Mechanisms of Autophagy Initiation.pdf

Mechanisms of Autophagy InitiationJames H.Hurley*and Lindsey N.YoungDepartment of Molecular and Cell Biology and California Institute of Quantitative Biosciences,University of California,Berkeley,Berkeley,United States;and Molecular Biophysics and Integrated Bioimaging Division,Lawrence Berkeley National Laboratory,Berkeley,United StatesAbstractAutophagy is the process of cellular self-eating by a double-membrane organelle,the autophagosome.A range of signaling processes converge on two protein complexes to initiate autophagy:the ULK1 protein kinase complex and the PI3KC3-C1 lipid kinase complex.Some 90%of the mass of these large protein complexes consists on non-catalytic domains and subunits,and the ULK1 complex has essential non-catalytic activities.Structural studies of these complexes have shed increasing light on the regulation of their catalytic and non-catalytic activities in autophagy initiation.The autophagosome is thought to nucleate from vesicles containing the integral membrane protein Atg9,COPII vesicles,and possibly other sources.In the wake of reconstitution and superresolution imaging studies,we are beginning to understand how the ULK1 and PI3KC3-C1 complexes might coordinate the nucleation and fusion of Atg9 and COPII vesicles at the start of autophagosome biogenesis.Keywordsvesicle;membrane remodeling;tethering complex;membrane fusion;nanoscale biology;ULK1;Atg1;Vps34;phosphatidylinositol 3-kinase;COPIIIntroductionMacroautophagy(hereafter“autophagy”)is the main mechanism used by eukaryotic cells to degrade cargoes that are larger than individual proteins.It is also the main mechanism for eukaryotic cells to replenish pools of biosynthetic precursors and energy sources by recycling cytosolic contents during starvation.Both the process of autophagy and its machinery is conserved from yeasts(S.cerevisiae and S.pombe)(1)to mammals(2).Autophagy can be either selective or non-selective(“bulk”).Selective autophagy removes and recycles harmful or simply unneeded materials from the cell.These include protein aggregates,damaged mitochondria,unneeded peroxisomes,excess ribosomes,ER and endosomes,lipid droplets,and intracellular pathogens(3,4).Failure to control the accumulation of any of these types of materials can lead to disease in humans.Excellent reviews cover the relationship of autophagy to neurodegeneration(5),cancer(6),aging(7),infection(8),and other diseases.Bulk autophagy is triggered by starvation,and is critical for*jimhurleyberkeley.edu.HHS Public AccessAuthor manuscriptAnnu Rev Biochem.Author manuscript;available in PMC 2018 June 20.Published in final edited form as:Annu Rev Biochem.2017 June 20;86:225244.doi:10.1146/annurev-biochem-061516-044820.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptmaintaining a cellular supply of lipids,amino acids,carbohydrates,and nucleotides.Selective and bulk autophagy are triggered by different signals.Yet,these diverse signals are thought to funnel into a single pathway that initiates the mechanical events and membrane remodeling needed to create the autophagosome.This review will focus on the conserved and common events that initiate both selective and bulk autophagy.Far more is known about bulk autophagy initiation.The review will focus mostly on data from bulk autophagy research,with the understanding that most of the findings probably apply to selective autophagy.Autophagy is initiated in yeast at a punctate structure called the Pre-Autophagosomal Structure(9)(PAS,also sometimes called the Phagophore Assembly Site;Fig.1).In mammals,initiation is associated with an endoplasmic reticulum(ER)subdomain enriched for the lipid phosphatidylinositol 3-phosphate(PI(3)P),known as the omegasome(10).From its inception at the PAS or omegasome,the phagophore elongates into a cup-shaped structure and begins to engulf cellular material.The membrane supply for phagophore growth can apparently be sourced to a variety of cellular reservoirs(11).In selective autophagy,the cargo itself templates the size and shape of the phagophore(4,12,13).In bulk autophagy,it is less clear how this occurs,but the actin cytoskeleton is involved(14).Finally,the cup closes upon itself.The narrow gap at the tip of the cup fuses,leading to the complete sequestration of the material inside.The outer membrane of the autophagosome then fuses with the lysosome(vacuole in yeast or plants)to form structure known as the autolysosome.At this stage,the inner membrane and all of its contents are degraded.This review will focus on the earliest steps in the process:the formation of the PAS in yeast and the formation of autophagy initiation sites in mammals,and the initial nucleation of the phagophore.The conserved machinery for autophagosome formation(1517)contains two major initiation complexes that are a central focus of this review:the ULK1 complex(known as the Atg1 complex in yeast)and the class III PI 3-kinase complex I(PI3KC3-C1)(Table 1).The sole