore showing phagophore (ERES) other the the from that study clear input however, for a is, receives It lipids 2010). with al., et provides line (Hailey in membrane 2013a), mitochondrial al., (Hamasaki 1). membrane et Box ER also mitochondria-associated (see connected 2008) the 2008; al., al., et et Sun (Itakura 2001; ATG14L yeast) al., and in et yeast) Atg14 Kihara in BARKOR; (Atg6 as 1 as known beclin known (also yeast), (also in p150 Vps15 with and in PIK3R4; complex 2008) Vps34 al., autophagy-specific et an PIK3C3; (Axe class formation forms as omegasome The for known 2008). required is al., also yeast) et (PI3KC3, (Axe PtdIns3-kinase ZFYVE1) III as known also PtdIns3 (DFCP1, the by of indicated. degradation are the autolysosomes to in leading cargo pathway. steps captured autophagic remodeling the and in sculpting organelles and Membrane structures Membrane 1. Fig. FP-oiieoeaoe pert ocnrt to near or at concentrate to appear omegasomes DFCP1-positive Endosome Lysosome Autophagosome Phagophore Autolysosome Phagophore Omegasome Membrane P bnigdul-YEcnann rti 1 protein double-FYVE-containing -binding o cargo

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r Membrane closure Phagophore elongation and detachment Membrane expansion Generation ofcurvedmembrane (recruitment ofcoreautophagyfactors) Nucleation Degradation fusion Membrane od ta. 03,wihmgtalweuiaino the of elucidation allow might which 2013), in al., different involved Koyama- 2013; the et al., proteins for function-dependent et Honda (Karanasios markers formation autophagy and phagophore provided of steps time- core has nucleation the the phagophore of among but elucidation to hierarchy expansion, difficult phagophore phagophore recent to been to the contributing has those contributing membrane It and as membranes 2E). nucleation implicated (Fig. between been and phagophore all al., differentiate the 2010) have et for 2013) Longatti al., al., sources 2013; et et al., Puri (Ravikumar et 2012; (Knævelsrud membrane endosomes plasma recycling the 2010), epciey(o eetrves e aaaie l,2013b; al., et Hamasaki activities, see 2011). E3-like al., reviews, and et Mizushima recent E2 E1, (for have respectively which ATG12–ATG5–ATG16L1/Atg12– complex, of the Atg5–Atg16 action the and modified through ATG3/Atg3 When LC3-II) covalently called ATG7/Atg7, (LC3-I). is becomes form protein lipidated (the LC3/Atg8 soluble PtdEtn with triggered, a as is healthy (also exists well-nourished autophagy -L2 a mostly -L1, In LC3/Atg8 [GABARAP, subfamilies. LC3 cell, GABARAP GATE-16)] the or as to to -C) belong referred and they homologs -B2 cells; Atg8 -B, mammalian seven (LC3A, in protein, identified Atg8 been one have only 2013b; has system yeast al., conjugation Although their et and proteins Hamasaki Atg8 see 2011). reviews, al., et the recent Mizushima in PtdEtn (for to can proteins LC3/Atg8 complex membrane of conjugation the the facilitate and then which conjugate, ATG10/Atg10, ATG12–ATG5/Atg12–Atg5 to binds and Atg16L1/Atg16 the respectively. ATG7/Atg7 activities, E2-like of and E1 action ATG5/have to the conjugated through is that Atg5 protein ubiquitin-like a is ATG12/Atg12 complex ATG12–ATG5–ATG16L1 The 2013; al., negatively et 2009). Dall’Armi Tooze, bulky see and reviews, its Simonsen recent (for through group structure head charged membrane might and influence WIPIs/Atg18 as also such factors downstream recruits which otisURG(t3 nyat,wihi novdin involved is which yeast), PtdIns3 PI3KC3/Vps34 II produces complex in The I and complex endocytosis. 2008), and (Atg38 al., maturation et autophagosome Sun UVRAG 2001; al., et contains (Itakura Kihara ATG14L/Atg14 2008; subunit al., autophagy-specific et the complex cells; contains distinct yeast two I and in mammalian engage in can complexes associated proteins PI3KC3/Vps34 the 1/Atg6 beclin and and kinase p150/Vps15 PI3KC3/Vps34 active catalytically complexes The Vps34 1 (for 2013). and al., PI3KC3 (AMPK) complex et The Wirth kinase rapamycin 2010; protein Mizushima, of see AMP-activated reviews, target recent the mammalian and these tightly the (mTORC1) are of and autophagy, by similarities of induction ULK regulated overall The upon conserved. activated the is are function their kinases