nvriyo osaz H88 rulne,Switzerland. Kreuzlingen, CH-8280 Konstanz, of University Peetades itcnlg nttt hra tteUiest fKonstanz, of University USA. the 06520, Switzerland. at CT Kreuzlingen, Thurgau Haven, CH-8280 Institute New Biotechnology Medicine, address: of *Present School University Yale Genetics, ae,C-06Bsl Switzerland. Basel, CH-4056 Basel, osaz Germany. Konstanz, aetn Spinnenhirn Valentina -targeted Salmonella decorates FAT10 modifier -like The ARTICLE RESEARCH eevd2 eray21;Acpe 5Spebr2014 September 15 Accepted 2014; February 27 Received ` (here 1 macroautophagy called process defense catabolic effective A also are mechanisms. pathogens and and of the nutrients nitrosative Effector compartmentalization cytosolic oxidative, the defend of on restriction rely invasion. The inducible, chemistries. and protonative cytokine infection pathogenic against often cell host are counteract which mechanisms, the to within autonomous cell replicate developed mechanisms and consequence cells a As eukaryotic cytosol. invade can Pathogens INTRODUCTION Xenophagy Autophagy, p62, FAT10, Ubiquitin, WORDS: KEY FAT10 (MAP1LC3B). on microdomains LC3B p62-positive marker with autophagy p62, colocalized the ubiquitin, with and decorated NDP52 simultaneously were cytosolic Typhimurium to recruited is before, pathogens Salmonella with been associate not has to but p62, shown adaptor autophagy the to binds which UBD), D, IFN characterized barely functionally xenophagy. and TNF the by that CALCOCO2) bacteria show as of we destruction Here, known the (also initiate which NDP52 of optineurin, (also SQSTM1), binding p62 like as and adaptors autophagy known ubiquitylation cell- of recruitment by by to leads bacteria galectin-8 counteracted cytosolic is of The decoration xenophagy. cells including mechanisms eukaryotic immune innate autonomous of invasion Bacterial ABSTRACT hra ooaiainwt D5 a nypril kinetic A partial. only was NDP52 with colocalization whereas htwr A1-eiin a ihrssetblt oorally bacteria. against defense to intracellular the role in susceptibility FAT10 a suggest for data our higher together, Taken FAT10. for wild-type were a that had FAT10-deficient inoculated were that vitro cells in overexpressing or FAT10-depleted in altered detectably replication not bacterial was Although bacteria p62. of of that decoration resembled transient, which FAT10, by only but early, an revealed analysis acsGroettrup Marcus uhrfrcrepnec ([email protected]) correspondence for Author iiino muooy eateto ilg,Uiest fKntn,D-78457 Konstanz, of University Biology, of Department Immunology, of Division 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,48–83doi:10.1242/jcs.152371 4883–4893 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. a idcbeuiutnlk oiirFT0(lokona ubiquitin as known (also FAT10 modifier ubiquitin-like -inducible uvvleprmnsrvae htNAP-rngncmice NRAMP1-transgenic that revealed experiments survival , S. yhmru nhmncls FAT10-decorated cells. human in Typhimurium yhmru atrata RM1tasei mice NRAMP1-transgenic than bacteria Typhimurium 2 oa raIfcinBooy iznrm nvriyof University Biozentrum, Biology, Infection Area Focal 1,3, n otiue to contributes and ` 1 es Farhan Hesso , 3 itcnlg nttt hra tthe at Thurgau Institute Biotechnology Salmonella 2, Typhimurium, 4 ,McalBasler Michael *, S. eatetof Department Typhimurium, c -and S. Salmonella 1,3 erdtv uoyooe.Teatpaooa pathway the form autophagosomal to organelles The fuses lysosomal autolysosome). and the sequestered endosomal degradative (where late maturation encloses with (iii) autophagosome and membrane, cargo), crescent double-membraned (where closure (a and elongation principle, initiation a (ii) formed), In (i) is phagophore involved: a deprivation. called and are nutritional homeostasis stages during energy three was levels restore Autophagy acid to non- 2005). amino a especially in al., 2004; manner, material et cytosolic al., specific bulk Ogawa degrade et to 2004; considered Gutierrez originally defense al., 2006; another et be al., to Nakagawa et shown been (Birmingham has mechanism autophagy) as to referred uohg aha o efsrigproe aebe evolved been have the purposes of self-serving components of for usage pathway the autophagy even or autophagy of suppression the form to organelles lysosomal and autolysosome. phosphatidylethanolamine. degradative endosomal late to with autophagosome closed the conjugated fuses the LC3- process, maturation paralogs initiates subsequent the the comprises