A Mutant O-Glcnacase Enriches Drosophila Developmental Regulators

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. O C D 1 aggregation, its prevents Ph of O-GlcNAcylation that shown been O-GlcNAcylated be to known protein (PcG) group comb Drosophila in (PREs) elements responsive thatshown havements employingStudieschromatinimmunoprecipitation experi (ChIP) cuticle embryonic the transformationshomeoticshowin and esis OGT/sxc homeotictransformations and transcripts, die at the late pupal pharate adult stage with distinct of sion fly fruit the is development metazoan and O-GlcNAcylation protein between pinning remain this largely unclear. animals in development onic by extension protein O-GlcNAcylation, has a critical role in embry OGT,demonstratedthat and been has approaches,it genetic using disorders neurodegenerative and disease vascular cardio cancer, diabetes, including pathologies a human of to number linked been have profiles O-GlcNAcylation protein specific responsestressdegradation, and trafficking proteinexpression, gene including process cellular stimuli cellular and statusnutrient alterations in responsiveto is that modification ible revers and dynamic pathway.a thetic O-GlcNAcylationProteinis biosyn hexosamine the through fructose-6-phosphate metabolite diphosphate (UDP)-GlcNAc, which is produced from the glycolytic OGA of nucleocytoplasmic proteins regulated by and two enzymes, OGT function duringembryonic development. developing the the enriched havewetrap, substrate a as mutant O-GlcNAcase bacterial inactive catalytically a Using tools. enrichment limited by and modification this of stoichiometry O-GlcNAcylated low the by of hampered is Identification samples biological understood. from proteins poorly are development embryonic to phenotypes these linking mechanisms underlying the and involvedproteins O-GlcNAcylated the of However,identities stage. the adult pharate the at die and tions gaster embryogenesis.The early for essential is O-GlcNAcylation protein revealedhavethat organisms model in It is cycled by the enzymes O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (O-GlcNAcase or OGA). Genetic approaches Protein O-GlcNAcylation is a reversible post-translational modification of serines and threonines on nucleocytoplasmic proteins. & A N developmental regulators A nature CH U U MR ell andMolecular K (R.W.). niversity ofGeorgia, Athens, Georgia, undee, ndrew ithya D An attractive model organism to begin to dissect the links links the dissect to begin to organism model attractive An C mutantO-Glc

aan MFvan 1 P . The donor substrate for protein O-GlcNAcylation is uridine rotein gene dues on target proteins, is a post-translational modification post-translational a proteins,is target on dues O-linked single ( acetylglucosamine a of addition the -GlcNAcylation, D OGT/sxc undergo developmental arrest at the end of embryogen of end the at arrest developmental undergo undee, E S 6 MIC T 7 elvan , T 8 hese authors contributed equally to thiswork. *e-mail: Drosophilamelanogaster P . The transcription factor polyhomeotic (Ph) is a poly a is (Ph)polyhomeotictranscription factorThe . supersex combs supersex Ferenbach hosphorylation and A L BIOLOGY U bt ean aenly otiue OT protein OGT contributed maternally retain but , Drosophila B K. iosciences ( 1 , 3 1 5 D and has been implicated in a broad range of range broad a in implicated been has and , 6 ivision of A ,

O R | Adv alten -GlcNAc is highly enriched at polycomb-at enriched -GlcNAchighly is itchie Williamson 3 . Flies lacking both zygoticlacking Flies . both and maternal O 1 1 embryo, identifying, among others, known regulators of regulators known others, among identifying, embryo, a and autophagy , -GlcNAc) to serine or threonine resi threonine or serine to -GlcNAc) IC 2 3– nce online public ( B , aM 6 U 1 iological sxc ; however, the mechanisms under mechanisms the however, ; T , 2 biquitylation Hox onia * B ), which encodes OGT, is a polycomb gene, whose null mutants display homeotic transforma homeotic display mutants null whose gene, polycombOGT, encodesa which is ), U ), . Flies that lack zygotic expreszygotic lack that Flies . SA ( N n ohr ee lses in clusters gene other and

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rug P harmacy andMedical Sciences, Faculty of , N 3 D D , Iva Hopkins- ewcastle-upon- 7 iscovery, School of aniel Mariappa I has It . [email protected] D β undee, - N 3 ------. D undee, number of limitations associated with these enrichment methods. methods. enrichment these with associated limitations of number mappingsiteO-GlcNAcylatedhowever,forare, substrates.There a anti- (WGA) agglutinin germ wheat chromatography affinity weak lectin and/orsuchas peptides, using labile which in fragmentation, (ETD) dissociation fer O-GlcNAcylated of mapping ref. in site (reviewed proteins and enrichment the for used andchemoenzymatic andlabelingapproachesmetabolic have been by substrates modified of Derivatization MS. using identified be can O fragmentation backbone peptide (CID) dissociation collision-induced standard pered by the fact that the proteins from O-GlcNAcylatedreporting study single a only is there tissues, and cells mammalian in proteinsO-GlcNAcylated of identification the on focused have studies proteomics many Although phenotypes. understanding of the mechanisms responsible for the the proteinsaid to modified the proteomics-basedidentificationof duringdynamic is Hox involved processes in addition in several in fly the to Ph-dependent autophagy development during stress temperature to homeostasis glucose-insulin like association the of have described of phenotypes opmental of the protein orderedassemblies functional, formationof the for required is and T -GlcNAc, enrichment of proteinsmodified is required before they pu yne, Identificationofnative O-GlcNAcylated proteins hamisby MS β gene repression. Our discovery that protein O-GlcNAcylationproteinthat discovery repression. Our gene -elimination followed by Michael addition of DTT (BEMAD) DTT of addition Michael by followed -elimination bli O 1 L 8 U N -GlcNAc antibody CTD110.6 (ref. CTD110.6 antibody-GlcNAc , strategies for the capture of native O-GlcNAcylated proteins U ife Sciences, s K. K. h avratilova 1 2 ed 1 5 , D 5 2 P I i teeoe la ta poen -lNclto is O-GlcNAcylation protein that clear therefore is It . , ivision ofGeneRegulation and resent addresses: 6

9 o , . Drosophila 1 OGT/sxc n 8 D . Additionally, given the substoichiometric nature of li avid GCampbell ne: 12June2017 | U Drosophila niversity of Drosophila Hox 3 L , Matthias -null mutants recapitulate some of the devel ife Sciences, S2cells, with no site assignments genes as candidate conveyors of OGTconveyors of candidate as genes O Ph 1 -GlcNAc moiety is labile and lost during 8 nl mutants -null C ). With the advent of electron trans electron of advent the With ). embryogenesis omplex D 1 undee, 9 or immunoprecipitation with the the immunoprecipitationwith or O 1 1 crain rhythm circadian ,

