© 2011 Nature America, Inc. All rights reserved. Ca Ca the (ref. ARL3 and 14) (ref. ARL2 GTPases Arf-like the proteins, including diverse several with interact to shown been has UNC119 nates in retinal photoreceptor inner segments and synaptic regions ortholog was discovered in the retina and to recognized be a chemosensation and behavior feeding locomotion, affecting mutation spontaneous a complex of neurons Utah, Utah, USA. Medical Institute, University of Utah, Salt Lake City, Utah, USA. Miller School of Medicine, Miami, Florida, USA. University, Piscataway, New Jersey, USA. USA. Science Center, Salt Lake City, Utah, USA. 1 of teome and prenyl-binding proteins components of the BBSome in vesicular trafficking and stabilizing tinues to emerge of an ever-increasing number of polypeptides involved disease.For both photoreceptors and transduction, ciliogenesis and cilia Defects in these trafficking pathways have been shown to impair signal nents, are transported to cilia by vesicular and intraflagellartogether mechanisms.with their G proteins and associated signal C. elegans or photoreceptors mammalian In neurons. sensory animal various in found are stimuli external to sensitive cilia primary Non-motile cilia. in sensory trafficking protein and mouse in both deletion UNC119 translocation. light-induced after segment outer to the back segment inner the from of diffusing capable complex T cavity.bound UNC119 hydrophobic T an acylated with of UNC119 G acylated and UNC119 between interaction a tight revealed calorimetry titration an immunoglobulin-like revealed resolution at 1.95-Å UNC119 of human structure transducin photoreceptor rod of the N terminus acylated the recognized We UNC119 that phyla. found other and vertebrates among expressed is widely UNC119 George Inana Yuanpeng Janet Huang Houbin Zhang sensory neurons UNC119 is required for G protein trafficking in nature nature Received 28 February; accepted 14 April; published online 5 June 2011; Department Department of Ophthalmology, University of Utah Health Science Center, Salt Lake City, Utah, USA. UNC119 is a 27-kDa polypeptide identified in the basal body pro body basal in the identified polypeptide is a 27-kDa UNC119 2+ channel Ca channel 4 Center Center for Advanced and Biotechnology Medicine, Department of Molecular Biology and Biochemistry, Northeast Structural Genomics Consortium, Rutgers 2+ 1 NEUR 9 -binding protein CaBP4, a modulator of the of modulator a CaBP4, protein -binding olfactory cells, light receptors (rhodopsin) or 0 . . C. reinhardtii C. 10 . Although expressed in multiple tissues, UNC119 predomi unc-119 C. elegans C. These These authors contributed equally to this work. should Correspondence be addressed to W.B. ([email protected]). OSCI 8 . Independently, a protein named retina gene 4 (RG4) 6 v , , Frank G Whitby 1.4 present in rod and cone synapses cone and rod in present 1.4 1 was first discovered in in discovered first was , EN 10 8 C , , Ryan Constantine and the mouse photoreceptor sensory cilium cilium sensory photoreceptor mouse the and E 6 , the flagellar rootlet of of rootlet flagellar the ,

2 advance online publication online advance 5 , Rab GTPases . 4 , , Rong Xiao C. C. elegans

maintenance a 5 N-terminal peptide at 2.0 Å revealed that the lipid chain is buried deeply into UNC119’s into deeply is buried chain lipid the that Å at 2.0 revealed peptide N-terminal Department Department of Biology, University of Utah, Salt Lake City, Utah, USA.

intraflagellar transport, including 3 odorantreceptors, evidence con Department Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, New York, a 7 , Erik , M Erik Jorgensen 7 (T a 3 Department Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah, USA. led to G protein mislocalization. Thus, UNC119 is a G UNC119 Thus, mislocalization. to G led protein , Arf and Arf-like GTPases -GTP, inhibiting its GTPase activity, thereby providing a stable UNC119–T a stable providing -GTP,activity, thereby GTPase its inhibiting C. elegans C. a 4 , , Gaetano T Montelione 1 ) subunit and and ) subunit 1 , often leading to severe , 2

transduction compo , 10 Naegleria gruberi Naegleria C. C. elegans unc-119

odorant , Sergey , VorobievSergey on the basis of of basis the on

9 voltage-gated voltage-gated Department Department of Neurobiology and Anatomy, University of Utah Health Science Center, Salt Lake City, 1 6

receptors, ; and the the and ; Caenorhabditis elegans Caenorhabditis doi:10.1038/nn.283 11– 5 15); 15); , 8 1 , , Christopher P Hill 7 3 4 - - - - . ,

as diverse as mouse and and mouse as diverse as models animal in trafficking protein G for required is UNC119 that found we knockouts, UNC119 Using UNC119. with contacts mate T the of residues acid amino six first the and proteins, related structurally for seen that of side opposite the from interior protein the Notably,enters ligand the the by formed cavity hydrophobic the β immunoglobulin-like an adopts UNC119 that revealed resolution ceptors and N acylated termini of G protein kingdom. animal the throughout functions multiple has and conserved highly is it that suggests protozoa lated and flagel even invertebrates of in vertebrates, UNC119 distribution half N-terminal its in located domains through homology Src them with interacting by receptor T-cell the and receptor interleukin-5 the with associated kinases tyrosine Src-type activate kidney and cerebellum glands, fibroblasts lung cells, T (eosinophils), leukocytes in model mouse transgenic a in dystrophy dominant an individual with late-onset dominant cone dystrophy also produced in the human identified zygous stop (K57ter) codon RIBEYE component ribbon ­synaptic -sandwich fold. The N-terminal peptide of T of peptide N-terminal The fold. -sandwich 3 We identified UNC119 as a lipid-binding protein interacting with with interacting protein lipid-binding a as UNC119 We identified detected been has UNC119 retina, mammalian the from Apart , , Yang Chen 4 , Cecilia D Gerstner , Cecilia 5 C. elegans 2 Graduate Graduate Program in University Neuroscience, of Utah Health G proteins ODR-3 and GPA-13. The crystal crystal GPA-13. The and ODR-3 G proteins a 3 , , Jayaraman Seetharaman peptides. The structure of co-crystals of co-crystals structure The peptides. 7 b , , Liang Tong olfactory neurons. The crystal structure at 1.95-Å -sandwich fold. Pulldowns and isothermal isothermal and Pulldowns fold. -sandwich α 6 peptide are buried in the core, making inti making core, the in buried are peptide Bascom Bascom Palmer Eye Institute, University of Miami C. elegans C. 1 a , , M Wayne Davis α 1 subunit cofactor essential for G for essential cofactor subunit 1 subunits present in mouse photore . UNC119 has also been shown to to shown been also has UNC119 . . 3 & Wolfgang Baehr 1 7 of photoreceptors. A hetero A photoreceptors. of β -sandwich fold of UNC119. UNC119. of fold -sandwich α inserts deeply into into deeply inserts