its During domain that share autophagosome and binding nascent adaptors the LC3 by This autophagy cargo of (LIR). known engulfment Zheng All region 2011; al., 2009). interacting et cytosol- Wild al., the 2009; al., to et et recruited (Thurston are pathogen functions, invading redundant have which not optineurin, but a and similar CALCOCO2) In as known compartment. SQSTM1), (also as endolysosomal known NDP52 (also p62 the adaptors autophagy the of step, second it integrity as general, the in pathogens surveys as vesicle-damaging serves for galectin-8, receptor lectin, One danger 2012). a al., et Thurston 2010; al., et ubiquitin-independent or diacylglycerol However, through 2004). targeting, and autophagosomal al., for et (Huettmechanisms host Perrin xenophagy proteasome ubiquitylated 2012; initiate al., to whether surface et the pathogens major their or whether on ubiquitylated accumulate debate cytosol: both of directly matter either for the a are still signal is in It degradation autophagosome. systems universal thereby proteolytic Ubiquitin the a specificity. substrate the by constitutes mediating in to performed role 2010). al., crucial allocated et are (Ponpuak level be that cellular the can at processes In machinery autophagy pathogens to 2009). immune products of al., specialized antimicrobial cytosol- niches of et delivery intracellular or (Kirkin the depolarized xenophagy, (aggrephagy) (xenophagy) of addition, microbes proteins elimination colonizing These aggregated the complexity. ribosomes regulate (ribophagy), (pexophagy), or peroxisomes turn (mitophagy), size intracellular mitochondria in their of by elimination processes unrestricted and capture components recognition, includes net Aichem Annette , saemcaim,lk viac fatpai capture, autophagic of avoidance like mechanisms, Escape a plays ubiquitin cargo, of capturing autophagosomal For b glcoebniglcis aeas enrpre (Shahnazari reported been also have lectins, -galactose-binding eitnei mice in resistance 3 lo Canaan Allon , 4 and 4883

Journal of Cell Science fxnpay upiigy uprieefc fatpayon autophagy of of effect replication supportive intracellular a Surprisingly, xenophagy. of A1 en ojgtdt h uohg dpo 6 (Aichem p62 adaptor autophagy the to al., conjugated is et 2006). being Lee al., FAT10 it et 2014; Zhang because al., antiapoptotic 2008; et al., carcinogenesis (Gao have et types Lukasiak in In to cancer 2003; implicated various 2008). suggested in is al., expressed been and et has (Kalveram functions FAT10 formation aggresome addition, NF- 2011), regulation and al., 2006), cycle et divergent 2010) al., Ren cell et 1999; and al., Ross partially stability et 2001; (Liu chromosomal al., many proapoptotic et on Raasi influence have in 1999; an to al., has et supposed involved FAT10 (Liu is functions discovery, be expression its FAT10 Since to processes. 2012). al., reported (Hipp et respectively been Rani Rpn1, 2004; and of al., Rpn10 binding et subunits through regulator conjugates UBL-UBA its 19S The the and the FAT10 of the 2009). of accelerates proteasomal isoform rate (Nub1L) al., 1 degradation long buster et ultimate the for NEDD8 Schmidtke with domain 2005; FAT10 directly substrates of of al., capable interaction et 2012). modified one (Hipp al., only degradation the covalently et is (Aichem FAT10 targeting ULMs, substrates the spectrometry, FAT10 Among mass identified has putative by enzymes group proteins including Pelzer our deconjugating FAT10-interacting 2007; Recently, and of al., identified. ligases hundreds been et E3 yet Jin not 2007; respectively Potential have al., USE1, 2007). et and al., Chiu UBA6 et 2010; al., enzymes constitutively et E2 the (Aichem no and by needs conjugated FAT10 E1 1999). and expressed al., activated et is Raasi but 1999; processing, al., et (Liu present are ecnaerpiae ihntemdfe hgsm,teso- the phagosome, modified the higher called A within 2006). replicates al., et percentage (Birmingham the cytoplasm the with reach bacteria infection ( During pathogen Typhimurium host immunity. intracellular a in as facultative autophagy mechanism escape of of importance defense number the The 2009). strengthens Levine, mechanisms and (Deretic pathogens by ARTICLE RESEARCH 4884 not is FAT10 1997; tissues, al., other et most IFN In (Bates cytokines pro-inflammatory 2008). the cells unless expressed al., dendritic during et induced activated is Lukasiak transcription Lukasiak of FAT10 2003; addition, al., maturation et In liver