-GlcNAccellularwith processes U d C niversity of o T D arbohydrate Research i : rost 1 undee, E

xpression, 0 2 O 1 . 1 0 , 0 1 0 . Other studies in flies flies in studies Other . -GlcNAc proteome of proteome -GlcNAc 3 ), have been employed been have ), article R FF signaling FGF , 1 3 1 , 6 4 8 U Drosophila melano Drosophila obert Gourlayobert led us to pursue the the pursueto us led

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© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. article ing downstream of the IL-1 receptor IL-1 the of downstream ing signal immune innate modulate to demonstrated been previously 1 Fig. Supplementary (TAB1; 1 protein binding factor growth transforming recombinant or unmodified with incubated and beads (Promega) HaloLink to coupled covalently was control negative a Halo-tagged O-GlcNAcylated proteins. down pull to trap substrate a as it using of feasibility the evaluate and human both O-GlcNAcylatedof detection proteins specific in probeforthe a as Cp that shown previously K in 4-methylumbelliferyl-GlcNAcincrease(4MU-GlcNAc; five-fold D401A mutant demonstrated loss of binding to the model substrate type enzyme), with negligible effect on the substrate impaired lytically in (8,100-fold decrease hydroxyl groups ( O6 and O4 its through site active the in GlcNAcmoiety anchor the of ing the for required bonding hydrogen in involved being as identified was hOGA) in Asp285 to (equivalentAsp401 and bond, glycosidic protonates the that acid catalytic the being as identified ysis number of conserved amino acids in the active site involved in catal enzyme bacterial the using OGA, of mechanism catalytic the of elucidation the on work earlier Our A tool for theenrichmentofO-GlcNAcylated proteins RE null phenotype. notypes as candidate conveyors of the phenonhomeoticand homeotic to links established proteinswith development.Wepreviouslyunknownofrange a reveal to identify the first O-GlcNAcylatedofproteins from that strate for used ofdetection the be peptides ( ( enzyme this of mutant inactive an that demonstrated possesses Werecently and (hOGA) OGA remarkableonhumanactivitycatalytic O-GlcNAcylated proteins human with similarity sequence OGAs, otic proteins/peptides. O-GlcNAcylated of enrichment native the for strategies novel for all recognize not does 110.6 CTD specificity, its of regardless that, possible is it II, polymerase against immunogen a specific from domain C-terminal the of RNA raised is Additionally,bait.it thata given as nonspecific somewhat to recognize terminal GlcNAc residues in other glycans anti- for affinity improved much possesses it Although GlcNAcfor affinity millimolar its chromatographyis affinity get glycans and other chemical groups. The drawback of using WGA which ods, can tolead the derivatization and enrichment of off-tar meth labeling metabolic and chemoenzymatic of true also is This identifications. false-positive eliminate to required are treatment appropriateof use conditions the phosphataseandcontrols as such reaction of optimizationrigorous and BEMAD, to susceptible also to addition In 2 Methods). Online sample with (see buffer mutant the from Cp

M OGA and 2,400-fold decrease in and2,400-fold decrease To this end, we first outcarried a proof-of-principle experiment. eukary the of ortholog bacterial a that We observed previously SU 2 OGA 3 O . In particular, Asp298 (equivalent to Asp175 in hOGA) was hOGA) in Asp175 to (equivalent Asp298 particular, In . L -GlcNAc antibody CTD 110.6, like WGA, has been shown been has WGA, like 110.6, CTD antibody -GlcNAc TS D401A D298N Fig. 1 Cp or the double mutant double the or lsrdu perfringens Clostridium Cp ), which retains which ), ability the to bind to O-GlcNAcylated OGA O a OGA ) with affinities down to the nanomolar range, could -GlcNAc, Cp Fig. 1 Drosophila O D298N OGA beads was achieved by boiling the beads beads the boiling by achieved was beads OGA -GlcNAc proteome associated with embryonic D298N Cp a is a powerful new tool for the enrichment the for tool new powerful a is , poen hs OGcAyain has O-GlcNAcylation whose protein a ), ). The D298N mutant of or the double mutant OGA O O i. 1 Fig. cell and tissue lysates tissue and cell -phosphate groups and groups -phosphate O -GlcNAcproteins k -GlcNAc sites. There, is thus a need need a thus is There, -GlcNAcsites. D298N cat comparedtype) wild to b Drosophila Cp (but not the binding-deficient the not (but and and Cp aJ ( NagJ 2 OGA as a model, revealed a revealed model, a as OGA Drosophila OGT/sxc 4 OGA . Elution of enriched TAB1 enriched of Elution . k upeetr Results Supplementary in vitro in Cp cat compared wild- the to D298N,D401A Cp β OGA embryos and use MS use and embryos atvtd iae 1 kinase -activated 1 G) sae 51% shares OGA), Cp 6 . Here,. we demon Cp O-GlcNAcylated OGA D298N 1 K 6 O OGA was OGA cata , we wanted to wanted we , M ) can be used used be can ) O -GlcNAc, the the -GlcNAc, nature , whereas the , butnot, the -glycans are -glycans 2 2 D298N,D401A , making it O 2 3 -GlcNAc catalytic . Having. ch 18 , 19 e N as as mic , 2 2 3 1 ------. . , atu a l

T hydrolyze Glc as ayellow withGlc cartoon, mutant trap for theenrichmentofO-GlcNAcylated proteins. Figure 1|Apointmutantof ments to determine differences between the binding to GlcNAc and mM) 2.7 used: tide 36 of affinity an with MSAN) binds to an (SYLN(GlcNAc) D cathepsin VTR) from derived peptide containing the of mM 2.5 to up when observed was binding able an an containing peptide backbone short peptide a sugar in the of conformation the that appears It D. cathepsin from derived peptide synthetic N-GlcNAcylated an described previously we assay polarization fluorescence a endo to bind by far-western blotting using obtained signal of tion (PNGase F) treatment of lysates does not result in any visible altera lysatesof of detector specific a specificity.It fromevidentourisprevious work that teins from cell/tissue lysates, we wished to further dissect its substrate proteins from more complex samples such as cell/tissue lysates. O-GlcNAcylatedof enrichment the forsuitable be might it that ing therefore sufficient for it to pull down the modified substrate, suggest ( an O-GlcNAcylatedin TAB1, occurred downbutunmodified not pull showing the that down pulling in successful was mutant, double Cp presence ofmodifiedbutnotunmodified manner by beads withsamplebuffer. with theantibodiesmentioned. down. mutant covalently to Halo O proteins andtherefore cannotactasasubstrate trap. ( biology i. 1 Fig. he doublemutant -Glc O re c Prior to applying O N GA - GcA-pcfc and -GlcNAc-specific -GlcNAc moiety affects affects moiety -GlcNAc N β 2 I 5 nput, flow-through andelutionfractions were blotted andprobed D Acylated b Cp C - was used ( used was