t r a 3 1 8 5 8 , Howard Howard Hughes a , . UNC119

20 1 -GTP -GTP 9 . Widespread Widespread . , the adrenal adrenal the , C I 1 gene of , 5 e l , 9 s  - - - -

© 2011 Nature America, Inc. All rights reserved. to T to antibody with Top, probed blot buffer). binding the in present (DT) NP-40 10 6, lane added; 10 5, lane control; GST 4, lane pulldowns; lysate retina 4–6, lanes (input); lysates retina mouse 1–3, (lanes pulldowns T non-acylated with interact not T to antibody with probed was Blot lysates). T bovine expressing cells HEK T bovine expressing cells HEK 4, 1 and lanes pulldowns; T T farnesylated (lane not 4), but 4 and 8, pulldowns lanes with GST-UNC119. Acylated T GST; with pulldowns control 7, 3 and lanes input; 6, 5 and 2, 1, Lanes T of T cone and rod with matched were red, in shown sequences, peptide Identified LC-MS/MS. by identified were GST-UNC119 with down pulled polypeptides 40-kDa The LC-MS/MS. ( GST-UNC119. by down pulled proteins the identifies asterisk An protein). fusion GST-UNC119 4, lane (GU); pulldown GST-UNC119 3, lane (GC); a control as GST with pulldown 2, lane (G); GST recombinant 1, (lane SDS-PAGE by analyzed were lysates retina bovine from polypeptides Bound experiments). independent four of T cone and rod 2 Figure nylated proteins participating in Fig. PrBP/ with similarity sequence and (RhoGDI, sequence identity with UNC119) and Rho-GDP dissociation inhibitor 1 similar structures, including PrBP/ www. ( stereochemistry expected and data ( this of revealing a narrow,edge deep cavity one penetrating the center at of this structure other each from apart splayed are loops ( proteins pattern key Greek a in ( fold strands, antiparallel four in arranged immunoglobulin-like an contains β 3GQQ) ID: (PDB resolution at 1.95-Å 57–237) of (residues human UNC119 structure The crystal The structure of human UNC119 at 1.95-Å resolution RESULTS t r a  GST-UNC119. with interact not does GCAP1 myristoylated Supplementary Fig. Supplementary 1a Fig. 1 Fig. α

-sandwich fold of -sandwich comprised two A search of the Protein Data Bank with the program DaliLite ( a and T and α α 2a

1c . Bottom, same blot probed with antibody to GCAP1. Note that that Note GCAP1. to antibody with probed blot same . Bottom, and (WT,1–4) lanes wild-type from ebi.ac.uk/Tools/dali ). When restricted to the region that folds into α ). PrBP/ ). a

C I

α β ). The atomic model is consistent with the crystallographic crystallographic the with consistent is model atomic The ). 7 α 1 1 and 2 Interaction of UNC119 with T with UNC119 of Interaction

1 β2 1 (G2A) expressed in HEK cells (lanes 1–3, input; lanes 4–6, 4–6, lanes input; 1–3, (lanes cells HEK in expressed (G2A) . Strands Strands .

Z β4

score 9, 10% overall identity) ( identity) overall 10% 9, score e l

β 6 β α 1 α

with GST-UNC119 (representative Coomassie-stained gel gel Coomassie-stained (representative GST-UNC119 with δ

2 2 in β C 5

is a prenyl-binding protein that interacts with pre with interacts that protein prenyl-binding a is

s µ

β g GST-UNC119 with 0.1% Triton X-100 and 0.1% 0.1% and Triton 0.1% X-100 with g GST-UNC119 β3 8 Fig. Fig. 1 β β9 3- β ), ), which is found in a large number of diverse

α lite/index.htm 4 and and 4

2 a ). ). In the UNC119 the structure, two sheets

P123 α γ (G2A); lanes 3 and 6, mouse retina retina mouse 6, 3 and lanes (G2A); α (lane 8). ( 8). (lane (G2A). ( (G2A).

