The Arf and Rab11 Effector FIP3 Acts Synergistically with ASAP1 to Direct

luuru,N 73,USA. 87131, NM Albuquerque, igWang targeting Jing receptor to ciliary ASAP1 in with Rabin8 synergistically acts direct FIP3 effector Rab11 and Arf The ARTICLE RESEARCH ß eevd5Spebr21;Acpe eray2015 February 3 Accepted 2014; September 5 Received the in components specific 1 of strict the retention periciliary of the and base The through barrier, admission the selective to diffusion 2006). the signal molecules by signaling by followed Reiter, of cilia, achieved associated transport is directed and membranes the sensory signals their the (Singla the of extracellular in compartmentalization and concentrated membrane of highly are range ciliary receptors which environmental wide machineries, transduction as sensory a function capture through cilia that a Primary of sensors properties organelle. by them organized membrane gives which separate plasma contiguous barrier, exquisitely surrounding diffusion are the periciliary the contain that from, separated cilia yet cells primary with, called eukaryotic projections membrane all Nearly INTRODUCTION Rhodopsin ciliary Rab11a, Cilium, WORDS: and during KEY assembly cascade ASAP1–Rab11a–FIP3 orderly Rab11–Rabin8–Rab8 the trafficking. the the receptor facilitating within of thus activation Rabin8 complex, for targeting regulator, the pocket shapes targeting and crucial interactions binding a rhodopsin–ASAP1 as guanine on Our functions impinges Rab8 RAB3IP). which FIP3 as the that known with implies (also Rab11a study Rabin8 and factor ciliary exchange ASAP1 nucleotide abolishes coordinates of FIP3 FIP3 interactions mislocalization. of the rhodopsin ablation causes ASAP1, phenotype and of the with targeting ternary lack Resembling the competes from from ASAP1. it and FIP3 resulting displaces Arf4–GTP ASAP1. and with ASAP1 complex and to binding Rab11a for through rhodopsin photoreceptor sensory interacts FIP3 the indirectly the rhodopsin of with (TGN), of out network trans-Golgi passage transport and its Golgi ciliary (GAP) During protein Arf4-dependent rhodopsin. GTPase-activating receptor and the the Arf Arf promotes in the the RAB11FIP3) and ASAP1 that as Rab11a show known of we (also activity Here, FIP3 unknown. effector human still Rab11 are of to cilia receptors range sensory primary of wide detailed targeting However, specific a for documented. mechanisms in molecular abundantly involvement been and has their biology ciliopathies that in importance now considerable disease gained have cilia Primary ABSTRACT Ato o orsodne([email protected]) correspondence for USA. *Author 87131, NM Albuquerque, Mexico, New of University eateto ugr,Dvso fOhhlooy nvriyo e Mexico, New of University Ophthalmology, of Division Surgery, of Department 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,17–35doi:10.1242/jcs.162925 1375–1385 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. 1 n uak Deretic Dusanka and 2 eateto elBooyadPhysiology, and Biology Cell of Department 1,2, * hti eesr o h otnosrpeiheto ROS of replenishment continuous phototransduction influx the membrane for associated high necessary exceptionally the is light the that the and houses Despite which machinery. (ROS), rhodopsin segment outer receptor organelle rod light-sensing the specialized as highly known the to rise gives cilium al., et that 2014). Sang 2009; al., manifestations al., et neural et Valente Gerdes of and 2007; 2011; al., obesity variety et kidneys, (Fliegauf defects a cystic tube ciliopathies, degeneration, as by retinal known characterized include disorders, are of and functions spectrum which these Garcia-Gonzalo wide of Disruption a 2010; 2010). underlies al., al., et Nachury et 2012; Reiter, (Emmer membranes ciliary 03 aeoae l,20;Wn ta. 02.AA1at sa as acts ASAP1 2012). al., et (Deretic, Wang 2009; cascade linking al., et ciliogenesis trafficking, Mazelova ciliary communication Rab11–Rabin8–Rab8 2013; in the promotes GTPases to that Rab Arf4 and Geng protein emerged Arf has scaffold the 2014; ASAP1 between essential GAP al., al., Arf et an et Ward The 2010; 2011). as al., Follit al., et destination et Knodler 2010; 2006; Westlake al., intracellular 2011; al., et Jenkins et 2006; specific al., et Follit this direct 2013; that to (Deretic, complexes trafficking delivery intracellular membrane as targeting ciliary well of as conservation motifs, high reveal targeting membrane ciliary of efficiency the transport. increase is receptor and complex directionality targeting the the ciliary Arf4-based provide to ciliary role the to physiological and complex In emerging motif another the VxPx Golgi Thus, the 2013). of of 2014). the al., al., et delivery from (Follit cilium et fibrocystin, delays receptor, (Lodowski reduction structures, sensory RTCs vesicular Arf4 to post-Golgi addition, ciliary in corresponding of the protein trafficking likely tenfold fusion the least rhodopsin at accelerates a enhances 2012). of al., motif et targeting VxPx Wang ciliary 2014; the other Deretic, among Notably, as and conserved are (Wang The they known receptors and motifs, 2012). sensory FR, targeting (also al., the ciliary and two et VxPx FIP3 least the Wang at 2009; encodes effector sequence al., rhodopsin Arf-Rab11 (GAP) et complex (Mazelova protein the targeting RAB11FIP3) GTPase-activating a and of Arf assembly ASAP1 the the to Rab11a, leads (Deretic containing This signal directly 2005). targeting al., and C-terminal rhodopsin et recognizes VxPx Arf4 the GTPase to (TGN), binds small network activated trans-Golgi and/or the rhodopsin Golgi where nascent the on The at 2014). formed location Deretic, are periciliary RTCs and (RIS) (Wang the the segment (RTCs) carriers to inner of transport rod delivered constituent the directly main in 2003; and the synthesized al., is Rhodopsin, et membranes, 2013). (Besharse ROS al., organism et the of Pearring lifetime maintained the is throughout compartmentalization functional their membranes, itntv ovrino h o htrcpo primary photoreceptor rod the of conversion distinctive A eetisgt notemlclrmcaim fciliary of mechanisms molecular the into insights Recent Xenopus os and rods, 1375