298 N | Adv p . h afnt of affinity The ). N (kDa) O O h MW b a Drosophila -acetylgucosaminidase (ENGase) activity, we performed performed we activity, (ENGase) -acetylgucosaminidase 55 55 N Cp 45 45 (binding) -GlcNAc moieties in lysates resulting from endogenous from resulting lysates in moieties -GlcNAc . GA GA D401 O IN em N O a -GlcNAc peptide derived from dHCF (VPST(GlcNAc) Unmodiﬁed Ac, therefore trapping nce online public , input;F D GA D 298 298

T IN T L AB1 A i N D N ink beads. Cp Cp c canbindto substrate proteins (substrate shown 298 ,

B FT Supplementary Fig. 2 D pull down Cp 401A OGA 1 was incubated withHalo- a O N Cp EL (no hydrolysis) S2 cells and embryos; peptide peptide andembryos; cells S2 OGA 1 butnotthecontrol probe asevidenced by the 6 GA T was includedto test thespecificity ofthepull l b Srae lso rsnne SR experi (SPR) resonance plasmon Surface . O , flow through; D298N 1 OGA D298N 6 T IN -GlcNAc in HEK293 cell lysates as well in well as lysates cell HEK293 in -GlcNAc D298N . To investigate whether D A O-GlcNAcylated 298 Cp N

B FT io P Cp 1 was pulleddown inan Ac depicted withpinksticks) butcannot N ull down usingthebinding-deficient OGA canbeexploited asasubstrate Cp E , D298N T D EL AB1 lutions were performed by boiling the OGA 401A l a Cp (no binding) Cp G-ciest-eedn manner OGA-active-site-dependent tion | og μ Cp IN D401A cannotbind to enrich for O-GlcNAcylated pro OGA M (highest concentration of pep of concentration (highest M OGA OGA O FT O D298N pull down -Glc EL y www.nature.com/naturechemicalbiology GcA mit cmae to compared moiety -GlcNAc T EL D298N, D401A , eluate; W A D298N

d D298N for glycosylated TAB1 was was TAB1 glycosylated for N Cp B Unmodiﬁed

IN o ). By contrast, 1 intheelutionfractions from Ac-modified proteins. OGA i TA : as a probe for detection for probe a as (no hydrolysis) FT idn, s o detect no as binding, Cp 1 O B1 0 D298N,D401A . D298N -Glc EL O 1 0 GA B 3 Cp ( WB: TAB1 RL2 WB: b O , western blot. 8 N a ) -Glc D / ) Acylated OGA 298 U n N O T Cp nmodified or c -GlcNAc he inactive -glycosidase F -glycosidase N N coupled h Cp OGA Ac-specific e N D298N m -GlcNAc- OGA b 1 6 i o , using , would D298N . 2 D298N 4 0 is is 4 ------

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. N OGT substrates, which are then chemoselectively enriched. Forty- enriched. chemoselectively then are which substrates, OGT of labeling metabolic the for used UDP-GlcNAcis of analog fied azide-modi cell-permeable a whereby approach, (TAS) substrate O study published previously a contrast, By method. wereproteinsamongenrichment the the thusvalidating identified, and H4 (ref. fide O-GlcNAcylated substrates, such as the histones H2A, H2B, H3 trol enrichment; (four-foldP enriched significantly were 859 which eluates, HeLaof the from identified were accessions protein 915 of downspull totalwere A from these MS. processedandtosubjected formed, including negative controls per using were downs pull replicate independent three enriched, teins for complexmethod samples. To O-GlcNAcylated the identify pro 5 Figs. the usingin blot (representative samples antibody of RL2 blotting western by visualized as proteins, O-GlcNAcylated of enrichment qualitative overall an in resulted the with performed thatnotbut Supplementary Fig. 4 inactive the to binding molar a G with Thiamet inhibitor OGA displacement the of by excess achieved was elution site, active the by specifically O-GlcNAcylatedcapturedproteins ( beads HaloLink to mutant control Halo-tagged with °C 4 at min 90 for incubated pull downs were also performed from HeLa cell lysates. Lysates were lished enrichment methods applied to lysates of a single cell line structures and would therefore preferentially bind to have poor affinity for terminal GlcNAc moieties on extended glycan ( (29 affinity lower 20-fold a with latter GlcNAc( to that component analyses ofproteins identifiedby O but notthecontrol mutant MS. ( proteins were concentrated usingaspinconcentrator andprocessed for achieved by usingamolarexcess ofthe down Cp ( Figure 2|Pull down ofO-GlcNAcylated proteins by nature CH were us by presentalso in that study identified proteins enriched significantly the of nine a Supplementary Fig. 3 Fig. Supplementary a Protein < 0.05) in the -Glc -GlcNAc modified proteins from HeLa cells using a tagging via via tagging a using cells HeLa from proteins modified -GlcNAc ) Schematicofthe atu O Fusion To determine how the substrate trap compares to previously pub tag GA mutantscovalently coupled to Halo Cp b G N O ) Protein OGA Acylated proteins detected intheelutionfractions. ( -Glc , P D298N Cp 6 ull down from re c Cp D298N , ugsig ht hs prah s sial enrichment suitable a is approach this that suggesting ), OGA E G OGA Eluted Substrate MIC Immobilized trapping N 2 D298N,D401A Acylated proteins. 9 A ); c-Rel L BIOLOGY Cp h Cp β em 1-4)GlcNAc revealed that revealed 1-4)GlcNAc OGA Cp Afﬁnity OGA Halo- resin b Fig. 2 Fig. Pull down O pull down ( Drosophila MW (kDa) 3 ). The pull down performed with 0 i GA ; CREB Cp ), suggesting that the mutantproteinwould the that suggesting ), D298N c Cp

Cp | Adv D298N,D401A OGA 2 a 95 55 75 170 130 7 D 44 26 35 ( O a 298 OGA mutant pull down compared to the con ). To ensure that the eluents contained eluents the that Toensure ). l b Supplementary Table 1 Supplementary GA x N a : E WB: enrichmentmethod.Halo-tagged – nce online public 3 lution ofproteins from thebeadswas embryo lysates using 1 D D298N ; CK2 298 D298N IN io O Cp (RL2) Supplementary Data Set 1 oaety ope t saturation to coupled covalently -GlcNAc N FT , D l OGA O 401A

mutant (with a (with mutant EL D298N, α og D401A GA inhibitor Fig. 2 Fig.