in Lee 2008). fetal 2006; al., reported al., spleen, et et like been (Canaan nodes organs lymph has lymphatic and secondary are expression and ubiquitin al., FAT10 thymus et (Bates in Constitutive residues conjugation substrate K63 of lysine 1997). C-terminal site the a and The as as well carboxylate K48 as 2014). glycine UBL-domains FAT10 K33, al., both in K27, et conserved to (Schmidtke linker corresponding two have with of short to connected formation assumed family a head-to-tail is it a protein the in Although the domains crystallized, of 1996). ubiquitin-like been al., member yet et not youngest (Fan has (ULMs) the modifiers represents ubiquitin-like UBD) D, mechanisms, targeting as identification. potential well with await as molecules, together adaptor Alternative recognition mechanisms. detection pathogen cytosolic only the remain target signaling (DAMP) whether pattern known of molecular yet danger-associated galectin-8-mediated not is modes it However, ubiquitin-based 2014). is al., et (Yu and proposed autophagy by targeted in efficiently used frequently is Typhimurium u ru a eetyivsiae ucinlapcsof aspects functional investigated recently has group Our A1 HAFajcn rncit1;as nw subiquitin as known also 10; transcript adjacent (HLA-F FAT10 Salmonella S. cnann aul SV.Cytosolic (SCV). vacuole -containing nvitro in Typhimurium), tde osuymcaitcaspects mechanistic study to studies Salmonella amnlaenterica Salmonella , 53%o h invading the of 25–35% k inln Gn tal., et (Gong signaling B a lobe recently been also has c n TNF and serovar S. a eeldahge ucpiiiyfor mice susceptibility NRAMP1-transgenic higher in a experiments revealed Survival xenophagy. A1 eewsdltdsgetn oefrFT0i the in FAT10 for role a suggesting bacteria. deleted against defense intracellular was FAT10 target that ubiquitin, found we to Indeed, cytosolic decorated FAT10 analogous degradation. we autophagosomal might, for cytokines pathogens FAT10 pro-inflammatory by that upregulated hypothesized and similarly FAT10 both are As function. adaptor p62 consequences p62 the prospective for interaction analyze this for This further of 2012). to al., us et adaptor (Aichem encouraged interaction finding non-covalent autophagosomal strong a a as UBA described efficient well as have p62, C-terminal We and FAT10 2007). an between its interaction al., covalent UBA-independent et with (Pankiv is combination substrates ubiquitylated In p62 2005; 2007). al., et domain, al., (Bjørkøy et autophagy substrate, by Pankiv autophagy LIR selective degraded a a continuously contains as being p62 fate adaptors, its determines autophagy that other domain Like 2012). al., et 02.Ti neato a ecniee sadrc interaction, direct a al., as et considered (Aichem be FAT10 can interaction and interaction noncovalent This p62 a 2012). adaptor demonstrated autophagy we the study, between previous a In Salmonella RESULTS Dpn ta. 09 hn ta. 09.W pcltdta the that been speculated We has 2009). remnants al., their et p62 or Zheng bacteria 2009; adaptor, cytosolic al., et of (Dupont autophagosomal capture the in an implicated as Functioning autophagy-targeted decorates FAT10 of decoration ubiquitin addition, In ( period. time Salmonella to Up that 1B). SHF2 (Fig. within total infection the of cells h of endothelial 3 bacteria 20% first GFP-positive vein the and during umbilical enumerated SHF2, were with human infected end, were green (HUVECs) this whether To tested SHF2 bacteria. We 6-phosphate. in glucose fluorescence to exposure SHF2 SCV in an GFP the of including Expression endomembranes, 1A). of (Fig. lumen the and cytoplasm from the promoter.excluded in 6-phosphate-responsive present (GFP–UhpT) exclusively glucose is metabolite a glucose protein This of control transport the a phosphate under generated hexose therefore GFP-tagged We pathogens. modified autophagosomal cytosolic in of involved be cytokine- degradation might the whether FAT10 to modifier as inducible hypothesis the Nevertheless, investigated 2012). further mCherry– al., we we et an (Aichem of However, protein targeting fusion exist. EGFP–FAT10 autophagosomal might demonstrate to targeting failed autophagosomal with FAT10 to and ubiquitin regard between with parallel a p62 that hypothesized First, recombinant we S1). Fig. of material (supplementary co-precipitation GST–FAT10 recombinant the by demonstrated H2idctsepsr fteebcei otecytosol. the We to with bacteria infected Lamp1. these cells of with in exposure decoration indicates