Cargo recognition and degradation by selective autophagy.pdf

SerieS|REVIEW ARTICLEhttps:/doi.org/10.1038/s41556-018-0037-zLife Sciences Institute and Department of Molecular,Cellular and Developmental Biology,University of Michigan,Ann Arbor,MI,USA.*e-mail:Klionskyumich.eduAutophagy is a highly conserved pathway in eukaryotes,involv-ing cellular recycling of multiple cytoplasmic components during standard physiological conditions and in response to different types of stress,such as starvation.Macroautophagy(hereafter autophagy)can be either non-selective or selective and involves the sequestration of cytoplasm within double-membrane vesicles termed autophagosomes.Upon maturation,autophago-somes fuse with the vacuole(in yeast and plants)or endosomes and lysosomes(in metazoans),leading to degradation of their cargo by resident hydrolases1,2.Cargo degradation produces molecular build-ing blocks such as amino acids,which are subsequently recycled back into the cytoplasm for reuse1,3.Whereas non-selective autoph-agy,a cellular response to nutrient deprivation,typically involves random uptake of cytoplasm into phagophores(the precursors to autophagosomes),selective autophagy is responsible for specifi-cally removing certain components such as protein aggregates and damaged or superfluous organelles4.Different studies have reported the selective autophagic degradation of several organelles,includ-ing mitochondria5,peroxisomes6,lysosomes7,endoplasmic reticu-lum(ER)and the nucleus8,under various conditions.Furthermore,autophagy selectively degrades aggregation-prone misfolded pro-teins and protein microaggregates implicated in the pathology of various neurodegenerative diseases9.In this Review Article,we address the principal mechanisms of selective autophagy in yeast and mammals,with an emphasis on mitophagy,which is the best-described type of selective autophagy to date.Cytoplasm-to-vacuole targeting pathwayThe cytoplasm-to-vacuole targeting(Cvt)pathway is a biosyn-thetic autophagy-related process specific to yeast,in which vacu-olar enzymes are transported from the cytoplasm into the vacuole utilizing the autophagic machinery.Among the enzymes that utilize the Cvt pathway are aminopeptidase I(Ape1),Ape4 and -man-nosidase(Ams1)10.Ape1 is first synthesized in the cytoplasm as an inactive proenzyme(prApe1).Following oligomerization,prApe1 is selectively recognized by the non-core,autophagy-related(Atg)protein Atg19,which functions as a receptor for Ams1,prApe1 and Ape4(refs 11,12).Once prApe1Atg19(the Cvt complex)is formed,Atg19 binds to the scaffold protein Atg11,which in turn directs the Cvt complex to the perivacuolar location termed the phagophore-assembly site(PAS).Here,autophagosomes and Cvt vesicles are formed in yeasts13,14;interaction of Atg19 with Atg11 is facilitated by Hrr25-dependent phosphorylation of the receptor15.After reaching the PAS,Atg19 interacts with the ubiquitin-like protein Atg8(ref.13).During autophagy and the Cvt pathway,Atg8 is covalently conju-gated through its C terminus to phosphatidylethanolamine(PE).Thus,Atg8PE is present on both the inner and outer membrane of forming autophagosomes16(Fig.1a).Atg8 has been implicated in phagophore expansion and autophagosome size regulation17.Atg19 binding to Atg8 therefore tethers the Cvt complex to the Atg8PE-conjugated sequestering vesicles.Once fully matured,Cvt vesicles fuse with the vacuole and deliver prApe1,which is then pro-cessed into its active form by resident hydrolases.Using the Cvt pathway as a model for selective autophagy,we can propose that although the core autophagy machinery directs phagophore membrane expansion and vesicle formation,cargo selectivity is achieved by a ligand receptor and a scaffold proteinroles taken