3 FT , results intheenrichmentof L 2 μ ; TAB1 (ref. ink beadswere usedto pull

Cp EL 2 D298N,D401A M compared to 590 to compared M 8 y Cp ( O c

Fig. 2 Fig. 136 b GA d Cp OGA and and 85 o a tion | Cp OGA D i Cp : 298 T Synapse Cell junction Extracellular matrix Membrane Extracellular region Macromolecular complex Organelle Cell part

1 OGA negativecontrol, hiamet G. 84 0 a D298N,D401A Cp N OGA 2 Supplementary Supplementary ), which retains which ), . . 31 2 7 1 ). We identified identified 199 identified O 0 www.nature.com/naturechemicalbiology D298N 4 K O 205 c 2 3 ); and OGT GA Cp D298N ) d Cp -GlcNAc. 8 of 688 nM; 688 of C 1 D298N / OGA binds the the binds D ellular n OGA 298 .

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. 2 4 the the from used next We E enrichment and identification of alarger number of substrates. for affinity better possesses WGA, unlike Also, method. one-step a is labeling—it in metabolic (for cell lines) or chemoenzymatic (for tissue samples) of tion using enrichment ( study each to unique being substrates of number large a with cells, HEK293 in study that in identified also were cells HeLa from us by identified O mapped study previous a which on lysates cell HEK293 denatured from BEMAD) to coupled approach capture chemoselective and labeling metabolic a (using proteins enriched significantly of 3.8% which on 2 Table ( proteins identi fied 859 the of 29 to mapped which identified, being peptides assignments). site ETD high-confidence 61 of total a in resultedanalyses with These (three analyses MS replicate three high-confidence 550 n sgaig aha ( pathway signaling Wnt the in functioning as (~1.6%) accessions protein mapped 2,044 of ment values are listed in enrich fold with alongapplied)protein multipleclassesfortesting ( overrepresentation significant of classified proteins in our data set and do not display a statistically kinases and phosphatases combined comprise only 5% (~2.5% each) 10%; to compared classes (16% general other in of proteins be than to frequently shown more been O-GlcNAcylated have kinases membranes, and synaptic mouse proteins In classified of T-relatedTaf6,Dp,includeand fkh proteinortholog). 4% (bynCand1, represent factors Transcription ( processing and transport RNA in involved are which of with 2,136 proteinsprotein class recognized hits) 2,044 of proteins 2,136; belonging ofto this out class, most (289 14% with identified, that nucleic acid binding proteins represent the largest protein class cellular GO applied) compartment terms are in detailed testing multiple for correction Bonferroni ( protein cell-junction a as 1 and proteins synaptic as 2 proteins, matrix extracellular or secreted as 116 proteins,membrane as with classified being proteins 84 cytoplasm), and nucleus the in complexes macromolecular andorganelle part, (cell nucleocytoplasmic are hits the of (678) ity major The obtained. were hits component cellular 881 and data, PANTHER,foranalysisgene ontology ofthe (GO) used was which down ( control the to compared down pull mutant were(four-fold;significantly enriched ( identified were ber) dant entries with unique Uniprot accessions contribute to this num A total of 3,558 protein accessions (isoforms of proteins and redun ortholog). Significantly enriched ( enriched Significantly ortholog). modomain containing homeotic protein female sterile (fs(1)h; Brd2 bro the is as present, also is HATEnok putative The present. are HDAC3and HDACsRpd3 the set, data the from absent are selves subunit 55-kDa andPtp4E,among others.Whereas histones them PP2A the include identified phosphatasesprotein while orthologs, Abl and Cdc2 Cdk7, Akt-1, the include identified kinases Protein 0 nrichment of -GlcNAcsites 4 aha aayi (efre uig ATE) dniid 33 identified PANTHER) using (performed analysis Pathway revealed PANTHER) using (performed analysis class Protein by recognized were enriched proteins 2,358 the of 2,044 O Drosophila -GlcNAc proteome responsible for the the for responsible proteome -GlcNAc Supplementary 3 Data Set O ; hs ersns .% f infcnl erce proteins enriched significantly of 3.3% represents this ); -GlcNAc moieties is required before enrichment, unlike unlike enrichment, before required is moieties -GlcNAc O -GlcNAc sites were mapped, and is comparable to the the to comparable is and mapped, were sites-GlcNAc

upeetr Dt St 2 Set Data Supplementary Supplementary Table 3 Table Supplementary 2 Cp 6 O embryo lysates in an attempt to begin to identify identify to begin to attempt an in lysates embryo . Interestingly, 373 significantly enriched proteins enriched Interestingly,significantly . 373 -GlcNAc proteins from Cp OGA OGA Supplementary Data Set 3 Set Data Supplementary i. 2 Fig. O D298N Supplementary Table 5 GcA ppie eune ace in matches sequence peptide -GlcNAc Supplementary Fig. 7 Fig. Supplementary D298N o nih -lNcltd proteins O-GlcNAcylated enrich to c O . infcnl erce ( enriched Significantly ). is n h fc ta n derivatiza no that fact the in lies -GlcNAc, potentially enabling the the enabling potentially -GlcNAc, i. 3 Fig. ). P Supplementary Table 4 < 0.05; Bonferroni correction Bonferroni 0.05; < P < 3.6 × 10 × 3.6 < a P ; < 0.05) in the the in 0.05) < ). The prime advantage of advantage prime The ). upeetr Tbe 5 Table Supplementary Drosophila and and Cp sxc OGA ). Examples of the the of Examples ). . article nl phenotypes. -null −4 ), of which 2,358 which of ), Supplementary Supplementary ) 2 1 . By contrast, By . D298N,D401A Cp Cp embryos O P OGA OGA -GlcNAc Fig. 3 Fig. 0.05; < . pull D298N D298N a ). ). 3 ------