fluorescence SHF2 continuous green Lamp1 that no to conclude least association therefore appreciable at any or exhibit staining not either with intact did correlated in that expression presence GFP their Furthermore, indicating SCVs. Lamp1, by surrounded clearly for marker 1D). a Lamp1, (Fig. for stained SCVs and SHF2 HeLa with addition, infected In 1C). were cells (Fig. ubiquitin with decoration minor a only , 5) ncnrs,GPngtv SHF2 GFP-negative contrast, In 35%). ora fCl cec 21)17 8349 doi:10.1242/jcs.152371 4883–4893 127, (2014) Science Cell of Journal Salmonella togycreae ihidcdGPexpression GFP induced with correlated strongly H2epessGPi h cytosol the in GFP expresses SHF2 Salmonella L34sri SF)ta xrse the expresses that (SHF2) strain SL1344 Salmonella Salmonella S. yhmru hc eetree for targeted were which Typhimurium hc i o xrs F were GFP express not did which a nue F expression GFP induced had atflyrprscytosolic reports faithfully S. Salmonella yhmru hnthe when Typhimurium S. Typhimurium Salmonella sidctv of indicative is Salmonella showed

Journal of Cell Science EERHARTICLE RESEARCH ihNP2 6,L3aduiutn(i.2) neetnl,we Interestingly, 2C). (Fig. ubiquitin and LC3 that p62, NDP52, revealed with Co-stainings infection subsequent SHF2 HUVECs. the FAT10-decorated in Raasi performed 1999; primarily we al., Therefore, experiments et (Liu 1999). HUVECs as al., like not et is cells, FAT10 primary of for induction as the strong that shown 2B). been previously (Fig. cells, has HeLa lines For it signaling. cell cells cytokine abnormal Cancer display types. can bacterial HeLa general cell in of two cytokine quantity these and between the after decoration in h 2A) FAT10 differences 24 (Fig. observed and we infection HUVECs Importantly, after in h 1 treatment at SHF2 decoration cytosolic using FAT10 of observe bacteria indeed cytosolic could to We recruited microscopy. To confocal is to marker. FAT10 xenophagy whether tempting analyze during a to is as performed we p62 it function possibility, this can proteins assess cytokines, FAT10 with both proinflammatory that As speculate by FAT10 interest. functional induced of of are be interaction might inflammation and upregulation B D A

1 GFP+ (% of total SHF2) 22212 121212 P 10 15 20 0 5 hours postinfection red 1 LAMP1 P 2 2 3 1 P Salmonella blue GFP 2 SCV C Autophagosome nvitro in dapi eeadtoal decorated additionally were Ub+ (%) 1 10 15 20 25 30 35 40 45 0 5 grey neto experiments infection intracellular extracellular Autolysosome 1 hours postinfection 2 2 1 merge+DAPI GFP+ SHF2 GFP- SHF2 3 2 hog bqiyain hrfr A1 eoainmight decoration FAT10 Therefore detection the ubiquitylation. especially mechanism, through defense cellular this escape autophagy. same is the for at SHF2 targeted are cytosolic time bacteria of these pool Remarkably, confocal certain FAT10. with a by decorated surface. that bacterial conclude detectable almost we the is summary, on place In present decoration already FAT10 takes are adaptors defined as microscopy, that also a assume The we as p62 minutes. Therefore, soon 2013). few and al., et a (Fujita ubiquitin simultaneously only with spans decoration beads, latex for cytosolic al., least of at Effectene-coated et timeline association, LC3B the (Tattoli and particular, recruitment cytosol In adaptor recognition, the 2009). al., into et entry Zheng 2012a; bacterial place takes after not induction rapidly Xenophagy but very 2C). FAT10 (Fig. for ubiquitin positive for positive were bacteria fraction autophagy autophagy-targeted small very an of a Only without present. (MAP1LC3B) bacteria LC3B or FAT10-decorated adaptor find not could oexnpaytree ahgn aeeovdsrtge to strategies evolved have pathogens xenophagy-targeted Some n bqii F2atbd) h ecnaeo bqii-otv (Ub+) antibody) ubiquitin-postive (anti-LPS of SHF2 percentage total The SHF2 for antibody). stained (FK2 and ubiquitin fixed and were as cells infected and were B HUVECs in (C) experiments. independent two in condition n el eefxdadsandfrLm1 nagdiae fthe image. of main images each Enlarged h below Lamp1. 