Journal of Cell Science elcnevd nti td,w hwta I3i nessential an is FIP3 Rab targeting. that membrane show are and ciliary we pathways of study, Arf trafficking regulator this distinct the In membrane, in conserved. ciliary between effectors well the their interactions to and TGN molecular GTPases the from that and endosomes imply surface, recycling the cell and of Rab11a the out ASAP1, to flow of membrane roles directing the prominent in ASAP1 2006). of The FIP3 with 2008). FIP3 maintenance along al., al., 2007), the et for al., (Inoue et in et (Horgan involved necessary (Eathiraj compartment is recycling endosomes FIP3 2005). is al., interphase, recycling et binding During Wilson to 2012; Rab11 cells al., recruitment daughter et cytokinesis, two Schiel of 2005; During separation al., the et to (Fielding leads 2014; that the Deretic, cytokinesis of and in (Wang ASAP1 Arf4 2012). assist on al., et the hydrolysis to controlling Wang GTP by be budding of RTC might of GAP timing stage initial Arf targeting the of the of function ciliary termination the stimulates Therefore, 2001). in 2008). domain al., al., BAR FIP3 et et (Inoue functions the Prekeris ASAP1 2004; Rab11 of to activity al., FIP3 of et of diversity Junutula Binding 2001; the to al., binding to et for (Hales contributing other Arf-Rab11 each thus Rab11- with of its the family Rab11, compete Through distinct that with the 2006). of proteins member interacts al., interacting a et is directly which Nie its FIP3, ASAP1 effector 2009; of al., domain, autoinhibitor and al., et an BAR tubulation (Jian et as membrane activity acting Che mediates GAP also 1998; which while domain 2004), al., homodimerization, (Bin/amphiphysin/Rvs) al., et BAR et a (Brown (Peter contains activity ASAP1 GAP 2005). its regulates oniec eetrfratvtdAf,aii phospholipids [PI(4,5) acidic 4,5-bisphosphate Arfs, phosphatidylinositol activated for and detector coincidence ARTICLE RESEARCH 1376 1A, (Fig. tips, membrane cilia ciliary merged insets). the the in in along arrows at yellow transport XY, detectable present its arrows, of also tubulin red; indicative was 1A, Rh–GFP–VxPx acetylated Rh–GFP–VxPx (Fig. 2012). images). with axoneme al., the colocalized et agreement within green) (Wang in targeting, study 1A, ciliary (Fig. previous on 1). our (Fig. effect siRNAs no with small FIP3 had different non-targeting siRNA two control Control with with or Rh–GFP– (si)RNA either expressing interfering transfected cells were IMCD3 VxPx transport, ciliary al., et rhodopsin Wang 2010; Williams, and Trivedi 2012; 2012). the kinesin-2 of al., heterotrimeric from conserved et the localization Rh–GFP–VxPx the using (Trivedi tip of the ciliary to recapitulate transport base ciliary ASAP1-dependent the cells medullary including and because hTERT-RPE1 rhodopsin, model inner Arf4 and this specific mouse selected IMCD3 We the cells. epithelial signal (IMCD) targeting in duct C-terminal rhodopsin VxPx collecting eight the hereafter) bovine of containing repeat we rhodopsin of (Rh–GFP–VxPx the rhodopsin, of by comprised acids of followed amino targeting protein eGFP, ciliary by define fusion in To followed 2012). FIP3 a al., of et expressed role Wang Golgi 2009; the precise al., to the FIP3 et and (Mazelova Rab11a TGN of and recruitment the and to Arf4–GTP leads with rhodopsin ASAP1 of interaction synergistic manner early FIP3-dependent The epithelial a kidney of in cilia cells primary IMCD the to targeted is Rhodopsin RESULTS n fteesnilrlso I3i nasiso,tels step last the abscission, in is FIP3 of roles essential the of One odtriewehrFP sa seta euao of regulator essential an is FIP3 whether determine To P 2 ,which ], sn netbihdmtoooy(eei n aeoa 2009). Mazelova, postnuclear and (Deretic retinal methodology RTCs, established fractionated and an fractions using Golgi/TGN-enriched we into (PNS) 666–756) trafficking, supernatant acids rhodopsin (amino in GST–FIP3C site. binding whereas Rab11 Schonteich the 2007), 2008; contained al., al., et (Inoue ASAP1, sites et binding the Rab11 encompassed and 441–756) Arf4/5/6 acids (amino GST–FIP3F1 hwdangtv orlto o hGPVP and Rh–GFP–VxPx for correlation negative a showed respectively; siRNA#2, and infcnl ifrn rmta on ncnrl ( controls in was found which that siRNAs, from FIP3 with different treated significantly cells in tubulin acetylated 0 fteclstasetdwt I3sRA1properly siRNA#1 FIP3 with ( transfected over little siRNA cells Rh–GFP–VxPx a control just the targeted with but of cilia, treated primary 20% cells to the Rh–GFP–VxPx localized of 80% Approximately I3adAA1fnto ln h aeclaypathway. ciliary that same notion the along the function supporting expressing ASAP1 2012), and cells in al., FIP3 ASAP1-depleted large, et the and (Wang cells of Rh–GFP–VxPx By IMCD3 that 1H). FIP3-depleted resembled (Fig. of greatly phenotype Rab11 the minimal of experiments had expression these siRNA the this on whereas effect to experiments), siRNA#2 separate by (three decreased was content hte I3adroosnaei h aecmlx we RTC complex, stimulates which same FIP3, of membranes the end budding cell C-terminal in the photoreceptor determine FIP3F1, are from To with pulldowns 2012). rhodopsin GST al., and et performed and Wang rhodopsin FIP3 2009; with al., whether interact et directly (Mazelova Rab11a FIP3 and Arf4 Golgi ASAP1, photoreceptor the of TGN out and passage and its Rab11a during through ASAP1 FIP3 with interacts indirectly Rhodopsin 1.60E and elgbeefc nteepeso fAA1(i.1) In 1F). ( having (Fig. siRNA with control ASAP1 with compared while treated of Rh–GFP–VxPx, siRNA#2, experiments), expression targeted FIP3 of the separate with 20–40% on experiments to (five effect content negligible protein levels FIP3 control decreased siRNA#1 with nes.Ipraty I3sRA i o fetclalnt in length cilia affect 3.8 (3.9 not cells did insets) right siRNAs IMCD3 left in FIP3 in arrows arrows Importantly, 1C, 1C, (Fig. insets). (Fig. accumulation periciliary base substantial ciliary and the from Rh–GFP–VxPx and of axoneme Rh– absence the of show complete localization the insets) a ciliary the in causing the GFP–VxPx, with in potentially arrow interfered (magnified siRNA#2 1C, one areas FIP3 (Fig. that boxed only cilium Two panel). the magnification, middle to lower FIP3 Rh–GFP–VxPx Rh–GFP– with at transfected of FIP3 localized cells localization right). seen several the the the Of siRNA#2 cilia on on green). 