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. article in the identification of a total of 52 high-confidence high-confidence ( 52 peptides of total a of identification the in in shown are peptides HexNAc two 6 Tables Supplementary ( assignments) site ETD with (32 high-confidence 268 of total the the examined next We Identification of huntingtin, for example, its affect interaction with hits like CBP pathogenesis of Huntington’s and Parkinson’s diseases. Mutations in many proteins involved in pathwaysthese are implicatedalso in the ( GTPase Rho by regulation cytoskeletal and apoptosis replication, pathway,DNA proteasome ubiquitin the as pathwayssuch involvedin are set data the in teins prodomino. Other helicase the and CK1 ortholog, mor the Rpd3, HDAC the ortholog), (CBP) protein (CREB-binding Neijre ferase Wnt signaling proteins identified are cadherin-87A, the acetyltrans 4 the in identified were matches sequence peptide HexNAc three ( proteins 43 of total represent themean. indicate theHexcase “s”and“t” ions are annotated. the spectrumwere filtered out.For clarity, onlyc[+1](red) andz[+1](blue) reduced speciesoftheprecursor andneutral losses associated withitin each from H from hostcell factor (H ( Data Set3 Cp identified proteins. host cell factor andnucleoporin153. example Figure 3 | Protein class grouping of proteins identified by b b a c

, O

c Intensity (counts) Intensity (counts) 1,000 1,000 1,400 1,400 1,600 1,200 1,200 ) GA Cp 400 400 800 800 600 600 200 200 m Nucleic acid binding E m / 0 0 xample z / =490.6022Th,MH+1469.7922Da OGA D z 200 300 250 350 =725.9014Th,MH+2,900.5839Da 500 100 298 150 E 600 50 Vt 496.07 619.27 I 0 TD fragmentation spectra for HexNAc modifiedpeptidesfrom z4 were usedasinputfor analysis on 513.27 c5 N c5 upeetr Dt St 4 Set Data Supplementary Hydrolase down versus control pulldown) provided in C S

PD Transferase D298N 583.40 F andnucleoporin153k ETD z5 Transporter 600 KP

758.27 Enzyme modulator z7 800 A pull downs we identified, in three experiments, a experiments, three in identified, we downs pull

fragmentation spectra for Hex Receptor U 844.47 FDLIIKPTVPVVsKPSTTDPIQSSK -nucleoporin153kDaA8JV18_DROME Ts T c7 niprot accessions ofsignificantlyenrichedproteins (in he sequence relevant to eachionisshown; lower 654.47 O

z6 TranscriptionOxidoreductase factor 700 C -GlcNAc proteins linked to development

859.27 Protease z8 upeetr Tbe 7 Table Supplementary F) ( Signaling molecule TS

1,000 Cytoskeletal protein O

1,047.47 z10 Extracellular matrix protein TVP b t IVPSVtASHSLR -hostcellfactorHCF_DROME GcA sts n nihd rtis In proteins. enriched on sites -GlcNAc ) andnucleoporin153( and and 800

817.40 Cell adhesion molecule P N Supplementary Fig. 7 Fig. Supplementary 1,144.47 z11 c6 O Transfer/carrier protein Ac-modified residues. -GlcNAc peptide sequence matches sequence -GlcNAc peptide 1,141.53 1,200 AS 7 Ligase c10 VV ; T famnain pcr for spectra fragmentation ETD ); 888.60 ( D 900

K Membrane trafﬁc protein c7 Supplementary Data Set 4 Set Data Supplementary a m m 1,240.67 a are shown. Signalsofcharged ) 958.33 / / c11 z z z7

P Phosphatase 1,272.53 rotein classes represented by z12 1,400 1,000 ta wr mpe o a on mapped were that )

i. 3b Fig. Calcium-binding protein 975.47

P Defense/immunity protein c8 V A sK NT HS ChaperoneKinase N 1,057.40 1,562.73 z8 Ac-modified peptides H 1,100 z13 1,600 . y otat only contrast, By ). 1,112.60 c S E , c9 ). c R database. Cell-junction protein . hs resulted These ). 1,144.53 O Supplementary 1,630.07 D z9 ne peptide c13 Isomerase ). Interestingly,).

VVP Lyase ata in 1,200 Cp P 1,199.73 Storage protein c10 nature 1,854.93 P Structural protein 1,800 OGA c15 O

1,241.27 Receptor protein L a z10 -GlcNAc

1,300 D298N

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Atx-2 and Iswi. includingdevelopment, system nervous in involved being as sified and wings to haltere-like structures legs prothoracic to antennae of transformation homeotic the ple, exam (for observed phenotypes the given flies, mutantOGT-null in affected clearly process a morphogenesis, and development age append with associated specifically those among LanA) and tay Gug, (if, four with morphogenesis, and development structure cal anatomi in involved being as mapped proteins 42 the of 19 rizes catego proteins O-GlcNAcylated these with associated processes understood. GO analysis using STRING to determine yet the biological not is proteins these on modification the of role the although organisms, other O-GlcNAcylatedin be to shown been previously reviewed (Atx2) recently (Nups; proteins pore nuclear the includes also set data Our evolution.across conserved are these of many and study, this in identified proteins O-GlcNAcylated fide bona of examples are UBA-domain-containingprotein) (lig; gerer lin domain-conand ortholog), (HDPTP)phosphatase proteintyrosine taining histidine (mop; myopic ortholog), atrophin Grunge (Gug; protein), (AUTS2-like Tay proteins. nucleocytoplasmic ments ( Cp either homozygous or heterozygous or homozygous either alleles lethal Recessive mop.(P element insertion) and Gug of modification of consequence decreased therefore and activity catalytic a OGT reduced was observed we interaction genetic potential any thatA.T.F. ensuredD.M.F.v.A., This and results). unpublished as allele, hypomorphic lytic cata OGT an used We performed. were experiments interaction genetic function, their mop,substrates,andGug affects OGT these respectiveaffect downstream signaling. Toll oogenesis during integrins lysosomes to receptor EGF ubiquitylated of transit for essential be to found in spectrum 8b Fig. fragmentation EThcD antibody; anti- an using modified being as verified orthogonally identified other the development.ofembryonicmultiple during Oneoutliningitsroles skipped signaling receptor EGF of ulation teashirt other the regulatorsofone offind to designed screen a in identified was that HexNAc peptides identified). Gug is a nuclear receptor co-repressor ( method 8a Fig. orthogonal an by status itsmodification confirming thereby antibody, control isotype an not but RL2, anti- the using lysates embryo from obtained immunoprecipitates in Gug from peptides O-GlcNAcylated tified iden also Wesubstrate. OGT genuine a as Gug establishes study this in confirmationSite-mapping substrates. OGT non-Ph the of of lack the with OGT activity associatedmay be downstream of hypo-O-GlcNAcylation phenotypes of one the of some that sibility being as study this in identified been have molecules cell-signaling and of sion toresponsible be posed for the in OGT of substrates prominent most biology re c OGA sxc Improper high-confidence the of majority The o netgt hw reduced how investigateTo 4 5 , Wnt/Wingless, H537A O 3 shows the EThcD fragmentation spectrum for one of the the of one for spectrum fragmentation EThcD the shows 5 ). A protein associated with intracellular vesicles, mop, was mop, vesicles, intracellular with associated protein A ). Supplementary Table 6 3

