Systematically defining selective autophagy receptor-specific cargo using autophagosome content profiling

•Protease protection coupled APEX2 proximity proteomics of autophagosomes•Identification selective autophagy receptor-dependent cargo•Ubl conjugation and protein aggregation only modestly drive cargo engulfment•The aggrephagy receptor TOLLIP is implicated in endosomal microautophagy Autophagy deficiency fed conditions leads to the formation inclusions highlighting contribution this lysosomal delivery route cellular proteostasis. Selective pathways exist that clear accumulated aggregated ubiquitinated proteins. Receptors for type (aggrephagy) include p62, NBR1, TOLLIP, OPTN, which possess LC3-interacting regions ubiquitin-binding domains (UBDs), thus working as a bridge between LC3/GABARAP proteins substrates. However, identity substrates redundancy related UBD-containing receptors remains elusive. Here, we combined labeling organelle enrichment with quantitative systematically map autophagic degradome targeted by under basal proteostasis-challenging human cell lines. We identified various substrates, some were differentially engulfed autophagosomal membranes via p62 respectively. Overall, resource will allow dissection proteostasis numerous individual Macroautophagy (hereafter autophagy) conserved degradation pathway. A broad range cytosolic constituents, including invading pathogens, damaged organelles or parts thereof, proteins, other macromolecules, are enclosed double-membraned vesicles, called autophagosomes, degraded upon their fusion lysosomes (Mizushima Komatsu, 2011Mizushima N. Komatsu M. Autophagy: renovation cells tissues.Cell. 2011; 147: 728-741Abstract Full Text PDF PubMed Scopus (3464) Google Scholar). Thereby, essentially contributes homeostasis organelles, metabolites well innate immunity. hallmark ubiquitin (Ub)-like ATG8 (hATG8) family members (MAP1LC3A, MAP1LC3B [LC3B], MAP1LC3B2, MAP1LC3C, GABARAP, GABRAPBL1, GABARAPL2) phosphatidylethanolamine (PE) inside outside incipient autophagosomes et al., Yoshimori T. Ohsumi Y. The role Atg autophagosome formation.Annu. Rev. Cell Dev. Biol. 27: 107-132Crossref (1953) major function hATG8-PE serve docking site regulatory components on concave forming (Birgisdottir 2013Birgisdottir A.B. Lamark Johansen LIR motif - crucial autophagy.J. Sci. 2013; 126: 3237-3247Crossref (485) These recognize distinct tether it engulfing (Stolz 2014Stolz A. Ernst Dikic I. Cargo recognition trafficking autophagy.Nat. 2014; 16: 495-501Crossref (717) Selectivity conferred 2 defining found all receptors: an LC3-interaction region (LIR) bind cargo-binding element can either be transmembrane (TM) segments protein-protein interaction scaffolds such Ub-binding (UBDs) (Rogov 2014Rogov V. Dötsch Kirkin Interactions ubiquitin-like form molecular basis autophagy.Mol. Cell. 53: 167-178Abstract (614) Through UBDs, bacteria, mitochondria, aggregates, modified Ub (Kirkin 2009bKirkin McEwan D.G. Novak 2009; 34: 259-269Abstract (943) Notably, engulfment also occur independent ubiquitin, through direct adaptor-mediated binding substrate (Jain 2010Jain Sjøttem E. Larsen K.B. Awuh J.A. Øvervatn McMahon Hayes J.D. p62/SQSTM1 target gene transcription factor NRF2 creates positive feedback loop inducing antioxidant response element-driven transcription.J. Chem. 2010; 