a and functional that the nor likely Atg17, it be Atg31, makes to complexes yeast and thought Atg29 of is yeast FIP200 homolog ATG101, of Although of orthologs ATG101. ortholog and human eukaryotes yeast FIP200 no higher ATG13, in are with Atg1 there complex of a homologs form functional and ULK1 are kinases Atg kinase) ULK2 (Unc51-like other and ULK of The release PAS. and the autophagy. recruitment from of proteins the induction in complex upon functions Atg31 a complex and This forms Atg29 Atg1 Atg17, kinase Atg13, with protein serine/threonine yeast complexes The ULK and machinery Atg1 autophagosomal The core The 1. Box ora fCl cec 21)18 9–0 doi:10.1242/jcs.141036 193–205 128, (2015) Science Cell of Journal P ttest fpaohr nucleation, phagophore of site the at

Journal of Cell Science COMMENTARY Cmeoe l,21;Kzo ta. 2014). modeling al., shape et membrane of Kozlov 2010; termed types al., two et be (Campelo The collectively remodeling. can membrane occurring transformations as those process to of as type referred This is such reactions. fission surfaces, and fusion membrane membrane during the new to leading as bilayer, of the literature of formation continuity the the sculpting; in of membrane distortions to transient or (2) referred generation is curvature alteration bending, membrane structural of bilayer. type in the This of disruption without functions changes shape (1) transformations: proposed with events. proteins modeling and 1 Table membrane-binding pathway. autophagy autophagy-related the in work lists at be to mentioned above likely 2012). the are of principles al., several detail, to et in thought (Stachowiak elucidated yet is effect not membrane crowding Although the a of by side curvature membrane-binding one generate on of density enrichment high the at proteins oligomeric Also, the scaffold. membrane the on membrane forming by or peripheral a geometry forces generated shape the certain that stabilize own create, their they effect addition, complexes on wedging depending In can, a proteins tilt. create to can phospholipids membrane core Partial hydrophobic the the kinases. into of of domains action protein PtdIns of the integration as or by insertion and such membrane, enzymes the sizes. head-group-modifying across different that flippases lipids of by are shuttle caused also sides actively be membrane two can lipids the of causes distribution on unequal groups An lipids head An the membrane 2014). if al., bending of et Kozlov distribution 2012; on Kirchhausen, asymmetric see 2013; depend reviews, al., recent to (for et thought figure) Derganc box is (see principles tubules different and several to vesicles and curvature organelles, yield to shape membranes cell of modeling the Generally, membrane action of protein generation by the curvature for Principles 2. Box ebae a neg w udmnal ifrn ye of types different fundamentally two undergo can Membranes A symmetric lipiddistribution Membrane scaffolding Membrane integration Membrane insertion Protein crowding Principle surfaces high densityonmembrane Protein enrichmentat lipid headgroups shapes associatingwith proteins withcurved Peripheral membrane proteins Integral membrane pathic helices proteins withamphi- Peripheral membrane Flippases Lipid kinases ieo hgpoenceto Ca ta. 09 auae al., et Ragusa N-terminus 2009; the in al., acids amino the positive et of to cluster recruitment (Chan a targeting/tethering Moreover, 2012). for nucleation autophagy essential phagophore be of early to appears site the it be as called might domain, This (EAT) is ortholog yeast 2A). its which (Fig. (and ULK 2013) Atg1), of al., domain C-terminal the et are by Wirth mediated ATG101] see and a partners review, (for nucleation RB1CC1) phagophore recent of ULK site complex the as at membrane mammalian associated the known to recruited (also the their FIP200 Firstly, and [ATG13, autophagy. kinases nucleation phagophore of (UNC51-like) to induction lead events we upon following studies, be biochemical the and that genetic to propose on and Based 1). imaging thought Table live-cell and/or (see recent membrane shaping membrane appropriate are mediate the to ability to that their targeting their domains for important membrane-interacting nucleation