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eferences

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46. 45. 44. 43. 42. 41. 40. 39. 38. 37. 36. to D.M.F.v.A. institutional affiliations. Correspondence and requests for materials should addressed be Nature remains neutral with regard to claims jurisdictional inpublished maps and available online at available inthe Any supplementary information, chemical compound information and source data are A The authors declare no competing interests. financial Competing financialinterests datathe and wrote manuscript the with input from authors. all MS data; D.M. analyzed genetics data; and N.S.,R.W., D.M., and D.M.F.v.A. interpreted T.A.molecular biology; and SPR; I.H.-N.performed N.S.,D.G.C., and M.T. analyzed experiments; D.G.C., and M.T. R.G. A.T.F. mass performed spectrometry; performed N.S., R.W. and D.M.F.v.A. conceived N.S.,R.W., study; the and D.M. performed A O. for Raimi help with protein purification. Research Grant.Royal Society We thank J. Peltier for help and with mass spectrometry D.M.F.v.A. M.T. by is funded aMRC grant (MC_UU_12016/5). R.W. by is funded a This work by is funded aWellcome Trust Investigator Senior Award (110061)to A 49. 48. 47. biology dditional information uthor contributions cknowledgments

re c Acad. Sci. USA Sci. Acad. for required is pathway 125 migration. and adhesion cell regulate to Rab4 with coordinates Development the by signaling enhance to pathway endocytic CiR. ofcorepressor a as acting Dev. Genes corepressors.receptor nuclear areproteinsAtrophin deacetylase-associating (2002). 45–56 processes. developmentalmultiple in corepressortranscriptional Drosophila in signaling receptor factor growth epidermal of regulation negative the (2002). in functions multiple C1. factor cell host proteinO-GlcNAcylated the of study assignments-a site proteinO-GlcNAc for essential breakthrough spectrometric mass the (ETD): dissociation cytokinesis. regulates phosphorylation in (2011). oncoproteinYorkie. the of regulator a as HD-PTP the identifies genessuppressor growth conditional Frizzled. of recycling endosomal (2014). 93–107 of Wnt/Wingless.trafficking endosomal in Myopicprotein Pradhan-Sundd, T. & Verheyen, E.M. The role of Bro1- domain-containing Bro1- of role The Verheyen,T.E.M. Pradhan-Sundd,& T. Maniatis,Endocytic & G.D. Chang, S., Kunes,Z.J., Chen, H.R., Huang, D.Y. Chen, the in Treisman, J.Y.,acts MyopicRoignant,J.E.& Wassef,G.I., M. Miura, Z. Zhang, HistoneTsai, C.C. & M. McKeown,J.L., Pitman, H., Rajan, Wang,L., T.Xu, & J. Lee, L., Xu, S., Zhang, to contributesAtrophin A. Baonza, & S. Kerridge, M., Freeman, B., Charroux, A. Erkner, transfer Electron A. Burlingame, & B. el, Affar S., Daou, S.A., Myers, Wang,Z. Liu, T.W.Liu, for screen A K.H. Moberg, & Veraksa,A. M., Tipping, M.M., Gilbert, enables Myopic-Ubpy-Hrs nexus The Verheyen,T.E.M. Pradhan-Sundd,& Drosophila melanogasterDrosophila , 4841–4852 (2012). 4841–4852 ,

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0 4 © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. auatrrs ntutos Bify Mge aoa Bas Poea were (Promega) Beads HaloTag Magne Briefly, instructions. manufacturer’s the per as (Promega) HaloTag Beads Magne to coupled and above described CpOGA Software). 2(BioLogic and Scrubber Data processing and analysis were performed using Analyser 2 (Sierra Sensors) surface. Streptavidin biotin-blocked and buffer of injections blank for enced refer double were data All s. 120 for dissociation and s 60 for measured was in concentration series adjusted specifically around their affinities. Association duplicates in injected compound each with Tween20), 0.05% NaCl, mM 150 30 of rate flow a at proteins captured over injected were Ligands RU. ~3,600–3,900 densities at Sensors) (Sierra instrument Mass-1 a of chip sensor amine capacity high on prepared before resonance. plasmon Surface K by fitting nonlinear regression curves with Prism (GraphPad) and converted to ofcompeting EC ligandsarbitrarily100%. wasas set The highest amount of fluorescent probe bound to min. 10 for (RT)temperature room at stand to allowed were Reactions ands. of nM 7 probe, fluorescent (receptor)/20nM nM 5 contained experiments ing bind competition for mixtures reaction depletion, receptor avoid to Briefly, Fluorescence polarimetry. plasmon resonance experiments were prepared previously as described surface and polarization fluorescence for used proteins Untagged use. until and snap frozen with a final concentration of 20% glycerol and stored at −80 °C TBS into1× dialyzed and columnvolumes. elutionwere Latepooled fractions 20 over mM) (0–500 imidazole of gradient linear a with eluted wereProteins buffer.lysis of volumes column 10 with washed was column buffer.The lysis NiSO with charged Sciences) Life Healthcare (GE column HisTrapHP a onto loaded and collected wereSupernatants °C). 4 atr.p.m. 15,000 min, (30 centrifugation by cleared was lysate the and kpsi) 20 at passes 3 (Avestin, disrupter cell flow continuous a using lysed were Cells 5 and PMSF mM 0.2 dine, at pH 7.5 (lysis buffer) supplemented with protease inhibitors (1 mM benzami r.p.m. 3,500 at (4 °C). Cell min pellets were resuspended 30 in 10–20 ml forof 50 centrifugationmM Tris, by 250 mM NaCl h 16 after harvested and IPTG OD an to grown amycin (LB-Kan) and at used to10 ml/L inoculate of fresh LB-Kan. were Cells containingmedium 50 Luria–Bertani in grown°C overnightat37 into transformed hexa-His-tagged C-terminally tagged, Proteinexpression and purification. obtained from DSTT, and Dundee were mycoplasma free. were lines Cell harvesting. beforeconfluence 80% to grownand dishes 10-cm CO 5% at air humidified with °C 37 at streptomycin FBS, 10% with supplemented Gibco) (DMEM; culture. Cell assay to quantify was used lysates. cell protein nm 660 Pierce or assay Bradfordbelow. described as prepared were this manner until enough material was obtained for further processing. Lysates in occasionsindependenton and over time Samplesprocessed. werecollected onated with bleach, snap ice frozenand in storeddry at −80 °C until werethey cages to represent three biological replicates. embryos were Collected dechori tions, flies were assigned from vials in a rack in random order to three separate were collected on apple juice agar plates at 25 °C overnight.h) For embryo collec (0–16 Embryos d. 10 every once vials flipping by maintained were stocks Drosophila ON doi:10.1038/nchembio.2404 supple buffer) (wash NaCl mM 150 7.5, pH Tris mM 50 with equilibrated d as described before as described L IN 1 6 . Briefly,. biotinylated proteins NeutrAvidinwerecaptureda on surface D298N E