by Atg19 and Atg11,respectively,in the Cvt pathway.Atg19 has a paralogue,Atg34(also phosphorylated by Hrr25),which functions as an Ams1-receptor during nitrogen starvation18.Other types of selective autophagy in yeast,such as mitophagy19 and pexophagy20,21,also rely on Atg11 as a scaffold for cargo delivery to the PAS.However,a counterpart to Atg11 has yet to be discovered in mammals.Similarly,most types of selective autophagy require binding of the cargo receptor to the core autophagy machinery.In the Cvt pathway,this process is illustrated by Atg19 binding to Atg8 through a specific WXXL motif found on the Atg19 C ter-minus,similar to that seen in mammalian SQSTM1(also known as p62)22,23.This interaction is evolutionarily conserved,as several proteins in yeasts and more complex eukaryotes contain Atg8-interacting motifs(AIMs)or LC3-interacting regions(LIRs),respectively.The AIMs or LIRs provide selective binding to yeast Atg8 or one of the members of the LC3/GABARAP family of Atg8 mammalian homologues24.Recently,a specific type of LIR called GABARAP-interacting motif(GIM)has been proposed,showing enhanced specificity to GABARAP versus LC3 family members25.Multiple examples of scaffold and receptor proteins will be show-cased as we discuss different types of selective autophagy(Table 1).AggrephagyThe selective degradation of protein aggregates by autophagy is known as aggrephagy.Multiple aggregation-prone proteins such as amyloid-(ref.26),HTT(huntingtin)27 and SNCA(synuclein alpha)28 are autophagy substrates.In yeast,Cue5 is a cargo receptor for the clearance of aggregation-prone polyglutamine(polyQ)-containing proteins.Cue5 possesses a ubiquitin-binding CUE domain and an Cargo recognition and degradation by selective autophagyDamin Gatica,Vikramjit Lahiri and Daniel J.Klionsky*Macroautophagy,initially described as a non-selective nutrient recycling process,is essential for the removal of multiple cel-lular components.In the past three decades,selective autophagy has been characterized as a highly regulated and specific degradation pathway for removal of unwanted cytosolic components and damaged and/or superfluous organelles.Here,we discuss different types of selective autophagy,emphasizing the role of ligand receptors and scaffold proteins in providing cargo specificity,and highlight unanswered questions in the field.2018 Macmillan Publishers Limited,part of Springer Nature.All rights reserved.NAture CeLL BioLoGy|VOL 20|MARCH 2018|233242| ARTICLE|SerieSNaTure Cell BIologyREVIEW ARTICLE|SerieSAIM,mediating the interaction between ubiquitinated cargo and Atg8(ref.29).Overexpression of TOLLIP,a Cue5 human homologue that also has a CUE domain,leads to degradation of polyQ protein aggregates in human cell lines30(Fig.1b).Ubiquitination of substrates has been demonstrated as a key mediator in the recognition and degradation of these proteins by selective autophagy31.At least three additional mammalian cargo receptors,SQSTM1(refs 16,23),NBR1(ref.32)and OPTN(ref.33),act as ubiquitin-binding proteins that Atg11prApe1Atg19Atg11Atg11Atg8Atg8AggrephagybYeastMammalsUbUbCue5Atg8Atg8TOLLIPNBR1WDFY3GBRPGBRPLC3SQSTM1NBR1cYeastMammalsUbPeroxisomePex3PpAtg30Atg36Atg8SQSTM1Atg11PpAtg8PpAtg8Atg8PEX14GBRPGBRPLC3LC3GBRPLC3LC3PEX5PEX2OPTNPpAtg11PpAtg11PpPex14PpPex3PpAtg37PEX5aThe Cvt pathway(yeast)Ams1,Ape4CvtcomplexAtg19Atg19Atg19Atg19Atg19Atg19Atg8Atg8Atg8Atg8Atg8ProteinaggregateAtg8Atg8Atg8Atg8PexophagyLC3LC3GBRPLC3LC3Atg8Atg8Atg8Atg8PpAtg30PpAtg8PpAtg8PpAtg8PpAtg8LC3Fig.1|the Cvt pathway,aggrephagy and pexophagy.a,In the yeast Cvt pathway,prApe1,Ape4 and Ams1 are synthesized in the cytoplasm.prApe1 oligomerizes into dodecamers and subsequent higher-order structures that are recognized by the receptor Atg19.Atg19,in