phagophore to for have crucial be to known factors the contribute of Several the nucleation be sources Phagophore on below. to discussed depending cargo as membrane expansion, the that, and/or of nucleation out different phagophore nature ruled the starvation-induced sequestered, and be undergoing signal cannot cells autophagy-inducing investigations in it most performed autophagy, as been However, have hierarchy. source membrane uhsos n h o l M1sosapa ob novdin involved be to ER appear transmembrane spots beclin-1-interacting VMP1 The all not formation. from why phagophore form and omegasomes or spots, phagophores such why understand to to required ATG core o early tempting characterization the of Further is recruitment proteins. by determines formation it that phagophore platform of date, recruitment site most a the as the to acts be trigger VMP1 identified to that to seems hypothesize factor it known membranes because upstream is and autophagosomal expression 2007) ‘temporally’ microtubule-associated al., the VMP1 of et As with (Ropolo as association 2013). autophagy 3) al., the known chain et facilitate (Molejon (also light ATG16L1 to 1 LC3 found and protein and also VMP1–beclin FIP200 The was ATG16L1 2013). al., subunit interaction et Nishimura complex 1 2013; al., ULK et interaction(Gammoh described recently the a by between mediated be of might hierarchy ULK1 functional with ATG5 the of recruitment synchronized in The complexes the formation. autophagosome PI3KC3 as and surprising ULK1 of rather the downstream placed is been has which complex 2013), ATG12–ATG5–ATG16L1 al., et (Koyama-Honda ULK1 with ER concomitant found structures VMP1-positive was to ATG5 recruited the 2A,B). be to (Fig. phagophore with the to (see recruited complex is complex 1) PI3KC3 ATG12–ATG5–ATG16L1 Box the the further might Finally, of 2013) membrane. al., association interaction et the (Molejon An 1 stabilize 2010). beclin and al., the VMP1 for et of between sites and (Matsunaga the autophagy to formation 1) in Box whichphagophore (see function subunits domain, PI3KC3 its ATG14L other N-terminal the for 2013). of recruitment essential its al., be in et to motif (Jao appears the ER-binding PAS of the an recruitment contains to for and Atg14 autophagy subunit for PI3KC3 required HORMA a is forms that Atg13 yeast domain of domain N-terminal PtdIns3 the that generate phagophorefound to to recruited is sites PI3KC3 nucleation Secondly, 2013). (Koyama- al., (VMP1) et 1 Honda protein to membrane targeted vacuole be protein to omegasomesspanning multi-membrane found ER-resident to been the ATG13 containing has structures pre-existing ULK1 of 2013). translocation al., the et for (Karanasios important and phospholipids acidic be with interact to to found been has ATG13 of ora fCl cec 21)18 9–0 doi:10.1242/jcs.141036 193–205 128, (2015) Science Cell of Journal h M1pstv tutrsis structures VMP1-positive the f P Fg A.Arcn study recent A 2A). (Fig. 195

Journal of Cell Science COMMENTARY 196 depletion the 2013). its al., of et and structures (Li geometry precursor autophagy omegasomes autophagosomal impaired unique and of to accumulation the further localizes the of might causes it generation (EMC6) as the phagophore, 6 to subunit contribute complex stric protein text. be see not r known membrane explanation, of might are be further depicted complexes to For events thought protein boxes. interactions the and protein–lipid gray that proteins and in noted of protein–protein indicated which be E, dissociation are In of should and shown. phagophore some It association are the endosomes, size. associations The of recycling membrane its reactions. only expansion and expand the clarity Golgi to for between ERGIC, the but phagophore synergy regulated, the at the indicate temporally from LC3 to studies originating of added recent tubules lipidation are as and the LC3, sequential, Vesicles in lipidated ar (E) resulting contain comple complexes (ATG3–LC3), (D). ATG12–ATG5–ATG16L1 already PI3KC3 LC3 phagophore the presumably and activated the of ULK recruits forming recruitment ATG12–ATG5–ATG16L1 The membrane by Bound (A) ph curved followed (C) 1. a ATG12–ATG5–ATG16L1. Table VMP1, denotes more see protein ‘:’ recruits