2 shown for for are stained 100) area and (MOI indicated fixed SHF2 with were infected cells independent were and two cells in HeLa condition (D) each experiments. for analyzed bacteria 1000 mean The microscopy. fluorescence total for stained mean and (GFP+) fixed GFP-positive h, of 3 fraction Salmonella to The were up antibody). HUVECs for (anti-LPS 6- (B) 100) SHF2 glucose SCVs. (MOI to damaged SHF2 exposure within with an or infected of cytosol indicative the is in SHF2 phosphate in lumen GFP the of from Expression excluded and the cytoplasm including the endomembranes, metabolite in glucose present This exclusively 6-phosphate. glucose is of control the under UhpT the of Schematic (A) 1. Fig. ora fCl cec 21)17 8349 doi:10.1242/jcs.152371 4883–4893 127, (2014) Science Cell of Journal 6 Salmonella ..i hw o tlat10 oa atraaaye o each for analyzed bacteria total 1000 least at for shown is s.d. Salmonella H2wsqatfe yfursec irsoy The microscopy. fluorescence by quantified was SHF2 htwr F+adGFP and GFP+ were that H2epessGPi h cytosol. the in GFP expresses SHF2 Salmonella Salmonella tan(H2 htepessGFP– expresses that (SHF2) strain 6 ..i hw o oa fa least at of total a for shown is s.d. cnann aul (SCV). vacuole -containing 2 a nmrtdby enumerated was 4885

Journal of Cell Science EERHARTICLE RESEARCH bqii eoaini neryeeti autophagosomal in 4886 event early an of is targeting decoration kinetics same Ubiquitin the with occur of Typhimurium targeting autophagosomal NDP52. and than decoration higher FAT10 but p62 than ubiquitin lower and factor FAT10 less correlation significantly a with Colocalization revealed but 3C). LC3B (Fig. and NDP52 p62 with with best colocalized fluorescent the FAT10-specific signal calculating The coefficient. by correlation signals NDP52 Pearson’s fluorescent p62, colocalizing with quantified Therefore 3B). FAT10 we (Fig. differences of marked showed colocalization LC3B or of ubiquitin degree bacterial the The representing microdomains coat. observe indeed could we IFN and TNF SHF2-infected, and of co-immunostainings performed therefore question, we this We address To 2011). microdomains. certain with al., correlates also et signal fluorescent FAT10-specific (Cemma the whether investigate to is other wanted bacteria cytosolic each to adaptors of two independent the segregated of recruitment rather the are that and signals cytosolic is fluorescent NDP52-specific instead immunostainings, of and but in that, homogeneous p62 decoration report not they the Moreover, is patches. in NDP52 that present and suggested p62 have with bacteria al. et Cemma microdomains less NDP52-positive significantly with but p62-positive with colocalizes (Deretic FAT10 autophagy evade to known 2009). is Levine, these that and target pathogen a to S2A,B), decoration ubiquitin FAT10 no Gram-positive for found on we However, mechanism particular. in backup pathogens a represent A FAT10 NDP52 CBGFP LC3B p62 red Ubiquitin a tetdHVC Fg A.Wt ofclmicroscopy, confocal With 3A). (Fig. HUVECs -treated Salmonella GFP GFP GFP .monocytogenes L. blue Fjt ta. 03 ut ta. 2012; al., et Huett 2013; al., et (Fujita GFP merge+DAPI splmnaymtra Fig. material (supplementary C B % 100 S. 20 40 60 80 0 red L A1 GFP FAT10 FA C FAT10

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Journal of Cell Science EERHARTICLE RESEARCH rtcinasy.W bevdrpdrpiaino SHF2 of replication rapid observed We Salmonella at assays. in gentamicin condition using box each by protection for The replication analyzed threshold. bacterial bacteria quantified above decorated FAT10 and correlation 190 HUVECs Pearson’s relative least immunostaine the the at of measure for shows colocalization min) to axis of and used ‘value’ (max Quantification was The whiskers (C) software *** software. and A. ImageJ percentiles) experiments. ImageJ in 75th of independent with pictures to plugin two recorded merged (25th coloc2 as in box The A, line median, FAT10. shows white in with plot along micrographs together is corresponding ubiquitin axis of and distance picture LC3B the merge signal; the fluorescent in of line intensity white the microdomains. along NDP52-positive scan with SHF2 less with but p62-positive infected with HUVECs correlates localization FAT10 3. Fig. A % of GFP+ SHF2 70 10 20 30 50 60 40 0 11,52 3 nuidcdcnrlclswti finfection. of h 6 within cells control uninduced in C AB Ubiquitin NDP52 LC3B p62