N phot δ α β

90 increased to 56% ( 56% to increased . . ( 7- δ

° ( β c β Z sheets ( sheets ) GST-UNC119 pulldown pulldown ) GST-UNC119 o α Supplementary Table 1 Supplementary 8 are linked by short short by linked are 8 b b

score 12, 24% overall amino acid transduction polypeptides. ( polypeptides.

e

) Identification of peptides by peptides of ) Identification β Gnat1 ) Specificity of retina lysate lysate retina of ) Specificity 6 d l ) returned a large number of number large a returned )

α ) GST-UNC119 pulldown of pulldown ) GST-UNC119

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1 β

7 β

Supplementary Figs. 1b 1b Figs. Supplementary Figs. Figs. 1a 8 −/−

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retinas (lanes 5–8). 5–8). (lanes retinas β4 g GST-UNC119 was was g GST-UNC119 β 5 1 , whereas RhoGDI α β

α , a α was pulled down b sheets, UNC119

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P108

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chains, which are important for targeting T for targeting are important which chains, acylated ously protein. prenyl-binding a not is UNC119 that confirming using negative were Pulldowns inactive state, GDP is bound to T to bound is GDP state, inactive ( f T of group of T acyl N-terminal the with interact would UNC119 that suspected we Given the structural similarity among UNC119, PrBP/ T with interacts UNC119 T to antibodies with probed Immunoblots and wild-type T with its interaction through plex T T subunits transducin the peptides matching the rod T rod the matching peptides GST-UNC119 bound specifically ( that kDa 40 polypeptide a approximately identified of we lysates, retina bovine From (LC- MS/MS). spectrometry mass tandem and chromatography liquid high-perform ance by photoreceptors, followed in pulldown protein UNC119 fusion GST of used we partners interacting identify To transducin- the with interacts UNC119 ties suggest that UNC119 might be a lipid-binding protein. and geranylgeranyl chains teins possess a pro Both (ref. 22). GTPaseCDC42 Rho ofthe function regulates the axis. vertical the around rotation a 90° after viewed UNC119 of 108–123 connecting strands loop the of structure The respectively. C, N and marked are C termini of the center at the cavity to the an opening create ( Nine 57–237). (residues UNC119 human of structure the of 1 Figure β T c a Fig. Fig. 2 Fig. T α 1– α γ The The glycine residue at the N terminus of rod T β β γ α G G Input 2 1

1 9), shown in red, create two two create red, in shown 9), WT retina . . To test UNC119 whether pulled down the heter 2 ih lnn, hc peet T prevents which alanine, with a b

3 2 ). Sequence identification by LC-MS/MS yielded numerous numerous yielded LC-MS/MS by identification Sequence ). ), both of which are involved in phototransduction. In the the In phototransduction. in involved are which of both ), Crystal structure of human UNC119. ( UNC119. human of structure Crystal G C advance online publication online advance α G down Pull- . . To test this hypothesis, we replaced G2 at the N 3 U GU

5 4 hydrophobic pocket capable of accommodating farnesyl Gnat1 2 * 5 , carrying either C12:0, C14:1, C14:2 or C14:0 side side C14:0 or C14:2 C14:1, C12:0, either carrying , 4 Gnat1 Input 20 30 40 50 70 −/− 7 6 –/ – (T retina G down Pull- 23 α α G β β , 16 8 Rod b 2 Cone knockout) mouse retina lysates ( U , but not with farnesylated T farnesylated with not but , 2 and 4 γ 1 . The aforementioned structural similari , yielding the heterotrimeric complex, complex, heterotrimeric the yielding , α T subunit and one matching cone T cone matching one and subunit β α T e d sheets that splay apart at one end to end one at apart splay that sheets α β α Gnat1 α 3 could not be resolved. ( T , permitting the interaction with with interaction the permitting , 1 α , pulldowns were repeated using using were repeated , pulldowns Mouse retina 2 1 Input Input G2A − 3 2 α /

− acylation. Recombinant Recombinant acylation. α eia yae ( lysates retina α mR 17 a nature nature α subunit to the outer segment outer to the ) Ribbon representation representation ) Ribbon 6 and T and 4 3 β α sandwich. The N The and sandwich. (G2) is heterogene T 5 4 G G α Pulldown Pulldown G2A NEUR γ o δ revealed that that revealed 6 5 trimeric com trimeric U and RhoGDI, β GU + γ strands strands mR b OSCI 6 ( ) Structure

terminus Fig. Fig. Fig. D SC389 UW101 SC389 40 35 32 28 24 20 16 12 80 EN

0 0 0 0 0 0 0 2 2 2 c c c C 2 α 6 ), ), ). ). ). ). E - - - - ­ .

© 2011 Nature America, Inc. All rights reserved. acylated ( revealed that the G N-terminal mouse of Alignment specificity. binding in tide ( that than of lauroyl was two weaker the T orders of magnitude roylated peptide derived from N-terminal ODR-3, but the interaction (0.7 ured ( tively K each exhibited tight toyl-GAGASAEEKH binding at a site single with myris and lauroyl- whereas UNC119, human recombinant with act T unacylated the that showed experiments in discovered mutation was originally (the transduction signal chemosensory in participates in expressed N-terminal T bovine N-terminal acylated the titrated two reactants enabling the resolution of two or more binding sites. We peptides with UNC119 ( mine the binding constant of acylated N-terminal G protein group as a mediator of the interaction between T ( T T acylated The N terminus of bovine T we added a synthetic lauroylated (C12:0) peptide corresponding to the protein, acyl-binding an is UNC119 that confirm Toindependently protein acyl-binding an is UNC119 specificity. of degree some requires UNC119 with interaction the that suggesting shown), not (data recoverin ( or GCAP1 N-myristoylated with interact to failed UNC119 T of ( assays T nature nature G to all not extend does Fig. 3 α α D b a We (ITC) to deter titration calorimetry then employed isothermal values of approximately 0.54 to UNC119 ( (G2A) did not interact with bovine GST-UNC119 in pulldown pulldown in GST-UNC119 bovine with interact not did (G2A) Kcal per mole injectant α µ K in in vitro –8 –6 –4 –2 mediates the interaction with GST-UNC119. Furthermore, Furthermore, GST-UNC119. with interaction the mediates M and 19 19 and M 0 a D Fig. 3 Fig. ). These results support the requirement for the N-terminal acyl Fig. 2 Fig. = 16.4 NEUR Fig. 3 Lau-GAGASAEEKH 0 C. elegans C. for farnesyl and geranylgeranyl side chains with PrBP/ with chains side geranylgeranyl and for farnesyl GAGASAEEKH b C. elegans C. d OSCI ). This dissociation constant is similar to those meas those to similar is constant dissociation This ). GST-Unc119 Retina lysate α c ± 0.4 ), suggesting that the acyl side chain attached to G2 G2 to attached chain side acyl the that suggesting ), ), indicating that the UNC119-G Fig. 3 3.0 peptide was able to competitively inhibit binding of bindingcompetitivelyinhibit to able was peptide Gna11-15 µ M, respectively) M, SC389 EN µ 0.8 Molar ratio M), M), further implicating the N-terminal residues a C α ODR-3 peptide (GSCQSNENSE). ODR-3 is is ODR-3 (GSCQSNENSE). peptide ODR-3 AWA, AWB and AWC and neurons olfactory ), whereas the unacylated peptide had no effect Fig. 3 E α (2-GAGASAEEKH-11) to the pulldown assay.