interactions, transmembrane where turn membrane the brackets, in containing square involved sites PtdIns3 in domains membrane (B) protein indicated curved For are to membrane. here species recruited the described protein initially near individual interactions or as protein–membrane depicted at or proteins, occurring cells. Participating protein–protein interaction mammalian illustrated. The are in shown. expansion biogenesis are phagophore autophagosome and complexes, in LC3 involved membrane-bound events of the accumulation for the model A 2. Fig. B D C DFCP1 :PI3P A ATG14L :membranecurvature DFCP1 PI3KC3 complexI P ATG14L P3)poue ymmrn-on IK3frhrehne ebaebnigadfrsapafr o h erimn fWP2 hc in which WIPI2, of recruitment the for platform a forms and bending membrane enhances further PI3KC3 membrane-bound by produced (PI3P) beclin1 :VMP1 beclin1 ATG12 VPS34 VPS15 ATG5 LC3 ATG5 :acidicphospholipids LC3 ATG16L1 WIPI2 LC3 LC3 LC3 ATG16L1 :WIPI2 WIPI2 :PI3P WIPI2 ATG3 : ATG12 curvature ATG3 :membrane ATG3 VMP1 ATG12–ATG5–ATG16L1 LC3 ATG12 complex ATG5 ATG16L1 ULK1 ULK1 :membranecurvature ULK complex ATG13 :acidicphospholipids ATG13 ATG101 FIP200 ATG12 From recyclingendosomes SNX18 :LC3/GABARAP (SNX18 dependent) opnnst ie fpaohr ulaincnact can nucleation 2010), phagophore Mizushima, autophagy and of (Itakura core activity PtdIns3 early PI3K sites the of independently of to recruitment components initial the Although PtdIns3 of role The ATG5 SNX18 :PI(4,5) E SNX18 : ATG16L ATG12–ATG5–ATG16L1 ATG16L1 :FIP200 LC3 ATG16L1 LC3 ATG9 P complex ora fCl cec 21)18 9–0 doi:10.1242/jcs.141036 193–205 128, (2015) Science Cell of Journal LC3 rdcinapast eipratfrterstable their for important be to appears production P 2 h ifrn tp tteatpayiiito ie edn to leading sites initiation autophagy the at steps different The

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Journal of Cell Science COMMENTARY

Table 1. Autophagy proteins with membrane-modifying or membrane-modeling activity Mammalian protein Yeast ortholog Protein function Membrane-active domain Proposed or putative membrane-active role References

ULK1/2 Atg1 Ser/Thr kinase C-terminal EAT domain Membrane targeting; curvature sensing; Chan et al., 2009; Ragusa et al., vesicle tethering 2012 FIP200a Atg17 Scaffold for Atg1/ULK complexes Elongated, crescent-shaped a-helical Vesicle tethering Ragusa et al., 2012 structure ATG13 Atg13 Scaffold for Atg1/ULK complexes N-terminal cluster of positively charged Membrane targeting Karanasios et al., 2013 amino acids ATG14L Atg14b Autophagy-specific PI3KC3/Vps34 C-terminal amphipathic helix within a Curvature sensing and stabilization Fan et al., 2011 complex recruitment subunit BATS domain PI3KC3 Vps34 Phospholipid kinase Kinase domain PtdIns phosphorylation (PtdIns3P generation) Petiot, 2000; Kihara et al., 2001 WIPI1/2 Atg18 PtdIns3P effector b-propeller structure Membrane targeting and insertion Krick et al., 2012; Baskaran et al., 2012; Watanabe et al., 2012 ATG3 Atg3 E2-like enzyme for Atg8/LC3– N-terminal amphipathic helix Curvature sensing Nath et al., 2014 PtdEtn conjugation ATG5 Atg5 Part of an E3-like ligase for Atg8/ Cluster of positively charged amino Membrane targeting Romanov et al., 2012 LC3–PtdEtn conjugation acids Bif-1 –c Membrane sculpting and protein N-BAR domain Membrane curvature sculpting and stabilization Takahashi et al., 2011 scaffolding SNX18 – Membrane sculpting and protein PX-BAR superdomain Membrane curvature sculpting and stabilization Knævelsrud et al., 2013

scaffolding doi:10.1242/jcs.141036 193–205 128, (2015) Science Cell of Journal Class II PI3K – Phospholipid kinase Kinase domain PtdIns phosphorylation (PtdIns3P generation) Devereaux et al., 2013 PI4K Pik1 Phospholipid kinase Kinase domain PtdIns phosphorylation (PtdIns4P generation) Wang et al., 2012 MTMR3 and Ymr1 Phospholipid phosphatases Phosphatase domain PtdIns3P dephosphorylation Taguchi-Atarashi et al., 2010; MTMR14 (Jumpy) Vergne et al., 2009 ATG9 Atg9 Integral membrane protein with Transmembrane domain Membrane wedging? unknown function VMP1 – Integral membrane protein with Transmembrane domain Membrane wedging? unknown function LC3 family proteins Atg8 PE-conjugated membrane protein Phospholipid anchor Membrane crowding?