1C, arrows effect the (Fig. YZ, similar to VxPx insets; a proximity in had close arrows siRNA#2 the XY in 1B, membrane (Fig. developed siRNA#1, into FIP3 that diverted arrows; with often projections transfected XZ, was cells red, In Rh–GFP–VxPx left). mislocalized 1B, the (Fig. on right). arrows tubulin the YZ, an acetylated with on detected arrows against accumulation cilia YZ, the antibody from insets; periciliary absent in largely its was arrows Rh–GFP–VxPx XY, included green, 1B, that (Fig. Rh–GFP–VxPx of oioaepooeetrboytei ebae involved membranes biosynthetic photoreceptor isolate To nlsso ie ooaiainb sn ero’ coefficient Pearson’s using by colocalization pixel of Analysis I3sRA1cue osdrbecagsi h localization the in changes considerable caused siRNA#1 FIP3 6 0.1 ora fCl cec 21)18 3518 doi:10.1242/jcs.162925 1375–1385 128, (2015) Science Cell of Journal nvitro in m 2 n 3.7 and m 6 o I3sRA1ad#,rsetvl)(i.1D). (Fig. respectively) #2, and siRNA#1 FIP3 for Mzlv ta. 09,o ihFPC(i.2A). (Fig. FIP3C with or 2009), al., et (Mazelova 6 6 0.1 0.3 m xnm ncnrlsRAversus siRNA control in axoneme m m nclstetdwt I3siRNA#1 FIP3 with treated cells in m P n P 5 5 5 5; 3.36E 8.29E 6 , s.e.m.). 2 0 fteclsproperly cells the of 20% 2 4 4 Fg E.Treatment 1E). (Fig. ) Fg G.FP protein FIP3 1G). (Fig. ) , 0 fcnrllevels control of 20% . 0 ftecells the of 60% P 5 9.75E 2 7

Journal of Cell Science xmndb muoltig G iir oaiaino hGPVP ncnrl n el rae ihFP iN# a eemndi he separat three in determined was siRNA#2 ( FIP3 experiment representative with a treated in cells ASAP1 and and FIP3 controls Student’s of in using mean expression analyzed Rh–GFP–VxPx The were of siRNA#1. data FIP3 localization The with Ciliary experiments. treated (G) cells immunoblotting. from lysate by the examined to compared were lysate control rsne stemean the as presented EERHARTICLE RESEARCH yrlsbeGPaao pNpt ciaeAf.GST– slowly pulldown Rab11, the Arf4. and in better the ASAP1 even activate performed with GST–FIP3C shorter with along to the rhodopsin, but supplemented GppNHp down pulled analog was FIP3F1 GTP whereas that Golgi/ hydrolysable the Golgi, Golgi/TGN from preparation pulldowns the the performed We in TGN 2B). in detected (Fig. RTCs were exclusively on Rab11 and nearly and ASAP1 present rhodopsin, was Arf4 Student’s using analyzed were data The in experiments. (arrows separate base sev ciliary mean the containing Rh–GFP– t the of and field a show proximity cilia a plane, the Data the in YZ shows from accumulates absent the panel Rh–GFP–VxPx is In middle left). Rh–GFP–VxPx the (red). The insets. on 5 cilium (C) the insets bar: in in the arrow). (arrows magnified Scale around left holes are right). black localized (red, areas as boxed is cilium ( appear The tips the arrows) that siRNA#2. cilia regions to (green, FIP3 the periciliary 0.9- juxtaposed Rh–GFP–VxPx with in confocal transfected is insets. and consecutive (green) cilia arrow) the Two Rh–GFP–VxPx proximal right in expressing siRNA#1. the (green, magnified at FIP3 projection are detected with areas membrane is transfected Rh–GFP–VxPx Tw boxed VxPx-filled (green) insets. microscopy. The the Rh–GFP–VxPx confocal plane. in expressing by XY channels cells analyzed the separate IMCD3 and in (B) shown tubulin and insets). (Ac) magnified manner.in acetylated is FIP3-dependent against area a boxed antibody in The with cells (yellow). stained IMCD3 fixed, epithelial 0.9- siRNA, kidney confocal of control consecutive cilia with primary transfected to were targeted GFP–VxPx is Rhodopsin 1. Fig. ooaiainaayi a efre ihnteclaadepesdb h ero’ ofiin ( coefficient Pearson’s the by expressed and cilia the within performed was analysis colocalization 6 ... *** s.e.m.; P 5 m ABadist nC,3 C), in insets and (A,B m 6 .08.()Teepeso fFP n a1 nteclstetdwt I3sRA2wseaie yimnbotn sabove. as immunoblotting by examined was siRNA#2 FIP3 with treated cells the in Rab11 and FIP3 of expression The (H) 0.0008). ... *** s.e.m.; 6 ... *** s.e.m.; m etosaepeetdi h Ypae hGPVP gen n ctltdtbln(y,rd ooaiei h iir axoneme ciliary the in colocalize red) (Cy3, tubulin acetylated and (green) Rh–GFP–VxPx plane. XY the in presented are sections m P , P .01.()Claylclzto fR–F–xxi otosadclstetdwt I3sRA1wsdtrie nfive in determined was siRNA#1 FIP3 with treated cells and controls in Rh–GFP–VxPx of localization Ciliary (E) 0.0001). 5 .03 F MD el eelsdadpoen eesprtdb D-AE he ifrn mut ftesiRNA the of amounts different Three SDS-PAGE. by separated were proteins and lysed were cells IMCD3 (F) 0.0003. m ist nAB,9 A,B), in (insets m t ts ( -test n 5 ,46clsi aheprmn,attlo 2clsfrec odto)adaepeetda the as presented are and condition) each for cells 22 of total a experiment, each in cells 4–6 3, t ts ( -test m C.()I ersnaieeprmn,R–F–xxaeyae-uui pixel Rh–GFP–VxPx–acetylated-tubulin experiment, representative a In (D) (C). m n 5 ,41 el nec xeiet oa f3 el o ahcniin n are and condition) each for cells 32 of total a experiment, each in cells 4–10 5, uldw r4drcl.Bt S–I3 n GST–FIP3F1 and GST–FIP3C Both to GST–FIP3C directly. down and pulled Arf4 GST–FIP3F1 the of down ability membranes, into TGN pull the and examined Golgi – the we from GST–FIP3F1 so down Very pulled TGN. than was and Arf4 Golgi readily little the at complex more targeting GST–FIP3C ciliary of – endogenous ability the incorporate reflected this to that reason We 2B). (Fig. ora fCl cec 21)18 3518 doi:10.1242/jcs.162925 1375–1385 128, (2015) Science Cell of Journal Xenopus n 5 2clsec o oto n I3sRA) tl control. Ctrl, siRNAs). FIP3 and control for each cells 12 n ua r4(i.2) lhuhFIP3C although 2C), (Fig. Arf4 human and A MD el rninl xrsigRh– expressing transiently cells IMCD3 (A) m etosaepeetdin presented are sections m neso the on insets rlcells eral n 5 o arrows )was 5) 1377 e he