subfamilies. and advance online publication online advance K Lau-GAGASAEEKH K Myr-GAGASAEEKH K Lau-GSCQSNENSE GAGASAEEKH + 1 1.2 D D D =0.54 =0.22 =16.4 b ). ITC measures the binding enthalpy of Gnaq 3 2 + + ± 0.28 Pellet 1.6

± ± ± + + + 2 0.28 0.14 3.0 4 subfamilies are not N-terminally . UNC119 also bound the lau the bound also UNC119 . µ C. C. elegans µ M and 0.22 2.0 µ µ M + + + + 5 4 M M α peptides and the acylated and acylated the peptides α Supernatant + peptide does not inter not does peptide ) α α c 6 + + + 8 GPA-13 ODR-3 Gnai Gnai Recov Gucala Gna11 Gnaq Gna15 Gna14 Gna13 Gna12 Gna1 Gnas Gnaz Gnai Gnat Gnat Gnat subunit interaction and UNC119. . Microcalorimetry . Microcalorimetry ± 0.14 2 3 1 3 2 1 + + + 7 unc-119 µ α M, respec

α peptides peptides Fig. 2 Fig. subunit α pep null null e δ ------)

lipid inserts into the hydrophobic cavity at the center of the Interpretation of our structural and biochemical results suggest site thatbinding lipid the is the cavity hydrophobic UNC119 The an through acyl chain does not to extend of all G subfamilies with UNC119 interaction at 2 therefore position be cannot myristoylated, Ca α (16.4 was ODR-3 binding (blue) more than one order of weaker magnitude K were fit peptides (green) to GAGASAEEKH a model and one-site bound with (red) and myristoyl- Lauroyl-GAGASAEEKH lauroyl-GSCQSNENSE. peptide with ODR-3 N-terminal titration blue squares, GAGASAEEKH; with myristoylated titration green triangles, GAGASAEEKH; non-lauroylated T N-terminal with G protein did peptide not acylated (lane 4). ( for (lane competed 3), binding but the non- that lauroyl-GAGASAEEKH of (lane 4); peptide lanes 2–4). 5–7, Note GAGASAEEKH supernatants (lane 3), and in of the of lauroyl-GAGASAEEKH presence non-acylated with GST-UNC119, in the of absence (lane 2), peptide the presence bead of pellets lanes that 2–4, lysates retina were glutathione incubated by T and an inhibition N-terminal acylated 3 Figure This structure is superimposable with that of the T elegans C. accommodated. the be with UNC119 of can structure isomorphous that an determined We chain also acyl of length the establishing thereby cavity, UNC119 the in the junction peptide-acyl define the of that location groups N-terminal of recognition specific provide interactions These molecule. water other H165, one and S218 Y194, includes H192, also that network hydrogen-bonding extensive of an part constitute Y220, and E163 with along molecule, water This ( molecule water buried a via NH A3 coordinates Y220 and NH, G2 bonds hydrogen E163 oxygen, lauroyl the with bonds hydro gen group hydroxyl Y131 The residues. UNC119 conserved by peptide. Remaining buried polar groups of the peptide are coordinated 3 distorted a of the formation through bonds hydrogen in participate groups chain main the of Most solvent. to exposed is chain side whose S6, of exception the The interactions. first six residues peptide lack polar side chains, with by specific satisfied were potentials hydrogen-bonding environment, hydrophobic a for expected As UNC119. of residues contacting the T of residues N-terminal the for conservation of degree high the rationalizing thereby ligand, the of shape the to complementary gen In highly is that ordered. a surface formed protein residues surrounding the eral, poorly are and UNC119 with contact peripheral 2c Fig. Supplementary are the do not exist in UNC119, but instead are found on the opposite edge of PrBP/ of sites lipid-binding ( site van der Waals forces, consistent with the properties of a lipid-binding mediate interaction the lauroylatedwith the T lined predominantly by hydrophobic residues (mostly Phe and Tyr)( that in the asymmetric unit of the containscrystal a similarly bound ligand lauroyl (C12) moiety ( 2.0 Å (PDB ID: showed3RBQ) that the pocket easily accommodates a The co-crystal structure of UNC119 with the lauroylated T Supplementary Table 2 Table Supplementary subunits, subunits, D 2+ h luol ru ad h frt i rsde o te T the of residues six first the and group lauroyl The values of values 0.54

β -binding proteins GCAP1, GCAP2 and recoverin. Peptides lacking Gly lacking Peptides GCAP2 GCAP1, and proteins recoverin. -binding uid n h hdohbc okt f N19 ( UNC119 of pocket hydrophobic the in buried Fig. 4 Fig. sandwich ( ± 3.0