Orthologs for which a membrane-active function has been described or proposed are shown in bold. For some mammalian proteins no obvious yeast ortholog exists (–). aMammalian FIP200 and Atg101 are viewed as functional counterparts to the yeast Atg17–Atg29–Atg31 complex without showing any sequence similarity (Mizushima et al., 2011). bYeast Atg14 appears to lack a BATS domain.cA distantly related protein is present in Schizosaccharomyces pombe and some other fungi, but not in Saccharomyces cerevisiae (Takahashi, 2009). 197

Journal of Cell Science hs T1Lhsbe on osnemmrn curvature membrane sense to found been a a has with create create line ATG14L can In to proteins. turn membrane-active this, seen in of binding which be the membrane, for can platform the clustered, in PtdIns3 bud Koyama- when cytosol-facing upon 2013; 2013). that, al., lipid disappeared al., et membrane punctae (Karanasios et ATG16L1 activity Honda PI3K and of ATG5 inhibition almost as ULK1, growth, phagophore all further and association membrane COMMENTARY 198 PtdIns3 for role possible a to addition In reaction lipidation LC3 the The of curvature membrane initial membrane. and phagophore elongation the facilitate amphipathic an through nipratgopo PtdIns3 of ( group 2B). PROPPINs (Fig. important membrane the to An PtdIns3 proteins autophagy curvature, of recruitment phagophore the initial for important a in act PtdIns3 specific they stimulate where to membrane recruited to ER are manner the complexes in synergistic ULK 2010). spots whereby al., and model membrane to a et PI3KC3 support complex Bartolomeo strongly is the studies PI3KC3 (Di these the AMBRA1 nucleation together, Taken phagophore of of of localization phosphorylation sites proper 2013) beclin-1-related for al., ULK1-mediated et important in (Nazio that dimerization molecule ULK1 and mediates (activating 1) beclin-1-interacting autophagy the not AMBRA1 that is note It to protein interesting 2012). is similar al., it have et but ATG17 mammalian functions, (Ragusa or proteins PAS ULK the whether known at curvature tethering membrane of vesicle stabilization and the to suggesting contributes Atg17 domains, that curvature-sensing (BAR) of Bin-Amphiphysin-Rvs it reminiscent inducing that and dimer forms showed crescent-shaped which Atg29–Atg31, a Atg17, with forms yeast in complex of role Atg1-interacting structure an important crystal an the plays Furthermore, subunit complex complex ULK1 ULK PtdIns3 the the PtdIns3 that to of and bind indicating 2013), phosphorylation to al., found by was et complex was ATG13 geometry- (Russell ULK1 PI3KC3 1 a Recently, the sense beclin in 2012). activate to liposomes al., to et appears to found further (Ragusa C- binds also manner it the for Atg1/ULK1 dependent as of Interestingly, curvature, important domain membrane curvature. be EAT phagophore and/or terminal of structures to stabilization recruitment curved its facilitate might which already 2011), al., et (Fan domain h ieo C rGBRPlpdto Fjt ta. 08)(see 2008b) al., et (Fujita lipidation ATG3 GABARAP determine or enzyme to LC3 of appears E2-like site of and the The the (PtdEtn) from 2011). transfer phosphatidylethanolamine GABARAP to al., the or et LC3 mediates forms activated (Ishibashi it complex ATG5–ATG12 although autophagy, ATG12–ATG5–ATG16L1 with in of complex involved isoform not an a ATG16L2, is from that absent ATG16L1 Interestingly, is identified. site involved been WIPI2-binding has sites FIP200 the this and from ATG5 different WIPI2- to is binding A that in 2014). ATG16L1 al., in et site (Dooley binding formation phagophore of sites to 3). Box (for see Atg18 2008) proteins, in al., WIPI involved homolog et on is (Krick which details yeast pathway Atg21, yeast (Cvt) as their cytoplasm-to-vacuole well the as and 2013), al., et proteins phosphoinositide- (Bakula domain (WIPI) repeat WD interacting mammalian the including eetsuysosta II ucin orcutATG16L1 recruit to functions WIPI2 that shows study recent A P rdcinadsaiiaina h phagophore. the at stabilization and production b poelr htbn polyphosphoinositides), bind that -propellers a hlxlctdi t -emnlBATS C-terminal its in located -helix P sa netdcone-shaped inverted an is P bnigpoen r the are proteins -binding P Krnso ta. 2013), al., et (Karanasios P sacn-hpdlipid cone-shaped a as P rdcinand production P facilitates ieyepaainfrti a rvddb tde nyeast, in studies by PtdIns3 provided that was that this indicating for showing 2010), explanation likely al., A et overexpression (Taguchi-Atarashi PtdIns3 with cells MTMR3 preventing in of seen by Interestingly, were 2009). phagophores act al., autophagosomes to et smaller Vergne investigated) that 2010; not al., autophagy et (WIPI2 (Taguchi-Atarashi WIPI1 of of identified regulators recruitment were negative MTMR3 MTMR14 and as 14) Both protein 3-phosphatases. (myotubularin-related phosphoinositide by regulated PtdIns3 o ;Fg C.A mhpti -emnlhlxi T3was ATG3 in helix N-terminal amphipathic An 2C). 