Journal of Cell Science EERHARTICLE RESEARCH 1378 was rhodopsin to GST–FIP3C more of ( access diminished 2D). times (Fig. the significantly GST–FIP3F1 GST–FIP3F1 the but six on than effect pulldown, little PNS had down Again, ASAP1 retinal of rhodopsin. Immunodepletion the pulled with from interaction rhodopsin reproducibly indirect FIP3, its GST–FIP3C recruits specifically allowing 2012), al., thus et (Wang Rab11a recruiting site. within binding al., is Rab11 et FIP3 the on (Inoue to site closer site binding 666–756, binding Arf4 acids the amino Arf5/6 that the indicates of This portion 2008). a only contains I31adGTFPCt uldw hdpi.Teadto of addition The rhodopsin. GST– down of pull capacity to GST–FIP3C the the and whether increase FIP3F1 determine would to Arf4–GppNHp wanted of we addition rhodopsin, to binds directly down associated pulls is rhodopsin. which indirectly and Rab11, ASAP1 FIP3C endogenous with to that binding indicate by binding rhodopsin data Rab11 the these contains and domain, domain binding ASAP1 the lacks esuh odtriewehrAA1 nadto to addition in ASAP1, whether determine to sought We eas I3i nw fetro ciae r4 which Arf4, activated of effector known a is FIP3 Because P 5 .1 Fg D.BcueFIP3C Because 2D). (Fig. 0.01) pNp SP n a1awr rsn Fg E,as 2E), (Fig. FIP3 When Arf4– present 2012). al., when were et (Wang formed Rab11a studies previous was and in A determined rhodopsin Arf4–GppNHp. ASAP1 and with GppNHp, rhodopsin 2001), not complex precipitate al., does et to specific FIP3 (Hales able Rab11b were Because and both 2E). Rab11a (Fig. between pulldown 6His–FIP3 discriminate GST–Rab11a/b and/or recombinant rhodopsin, ASAP1 performed purified Arf4–GppNHp, we using experiments complex, targeting Arf4 activated diminished. exogenous greatly with is endogenous interaction the its to bound FIP3 complex, is targeting that the FIP3 When indicate into complex. incorporation data targeting with ciliary These concomitantly 2D). Arf4 (Fig. activated exogenous down recruits the pulled GST– of amount was following minimal Arf4 minutes a 30 only However, GST–FIP3C, at 2D). or added FIP3F1 (Fig. PNS was retinal increased Arf4–GppNHp the but when from GST– rhodopsin, Arf4 of with of pulldown interact ability their to the GST–FIP3C increase or not FIP3F1 did Arf4–GppNHp exogenous ofrhreaieteivleeto I3i h ciliary the in FIP3 of involvement the examine further To ora fCl cec 21)18 3518 doi:10.1242/jcs.162925 1375–1385 128, (2015) Science Cell of Journal muoltig sindicated. as by immunoblotting, analyzed to were bound beads Proteins glutathione length). (full 6His–FIP3 length) and/or (full ASAP1 Arf4–GppNHp, rhodopsin, bovine purified with incubated were GST–Rab11b mean using analyzed Student’s were data separate The three experiments. in quantified was bound rhodopsin and indicated, were as Proteins immunoblotted indicated. as the PNS, to control added samples, was some Arf4–GppNHp In recombinant PNS. ASAP1- retinal or frog control depleted the with either were incubated GST–FIP3C and immunoblotting. GST–FIP3F1 by (D) detected was Arf4 bound with purified incubated either GST– were (C) GST–FIP3C control. and Ctrl, FIP3F1 TGN; and G/ Golgi indicated. TGN, as immunoblotting, were by proteins analyzed retinal fusion interacting GST and as proteins expressed amino were (C; 666–756) FIP3C acids and (F1; 441–756) FIP3F1 acids (B) amino indicated. binding are Rab11 (BD) and domains Arf ASAP1, FIP3. of structure of TGN. out and passage Golgi its the during through Rab11a FIP3 and with ASAP1 interacts Rhodopsin 2. Fig. 6 ... * s.e.m.; t ts n r rsne sthe as presented are and -test Xenopus P 5 .1 E S–a1aor GST–Rab11a (E) 0.01. A h schematic The (A) rhmnAf,and Arf4, human or

Journal of Cell Science SP id hdpi n I3i mutually a in manner FIP3 exclusive and rhodopsin cooperative binds ASAP1 their complex. rhodopsin-targeting the of indicating of assembly specificity 2E), the (Fig. in function the ASAP1 Arf4–GppNHp pulldowns and FIP3 of both with individual observed recruitment those together, to the compared increased and were added Rab11a with were interaction ASAP1 and ARTICLE RESEARCH httebnigstsfrroosnadFP nteAA1BAR ASAP1 the on FIP3 suggesting and 3B), rhodopsin (Fig. for nM BAR-PZA sites to 60 binding FIP3F1 the nM that of 15 competed binding of rhodopsin binding Similarly, the the 3B). al., (Fig. et competed BAR-PZA Wang to 2009; rhodopsin substantially al., FIP3F1 et (Mazelova 2012). ASAP1 budding full-length We the RTC 3A). than stimulates (Fig. ASAP1 it of vitro because domain 6His–BAR-PZA BAR, repeat to the ankyrin chose FIP3F1 of and composed GAP and protein examined PH, rhodopsin we recombinant purified this, a of test 6His–BAR-PZA, To binding al., and ASAP1. simultaneous et to rhodopsin the Mazelova binding whether 2008; for establish compete to al., FIP3 wanted et we (Inoue to therefore, mapped domain been 2009); has BAR FIP3 ASAP1 and rhodopsin the both for site binding The n smr fiin nroosnAf–T pulldowns rhodopsin–Arf4–GTP in efficient more is and in yrlsso r4b SP,adteei froosnfrom rhodopsin GTP of exit controls the TGN. and likely and ASAP1, Golgi by complexes the dynamic Arf4 a on these that hydrolysis and FIP3, between a with form or equilibrium can rhodopsin Collectively, ASAP1 with Arf4–GTP-bound 3C,D). either the (Fig. complex that BAR-PZA complex indicate of data this association these displaced the within Arf4– on Arf4–GTP and effect FIP3F1 negligible with BAR-PZA a overlapping. with had complex but partially GTP, ternary the least from at rhodopsin are domain eeaeabnr upti h omof form the in reactions output unique binary enzymatic a generate of to – series probes oligonucleotide attached labeled a fluorescently with hybridization covalently through dualand – antibodies the that on secondary oligonucleotides antibodies based primary two two is by PLA complex and protein Briefly, target 2012). a of al., recognition et vertebrate of (Wang analysis the retinas for previously FIP3 modified in we complex which examined (PLA), targeting ciliary we established the the experiments, using photoreceptors, and rhodopsin binding with our interactions TGN corroborate and Golgi the To RTCs at on FIP3 to not proximity but close in is Rhodopsin ora fCl cec 21)18 3518 doi:10.1242/jcs.162925 1375–1385 128, (2015) Science Cell of Journal hwtemean the Data show experiments. separate were three Arf4 in and quantified FIP3 rhodopsin, Bound GST–FIP3F1. (D) of concentrations increasing followed by Arf4–GppNHp, and rhodopsin purified bovine with incubated Recombinant was (C) 6His–BAR-PZA control. Ctrl, indicated. (IB) as Ni- immunoblotted to were bound beads Proteins NTA–agarose versa. vice or GST– FIP3F1, of concentrations increasing by rhodopsin followed bovine purified with incubated was 6His–BAR-PZA indicated. Recombinant are (B) (BD) ASAP1. domains and binding FIP3 Mutual of structures Schematic manner. (A) FIP3 exclusive and mutually rhodopsin a to in binds ASAP1 3. Fig. nsitu in 6 s.e.m.. , rxmt iainassay ligation proximity 0.5 m os hc are which dots, m 1379