embryos. ME HeLa cells were cultured in Dulbecco’sin culturedwere cells HeLa Eagle’s modified medium ul downs. pull THODS 600 Cp . coli E. of 0.6–0.8, transferred to 18 °C and induced with 250 with induced and °C 18 to transferred 0.6–0.8, of OGA 1 mro from Embryos 6 . All experiments. All were intriplicate. performed L1Gl (E) Ls cls Aiet. el were Cells (Agilent). cells pLysS (DE3) BL21-Gold D298N Experiments were performed as beforedescribed Halo-tagged μ μ M leupeptin), DNAse and lysozyme before lysis. before lysozyme andDNAse leupeptin), M l min l Experiments were performed as described described as performed were Experiments (receptor) rangeanda ofconcentrations oflig −1 Plasmids containing Halo-N-terminally in running buffer (25 mM Tris pH 7.5, TrispH mM (25 buffer running in w1118 Cp Cp G (mn ais 168 were 31–618) acids (amino OGA G poen wr prfe as purified were proteins OGA idtp fis ee sd Fly used. were flies wild-type L guaie ad penicillin– and -glutamine, 4 Cp and pre-equilibrated with pre-equilibrated and 50 OGA values werevalues determined 2 . Cells were plated on plated were Cells . D298N in the absence μ Cp g/ml kan g/ml OGA 1 6 . μ WT M 1 6 ------.

buffer (50 mM Tris pH 7.5, 1% NP-40, 0.5% sodium deoxycholate, 0.1% 0.1% deoxycholate, sodium 0.5% SDS, NP-40, 150 mM NaCl, 1% 2 mM EDTA7.5, and 50 mM pH NaF). For Tris each replicate mM experi (50 buffer RL2 (ab2739; Abcam; 1:3,000 or 1:1,000). anti- and 1:5,000) Signaling, Cell Anti-TAB1were(C25E9; used fractions were subjected to SDS–PAGE and western blotting. The antibodies ‘input’The upvolumemadewere toa fractions also of 200 using 200 collected, beads washed three times with wash buffer and bound protein eluted of200 Cp of a 20% slurry of HaloLink beads coupled to saturation with loading as input and one each of the remainder of the two ontoloaded 50 (containing3 umes ponentsexcept for UDP-GlcNAc. Thereactions were split fourin vol equal of UDP. ‘Unmodified TAB1’ mM was 20 the of product of reactionsconcentration containing final a all of com addition the by stopped was reaction The UDP-GlcNAcmM volume30 10 ofand final hOGT a in wasperformed by incubating 24 freshly for eachexperiment. Halo- ice. on stored and buffer wash with sively were coupled to saturation with beads for tagged the of capacity binding The buffer). (binding Tween-20 0.05% with mented with 200 either with downs pull Cp out carry to half in split were lysates protein ment, pooled and inaSpeedVac dried pooled and stored at −80 °Cuntil MS analysis. the gel pieces using 50% acetonitrile containing 2.5% formic acid. Peptides were taining liberated peptides was then collected and more peptides extracted from ml of triethylammonium bicarbonate buffer at 30 °C for 16 h. The solution con subjected to enzymatic digestion using trypsin (MS grade, Promega) at 5 triethylammoniummM 25enough bicarbonatewith andcoverbuffertothem incubatingforatmin 10RT acetonitrile.100%in pieceswereGel swelledthen bufferwashanddehydratedwithtimes gelpieceswerewashedseveral then by The dark. the in RT at min 30 for buffer bicarbonate ammonium mM 50 in made iodoacetamide mM 50 adding by alkylated RT,at then min 20 for nate ammoniumbicarbomM 50 in made DTT mM 10 in pieces gelincubatingby ammoniumperformedbicarbonateIn-gel buffer).reductionwas (wash buffer rinsed with 50% acetonitrile and subsequently with 50% acetonitrile in 50 mM pel. The excised gel pieces were de-stained until colorless using 50% methanol, scal clean a using cubes mm 1 into diced further then and sections cm 0.5 × cm 0.5 to up into excised was gel the on lane Each (VWR). water MS-grade containers with InstantBlue (Expedeon) Coomassie stain then de-stained using precastNuPAGE Bis–Tris4–12% plastic(Invitrogen)gelsclean in stainedand spectrometry. mass forpreparation Sample forbelow.essed MSas described in min DTT.mM procwereEluates200 and SDS 5 containingTris4% 6.8 mM pH 50 for beads the boiling by eluted and wash per 20 Tween 0.02% ing immunoprecipitates were washed times several with 500 coupledRL2/IgG1(5 freshly with bated manufacturer’s the per andincu collected throughwas flow Theinstructions. (Cell Signaling; G3A1) antibody bound to Protein G dynabeads (Invitrogen) as 5 with °C 4 at h 3 forincubated were above immunoprecipitation. RL2 were performed in triplicate. western blotting and the rest prepared for MS analysis as below. Experiments a using concentrated molecular-weightconcentratorspin kDa cut-off 10 ~2 and were eluents The G. Thiamet mM 3 with plemented forby2× at250 withmin °C incubating30 4 beads the eluted were proteins Bound buffer. wash with extensively washed beads the or Drosophila TAB1experiment, pull-down the For OGA Cp OGA OGA μ D298N,D401A l made up with binding buffer for 1 h at 4 °C. The flow through was was through flow The °C. 4 at h 1 for buffer binding with up made l D298N μ μ l of settled HaloLink beads coupled to saturation with D298N, D401A l l of 10 mM Tris pH 6.8, 4% SDS, 200 mM DTT by boiling for 2 min. or the control embryo lysates and HeLa lysates were prepared with RIPA with prepared were lysates HeLa and lysates embryo Cp . The incubation with beads was performed in a total volume OGA OGA was determined to be 8 mg/ml of settled beads. Beads for 90 min at 4 °C. The flow through was collected and and collected was through flow The °C. 4 at min 90 for μ g of total TAB1 each), of which two were retained for for retained were two which of each), TAB1 total of g Cp 5 mg of embryo lysates prepared as described described as prepared lysates embryo of mg 5 OGA Cp μ OGA OGA for 90 min at 4 °C then washed exten g(18.2 D298N,D401A μ g) dynabeads overnight at 4 °C. The °C. 4 overnightatdynabeads g) in vitro in μ μ M)of TAB1 with 10 Samples were run halfway down halfway run were Samples . 7 mg of lysates were incubated incubated were lysates of mg 7 . g of RL2 or mouse normal IgG1normal mouse or RL2 of g Cp O-GlcNAcylation of TAB1 of O-GlcNAcylation nature CH OGA beads were preparedwere beads OGA μ μ l of 1× TBS contain μ l washbufferl sup l for 3 h at 37 °C. °C. 37 at h 3 for l μ E μ Cp l andl 8 MIC g set aside for for aside set g Cp OGA μ A g(4.1 O L BIOLOGY OGA -GlcNAc -GlcNAc μ D298N l of all all of l μ g per μ D298N M) M) or μ ------l