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Fig. material (supplementary mice C57BL/6 in load bacterial enhanced FAT10 significantly from days, a 14 nodes After lymph 6A–D). detect increased (Fig. mesenteric organs burden could tested all lymph we bacterial in we mesenteric days infection The 14 liver, 6A–D). to after 4 (Fig. spleen, days from cecum in 14 and load and nodes bacterial 4 transgenic the NRAMP1 At To longer more-resistant analyzed FAT10 model. days. a the over mouse used few of survival we C57BL/6 a and time effect weight within of body period different die any load, mice to bacterial mask monitor infected mice because could C57BL/6 deficiency pathogens inbred FAT10 of intracellular a had susceptibility or with extreme FAT10 them infected for orally wild-type and knockout, were that mice Our of C57BL/6 effect hypothesis functional disease. a our show intracellular not systemic on far expression FAT10 so severe extraintestinal could setup to experimental causing access gain to tissues, susceptible bacteria strains mouse infection, in or Typhimurium fever typhoid with humans In NRAMP1 in deficiency FAT10 bacterial on expression FAT10 of effect IFN replication. significant a of show to action or able bactericidal down cells. the knocking FAT10-overexpressing TNF by in either as confounded by well However, as origin FAT10, different out knocking of cells primary ifrneo ia iesi vre olwn acnavirus FAT10 Vaccinia no and following addition, wild-type ovaries comparing In in detectable S2C). titers were Fig. infection viral material of (supplementary difference of liver replication bacterial in on However, FAT10 of 6B). influence (Fig. mice type ucpiiiyto susceptibility rngncwl-yeadFAT10 and wild-type transgenic utemr,w efre he needn survival independent three performed we Furthermore, a htaenee o h nuto fFT0 ewr not were we FAT10, of induction the for needed are that 2 / 2 Salmonella ie oe uvvlo h FAT10 the of survival lower a mice, S. yhmru ihtoFLAG–FAT10-expressing two with Typhimurium nvivo in S. Typhimurium yhmru nclsadcnrbtsto contributes and cells in Typhimurium S. yhmru neto ihNRAMP1 with infection Typhimurium egnrtdNRAMP1-transgenic generated we r/r 2 2 / / 2 nvivo in iervae higher a revealed mice 2 Salmonella ie hsrsle nahigher a in resulted This mice. ieta o idtp mice wild-type for than mice 2 / 2 2 / Salmonella 2 iesoe tendency a showed mice netosrvae no revealed infections scmae owild- to compared as elcto.T test To replication. .monocytogenes L. 2 / L34 The SL1344. 2 iewas mice nvitro in c 2 and / S. 2 nte aallt h ito omnpoete n hti that is add that and we properties study, common this of In list the p62- striking the 2012). in to al., colocalize Another parallel and et another p62 2008). (Aichem to bodies bind al., modifiers containing both through et that is to - them (Kalveram parallel the shuttles to binding center which along through organizing motors 6 dynein mediated to (HDAC) linkage is deacetylase localization histone this modifiers, sebeit hisi re otre o proteasomal for target to order to in needs chains Ubiquitin the into under cytokines. are al., that pro-inflammatory assemble et and bacteria of Lukasiak by in invaded on 1997; influence switched been al., selectively have therefore that et is tissues system (Bates FAT10 cells The 2008). dendritic striking exist. mature the also IFN in in whereas appreciate expressed ubiquitously, which only expressed To is to is FAT10, FAT10 reveals, p62. name important and its to as is ubiquitin Ubiquitin, bind it both between that issue, differences proteins this family discuss coating how ubiquitin to from as two benefit question might with the defense pose antibacterial needed. study intracellular this are the of characteristics results similar the such particular, the In with into modifiers two vacuole 4). why its and 3 from (Figs release cells infected upon of cytoplasm FAT10 and ubiquitin 02.Hwvr eddfn htFT0adp2ascae on associated p62 and FAT10 al., that et find to xenophagy-targeted did mCherry– (Aichem localize we did proteins not However, 2012). by did mCherry–EGFP–ubiquitin fusion p62, and the degraded to EGFP–p62 when non-covalently is autolysosomes binding acidifying protein, ubiquitin, of fusion spite like mCherry–EGFP–FAT10 in a we FAT10, However, p62, to autophagy. non-covalently that and covalently autolysosomes, hypothesized binds in FAT10 degraded that be and can probably An mammals. which system). of aggresomes, pathogens immune certain FAT10 against evolved adaptive mechanism has (i.e. defense FAT10 that the a mammals be as therefore than in would hypothesis later exists attractive FAT10. only even by which evolved the than than system, older ubiquitin much evolutionarily FAT10 modifies by is system FAT10 modified ubiquitin the If Finally, are 2013). proteins al., substrates to different et of compared surface (Merbl FAT10 the when on ULMs proteins that substrates five ubiquitin other revealed from the