Journal of Cell Science nvrert htrcpos eas fteetariaiyhigh extraordinarily the of because complex photoreceptors, targeting ciliary vertebrate the in within interactions of analysis the for interactions transient and stable of situ detection the for PLA suited so well interactions, protein–protein is of ( limits the proximity within PLA is close the distance when in only possible are is probes signal positive the Because 2011). (So microscopy by visualized ARTICLE RESEARCH odr elwdse ie eact h odro h lisi n yi ein fteRS hc sdtcal nteDCiae e oswti the within 7 dots bars: Red Scale 1380 image. sites. DIC interaction the RTC as in counted detectable were is ellipsoid which the RIS, syna in the Sy, those of whereas nucleus; sites, regions N, interaction myoid junctions; TGN and adherens the or ellipsoid and AJ, from Golgi photoreceptors bud the (m). the individual that a of mitochondria outline as RTCs lines of border counted with RIS. dashed Diagram the were the filled White of (G) myoid demarcate panel. (‘E’) (M) RTCs. this lines region region in on myoid dashed ellipsoid summarized sites the Yellow is the in interaction border. methodology through localized quantification indicate are (arrow), The Arrows (G/TGN) PLA. cilium Rab11–FIP3. TGN Rhodopsin–ASAP1 the (F) the (H) and to and Golgi exp marke targeted ASAP1–FIP3 The representative (E) trans-Golgi are ROS. a for the a TGN from elaborates repeated (green), cilium data inhibite was anti-Rab6 Primary The is experiment and cell. retinas. synthesis same photoreceptor (red) (Cyclo)-treated protein The 11D5 cycloheximide which PLA. mAb and junctio in by anti-rhodopsin (Ctrl) adherens retinas detected with control of Student’s in in double-labeled comprised using detected quantified were (OLM, analyzed are were that membrane were (dots) sites retinas limiting signals interaction show fluorescent outer rhodopsin–FIP3 B Red the No and (C) indicates (B) A line (blue). TGN. in dotted and TO-PRO-3 Insets The with Golgi cycloheximide. DIC. stained the by at were visualized (N) predominantly were Nuclei rhodopsin sections to Retinal proximity retinas. in control is FIP3 4. Fig. interactions Rhodopsin–FIP3 4G). Fig. schematic the cell, rhodopsin–FIP3 see photoreceptor (M; detected the region myoid of we 4A, photoreceptor (Fig. the 2012), area in Golgi dots) al., the red in exclusively et almost PLA Wang by interactions 2009; al., et 2012). al., et Wang 2012; al., markers et organelle (Blasic specific and staining nuclear microscopy, (DIC) contrast interference exquisitely differential by identified readily retinal these is single which a layer, in within organized and well-aligned are flow that cells polarized membrane ciliary unidirectional DFHadist nAB.()Rdfursetsgas(os eecutdadtedt rmarpeettv xeiet( experiment Student’s representative using a analyzed from data RIS, the the and counted within were sites (dots) interaction signals fluorescent total Red of (I) A,B). in insets and (D–F,H sn rvosycaatrzdseii niois(Mazelova antibodies specific characterized previously Using tedgnu rti ees L spriual appropriate particularly is PLA levels. protein endogenous at t ts ( -test drege l,20;Tiiif tal., et Trifilieff 2006; al., et ¨derberg , n 5 0n) h aia detected maximal the nm), 40 0 n r rsne stemean the as presented are and 30) t ts ( -test n 5 in 2 n rsne stemean the as presented and 62) 6 ... *** s.e.m.; ie otiigAA1roosno SP+I3o RTCs of on 30% ASAP1+FIP3 to or close interaction ASAP1+rhodopsin where of containing proportion RTCs, equal sites The on localized. are persist sites interaction with Rab11 interactions FIP3 and whereas nearly TGN, FIP3 ASAP1 and of vicinity Golgi the the in at is exclusively rhodopsin Thus, 4I). only whereas (Fig. also TGN, RTCs and on Golgi is that the which at showed are 2012), sites interaction 4H, al., Fig. et in using (Wang quantification, detailed PLA methodology 4H). established (Fig. an 2012) previously al., as et RTCs, (Wang and ellipsoidreported TGN Golgi, the the sites in between interaction distributed Rhodopsin–ASAP1 RTCs, were arrows). on 4E,F, FIP3 Fig. (E; ASAP1 with region 4D), colocalized (Fig. also TGN Rab11 and specifically were Golgi the and the sites from at FIP3 interaction only exit rhodopsin–FIP3 concentrated Thus, highly Whereas the TGN. at insets). and rhodopsin Golgi synthesized in newly arrows with 4B,C), interacts the (Fig. 4A,B, and (Fig. membranes cycloheximide biosynthetic Golgi the with from rhodopsin treatment clears which by abolished were A hdpi–I3itrcinsts(e os eedtce yPAin PLA by detected were dots) (red sites interaction Rhodopsin–FIP3 (A) P , ora fCl cec 21)18 3518 doi:10.1242/jcs.162925 1375–1385 128, (2015) Science Cell of Journal .01 D hdpi–I3itrcinsts(e os were dots) (red sites interaction Rhodopsin–FIP3 (D) 0.0001. 6 ... ** s.e.m.; P n , 5 .0,* 0.001, , )wr xrse sapercentage a as expressed were 3) 0 fterhodopsin–FIP3 the of 90% P , 0.01. m AB,5 (A,B), m rmn ( eriment , pse. dby 0 are 10% RIS/ROS ns). n r. 5 m the 3) m