turn,binds the scaffold protein Atg11,forming the Cvt complex.Ams1 and Ape4 also oligomerize and bind Atg19.Atg11 brings the Cvt complex to the PAS,where Atg19 binds Atg8PE,tethering the Cvt complex to the phagophore.b,In both yeast and mammalian aggrephagy,protein aggregates are ubiquitinated(Ub)and subsequently recognized by cargo receptors.In yeast,Cue5 links the ubiquitinated aggregates to Atg8PE.During mammalian aggrephagy,TOLLIP,SQSTM1,NBR1 and OPTN tether the ubiquitinated aggregates to the phagophore by binding LC3/GABARAP family members.WDFY3 has been described as a scaffold for SQSTM1-dependent degradation.c,In S.cerevisiae pexophagy,Atg36 functions as a receptor,linking peroxisomes to the phagophore by binding Pex3 and Atg8PE.In P.pastoris pexophagy,PpAtg30 acts as a receptor by linking PpPex3 and PpPex14 to PpAtg8PE.Atg11 functions as a scaffold for both S.cerevisiae and P.pastoris.The current model of mammalian pexophagy involves the E3-ubiquitin ligase PEX2-mediated mono-ubiquitination of PEX5,which in turn is recognized by receptors SQSTM1 and NBR1,tethering peroxisomes to the phagophore.PEX14 has also been reported to link peroxisomes to the phagophore by directly binding LC3 family members.2018 Macmillan Publishers Limited,part of Springer Nature.All rights reserved.NAture CeLL BioLoGy|VOL 20|MARCH 2018|233242| ARTICLEhttps:/doi.org/10.1038/s41556-018-0037-zSerieS|REVIEW ARTICLENaTure Cell BIologymediate the interaction between ubiquitinated proteins and the core autophagy machinery.All three receptors possess LIRs and ubiquitin-binding domains,thus working as a bridge between LC3/GABARAP family members and ubiquitinated substrates16,2329,3134.The nucleocytoplasmic shuttling protein WDFY3(also known as ALFY)has been proposed as a scaffold in aggrephagy35.While unable to directly interact with ubiquitinated substrates,WDFY3 binds the core autophagy protein Atg5,the cargo receptor SQSTM1(ref.36),GABARAP subfamily members37,and phosphatidylinosi-tol-3-phosphate(a prominent lipid in the regulation of autophago-some membrane formation)38.WDFY3-depletion hinders clearance of aggregated polyQ proteins.The latter observation,in conjunction with its high number of interacting partners,suggests that WDFY3 is an important scaffold protein in SQSTM1-dependent degrada-tion of ubiquitinated aggregates by selective autophagy.Ubiquitination plays an important role not only in substrate recognition and degradation by the ubiquitin-proteasome system(UPS),but also by selective autophagyraising a set of questions regarding the hierarchy between these two degradation pathways.It has been proposed that protein aggregates that cannot be degraded by the UPS(for example,due to size)may be cleared by autoph-agy39,40.At the same time,the lysine residues used for linkage,as well as the length and nature of the ubiquitin chains,have been proposed as a mechanism to select which degradation pathway is chosen39.However,a recent paper by Lu et al.emphasizes the role of recep-tor oligomerization over the type of ubiquitination in selecting a degradation pathway41.This finding agrees with data showing the importance of Cue5 and SQSTM1 oligomerization in their associa-tion with the phagophore39,42.Thus,both autophagy and the UPS provide dynamic alternatives to different cellular challenges.PexophagyPexophagy is the selective removal of peroxisomes.Pexophagy has been mostly studied as a pathway for the removal of superfluous organelles in various fungi43.Incubating these fungi in oleic acid or methanol leads to peroxisome proliferation;following a shift to a preferred carbon source such as glucose,the excess peroxisomes are rapidly degraded through pexophagy43.Similar to other types of selective autophagy,cargo