FAT10 most of the substrates and differed conjugation NEDD8, ISG15 SUMO3, and the SUMO2 UFM1, SUMO1, ULMs of the and analysis ubiquitin comprehensive microarray recent signal, a protein Strikingly, amenable degradation pathogens. therefore by and intervention irreversible for reversible would an is This polyubiquitylation 2005). as al., whereas its et with ‘FAT10ylation’ (Hipp lived along short render degraded hence therefore probably is is is and and substrate FAT10 proteasome whereas the (Hipp recycled lived, at and proteins long degradation off such substrate cleaved is to identify ubiquitin prior to Consequently, 2005). efforts date al., considerable et to of described whereas ubiquitin. spite been ubiquitylation, in not by counteract with have that enzymes enzymes could action 2012) FAT10-deconjugating deubiquitylating Chen, they FAT10 encode and (Jiang pathogens be as with to several formation need interfere Likewise, chains ubiquitin-like chain chain FAT10 cannott for no two suppressing need As bacteria no 2012). its with al., formed, signal et degradation with (Rani a formation as FAT10 serves domains whereas degradation, ao,esnilyuasee,qeto nFT0booyis biology FAT10 in question unanswered, essentially major, A eas ehdpeiul on htFT0i oaie in localized is FAT10 that found previously had we Because amnlaenterica Salmonella ora fCl cec 21)17 8349 doi:10.1242/jcs.152371 4883–4893 127, (2014) Science Cell of Journal S. yhmru ti ok.We we When work). 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NDP52, FAT10 cytosolic and p62 that with that bacteria, decorated reason simultaneously SHF2 given to are GFP-positive they But exclusively and microscopy almost (LAP). electron targets phagocytosis FAT10 be LC3-associated would exclude targeting autophagosomal ARTICLE RESEARCH bqii n A1 eoainfrteitaellrdefense intracellular the , for decoration against FAT10 cellular and with 2012). ubiquitin done al., et have (Aichem we previously the FAT10-decorated substrates as conjugation recover into spectrometry, SHF2 FAT10 mass to insight by to pertinent proteins provide be recruitment cannot will Salmonella FAT10 UBA6 it S3B). 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ARTICLE RESEARCH 4892 wild-type Salmonella used we assay infections protection For gentamicin and strains Bacterial FAT10 primers: following DNA the Start with Fast (Roche) LightCycler (5 Kit a mouse I and Green with (Roche) SYBR amplification instrument Promega, Master PCR cycler for kit; used light transcription was the cDNA reverse The followed (Promega Germany). cells Mannheim, from transcription Qiagen) kit, reverse mini by (RNeasy extracted was RNA Total RT-PCR real-time Quantitative 2 knockdown siRNA 10 Germany), mM 1 Mannheim, 7.5, (Roche, pH cocktail Tris-HCl 1 inhibitor NP40, mM 1% protease [50 SDS, buffer free 0.1% NaCl, lysis mM Ripa 150 in EDTA, lysed were Cells Immunoblotting cnigmcocp Cr es,Jn,Gray sn 63 a using Germany) Jena, Zeiss, laser- (Carl confocal 510 microscope LSM a scanning with analyzed and acquired were with Images slides USA). glass Birmingham, SouthernBiotech, on Flouromount-G, mounted (DAPI were Coverslips medium temperature. out mounting room carried were at incubations h All 1 gelatin. for 0.2% in diluted were antibodies All e,Getne,Gray n e yoie eeaddafter added with for lysed were infected and harvested cytokines either or were cells analysis new h, 24 immunoblot and and 12 Germany) After transfection. 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SL1344 Basel, (strain of Plasmid Typhimurium University Kasper, GFP–UhpT Christoph and the Arrieumerlou SHF2 generate with To transformed SHF2). (strain phosphate nuae o 0mn(E23 Es r6 i HVC,peritoneal 1000 (HUVECs, min at 60 min or 37 5 respective MEFs) at (HEK293, the for macrophages) min with centrifuged 30 infected for (MOI), were incubated plates infection 24-well of in multiplicity cells and PBS, with S14 n H2 n 100 and SHF2) and (SL1344 osaz 5B/ idtp CalsRvrLbrtre)and Laboratories) River of University (Charles the of wild-type facility animal FAT10 C57BL/6 the from purchased Konstanz. were mice All and Mice ihm . ezr . uaik . avrm . hpad .W,Rn,N., Rani, W., P. Sheppard, B., Kalveram, S., Lukasiak, C., Pelzer, A., Aichem, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.152371/-/DC1 online available material Supplementary material Supplementary Konstanz. Chemical of School University Graduate the the the and at from Foundation; stipend Biology Velux a