UNC119 is an acyl-binding protein. ( is UNC119 protein. an acyl-binding D- luolGCSES lgn (aa o shown). not (data ligand lauroyl-GSCQSNENSE ODR-3 C. C. elegans c µ α and and M). M). ( peptide (lauroyl-GAGASAEEKH); black circles, titration with titration black circles, (lauroyl-GAGASAEEKH); peptide α subunit N-terminal peptides. Red symbols, titration with Red titration symbols, peptides. N-terminal subunit Fig. 4a ± c Supplementary Fig. Supplementary 0.28 ) Alignment of N-terminal peptides of ) peptides of mouse G Alignment N-terminal protein G protein Fig. , µ b ), whereas peptide residues 7–10 make only only make 7–10 residues peptide whereas ), M M and 0.22 and and and 4 ) in a very specific fashion, as each molecule 10 δ - and RhoGDI ( RhoGDI and α α Supplementary Supplementary Fig. 2a Supplementary Fig. 3 Fig. Supplementary subunits GPA-13subunits and and ODR-3, helix by the first six residues of the the of residues six first the by helix b ) ) ITC. Human was UNC119 titrated ± 0.14

α 2c peptide (lane 1, peptide lysate; retina ). Notably, entrances to the the to Notably,entrances ). a µ ) ) GST-UNC119 pulldowns M, M, respectively. Lauroyl– Supplementary Fig. 1b Fig. Supplementary α peptide primarily via primarily via peptide α t r a ligand, but with a , ). The cavity is is cavity The ). b α ). . i. 4 Fig. α β C I sandwich. α peptide at peptide peptide Fig.

e l α c and and and and

4 c s ). ).  - - )

© 2011 Nature America, Inc. All rights reserved. were effective in solubilizing T in were solubilizing effective Collectively, these results indicate that neither GTP nor UNC119 alone nearly completely in inhibited the of presence recombinant UNC119. T However, min. per transducin of mol per reconstituted GTPase activity, yielding a ( membranes ROS the in ducin trans of traces remaining the from originating activity GTPase low system. Depleted ROS membranes containing rhodopsin exhibited very GTPase activity of T UNC119–T the of formation the for necessary ( sta formed T with UNC119 complexes ble that indicated Pulldowns assays. for pulldown used and respectively, retinas, dark-adapted and light-adapted T dissociation. T and membrane and complex for the exchange of GDP/GTP disruption requiring rhodopsin, to bound tightly is GTP of absence the in buffers (hypotonic) salt low by dark the in disrupted be can membranes to T with UNC119 of interaction the investigated we membranes, from dissociation. for T essential of disruption and exchange GTP/GDP that suggesting (ROS) membranes by UNC119 only in the presence of GTP ( of GTP. absence and T T of extraction the examined we ated, branes under isotonic conditions where it is membrane firmly associ mem from transducin of dissociation facilitates UNC119 investigate Towhether pocket. hydrophobic its into chain acyl the binding T of association membrane disrupt may UNC119 that T implies acylated the with UNC119 of structure co-crystal The interactions transducin-membrane regulate UNC119 and GTP ity and absence of amino acid residues relevant for optimal binding. less-ordered peptide structure, consistent with the weaker binding affin ( PyMOL with gray.created dark were colored Figures are residues attached the and is green chain in yellow, lauroyl shown the are residues UNC119 stereochemistry. bonding hydrogen appropriate satisfied Å and 3.2 was or less unit asymmetric in the molecules six all for length bond of the average if the included were bonds Hydrogen UNC119. penetrate T the to which depth the limits lines) dashed (black network hydrogen-bonding The ligand. lauroyl-GAGASAEEKH the with interacting individual the and axis vertical the around rotation 90° gray.in dark modeled are In peptide of the acids amino ten the and in green is shown chain cavity. lauroyl The hydrophobic 4 Figure t r a  T extract to of UNC119 ability Fig.

α a To determine whether UNC119 binding to T to binding UNC119 whether determine To T extract not does UNC119 that demonstrate To -GTP and T and -GTP

5 α

b β1

C I -GDP–T

), confirming that disruption of T of disruption that confirming ), β α 7 The lipid-binding pocket of UNC119. ( of UNC119. pocket lipid-binding The β2 1 β4 e l C α s β -GDP–T γ β containing retinal lysates were prepared from from prepared were lysates retinal containing 3 α , , we measured GTP hydrolysis in a reconstituted

α β9 α -GTP ( -GTP could be extracted from rod outer segments segments outer rod from extracted be could α 2 β γ N . . 2 In vitro In α α 7 Fig. 5 Fig. Fig. 5 Fig. . In contrast, in the light, transducin transducin light, the in contrast, In . from membranes. The light-adapted -GTP from membranes and stabilize stabilize and membranes from -GTP

90 b

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β -GTP inhibits the the inhibits -GTP 8 β strands are labeled labeled are strands α

α β7 1 -GDP–T α α α α 2 β4 -GDP–T -GDP–T -GDP–T

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β 1–9 in yellow. 1–9 ( partially retained in the inner segment and the outer nuclear layer layer ( nuclear outer the and segment inner the in retained partially tion in polypeptide trafficking to olfactory cilia and investigated the the investigated and cilia olfactory to trafficking polypeptide in tion commissures neuron motor larvae dauer form to inability behavior, altered in feeding plex including defects phenotype, chemosensation, in discovered Originally in mistraffic GPA-13 and ODR-3 Discussion). (see isoforms UNC119 redundant to may attributable be slow degeneration and resulting the calization dark-adaptation prolonged ( following membranes segment inner T of amount substantial a but dark-adaptation, of h 3 after photoreceptors wild-type in plete h; (0–24 dark-adaptation and wild-type exposed we Unc119 diffusion, by transducin on of deletion return UNC119 the of effect the determine To transport. driven motor- molecular for ATP, required of is depleted which eyecups in diffusion by restricted dark-adaptation prolonged during hours in segments minutes in T light, intense Under mechanism. desensitization and part light-adaptation a of as exposure light intense during in-bulk segment inner the to localization. its on effect no have to appeared deletion transport vesicular either ( T retina, type postnatally months 6 beginning degenera tion photoreceptor progressing slowly a develops but life, in The trafficking transducin affects mouse in deletion UNC119 Discussion). (see of transducin translocation light-induced the for important be may UNC119 that suggests GTP Fig. Fig. Fig. A fascinating property of transducin is its observed translocation translocation observed its is transducin of property fascinating A F177 c Unc119