Journal of Cell Science EERHARTICLE RESEARCH hdpi;T,tasto oe F rniinfiber. Rh, transition (TC). TF, carriers zone; transport on transition Rab8 GEF TZ, of Rabin8 rhodopsin; activation extended subsequent the of the the positioning for binding at the domain the optimizing complex constrains likely targeting TGN, FIP3 ASAP1–Rab11a–FIP3 and that the Golgi suggests within study Rabin8 Our for taking cilia. site likely to interactions route molecular en of place sequence the summarizing diagram xeiet.Dt r rsne stemean separate the three as in presented quantified are was Data Rab11a experiments. by (D) detected indicated. FIP3F1. were as without beads immunoblotting, or Ni-NTA–agarose with to Rab11a, bound recombinant Proteins with incubated were control, a uigRCbdig SP csa cfodta eristo recruits that scaffold a as acts ASAP1 budding, RTC During ASAP1– the complex within targeting Rabin8 for Rab11a–FIP3 pocket binding the shapes their FIP3 PLA. but by detection FIP3, of range and large the rhodopsin outside single contain be a might might placement Alternatively, RTCs FIP3. on with complex other with the one complexes: and two rhodopsin between partitions ASAP1 that suggests the as presented was are beads Data anti-Myc experiments. mean to separate bound five ASAP1 in by (B) quantified detected indicated. without were as or proteins (IB), with Bound immunoblotting ASAP1, FIP3F1. recombinant untagged with or incubated the 6His–FIP3 were with Rab11a control, and a as ASAP1 of Rabin8. interactions GEF the Rab8 increases FIP3 5. Fig. neatosaddslcsroosnfo h enr complex ternary the from rhodopsin displaces ciliary and of rhodopsin–ASAP1 regulator controls interactions essential FIP3 an that is demonstrate FIP3 We that trafficking. show we study, this In DISCUSSION ( interaction ASAP1–Rabin8 increased 2.85E of significantly pulldowns 6His– FIP3F1. Myc–Rabin8 or 6His–FIP3 FIP3 prebound performed the without or We with ASAP1, ASAP1. displaces determine or from to with, cooperates FIP3 wanted Rabin8 trafficking we Because ciliary 2011). FIP3, in al., whether of et Westlake downstream 2007; functions al., Moritz Rabin8 2010; et al., Nachury et ciliogenesis essential 2001; Knodler 2002; al., and are al., et membrane et which Hattula plasma 2012; of al., the et factor both (Feng with 2012), exchange fusion al., nucleotide of et regulators guanine (Wang its Rabin8 (GEF) and Rab8–GDP RTCs nrae a1aRbn neato ( interaction RTC stimulates Rab11a–Rabin8 that increased Rab11 of effector an budding as functions FIP3F1 chose between it We FIP3F1. because association prebound the without the or pulldowns with 6His–Rabin8 examine Rab11a, performed of to We Rab11a. wanted and FIP3 next Rabin8, effector we an is Rab11, FIP3 Because of Rabin8. to separately bind FIP3 ASAP1 that suggesting and 5A), (Fig. FIP3F1 and 6His–FIP3 down pulled ( eetrtreig ossetwt h eut forstudy. ciliary our the of results regulates within the with interactions consistent that targeting, molecular receptor complex of sequence ASAP1–Rab11–FIP3–Rabin8 the recruitment ciliary the represents in enhancing spatiotemporally activation the Rab8 by to restricted stringency that additional site imparts notion FIP3 binding Rabin8, the of Rabin8 support complex. the targeting data ciliary shapes the ASAP1 Our within ASAP1 FIP3 both and Rab11 to that by formed binding suggests Rabin8 Rab11 in and FIP3- the increase Thus, 2012). threefold to al., mediated separately et Rab11a (Wang and bind Rabin8 ASAP1 to Rab11a that independently bind established and previously have FIP3 Rab11a We of that Rabin8. absence suggesting the in 5C), FIP3F1 (Fig. down pulled also 6His–Rabin8 P 5 .1) oevr nteasneo SP,Myc–Rabin8 ASAP1, of absence the in Moreover, 0.016). 6 2 ... *** s.e.m.; 3 ora fCl cec 21)18 3518 doi:10.1242/jcs.162925 1375–1385 128, (2015) Science Cell of Journal i.5,) I31hdasmlrbtsalreffect smaller but similar a had FIP3F1 5A,B). Fig. ; nvitro in P , A y–ai8budt niMcbas rMyc–GST or beads, anti-Myc to bound Myc–Rabin8 (A) .01 ** 0.0001, Mzlv ta. 09.FPF significantly FIP3F1 2009). al., et (Mazelova P 5 .0.()6i–ai8 r6i–F as 6His–GFP or 6His–Rabin8, (C) 0.005. rnpr.Fg Eschematically 5E Fig. transport. 6 P ... ** s.e.m.; 5 5.65E 2 P , 3 i.5C,D). Fig. ; .0.()A (E) 0.005. 1381 P 5

Journal of Cell Science rfikn hog h eyln nooe Peei ta. 2000) al., et membrane (Prekeris plasma endosomes 2005). of recycling al., regulation the the et through trafficking Wilson in implicated 2009; been al., has et Rab11 Mazelova 2006; al., et (Eathiraj the Thus, homeostasis. their 2010). on al., influence powerful vertebrate et has in trafficking photoreceptors Goldstein ciliary during 2011; Rabin8 of visual regulation al., of precise loss et severe (Berta by characterized function a is which in congenital (LCA), Leber result ciliopathy amaurosis which human the in to mutations corresponding condition a gene, as degeneration called identified was retinal (also NDR2 canine Recently, NDR2 2013). al., kinase et specific (Chiba serine/threonine STK38L) by membrane a the and to recruited phospholipids is Rabin8 progress cilia. they the as might Rab11a towards and Rabin8 ASAP1 of with Phosphorylation interactions its TGN. modulate and rhodopsin–Arf4–ASAP1 Golgi early the ASAP1 at the GTP complex with terminates traffic. ciliary Arf4 interactions of on direction sequential hydrolysis the the and progression FIP3 that cargo control that indicate shows data study our current Our ASAP direct 2012). increases al., significantly et (Wang interaction trafficking. ciliary facilitate to region polymerization periciliary actin the of control in the include of might role bridge ASAP1 the and 2010), FIP3 al., connecting et blocking (Kim Because ciliogenesis the 2009). facilitates al., assembly actin et of (Jing migration and cell telophase, severing 2012), cancer al., breast late et in the (Schiel cytokinesis in during for cells both daughter between essential cytoskeleton in actin role is important the which its of in with regulation involved is keeping FIP3 the 2000). in al., et 2012), (Randazzo primary dynamics al., cytoskeleton to proximity et in (Wang the protrusions causes cilia actin-based which the ASAP1, of of the depletion formation the resembles from closely Rab8 that and – fusion. recruitment RTC maturation Rabin8 for preparation to in RTC budding, activation RTC FIP3-assisted during of its also