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Phosphorylation phagophore. Atg18 al., the et of to Atg9 (Rieter protein autophagy transmembrane and PAS PtdIns3 the with autophagy- association these how known not was PtdIns3 it specific PtdIns3 membranes As 2012). al., cellular et Watanabe 2012; al., et aulrmmrn hr trgltsvcoa opooy(Efe morphology 2007). vacuolar al., regulates et it where membrane vacuolar n eetsrcua nlssso htte aeteaiiyto ability autophagosome their 2007), the in have of PtdIns3 al., motif they two et sites that with show Proikas-Cezanne the interact analyses 2010; structural to al., recent and et recruited (Polson are formation proteins proteins WIPI WIPI The 3. Box PtdIns3 P P . 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Journal of Cell Science uvtr,freape ywdigo h ebae ti also is It membrane. the of wedging phagophore by of formation example, it the for that system in curvature, possible participates is model indirectly being it or but protein, and/or known, directly membrane not integral is methodological multi-spanning ATG9 a of to by function exact contrast explained The differences. the in could be with differences these is transiently but also 2012), al., This only et (Orsi associates phagophore 2012). forming which al., ATG9, et mammalian (Yamamoto membrane COMMENTARY 200 and of formation production the through to curvature, contribute phagophore PtdIns3 might of complexes stabilization PI3K ULK III class curvature the and the above, discussed is As of obtained? how phagophore fusion and the and of phagophore, tethering the the to in membrane involved incoming mechanisms membranes the phagophore are incoming What of fusion and Tethering phagophore. the forming facilitate the might to proteins membrane interaction homolog of the Atg8 recruitment Thus, with 2012). 2012; 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been as al., RAB11 all et However, have and Longatti 2013) 2010) 2013; Ja al., al., al., et et 2004; et Puri Knævelsrud Tabata al., 2008; 2010; al., et al., RAB5 et et (Fader (Gutierrez 2014), Pankiv RAB7 2004; al., al., 2008), et et al., (Talaber et The RAB4 (Ravikumar autophagy. proteins understand in RAB fully events to endosomal needed fusion are been membrane studies also further RAB33B-mediated (Itoh has Thus, lysosomes 2011). with but al., autophagosomes 2008), et of and al., fusion the et ATG16L1 in (Itoh implicated with lipidation interacts The LC3 manner. modulates RAB33B TRAPP-dependent likely a protein is autophagosome in Golgi-resident it phagophore in 2010) growing the membrane al., provide implicated to et also in vesicles Zoppino COPII-coated been 2013; involved ERES-derived that al., have ER et are functional (Graef (ERES) and complexes biogenesis RAB1 sites as TRAPP but exit cells, whether mammalian It in known 2013). autophagy al., complex not et protein (Tan elongation is coat and/or formation the membrane TRAPPIII phagophore provide of might for the vesicles of subunit COPII Interestingly, that localization effector, a suggesting 2010). conserved (COP)II, proper with al., for Ypt1 et interacts the required (Yen complex is another Atg9 Moreover, and and complex, PAS Atg8 vesicles. the (COG) to Atg9 localizes Golgi of oligomeric tethering al., activated facilitate et might the (Wang Ypt1/RAB1 the PAS and and the TRAPPIII Atg17, to that Atg1 suggesting by 2013), of PAS recruitment the the stimulates to Ypt1 recruited is TRAPPIII eea ebr fteRB(a-eae rti)fml of family protein) (Ras-related RAB the of members Several ora fCl cec 21)18 9–0 doi:10.1242/jcs.141036 193–205 128, (2015) Science Cell of Journal ´ iand ri ¨ger

Journal of Cell Science ovxsd ftedul iae.Mroe,a tEn h target the PtdEtn, the as and Moreover, bilayer. concave double the the the a of between by side caused LC3-II convex as tension of membrane scenario, envisioned distribution of a result unbalanced such be the In group). be head might indeed to large curvature very leads can a that with lipid (LC3-II effect membrane the crowding bending at a accumulation create membrane and might lipidation surface LC3 membrane autophagosome. geometry the the forming of in participate themselves proteins homolog COMMENTARY rpe rudtebasi h rsneo t1 n that and Atg19 closely of membrane presence GUV the in Atg8-positive beads the it the beads, around that pro-peptide wrapped prApe1 found and was (GUVs) Atg8- vesicles one unilamellar only an contains Using its (Sawa-Makarska Atg34 in motif. cargo motifs binding paralog membrane Atg8-binding its the multiple the Cvt whereas contains of C-terminus, in Atg19 the 2014). interaction Atg8 that al., the et with found phagophore facilitates Atg19 study to prApe1 receptor recent contribute cargo A cargo receptors vesicle curvature. the that autophagy and likely is bound expansion it its autophagy, selective and to comes it autophagy When selective indeed in membrane modeling proteins Membrane cell. phagophore the purified whether in determined operate with outer that be mechanisms is