6f 6 6 Y220 M161 b a α , −/− ), whereas in the dark-adapted dark-adapted the in whereas ), ). Transducin is thought to arrive at the outer segment by segment outer the at arrive to thought is Transducin ). h -GTP and -GTP T D8 , F175 advance online publication online advance j mice to intense light for 60 min followed by prolonged prolonged by followed min 60 for light intense to mice H192 F8 30 7 ). The partial efficacy of UNC119 deletion on T on deletion UNC119 of efficacy partial The ). −/− 8 , S218 3 α c 1 ) Stereoview of UNC119 residues and key water molecules molecules water key and residues of UNC119 ) Stereoview N230 by diffusion by was detected almost exclusively in the outer segment segment outer the in exclusively almost detected was mouse shows no obvious retinal degeneration early early degeneration retinal obvious no shows mouse K107 Y194 E163 F196 β 2 γ 9 translocate individually to the inner segment to segment the inner individually translocate , as transducin does , return to does as outer transducin segments H165 C. elegans C. Fig. 6c Fig. 2 V129 α 8 3 r asv diffusion passive or 2 3 remained associated with with associated remained Y131 . Both T Both . 3 . We suspected G protein participa protein G suspected We . F137 F133 – α k , , peptide in the UNC119 UNC119 in the peptide ). Return of T of Return ). F177 unc-119 unc-119 α 1 Y220 M161 2 and T and 8 . In the dark-adapted wild- dark-adapted the In . and excessive branching of branching excessive and D8 b Unc119

, UNC119 is viewed after a after is viewed , UNC119 F8 F175 H192 7 nature nature 8 mutants exhibit a com a exhibit mutants S218 olfactory neurons olfactory β N230 γ K107 return to the outer outer the to return Y194 −/− α α E163 2 F196 was nearly com nearly was peptide can peptide 9 eia T retina, NEUR , and UNC119 UNC119 and , 3 0 , presumably presumably , H165 Unc119 OSCI V129 α Y131 mislo α F133 F137 EN was was −/− C E - - - - -

© 2011 Nature America, Inc. All rights reserved. mutant ( mutant ( wild-type both of cilia the in present GPA-13was although Correspondingly, worms. the in ODR-3 of mislocalization revealed ODR-3 ( ( found in the dendrites and cell bodies being amounts trace only with cilia, the to predominantly localized AWA in neurons AWC transduction and sensory signal duction, ODR-3, predicted to be acylated at G2, is required to mediate Although several G proteins participate in chemosensory signal trans of ­consequences the containing rhodopsin and a trace of transducin (only T (see inset). Inset, SDS-PAGE of purified native transducin (only T addition of UNC119 (green) reduced the activity nearly to baseline. Baseline activity was caused by a low amount of T the reconstituted system (red line) corresponded to a rate of 1.5 mol GTP per min. Addition of bovine serum albumin (light blue) had little effect, whereas activity of purified T GST-UNC119 binds T proteins (supernatant) were analyzed by western blot using antibody to T The proteins pulled down by GST or GST-UNC119 (pellet) and unbound retinas of a dark-adapted mouse were used for pulldown assays, respectively. retinas of a light-adapted mouse (2,000 lx) and hypotonic supernatants from light-adapted and dark-adapted mouse retinas. PBS/GTP supernatants from elutes only in the presence of UNC119 and GTP. ( centrifugation. T soluble proteins (S) were separated from membrane-bound proteins (P) by GST, mUNC119, GST and GTP, or mUNC119 and GTP, respectively. The lysates in 1× phosphate-buffered saline (PBS) were incubated with either to 10,000 lx over 20 min, driving transducin to the inner segments. Retina ( Figure 5 nature nature Fig. 7 Fig. Fig. 7 Fig. a ) Extraction of T b a

and and and UNC119 interacts with T NEUR Fig. 7 Fig. 24 6 3 0 h Supplementary Fig. 4b Fig. Supplementary i g e c a Supplementary Fig. 4a Fig. Supplementary OSCI α d k was detected by western blot using antibody to T and and α from membranes by UNC119. Live mice were exposed Percent total signal αβγ Fig. 7 Fig. 10 20 30 40 50 60 70 80 EN α 0 WT unc-119 -GTP (left), but not T in the presence of ROS membranes. The activity of Supplementary Fig. 4d Fig. Supplementary WT C E

0 c and and h advance online publication online advance Dark-adapted deletion in deletion α Supplementary Fig. 4c Fig. Supplementary 3 -GTP and inhibits GTPase activity. h b f d h j ) with antibody directed against against directed ) antibody with 3 5 ) and and ) . . Immunolabeling of wild-type 6 α C. C. elegans Unc119 h -GDP–T Unc119 unc-119 b ) ADF, ASH and AWC and ADF, ASH ) ) Pulldown assays with 24 –/ –/ – βγ – α h KO WT GNAT1 10 GNAT1 is visible, T (right). ( 10 olfactory neurons. olfactory µ µ unc-119 m mutant worms worms mutant m ) and and ) IS IS IS IS ONL OS ONL OS ONL ONL OS ONL OS IS OS 3 4 c . ODR-3 is is ODR-3 . α

) GTPase and T α β mutant mutant unc-119 . T co-migrates with rhodopsin).