received the V.S. 1517/10-1]; Foundation. [grant GR Novartis Foundation and Research 1517/2-4 German the GR from numbers grants by supported was study This Funding generated A.C. studies, infection FAT10 interaction p62-FAT10 mouse performed performed the A.A. M.B. wrote experiments, bacteria, and SHF2 experiments generated H.F. the manuscript, interpreted and performed designed, V.S. contributions Author interests. competing no declare authors The interests Competing the of Center Bioimaging Konstanz. the FLAG– of at of University performed contribution was the Microscopy for of transfectants. acknowledged (University FAT10 is Lukasiak Germany) Sebastian Konstanz, studies. Konstanz, interaction Switzerland) FAT10–p62 Kreuzlingen, with bacteria Thurgau, help SHF2 Institute for of (Biotechnology generation the Catone Switzerland) during Nicola Basel, support and and Basel, advice of for University acknowledged and Biozentrum, are Arrieumerlou from Cecile (all Bumann, the Kasper Dirk for Christoph mice. Borstel transgenic the Leibniz-Zentrum NRAMP1 for of the contribution CT) and Haven, mice FAT10-knockout New of University, (Yale contribution Weissman Sherman thank We Acknowledgements xeiet,adrfndtemanuscript. the refined and experiments, 25 100 with n g-ace iewr atdfr4hfloe yoa aaewith gavage oral by followed h 4 sex for experiments, fasted were enumeration mice (CFU) age-matched unit and colony-forming and survival 1.6 RM1tasei 5B/ idtp n FAT10 and generate type to wild Borstel) C57BL/6 (Forschungszentrum transgenic mice NRAMP1 transgenic NRAMP1 13)i rs BL o t civ notcldniya 600nm at density optical an achieve [to h 3 for LB-LS (OD fresh in (1:33) lts ttsia nlsswspromdwt h rpPdInStat Regierungspra GraphPad of Board protocols the Review to the according with by performed agar were approved performed experiments LB-LS animal was streptomycin All program. analysis on body plated Statistical when and plates. killed diluted (Kinematica, were homogenizer Switzerland), PT2100 Polytron Luzern, mice harvested, a were using and organs PBS enumeration, in monitored CFU homogenized For was 20%. exceeded mice loss weight all of weight ltdi rpiae nsrpoyi BL grpae.Statistical plates. were agar program. dilutions InStat LB-LS GraphPad Serial the streptomycin X-100. with on performed Triton was triplicates 1% analysis in lysed containing in were PBS plated cells cold bacteria, intracellular ml 0.5 enumerate To gentamicin. ml cmdk,G n retu,M. Groettrup, and G. Schmidtke, 6 m 600 /lgnaii HVC)fr3 i el eeclue n25 in cultured were cells min 30 for (HUVECs) gentamicin g/ml 10 2 / 2 ) 8 2 . ora fCl cec 21)17 8349 doi:10.1242/jcs.152371 4883–4893 127, (2014) Science Cell of Journal / CFU 2 ie n ..sprie h rjc,dsge n nepee the interpreted and designed project, the supervised M.G. and mice, m ]pirt neto.SF atraclue eewse twice washed were cultures bacteria SHF2 infection. to prior 1] /lgnaii HK9,MF,prtna arpae)or macrophages) peritoneal MEFs, (HEK293, gentamicin g/ml nvivo in ie(aane l,20)wr necosdwt C57BL/6 with intercrossed were 2006) al., et (Canaan mice S. yhmru S14)i 100 in (SL1344) Typhimurium infections ˚ .Floigtowse ihPSadincubation and PBS with washes two Following C. m /lapcli SF)adsub-cultured and (SHF2) ampicillin g/ml 21) S1i ipcfcconjugating bispecific a is USE1 (2010). Salmonella sdu Freiburg. ¨sidium m m B.Tebody The PBS. L /lstreptomycin g/ml 2 L34was SL1344 , / 2 ie For mice. g and m g/ S.

Journal of Cell Science uire,M . atr .S,Snh .B,Tyo,G . oob,M .and I. M. Colombo, A., G. Taylor, B., S. Singh, S., S. Master, G., M. Gutierrez, D., C. Cohen, V., Nair, A., Snyder, J., Leventhal, B., Wang, A., Canaan, P., Gong, G. C. Lee, and J. Ren, B., W. Teo, J., Zhuo, S., S. Theng, Y., Gao, T., Umemoto, Y., Osada, M., Nakaoka, A., Tanaka, T., Itoh, E., Morita, N., Fujita, M. S. Weissman, and G. G. Lennon, C., D. Schwarz, S., Parimoo, W., Cai, W., Fan, T., G. Nhieu, Van B., Freche, I., Paz, J., Bertout, S., Lacas-Gervais, B. N., Dupont, Levine, and V. Deretic, ut,A,Hah .J,Bgn . as,S . at .A,Va,J M., J. Vyas, A., L. Baxt, O., S. Sassi, J., Begun, J., R. Heath, A., Huett, hu .H,Sn .adCe,Z J. Z. Chen, and Q. Sun, H., Y. Chiu, H. J. Brumell, and K. K., P. Kim, Castille, M., L., Cemma, S. Gamfi, I., Lazar, J., Lian, J., C. Booth, X., Yu, A., A., Canaan, Overvatn, M., Perander, H., Outzen, A., Brech, T., Lamark, G., Brumell, Bjørkøy, and T. Yoshimori, A., M. Bakowski, C., A. Smith, L., C. 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