observations to reflect remains the Atg16–Atg12–Atg5 interesting It 2011). why these Atg8–LIR-sequence on al., to Atg12, et through (Mizushima to found as Atg8 adaptors explanation of exclusively the binding an cargo the of providing outcompete side or the would convex interactions to binding the cargo side, concave at the leading At of scaffold 2014). al., interaction, ‘coat-like’ et (Kaufmann phagophore a Atg8–Atg12 membrane the of specific to assembly complex a Atg12–Atg5–Atg16 also it through formed, the study, is Another Atg8 anchor lipidated 2012). vesicles once can al., that showed et Atg9-containing proteins, Yamamoto yeast for using of 2010; fusion al., suggested early et the as al., (Mari in work et vesicles, at (Romanov be incoming might Atg8 have mechanism of a to Such independently 2012). tethering vesicles the shown mediate membrane to ATG12– was and of lipids, essential charged Atg5 negatively the for Yeast affinity of complex. functions ATG5–ATG16L1 additional suggested Weidberg 2008a; have al., et is (Fujita that family 2010). membrane reaction LC3 al., fusion the et 2010). the seal al., in to implicated et required been (Weidberg also process have the proteins of a important at stage is functions LC3 later GABARAPL2 that whereas found elongation, homolog study phagophore Atg8 one for individual but the autophagy, of the in Little roles 2004). proteins by specific al., the cytosol et about the (Kabeya known ATG4 to is protease recycled cysteine is the of LC3-II membrane action together convex 2014). LC3-II outer autophagosome whereas the degraded, al., the eventually on are inside et cargo and trapped receptors (Rogov to with are bind proteins that that sequences GABARAP molecules LIR and possess all LC3 that identified receptors of cargo-specific been curvature such have the Several influence phagophore. concave LC3-mediated also expanding might the 2008). the phagophores to al., the et receptors of autophagosome experiments (Xie autophagy surface the the affected cargo-bound in manipulated, be of to further to recruitment was Indeed, found Atg8 possibly might was of size. size and it level the curvature autophagosome lipid, where cone-shaped membrane of a the regulation is to lipidation, contribute LC3 of nvitro In tde ihlpsmsadprfe uohg proteins autophagy purified and liposomes with studies nvitro in eosiue ytmwt giant with system reconstituted AAA aiiae h erimn fL3 oAF and to ALFY ALFY to of LC3B Binding 2014). of al., recruitment et the (Lystad facilitates LIR than p62 proteins GABARAP a GABARAP with the adaptor for motif of affinity LIR that likely of an p62-interacting higher has tenfold This WDFY3) The than sequestration as greater known 2005). aggregates). p62-mediated (also ALFY protein al., protein exclusive (e.g. self- et the PB1-domain-mediated cargo (Bjørkøy to to p62 owing contributes of of high, ubiquitin-binding avidity or the is polymerization 2014), LC3 the al., and interaction et of SQSTM1) (Maruyama binding the low as the domain rather known is although (also GABARAP LIR p62 that receptor the note autophagy to between interesting affinity is It autophagy. not but selective for prApe1. of sufficient sequestration is Atg34 exclusive chimera) in Atg19–Atg34 site apposition to Atg8-binding the one Atg19 (or membrane–cargo whereas of molecules, close binding Atg8 the several which requires autophagy’) in normal ‘exclusive to (termed model nearly authors the was a leading processing suggest autophagy, processing cargo prApe1 bulk prApe1 rapamycin-induced contrast, prApe1 the upon In to support binding one intact. although to with was pathway, Cvt unable protein Atg8-binding the the Atg19–Atg34 was through and chimeric site Atg8 A Atg8-binding of Atg19. density of the activity with correlated this hgpoe novdi eetv uohg lonucleates be also of to autophagy membrane selective remain omegasomes. phagophore ER-associated in from questions the involved LIR-containing whether key phagophores a including Several and expansion and addressed, phagophore sequestration. further protein for cargo platform Atg8 initial a an forms an that receptor suggest family studies between Atg8 these together, other interaction Taken of 2012). recruitment al., et the was cytosolic for LC3C to required with members be CALCOCO2) autophagy as to ubiquitin-binding known found the (also of NDP52 interaction high-affinity receptor this, with autophagy. a line exclusive for that In required is indicating interaction protein further LIR–Atg8 structures, p62-positive nooefso.I sitrsigt oeta eea ESCRT autophagosome– several that or al., note being to et ESCRTs closure interesting PtdIns3 to Rusten are is due subunits It 2007; autophagosome is this fusion. al., whether endosome for autophagosomes clear et not of Lee is required it 2007; accumulation but al., 2007), regulatory an their et of causes as (Filimonenko well Vps4, as subunits, the ATPase shown membrane ESCRT have of The of studies cleavage Several depletion facilitate 2010). 