285: 22576-22591Abstract (876) Scholar; 2010Komatsu Kurokawa H. Waguri S. Taguchi K. Kobayashi Ichimura Sou Y.S. Ueno Sakamoto Tong K.I. al.The activates stress responsive Nrf2 inactivation Keap1.Nat. 12: 213-223Crossref (1446) Scholar) vesicles (Maejima 2013Maejima Takahashi Omori Kimura Takabatake Saitoh Yamamoto Hamasaki Noda Isaka sequesters control biogenesis kidney injury.EMBO J. 32: 2336-2347Crossref (279) Princely Abudu 2019Princely Pankiv Mathai B.J. Håkon Lystad Bindesbøll C. Brenne H.B. Yoke Wui Ng Thiede B. Mutugi Nthiga al.NIPSNAP1 NIPSNAP2 Act “Eat Me” Signals Mitophagy.Dev. 2019; 49: 509-525.e12Abstract (55) Scholar), Due similarities, 6 soluble grouped together SQSTM1-like (SLRs) (Deretic, 2012Deretic immunity paradigm: expanding scope repertoire pattern receptors.Curr. Opin. Immunol. 2012; 24: 21-31Crossref (216) founding member SQSTM1 (alias p62) highly similar NBR1. Both harbor so-called canonical motifs core sequence [W/F/Y]0-X1-X2-[L/V/I]3 2009aKirkin Bjørkøy G. Nunn J.L. Bruun Shvets Clausen T.H. Wild P. al.A NBR1 substrates.Mol. 33: 505-516Abstract (790) 2007Pankiv Brech Outzen binds directly Atg8/LC3 facilitate aggregates 2007; 282: 24131-24145Abstract (3067) interact PB1 (Lamark 2003Lamark Perander Kristiansen Michaelsen Interaction codes within mammalian Phox Bem1p domain-containing proteins.J. 2003; 278: 34568-34581Abstract They share Ub-associated domain (UBA) help peroxisomes (Deosaran 2013Deosaran Hua R. Sargent Wang Kim Jauregui Law Lippincott-Schwartz al.NBR1 acts peroxisomes.J. 939-952Crossref (211) 2008Kim P.K. Hailey D.W. Mullen R.T. Ubiquitin signals peroxisomes.Proc. Natl. Acad. USA. 2008; 105: 20567-20574Crossref (409) NDP52 CALCOCO2) TAX1BP1 have architecture containing atypical c-type LIRs zinc fingers (ZFs) (Fu 2018Fu Liu Xie X. Hu Pan L. Mechanistic insights into interactions NAP1 SKICH TAX1BP1.Proc. 2018; 115: E11651-E11660Crossref (18) eliminate Heo 2015Heo J.M. Ordureau Paulo Rinehart Harper J.W. PINK1-PARKIN Mitochondrial Ubiquitylation Pathway Drives Program OPTN/NDP52 Recruitment TBK1 Activation Promote Mitophagy.Mol. 2015; 60: 7-20Abstract (445) Lazarou 2015Lazarou Sliter D.A. Kane L.A. Sarraf S.A. Burman Sideris D.P. Fogel A.I. Youle R.J. kinase PINK1 recruits induce mitophagy.Nature. 524: 309-314Crossref (1284) 2020Sarraf Shah H.V. Kanfer Pickrell A.M. Holtzclaw Ward M.E. Loss TAX1BP1-Directed Results Protein Aggregate Accumulation Brain.Mol. 2020; 80: 779-795.e10Abstract (20) von Muhlinen 2012von Akutsu Ravenhill Foeglein Á. Bloor Rutherford T.J. Freund S.M. Komander D. Randow F. LC3C, bound selectively noncanonical NDP52, required antibacterial 48: 329-342Abstract (204) serves SLR mitophagy, xenophagy, its phosphorylation (Heo carries (Wild 2011Wild Farhan Wagner Rogov V.V. Brady N.R. Richter Korac Waidmann O. Choudhary al.Phosphorylation optineurin restricts Salmonella growth.Science. 