termination transition but activity, GAP functional ASAP1 only not control SP–I3itrcin Ioee l,20) stePI(4,5) the plasma As PI(4,5) RIS 2008). al., However, target et the 2004). (Inoue to al., interactions fusion ASAP1–FIP3 et RTC (Deretic for membrane preparation in Rab8 hdpi iir agtn ope Wn n eei,2014). Deretic, and (Wang the complex in PI(4,5) Inoue of ASAP1 targeting assembly 2005; assist the ciliary might al., of FIP3 rhodopsin stage et Thus, Arf4-dependent 2009). first, (Che al., the et terminating ASAP1 Jian of 2008; al., activity et GAP the regulate SP n a1ai h tpieasml fteciliary the of assembly assists stepwise that the component in ciliary a crucial Rab11a in as a step and is acts next downstream ASAP1 FIP3 the accordingly their to Therefore, which progression targeting. with Rabin8, for GEF ASAP1 detector links coincidence Rab8 the and on increases likely the FIP3 Rab11a hydrolysis partner, that that GTP show of we property with interactions Furthermore, ASAP1. a RTCs by into Arf4 ASAP1, incorporation and rhodopsin Arf4–GTP with ARTICLE RESEARCH 1382 PI(4,5) of to this regulation route site, the en fusion interaction the Consequently, ASAP1–FIP3 reach cilia. RTCs diminish as gradually increase might to appears content PI(4,5) and FIP3 by Conformational induced 2009). al., ASAP1, et in (Mazelova GAP a Arf4 changes as of functions effector which ASAP1, an with and interact to ability cilia. its of the with to succession progression ordered cargo direct the that regulates GTPases small which complex, targeting a1–I3itrcin r motn nalFP functions FIP3 all in important are interactions Rab11–FIP3 ASAP1–rhodopsin direct increases significantly Arf4 Activated phenotype a produces cells IMCD3 from FIP3 of depletion The h euaoyrl fFP nclaytafcigi associated is trafficking ciliary in FIP3 of role regulatory The P 2 n hshioiiebnigpoen oprt with cooperate proteins phosphoinositide-binding and –ai8itrcin Collectively, interaction. 1–Rabin8 P 2 eesmight levels P P 2 2 binding, inhibits P 2 ih eivle nrcutn n oiinn ciae Arf4 late activated during positioning FIP3 and that midbody recruiting suggest in the data Our involved to 2007). be al., might recruitment et (Schonteich Arf6 is for telophase FIP3 complex. responsible for targeting not ciliary the is required into Arf4 FIP3 of activated recruitment was that the that interactions suggests finding rhodopsin–FIP3 our It increase Thus, surprising. not 1999). al., did et Arf4 Shin activated 2007; al., et Schonteich to in compared trafficking. function ciliary Rabin8, in cooperative activation Rab8 for their and not recruitment affinity of Rabin8 are greater indicative interactions affinities, a these individual ASAP1–Rab11a–GTP– has that the complex contrary, shows FIP3 the study On exclusive. Our mutually Knodler 2010). 2006; al., al., et (Eathiraj et Rabin8 and FIP3 with both interacts be Rab11–GTP the could complex. ASAP1–Rab11–GTP–FIP3 in the Rab11–GDP resulting of formation GEF, TGN, al., vertebrate yet-to-be-identified the et a at Wang by activated that, 2009; hypothesize al., We et 2012). (Mazelova Rab11–GDP or GTP–FIP3 PI4K like that, al., observed complex et We ternary (Inoue 2008). ASAP1 A and FIP3 2014). Rab11–GTP, between formed al., be et can (Burke Rab11–GDP or complex PI4K that 4- phosphatidylinositol by recruited kinase is it where TGN, the through or oeta novmn ncloahe n te ua diseases. human other and ciliopathies with in receptors, involvement signaling potential membrane of ciliary compartmentalization of and regulator indispensable expansion an here as presented FIP3 data implicate the Nevertheless, and coat. architecture putative this the of about function investigation information Future invaluable trafficking. provide al., TGN-to-cilia should et protein regulates Nie a comprise 2006; that might al., together coat form et they (Eathiraj complexes Rab homodimers the 2006), and as act Arf ASAP1 of FIP3 Because and activation cargo. ciliary successive of movement forward the the with couple GTPases 2001; to al., 2007). Rab11a al., et and et Yoshimura Moritz 2012; 2010; al., et al., Wang et 2007; al., Knodler polarized et 1995; Nachury of al., stages et final (Deretic ciliogenesis and the carriers ciliary-targeted of regulates fusion traffic, membrane Rab8 membrane, activated plasma periciliary renders the because Rab8 with fusion (Wang of for budding competent activation RTC RTCs Rabin8-mediated of Rabin8 2014). stage Deretic, post-Arf4 FIP3. later and the for and prevent necessary Rabin8 might as is complex, Rab11a targeting ASAP1 ciliary the of into with inclusion conformation premature its active complex the Rabin8 catalytically for in its the preference to exposes binds ASAP1 also Rabin8 rhodopsin that 2008), active conclude al., we with activating so et an site, Arf4 (Inoue binding causes ASAP1 which on in binding, hydrolysis change FIP3 RTCs. GTP by into ASAP1 couple packaging from might rhodopsin of FIP3 Displacement GTP blocking Arf4. to by binding on RTCs Rhodopsin hydrolysis into 2009). packaging its al., allows et possibly Mazelova ASAP1 complex 2008; the al., transport. et of ciliary (Inoue part to directionality a recruit impart is to to FIP3 important recurring ability be when support might diminished Arf4 to activated the likely additional However, complex, biogenesis. targeting carrier ciliary the into idgf fDvdWlim Uiest fClfri,LsAgls CA). Angeles, Los California, of a (University was Williams David rhodopsin–eGFP of bovine gift kind encoding vector expression Mammalian Materials METHODS AND MATERIALS I3i loakonAfefco Hcsne l,2003; al., et (Hickson effector Arf known a also is FIP3 nsmay eso htFP cssnritclywt ASAP1 with synergistically acts FIP3 that show we summary, In ASAP1 of domain BAR the to bind FIP3 and rhodopsin Both b ora fCl cec 21)18 3518 doi:10.1242/jcs.162925 1375–1385 128, (2015) Science Cell of Journal (PI4K b a iutnosyrcutteRab11–GTP–FIP3 the recruit simultaneously can b d ra ta. 04.Arcn td a shown has study recent A 2004). al., et Graaf (de ) b SP lorcut Rab11– recruits also ASAP1 ,