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. 2.0 (Thermo) using Mascot 2.4.1 (Matrix Science), and searches were against against were searches and Science), Discoverer (Matrix 2.4.1 Mascot using Proteome (Thermo) 2.0 by performed was assignation fragment peptide and peptides HexNAc for analysis file Data ms. 150 of time injection maximum scan range of 30,000, (resolution orbitrap in the HexNAcHex and detected ion, 366.1396 the of presence the by or ETD for as triggered were reactions EThcD ms. 105 of time Ion Trap, the injection in maximum AGC Targetand detected 10,000 m/z with ions product of presence the by triggered were analyses MS2 ETD ions. 1 using trap ion linear of time 250 ms the and of AGC a injection with 100 maximum setting microscan, in was detected were energy ions fragment collision and 30% normalized to HCD set ions. 400,000 of setting AGC and 120,000 of range (scan resolution 60%, of level RF S-lens with were orbitrap spectra the in scan acquired full MS acquisition. MS/MS and MS automatically between mode, switching data-dependent the in acquired were Data 90:3. 80:3, 79.5:99, 99, 74.5: 74:40, 55:25, 5:3, 0:3, (%); B Solvent (min): Time follows: as acetonitrile, 0.08% formic acid, 3% DMSO in H A (Solvent B Fisher,Solvent 3% was 2% acetonitrile, 0.1% formic of acid, 3% DMSO (Thermo in H nl/min 300 column of flow EasySpray a with cm equilibrated 50 ES803) a on separated then 164564) a on 100 trapped × cm nanoViper2 Trap were column, Peptides injected. were samples samples/HeLa embryo pled to a U3000 RSLC HPLC (Thermo Scientific). 50%/10% of the a on LC–MS/MS Fusion by ion trap-orbitrap hybrid mass spectrometer (Thermocou Scientific) performed was immunoprecipitates) RL2 for EThcD analysis. data and spectrometry Mass nature 204.0867 (HexNAc oxonium) and/or 138.0545 (HexNAc fragment) and and fragment) (HexNAc 138.0545 and/or oxonium) (HexNAc 204.0867 ch m/z e mic m/z 120–2,000) using 120–2,000) 1 AGC microscan, setting of 300,000 ions and a 400–1,600), with a maximum ion injection time of 50 ms, ms, 50 of time injection ion maximum a with 400–1,600), l

biology μ m C18 5 C18 m HCD- and ETD-MS analyses (or (or analyses ETD-MS and HCD- 2 O). The elution gradient was was gradient elution The O). μ m 100 Å (Thermo Fisher, (Thermo Å 100 m 2 O; Solvent B was 80% 80% was B Solvent O; Drosophila -

in this published article (and its supplementary information files). Raw data Raw files). information supplementary its (and article published this in Data availability. microscope. E24HDdissection with aLeica The wings were then mounted in h. DPX Mounting24 medium (Sigma) for and isopropanolimaged into them transferring and flies the from wings whole dissecting by made werepreparations Wing microscope. SMZ Motic a using assessed were genotypes various the of flies of respectively.phenotypes Wing with crossed were virgins or heterozygotes Cr control virgins were crossed with mutantCRISPRtoderive the control flies control)(Cr stock.To derivedouble the to similar chromosome X containing Vasa::Cas9 the eliminate to stocks Vasa::Cas9 stock used for the CRISPR injections were crossed with the balancer A.T.F.(D.M., editing D.M.F.v.A,gene andBL51323 The results).unpublished hypomorphic catalytically Drosophila Drosophila proteins were using identified atwo-tailed significant package; software Perseus the using analyzed and was further data 1.5.1.7 MaxQuant using performed was analysis abundance Protein (S/T)). HexNAcand phospho(S/T) dioxidation(oxidation(M), modifications (M), variable and (C)) (carbamidomethyl fixed for made was Allowance priate. Uniprot_DROMEUniprot_HUMAN the approthe oras database database on reasonable request. analyzed during the current study are available from the corresponding author sxc H537A /sxc Stock Centre: Stock genetics. H537A All data generated or analyzed during this study are included ;mop The following fly stocks were obtained from Bloomington T482 Gug /TM6 Gug sxc 03928 H537A 03928 flies. Toderive flies. /TM3, Sb /TM3, /TM3, Sb /TM3, le wr gnrtd sn CRISPR–Cas9 using generated were flies 1 , 1 t , -test ( Ser Ser 1 Gug and 1 sxc or P doi:10.1038/nchembio.2404 <0.05). H537A 03928 mop mop or /sxc T482 T482 mop H537A /TM6B, Tb /TM6B, /TM6B, Tb /TM6B, T482 ;Gug Cr control Cr 03928 1 1 /TM6 flies, flies, . The . -