333: 228-233Crossref UBDs: ZF UBD ABIN NEMO (UBAN) 2013Korac Schaeffer Kovacevic Clement Jungblut Behl Terzic Ubiquitin-independent clearance aggregates.J. 580-592Crossref (203) Lastly, contains CUE putative (Lu 2014Lu Psakhye Jentsch Autophagic polyQ mediated ubiquitin-Atg8 adaptors CUET family.Cell. 158: 549-563Abstract (214) poorly defined. While SLRs associated these largely Furthermore, do not know whether exclusively challenges unperturbed conditions. Finally, degrading different unexplored. To address questions, inventory SLR-specific manner proteostasis-disturbing approach manipulation regulators complementarily performed neighborhood organellar further explore therewith potential new across functions compartments. Using biochemical biological methods, validated number candidates bona fide subsequently demonstrated In addition, our profiling revealed small fraction depends hATG8 membrane majority unaffected perturbations, while substantial amount additional pathway identify SLRs, generated panel HeLa expressing TAX1BP1, N-terminal engineered peroxidase (Hung 2016Hung Udeshi N.D. Lam S.S. Loh K.H. Cox K.J. Pedram Carr Ting A.Y. Spatially resolved proteomic mapping living APEX2.Nat. Protoc. 2016; 11: 456-475Crossref (186) myc epitope tag (Figures 1A S1A). myc-APEX2-LC3B-expressing used controls. Treatment biotin-phenol (30 min) subsequent pulsing H2O2 (1 triggers biotinylation fusions. specifically enrich biotinylated baits following BafilomycinA1 (BafA1) treatment, incubated homogenates proteinase K, digests protected intact (Figure 1A). Immunoblotting confirmed fusions, endogenous partly from TM ATG9A, harbors antibody epitope, was 1B). Only when protective lysed Triton X-100 did K completely homogenates. Confocal microscopy pairwise localization baits, LC3, biotin-positive puncta, turn partially colocalized marker LAMP1 BafA1 treatment 1C S1B). Moreover, fusions generate electron (EM) contrast structures BafA1-treated subjected post-fixation 3,3-diaminobenzidine (DAB) 2A). results indicate APEX2-SLR chimeras where they biotinylate destined degradation.Figure 2Identification protease-protected SLRsShow full caption(A) Electron micrographs APEX2-SLRs -LC3B cells. Following fixation, DAB before standard embedding ultrathin sectioning. Scale bars, 1 ?m.(B) Workflow APEX2-based content profiling.(C) Pearson’s correlation 3 experiments depicted (B).(D) Bar graph membrane-protected streptavidin-enriched indicated DMSO whose abundance increased marked orange sensitive.(E F) Volcano plots (D) labeled APEX2-LC3B (E) APEX-SLRs (F). significantly enriched highlighted (p ? 0.05, Student’s t test). Known shown italics.See Figure S2.View Large Image ViewerDownload Hi-res image Download (PPT) (A) ?m. (B) profiling. (C) (B). sensitive. (E italics. See S2. Before lines, improved throughput implementing tryptic in-solution processing mass spectrometry (MS) samples. specificity treated presence membrane-lysing detergent, substituted MS-compatible RAPIGest, had no effect S2A). test protocol autophagy-unrelated protein, chose TMEM106B, C terminus predominantly locates endosomes lysosomes. C-terminally APEX2-tagged TMEM106B S2B) them labeling. TMEM106B-APEX2 concomitant S2C), markers EEA1 LAMP1, respectively S2D). For TMEM106B+ compared empty TMEM106B-APEX2-expressing BafA1/Torin1. Upon labeling, homogenized followed digestion absence RAPIGest. Proteolyzed material cleared centrifugation samples denaturing lysis, streptavidin pull-down, digest, MS analysis S2E). coefficients minimum 0.97 high reproducibility triplicates S2F). excluding protease-resistant (found RAPIGest-treated sample; Table S1), statistical total 262 luminal S2G; S1). Gene Ontology (GO) shows prominent terms reflecting S2H). findings provide confirmation allows robust identification