Journal of Cell Science n nuae t37 medium pre-warmed at trypsinized in incubated were (Costar) plates Cells and culture (GIBCO). six-well medium serum to DMEM/F12 transferred bovine from in and fetal obtained flasks 10% culture cells with Falcon IMCD-3 T75 (ATCC) Mouse on control. plated a were ATCC as used was Dharmacon AGAUU-3 Aesa isine) abtat-Hs(ou ilgcl) and Biologicals); (Novus anti-6His anti-GST rabbit goat Biosciences); (Sigma); Cruz tubulin (Amersham (Santa anti-acetylated anti-Rab11 polyclonal mouse goat Biotechnology); and 2000); anti-Rab6 al., et polyclonal (Randazzo rabbit anti-ASAP1 anti- and anti-Rab11 polyclonal rabbit and 2009); (Deretic Biosciences); anti-Rab8 (BD 11D5 al., anti-ASAP1, monoclonal (mAb) et 1991); antibody (Mazelova Papermaster, monoclonal anti-Arf4 C-terminus were polyclonal study rhodopsin Western this rabbit (Case in used follows: Palczewski antibodies Kris as primary of The gift OH). University, a Reserve was rhodopsin bovine Purified ARTICLE RESEARCH H74 5 MNC,5m MgCl mM 5 NaCl, mM 150 500 7.4, in (5 pH fractions alone retinal GST frog or with proteins fusion GST assay Purified pull-down protein fusion GST 5 Scientific was Thermo siRNA#1 from for GGCAGAACUA-3 sequence obtained target were The Dharmacon. oligonucleotides siRNA siRNA FIP3 and FIP3 rhodopsin–GFP–VxPx Bioscience. of Kit Olink transfection Red Double from was Rabbit/Mouse nm) II 634 Duolink emission, nm; kind (NCI/NIH). 6His– 598 were Randazzo CO). (excitation, anti-ASAP1 Colorado, Paul polyclonal of of (University rabbit gifts vector, Prekeris and pGEX-KG Rytis 6His–ASAP1 of GST–FIP3, of BAR-PZA, the (University gifts anti-FIP3, in kind Peranen polyclonal were GST–Rab11b, Johan rabbit GST–Rab11a, of and GST–FIP3F1, gifts Goat Finland). were al., Helsinki, Myc–Rabin8 et (Hattula and anti-Rabin8 polyclonal 2002) Rabbit (Qiagen). anti-6His mouse eewse i ie ihrato ufr on rtiswr eluted were proteins Bound buffer. reaction 20 with in times six washed were .%BA ntepeec rasneo 5 of absence or presence the 100 in BSA) 0.1% S uinpoen eeimblzdo glutathione–Sepharose-4B on immobilized were (30 beads proteins fusion GST LSRaet Ivtoe)floigtemnfcue’ protocol. manufacturer’s the following 2 and (Invitrogen) Briefly, (rhodopsin– LTX rhodopsin Lipofectamine Reagents of with acids PLUS performed amino was C-terminal Transfection eight GFP–VxPx). of followed room repeat eGFP the by at followed by mammalian rhodopsin pcDNATM3.1 hours. bovine minutes a 5 encoding with 20 vector for Lipofectamine expression transfected incubated transiently and for then diluted cells were to incubated Cells added OPTI-MEM and was and complex medium This duplexes combined temperature. reduced-serum were siRNA the RNAiMAX Diluted of (6 ml OPTI-MEM. 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O’Brien, O., Goldstein, S., Genini, K., Boesze-Battaglia, I., A. months. Berta, 12 after release References for PMC in Deposited Center. Institute Cancer Cancer UNM National Center the Foundation, EY- National and Science number by National [grant supported Resources, Health is Research of Facility for Institutes Microscopy National Fluorescence the UNM by 12421]. supported was work This Funding manuscript. the wrote all and perfomed analysis J.W. quantitative methods. the the performed wrote D.D. and experiments. experiments the planned authors Both contributions Author interests. financial or competing no declare authors The interests Competing reagents. of Randazzo gifts Paul generous Prekeris, their Rytis for Peranen, Williams Johan David Palczewski, and Kris Drs thank We Acknowledgements first were eyecups experiments, 25 without some or with (100 retinas. In and hours overnight 5 agarose. frog for paraformaldehyde incubated 5% 4% dark-adapted in with on fixed embedded were performed eyecups was Isolated microscopy Confocal assay ligation Proximity grs ed eete ahdsxtmswt ecinbfe.Bound buffer. 20 reaction above. in as with analyzed times eluted six were washed proteins then were beads agarose onesandwt h ula ti OPO3adeaie by examined and TO-PRO-3 stain nuclear microscopy. the confocal with counterstained SP n HsFP,o I3,wr de oMyc–Rabin8 to 4 at added above, were as (30 buffer, beads FIP31, reaction in anti-Myc–agarose or incubated the on 6His–FIP3, immobilized and ASAP1 ape n nuae o ora 37 at hour 1 for incubated and samples oyeaeslto o 0 iue t37 at minutes 200 for solution Polymerase ed eewse i ie ihrato ufrcnann 5mM 15 containing buffer reaction 20 in with eluted were times proteins six Bound imidazole. washed were beads eobnn I3F rwt ie muto I3F n different 20 and of of FIP3-F1 presence amounts of the imidazole. amount different a in fixed mM rhodopsin and with 15 a of rhodopsin with amounts either containing bovine or incubated FIP3-F1 purified PBS recombinant then of with were amount once fixed proteins His-tagged washed Immobilized and hour 1 ufradaaye sabove. as analyzed and buffer et,poieainadfraino yrdrdScn htrcposi the in photoreceptors rod/S-cone hybrid retina. mutant of STK38L formation degenerating and proliferation death, A ´ ,Aln,G . eta,W .adAure .D. 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Journal of Cell Science iko,G . ahsn . ig,B,Mir .H,Fedn,A . Prekeris, B., A. Fielding, H., V. Maier, B., Riggs, J., Matheson, R., G. Hickson, Pera and A. Arffman, J., Furuhjelm, K., Hattula, ae,C . rnr . od-edro,K . on .C,Hry D., Hardy, C., M. Dorn, C., K. Hobdy-Henderson, R., Griner, M., C. Hales, M. G. Acland, and D. G. Aguirre, V., A. Kukekova, O., Goldstein, eei,D. Deretic, Geijsen, K., T. Schulz, M., Deneka, A., R. Dijken, van T., W. Zwart, P., Graaf, de eds .M,Dvs .E n asns N. Katsanis, and E. S. E. Somlo, Davis, and M., S. J. Shibazaki, Y., Gerdes, Cai, X., Tian, Z., Yu, D., Okuhara, L., Geng, F. J. Reiter, and R. F. Garcia-Gonzalo, ahrj . iha . rkrs .adLmrgt .G. D. Lambright, and R. Prekeris, A., Mishra, S., Eathiraj, Arendt, and A. P. Hargrave, V., Morel, N., Ransom, H., A. Williams, D., Deretic, B. E. Turco, de Rodriguez F., Jackson, N., Parkins, V., Traverso, D., Deretic, Deretic,D.,Huber,L.A.,Ransom,N.,Mancini,M.,Simons,K.and S. D. Papermaster, and D. Deretic, J. 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