2012). subsequent that Hanson, and and proteins al., (Hurley cytosol neck the bud associated et from away (Rusten complex their budding sorting (ESCRT) and endosomal transport the ESCRTs upon on for formed rely required and all (MVB) budding viral which bodies membrane, cytokinesis, endosome multivesicular most early the of from of closure invagination that different is phagophore opening, the to its of narrow topology similar The that a events. fusion of membrane a infers other fusion is phagophore the which The in involves membrane. involved structure, phagophore mechanisms the the double-membraned of about known closure is the anything, if closure Little, phagophore during dynamics Membrane ebaeadt erqie o h ia lsr fthe of closure of regulation final tight the A for autophagosome. phagophore an the required form from be ATGs to to core phagophore early and the membrane of dissociation the PtdIns3 iia rnil slkl oapyt te om fselective of forms other to apply to likely is principle similar A P ora fCl cec 21)18 9–0 doi:10.1242/jcs.141036 193–205 128, (2015) Science Cell of Journal hshtss(TR,Yr)wsfudt mediate to found was Ymr1) (MTMRs, phosphatases P bnigpoen.PtdIns3 proteins. -binding Salmonella yhmru vnMuhlinen (von Typhimurium P unvrb the by turnover 201

Journal of Cell Science jrø,G,Lmr,T,Beh . uzn . eadr . vran A., Overvatn, M., Perander, H., Outzen, A., H. Brech, T., J. Lamark, Hurley, G., and Bjørkøy, E. Boura, J., M. Ragusa, S., T. Baskaran, Proikas-Cezanne, and Z. Takacs, D., Bakula, x,E . akr .A,Mnfv,M,Cada . oeik .L., H. Roderick, P., Chandra, M., Manifava, A., S. Walker, L., E. Axe, Jain, B., K. Larsen, B., A. Birgisdottir, M., K. Torgersen, T., Lamark, A., E. Alemu, a pclt htECT r erie ocoigphagophores closing PtdIns3 to one with recruited interactions Thus, are through ESCRTs 2010). that al., speculate et can (Taguchi-Atarashi size autophagosome PtdIns3 COMMENTARY 202 Z. Elazar, and A. Abada, References of Council Research the and Society Cancer Norwegian the Norway. by funded is A.S. Funding interests. competing no declare authors The interests figures. the Competing with help for Knudsen S. V. are Carina thank phagophore We future near Acknowledgements of biology. closure the membrane in final in principles Investigations of novel biogenesis the reveal to execution and regulated? is likely properties and How and membrane fusion? regulation be and the the mediated to are tethering in What remain from? membrane coming involved questions membrane the mechanisms fundamental mostly is and Where still several answered. are species determining and size in lipid autophagosome involved unknown constitutive and are curvature the that phagophore proteins However, modeling the years. membrane during few immensely increased last in has involved events biogenesis modeling autophagosome membrane the of understanding Our remarks Concluding oe,ls fAg,wihfrsacmlxwt h PtdIns3 the with complex a forms a which such Atg2, with line of In loss closure. model, phagophore final the controls opening PtdIns3 lae f h ue ebaeb h ciiyo h Atg4 the of are activity being the membrane, by Atg8, phagophore 2000). al., membrane the and et outer (Kabeya of LC3 protease the sides off proteins both cleaved ubiquitin-like to before the such conjugated recycled with Consistent model, pathway, this. is endocytic address to the a ubiquitin needed in are whether case studies future the and known is as al., not closure, et autophagosome is (Katsiarimpa and vacuole It the markers in 2013). bodies autophagosome autophagic accumulate fewer have mutants enzyme VPS2.1 deubiquitylating in and the autophagy vacuolar for with recent important subunit is (VPS2.1) ESCRT-III a AMSH1 2.1 the subunits and of sorting 2011), ESCRT interaction protein Piper, autophagosome that Several and found inhibit (Shields 2012). study ubiquitin to al., with found et interact was (Velikkakath Atg18 closure protein binding grgtsdgae yatpayadhsapoetv feto huntingtin- on effect protective a has death. T. and cell autophagy induced Johansen, by degraded and aggregates H. Stenmark, longevity. and diseases autophagy-related eonto fpopaiyioio -hsht yPOPN nautophagy. in PROPPINs by Cell 3-phosphate Mol. phosphatidylinositol of recognition reticulum. endoplasmic 3- the phosphatidylinositol to in connected T.Biol. enriched dynamically N. and compartments Ktistakis, phosphate membrane and from G. formation Griffiths, A., Habermann, motifs. (LIR) requirements T. sequence region complex: LC3-interacting Johansen, ULK for the and of assembly V. for scaffolds Kirkin, as act A., proteins Øvervatn, H., Olsvik, A., uohgsm biogenesis. autophagosome 182 P P 685-701. , 47 eesi h ihycre ebaea h remaining the at membrane curved highly the in levels eesa h hgpoeas per oregulate to appears also phagophore the at levels 339-348. , .Cl Biol. Cell J. 21) etn ed o ulig inln and signaling building: for ready Getting (2014). MORep. EMBO 171 603-614. , .Bo.Chem. Biol. 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