transient endomembrane vesicles. Next, -LC3B-expressing grown same procedure described 2B). As control, included APEX2-LC3B-expressing Therewith, 181 contaminants excluded (Table With good 2C), detected 500 900 K-protected S2I). Statistical 279 non-redundant BafA1-sensitive (68 LC3B, 88 122 148 19 51 57 TOLLIP) 2D–2F; defined basal, housekeeping autophagy. Among those profiled hATG8s, receptors, known (ULK1, RB1CC1), established (KEAP1, FTL), but several candidates. Intriguingly, already been (TBK1, TNFAIP3, VCP, TBC1D15, UBQLN2, VAPA, VAPB, PRKCI), others linked (PRKAR1A) 2F). contrast, unveiled quite Apart autophagy-related TMEM59. Consistent coordinating compartment (Jongsma 2016Jongsma M.L. Berlin Wijdeven R.H. Janssen G.M. Garstka M.A. Mensink van Veelen P.A. Spaapen R.M. Neefjes An ER-Associated Defines Endosomal Architecture Controlled Transport.Cell. 166: 152-166Abstract (101) mediating endocytic ubiquitylated cargoes (Katoh 2004Katoh Shiba Mitsuhashi Yanagida Takatsu Nakayama Tollip Tom1 complex recruit ubiquitin-conjugated onto early endosomes.J. 2004; 279: 24435-24443Abstract (108) Xiao 2015Xiao Brannon M.K. Zhao Fread Ellena J.F. Bushweller J.H. Finkielstein C.V. Armstrong G.S. Capelluto D.G.S. Modulates Binding Phosphatidylinositol 3-Phosphate Coupled Folding Mechanism.Structure. 23: 1910-1920Abstract (17) Yamakami 2003Yamakami Yokosawa Tom1, VHS interacts tollip, clathrin.J. 52865-52872Abstract (103) involved processes (PDCD6IP, PDCD6, HGS, STAM, ITCH, RAB5C). none factors endomembranes. links (SCAMP3, TFRC, NDFIP1, SLC2A1, ATP1A1, LPL) quality datasets. First, current earlier version (Le Guerroué 2017Le Eck Jung Starzetz Mittelbronn Kaulich Behrends Autophagosomal Content Profiling Reveals LC3C-Dependent Piecemeal Mitophagy Pathway.Mol. 2017; 68: 786-796.e6Abstract (56) considerable overlap 15 whereas 53–56 uniquely workflows. Importantly, far larger method (16 versus 3) S3A). Second, very few overlapped recent BirA-LC3B MS-based secretory (Leidal 2020Leidal Huang H.H. Marsh Solvik Zhang Ye Kai Goldsmith J.Y. Y.H. LC3-conjugation machinery specifies loading RNA-binding extracellular vesicles.Nat. 22: 187-199Crossref (115) S3B), suggesting mainly capture en Closer inspection each except OPTN shared at least smaller portion single bait. ratio unique about 3A). Examination relative trend. average candidates, lesser extent showed biggest 3B). Autophagy-related 4 GABARAPL2, ferritino- ER-phagy NCOA4 CCPG1, FASN, FTL, KEAP1, TBK1, CLTC, RAB1B, RAB5C, RAB7A S3C). Remarkably, experiments. Analysis topology large APEX2-p62, -NBR1, -NDP52, represented peripheral (PMPs) 3C). APEX2-OPTN, -TAX1BP1, -TOLLIP, (TMPs) among expense GO adherens-, ATP/GTP-, folding-related besides expected associations 3D). Literature curation involvement even broader annotation 3E). Given dataset minimal survey exosomal secretion caught attention Manual (SDCBP, PDCD6IP, SDC4, ATP1A1) actually part (ITCH, SDC2) association S3E). LC3B GTPases This group represents intriguing many processes, maturation nutrient signaling, mitochondrial cytoskeletal dynamics, regulated GTP-dependent manner. Since occurred asked bait engulfment. this, second-round without S4A). experiment carried out BafA1. served correlations above 0.9 replicate S4B). stringent filtering against controls BafA1-dependent changes, betwee