conserved transmembrane protein in the core machinery,Atg9,is also closely connected to initiation.The PI3P binding WIPI 14 proteins and the Atg8-family and Atg12 conjugation systems(Atg3,Atg4,Atg5,Atg7,Atg10,Atg12,Atg16,LC3s,and GABARAPs;Table 1)function downstream and drive phagophore elongation.Conjugation of Atg8/LC3 family proteins to phosphatidylethanolamine,known as“LC3 lipidation”for short,is the hallmark downstream reaction driven by these proteins.These Atg proteins were discovered for their roles in autophagy and are primarily dedicated to this function,although“moonlighting”roles in non-autophagic functions have been reported(18).Multipurpose membrane trafficking factors also have essential roles in the pathway.In many cases,these roles are also conserved from yeast to humans.These factors include the coat complexes COPI and COPII,the vesicle-and organelle-identifying RAB GTPases,and SNARE proteins of vesicle fusion(1921).In selective autophagy,a variety of adaptor proteins link cargoes to the autophagy machinery,including p62,Optineurin(OPTN),and NDP52(3,4,22).Hurley and YoungPage 2Annu Rev Biochem.Author manuscript;available in PMC 2018 June 20.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor ManuscriptThe ULK1/Atg1 ComplexAutophagy initiation starts with the activation of the ULK1 complex(Atg1 complex in yeast)(2325).ULK1 is part of a family of kinases ULK14 in humans.Isoform ULK1 is the most important of these in autophagy.In some cells lines it is necessary to block both ULK1 and ULK2 to completely shut down autophagy,however ULK2 is less characterized and will not be discussed further.The ULK1 complex consists of ULK1 itself,and the non-catalytic subunits FIP200,ATG13,and ATG101(2628)(Fig.2B).FIP200 is a large predicted coiled coil protein involved in scaffolding(29).ATG13 and ATG101 contain HORMA(Hop/Rev7/Mad2)domains(30)which heterodimerize with each another(3133).ATG13 contains a long IDR(intrinsically disordered region)following the HORMA domain,and the C-terminal part of its IDR contains motifs that bind to the C-terminal EAT/tMIT domain of ULK1(34).The budding yeasts organize their Atg1 complexes in a uniquely complicated way.The Atg1 subunit itself is organized into the same domain structure as ULK1.Atg13 is conserved,but ATG101 is absent from budding yeasts.FIP200 is replaced by two scaffolding subunits:Atg11,which functions in selective autophagy(35),and Atg17,which functions in bulk autophagy(36,37).Atg17 in turn co-assembles with two smaller subunits,Atg29 and Atg31(38,39)(Fig.2A).The large scaffolding subunits Atg11 and FIP200,whose structures are unknown,are similar to each other in size and predicted helical content.Their C-terminal domains are homologous,suggesting a common function.Atg17 is smaller than Atg11 and FIP200,and aside from a similar helical content,has little sequence similarity with Atg11 and FIP200.It is often stated in the literature that Atg17 and FIP200 are orthologs.It is important to bear in mind that this inference,while reasonable,is based on functional parallelism,not detailed sequence or structural similarity.ULK1/Atg1 kinase activation and inactivationULK1/Atg1 is activated in at least three ways upon autophagy induction,and all three are essential.Protein kinase activity needs to be switched on,the active kinase needs to be recruited to the PAS,and essential though still vaguely definednon-catalytic scaffolding activities must be turned on.Autophosphorylation of the kinase domains activation loop at Thr180 of ULK1(40,41)(Thr226 in yeast Atg1(42,43)is essential for activation.Autophosphorylation is promoted by conditions that induce autophagy and by co-assembly with other subunits of the complex(42,44,45).This co-assembly in turn increases the local concentration of Atg1 molecules and promotes their mutual autophosphorylation.This can occur both in selective autophagy under nutrient-rich conditions(44)and in starvation(45).Following activation,ULK1(and PI3KC3-C1)can be ubiquitinated by the Cul3-KLHL20 ligase complex and degraded(46),thereby switching off the autophagy initiating signal.How does starvation trigger Thr180 phosphorylation of ULK1?Autophosphorylation is usually promoted by the dimerization or higher-order oligomerization of kinases.The C-terminal EAT domain of Atg1 dimerizes in isolation(34,39,47),however,full-length Atg1 is reportedly a monomer in the absence of other subunits(48).It is currently not clear whether Atg1 undergoes regulated dimerization via its EAT domain under some conditions.Hurley and YoungPage 3Annu Rev Biochem.Author manuscript;available in PMC 2018 June 20.