selectivity is provided by receptor pro-teins;in yeast,this role is taken by Pichia pastoris Atg30(PpAtg30)20,and by Atg36 in Saccharomyces cerevisiae44.Both Atg36 and PpAtg30 tether peroxisomes targeted for degradation to nascent phagophore membranes by linking Atg8 to peroxisomal membrane proteins,with Atg36 binding Pex3 and PpAtg30 binding both PpPex3 and PpPex14(refs 20,21).Phosphorylatable variants of the classical AIMs have been reported for both Atg36 and PpAtg30.However,disrup-tion of these AIMs only delays pexophagy rather than abrogating it45.As previously mentioned,Atg11 is required for pexophagy46.PpAtg37 is an integral peroxisomal membrane protein specifically required for pexophagy in P.pastoris.During pexophagy,PpAtg37 is necessary for phagophore formation,as PpAtg37-null cells fail to recruit PpAtg11 to peroxisomes47.In contrast to yeast,no pexophagy-specific cargo receptor has been described in mammals.Rather,mammalian pexophagy relies on the ubiquitination of peroxisomal proteins and their recogni-tion by SQSTM1(ref.31)and NBR1(ref.48).Initially,it was reported that PEX3 overexpression leads to peroxisome ubiquitination and pexophagy induction49.However,blocking PEX3 ubiquiti-nation does not prevent pexophagy and this study did not deter-mine the specific peroxisomal proteins targeted for ubiquitination.Subsequently,two studies identified PEX5 mono-ubiquitination as the cargo signal for peroxisome degradation50,51.PEX5 is a cytosolic protein that shuttles between the peroxisomal membrane and the cytosol in a ubiquitin-dependent manner52.Accumulation of mono-ubiquitinated PEX5 on the peroxisomal membrane,which is unable to shuttle back to the cytosol,triggers pexophagy50.Furthermore,in response to reactive oxygen species(ROS),PEX5 is phosphorylated and subsequently mono-ubiquitinated,which leads to pexophagy induction in a SQSTM1-dependent manner51.A recent study has indicated that the peroxisomal E3-ubiquitin ligase PEX2 is respon-sible for PEX5 ubiquitination53.These data suggest a model in which mammalian pexophagy is dependent on membrane accumulation of ubiquitinated peroxisomal proteins,such as PEX5,that are rec-ognized by the ubiquitin-binding receptors SQSTM1 and NBR1,which in turn link the target peroxisomes to LC3/GABARAP-bound sequestration membranes(Fig.1c).However,this simple model Table 1|Selective autophagy ligands,receptors and scaffolds in yeast and mammalsProcessorganismLigandreceptorScaffoldCvt pathwayYeastprApe1,Ams1Atg19Atg11AggrephagyYeastProtein aggregates(Ub)Cue5PexophagyMammalsSQSTM1/p62,NBR1,TOLLIPWDFY3/ALFYYeastPeroxisomes(Pex3,PpPex14,PpPex3)Atg36,PpAtg30Atg11,PpAtg17MammalsPeroxisomes(Ub)SQSTM1/p62,NBR1MitophagyYeastMitochondriaAtg32Atg11MammalsSQSTM1/p62,BNIP3L/Nix,OPTN,FUNDC1,PHB2ReticulophagyYeastEndoplasmic reticulumAtg39,Atg40Atg11MammalsRETREG1/FAM134BNucleophagyYeastNucleus,nuclear Nvj1 portions(PMN)Atg40,Vac8(PMN)LysophagyMammalsLysosomes(Ub)SQSTM1/p62XenophagyMammalsBacteria(Ub),virusesSQSTM1/p62,CALCOCO2/NDP52,OPTNLipophagyMammalsLipid dropletsYeastFerritinophagyMammalsFerritinNCOA4GlycophagyMammalsGlycogenSTBD1Ub,ubiquitinated.2018 Macmillan Publishers Limited,part of Springer Nature.All rights reserved.NAture CeLL BioLoGy|VOL 20|MARCH 2018|233242| ARTICLE|SerieSNaTure Cell BIologyREVIEW ARTICLE|SerieSfails to answer several questions.From a mechanistic perspective,how does PEX5 ubiquitination at a specific site determine whether the protein shuttles into the peroxisome or is directed to protea-somal degradation?Are there distinct mechanisms involving ROS-and amino-acid-starvation-induced pexophagy?Regarding this last point,other studies have reported that the peroxisomal membrane protein PEX14,which acts as a docking factor for PEX5,can dire