α α

β . subunits are shown), recombinant human UNC119 and depleted ROS membranes -

phology. To exclude this possibility, we crossed transgenic worms worms transgenic crossed we possibility, this exclude To phology. mor neuronal abnormal from indirectly arise could worms mutant worm. mutant the neurons chemosensory included three independent measurements. Error bars denote means means denote bars Error measurements. independent three included bar Each software. ImageJ using quantified was signal Fluorescence of dark-adaptation. at start 0, 3, 24 6, h and after fluorescence segment ( segment. outer OS, layer; nuclear outer ONL, to the returning from part T to T antibody with 3 for h ( adapted ( of T antibody. presence the Note to T antibody with probed were sections and ( exposure. light 6 Figure phenotype. wild-type the of restoring capable was neurons these in UNC-119 of supplemental transgenic also detected in the cilia of transgenic worms ( to with antibody ODR-3. of levels labeled also ODR-3Increased were were worms transgenic AWC neurons, and ADF, ASH in expressed ( nontransgenic worms transgenic the of cilia the in reactivity immuno GPA-13 in increase an showed GPA-13 to antibody with ADF,presumably neurons, sensory ASH and AWC ( worms. GFP::UNC-119 expression was found in three pairs of amphid GFP introduced we neurons, amphid in stability protein G restore could integrity. cellular by disrupted in and GPA-13 observed levels protein however,ODR-3was normal, it that improbable the decreased seems the in AWCneurons of morphology ciliary the As was slightly different from that in wild types ( in ending AWCsensory to those in wild ( types neurons in the onto neurons AWC AWA,or AWB in GFP expressing specifically mouse retinas b a c Light-adapted α T T – PBS/GTP Reduced amounts of ODR-3 and GPA-13 present in the cilia of of cilia the in present GPA-13 and ODR-3 of amounts Reduced To determine whether expression of transgenic, wild-type To wild-type of expression transgenic, whether determine slowly returned to the wild-type outer segment, but is blocked in is blocked but segment, outer wild-type to the returned slowly α α j ) Mice were first exposed to intense light for 60 min, then dark- then 60 min, for light to intense exposed first were ) Mice unc-119 Unc119 ø P S

UNC-119 driven by driven the UNC-119 GST

Slow return of transducin to the outer segment after intense intense after segment outer to the of transducin return Slow GST-UNC119 UNC119 +GTP GST +GT UNC119 GS unc-119 unc-119 − mutant background. Ciliated endings of AWA endings Ciliated mutant background. and AWB mouse retinas Dark-adapted /

− GST T hypotonic retina. Mice were dark-adapted for at least 12 h. Frozen 12 h. Frozen at least for dark-adapted were Mice retina. a e unc-119 , , α b GST-UNC119 f ) Localization of T ) Localization and FITC-linked secondary antibody. that Note secondary FITC-linked and ), ), 6 h ( P mutants ( P S mutant worms ( worms mutant Supplementary Fig. 4e Supplementary 3 Unc119 uat ( mutants g 5 , unc-119 , it was downregulated and mislocalized in in mislocalized and downregulated was it , h c

) and ) 24 h and ( pmoles GTP per aliquot 100 150 200 Supplementary Supplementary Fig. 4f 50 α gpa-13 in dark-adapted inner segments. segments. inner in dark-adapted −/− T T α β αβγ mutants ( mutants outer segment. IS, inner segment; segment; inner IS, segment. outer α 0 i. 7 Fig. (green) in dark-adapted wild-type wild-type in dark-adapted (green) Transducin α Fig. 7 Fig. still attached to the membranes and FITC-linked secondary secondary FITC-linked and promoter into i

, UNC119 j unc-119 ). Frozen sections were probed probed were sections ). Frozen 5

f ROS ). Because ODR-3 is also also is ODR-3 Because ). k h Time (min) ) Quantification of inner of inner ) Quantification ). Transgenic expression expression Transgenic ). Supplementary Fig. Supplementary Supplementary Supplementary Fig. 4i Fig. Fig. 7 , g mutants were caused mutants were caused ), ), but the shape of the 10 Fig. 7 Fig. t r a g , Fig. h ) unc-119 unc-119

relative to non ) ) were identical 1 5 e

ROS/T ) relative to to relative ) 7 hUNC119 ROS/T ROS GT C I ). Labeling ). Labeling ROS/T P unc-119 mutant mutant mutant

αβγ

e l αβγ ± αβ /BSA s.d. /

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© 2011 Nature America, Inc. All rights reserved. to GST-UNC119, GST-UNC119B pulls down T down GST-UNC119,GST-UNC119Bpulls to mosome 17) is 61% similar and 52% identical to UNC119 and, similar the by UNC119B encoded UNC119B, isoforms. UNC119 of redundancy the the in seen effect partial The intact. largely transport post-biosynthesis leaving dark-adaptation, complete after even segment inner the in transducin of retention tial ( reticulum endoplasmic the at proteins G erotrimeric G Acylated transferase. acyl reticulum–resident endoplasmic unidentified an by acylated or acylated co-translationally either are subunits protein G trafficking G protein and Unc119 post-trans modification. the to lational adjacent on immediately part in sequence based acid is amino the proteins lipidated with specificity interaction ( limit T the to that which depth the interactions extensive the with along data, These cavity. T of acids amino six first the by occupied completely is pocket UNC119 the of opening presumably (and PrBP/ RhoGDI the in which pocket through hydrophobic the entrance enters lipid The structure. UNC119 the in seen ( divergent Fig. ( superimposed are structures two the when nicely form bound GDP inactive their in G proteins small these trapping thereby in pocket, its anchors prenyl their for environment hydrophobic a providing membranes from CDC42 and Rho Rac, geranylgeranylated minally photoreceptors in proteins prenylated of transport the in cofactor important an and protein prenyl-binding PrBP/ in 1KSH) seen ID: that to (PDB similar is UNC119 human of structure tal immunoglobulin The DISCUSSION ( control mutant gpa-13 The localization. ODR-3 and GPA-13 restores in evident is axons and ( ODR-3 to ( mutant. 7 Figure t r a  lysate and (R.C. W.B., unpublished results). Fig. Fig. 6 Fig. 4 Fig.