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor ManuscriptULK1 is bridged by ATG13 to the scaffolding subunit FIP200(2628).In yeast,Atg13 bridges to Atg11(49)and Atg17(36,37).While the oligomeric state of FIP200 and Atg11 is unknown,Atg17 is a constitutive dimer(38,39).The recruitment of ULK1 to FIP200 via the intermediation of ATG13 could in principle be a mechanism for ULK1 trans-autophosphorylation.Whether and how starvation regulates formation of the Atg1 complex has been intensively investigated,yet consensus has been elusive.mTORC1(TORC1 in yeast)is a master regulator of cell growth and metabolism,and its inactivation in response to amino acid depletion is a major trigger for autophagy(50).In the canonical model of the process in yeast,Atg13 is phosphorylated by TORC1 under non-starved conditions(51).The extensive phosphorylation inhibits the assembly of Atg13 with Atg1,and Atg13 with Atg17,by introducing steric and electrostatic repulsion into the binding sites on Atg1 and Atg17(34).In a contrasting report,it was found that the Atg1 complex is assembled constitutively in yeast in both nutrient-rich and starved conditions(52).Of 51 reported phosphorylation sites within yeast Atg13,six fall within the crystallographically defined Atg1-binding site.Mutation of all of these sites from Ser to the phosphomimetic Asp only reduces Atg1 binding by a factor of three(34).The effect of Atg13 phosphorylation on Atg17 binding may be larger.Two of the 51 reported phosphorylation sites occur in the Atg17 binding site of Atg13.Phosphomimetic mutations in these two residues substantially disrupt binding(34).Phosphoregulation at the level of the Atg17-Atg13 interaction thus seems to lead to bigger affinity changes than for the Atg1-Atg13 interaction.FIP200 and Atg11 are less tractable biochemically than Atg17,and little is known at the quantitative and structural level about how ULK1 and Atg1 association with these two scaffolds is regulated.It is often stated that ULK1 is assembled constitutively in mammalian cells(2629),yet on the other hand,the mammalian ATG13-ATG101 subcomplex appears to have autophagic functions that are independent of the ULK1 complex(53).Other regulatory mechanisms function in parallel to,or even antagonize mTORC1/TORC1 regulation(Fig.2C).AMPK(AMP-activated protein kinase)upregulates autophagy in response to energy depletion as detected by an increase in cytosolic AMP(54).AMPK directly phosphorylates ULK1 at multiple sites in its central IDR(40,5559),leading to its activation.The details of how these IDR phosphorylation sites communicate with the catalytic domain remain to be elucidated.In selective autophagy,ULK1 must be locally active even under fed conditions when mTORC1/TORC1 is also active.Relatively little is known about how the ULK1 complex is sheltered from inactivation under these conditions.Huntingtin,the protein product of the gene mutated in Huntingtons disease,interacts with ULK1 and has been proposed to have such an ULK1-shielding function(60)ULK1 recruitment to initiation sitesRecruitment of the ULK1 complex to sites of autophagy initiation is the second regulated step in its activation.In bulk autophagy in yeast,PAS recruitment is regulated at the level of Atg13 phosphorylation.Atg17,along with its accessory proteins Atg29 and Atg31,is the first protein to arrive at the PAS is yeast(61),which set the stage for the recruitment of Atg13 and Atg1 as described above.The EAT domain of ULK1,the locus for ATG13 Hurley and YoungPage 4Annu Rev Biochem.Author manuscript;available in PMC 2018 June 20.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptbinding,is essential for its recruitment to the sites of phagophore initiation in human cells(62).This suggests that the principles for recruitment are similar in this respect in yeast and mammals.A number of other protein-protein interactions influence ULK1 localization to the site of phagophore initiation.The LC3 family proteins bind to LIR/AIM motifs in both human and yeast ULK1/Atg1(52,63)and in human ATG13(63).LC3 conjugation is thought to occur downstream of ULK1 activation,however,and the LIR/AIM motifs seem likely to be involved in later events in autophagosome biogenesis.The yeast Atg1 complex binds to Atg9 through a direct interaction between the HORMA domain of Atg13 and the N-terminal soluble domain of Atg9(64),and perhaps also through Atg17(48,65).Yeast Rab1(Ypt1)is a small G-protein better known as a regulator of ER-Golgi and intra-Golgi traffic,binds to Atg1 and helps recruit it to the PAS(66).Ypt1 is activated and recruited to the PAS by the TRAPPIII complex,which binds to Atg9(67)and Atg17(66).TRAPPIII is also implicated in mammalian autophagy(68,69).C9orf72 is a protein that is mutated in the most common hereditary forms of amyotrophic lateral sclerosis(ALS)and frontotemporal dementia(FTD).C9orf72 was very recently shown to be important for the RAB1A dependent recruitment of ULK1 to the sites of phagophore initiation in human cells(70).C9orf72 contains a DENN domain,and in many cases DENN domain proteins act as RAB guanine nucleotide exchange factors(GEFs).Apparently the C9orf72 DENN binds to RAB1A but lacks GEF activity,making C9orf72 a RAB1A effector rather than a RAB1A GEF.It has been proposed that impairment of ULK1 recruitment to autophagy initiation sites in C9orf72 m