c a

2a δ b promoter in ADF, ASH and AWC in in AWC ADF, and in ASH promoter c ), whereas the the whereas ), ) is located at the opposite edge of the the of edge opposite the at located is )

C I ) ) indicate that of the deletion in to UNC119 mouse leads par and and a Mislocalization of the G proteins ODR-3 and GPA-13 in a a in GPA-13 and ODR-3 proteins G the of Mislocalization – gene located on human chromosome 5 ( a d , WT α b ) Wild-type ( Wild-type ) upeetr Fg 2b Fig. Supplementary e l ) and GPA-13 ( GPA-13 and ) and prenylated G prenylated and Supplementary Fig. Supplementary f , h s ) were labeled with GPA-13 ( GPA-13 with labeled were ) 2 2 3 2 and RhoGDI (PDB ID: 1DOA) ID: (PDB RhoGDI and . The The . α unc-119 β with the acyl chain deeply inserted into the the into inserted deeply chain acyl the with β a strands of RhoGDI and UNC119 are more more are UNC119 and RhoGDI of strands b d , -sandwich fold demonstrated by the crys the by demonstrated fold -sandwich α c ) and and ) peptide can penetrate the UNC119 cavity cavity UNC119 the penetrate can peptide β c , strands of PrBP/ of strands d ). Mislocalization of ODR-3 and GPA-13 to the olfactory cell bodies bodies cell olfactory the to GPA-13 and ODR-3 of Mislocalization ). mutants. ( mutants. UNC119 β unc-119 unc-119 γ most likely combine to form het form to combine likely most

2a , ). Important differences are are differences Important ). b −/− GPA-13 ), support the assertion that that assertion the support ), unc-119 mutant ( mutant ODR- e 10 – mouse is likely a result of result a likely is mouse 5 h unc-119 . RhoGDI extracts C-ter extracts RhoGDI . µ ) Cell-specific rescue of of rescue Cell-specific ) m 3 e , f β δ ) or ODR-3 antibodies ( antibodies ODR-3 or ) α mutants. The transgenic ( transgenic The mutants. and UNC119 align align UNC119 and -sandwich fold. The The fold. -sandwich b g e from bovine retina retina bovine from UNC119 , d Pgpa-13::gfp::unc-119 gene fused with GFP was driven by the the by driven was GFP with fused gene Pgpa-13::gfp::unc-119 ) ) Supplementary Supplementary C. elegans C. 2 2 2 8 . PrBP/ . . Our results results Our . is on chro were stained with an antibody antibody an with stained were C. elegans unc-119(ed3) elegans C. δ is a is unc-119 δ ------

g , h h f ). only 27% overall sequence identity, with sequence similarity at the the at similarity sequence with identity, sequence overall 27% only Fig. 5b ( rhodopsin factor exchange guanine the of absence the In rhodopsin. of devoid are which membranes, segment inner to activity hydrolyzes GTP, permitting heterotrimeric ducin subunits are presumed to recombine after T following light-induced translocation ( UNC119/T T of dissociation the that suggesting elutes T UNC119 that We segments. determined inner the in sink switchable T of return the for responsible is diffusion ‘restricted’ which in model function not do motors molecular which ATP,without of depleted eyecups in described been previously has phenomenon same This mice. wild-type in hours in segments outer the to return ment membranes T for both affinity its reduces which phosducin, T for min 12 approximately of a with diffusion passive by ment T causing T on exchange GTP/GDP triggers rhodopsin of Light-activation diffusion by transducin of translocation Light-induced ( calorimetry titration by isothermal shown as UNC119, ( tide T the with acids amino ten of three only N-ter shares ODR-3 peptide minal The limited. very being acids amino 50 N-terminal the outer segment is severely affected in a PrBP/ a in affected severely is segment outer the T cilium, depositing T connecting and segment inner the through passively diffuses likely it return rates return to the outer segment, which agrees with experimentally observed dictates that it will take approximately 10 return to the outer segment ( for step rate-limiting the is event this that suggest constant rate slow Both the GTP requirement for T e α α , We propose that the restriction is provided by an enzymatically enzymatically an by provided is restriction the that propose We g in in -GTP solubilization, PrBP/ solubilization, -GTP and T and ) and and ) Fig. C. elegans C. unc-119 unc-119 UNC119 ), GTP/GDP exchange is very slow (rate constant = 10 Unc119 α unc-119 bound to membranes only in the presence of GTP ( β

α advance online publication online advance 3 γ α c -GTP and T and -GTP 31 to the outer segments is plausible. is segments outer the to ), resulting in a lower affinity interaction with human human with interaction affinity lower a in resulting ), -GTP complex formation. On arrival at the inner segment GPA-13 ODR- , 3

10 2

. . On solubilization of the UNC119/T

µ

m 3 3 7 . . On return to the dark, both T α directly at discs in the outer segment. Following 7e under ( promoters intrinsic UNC119 their of control the transgenic expressing by restored was GPA-13 and ODR-3 of zation ( cells amphid ADF and ASH AWC, AWB, AWA, in expressed are which GPA-13, and ODR-3 proteins G the of mistargeting and mislocalization the tors. photorecep vertebrate to similar phology mor overall an with cells ciliated polarized molecules ble amphid neurons to odorants and detect solu worms tion have been in reported unc-119 knockout in chemosensa as defects neurons, olfactory on We focused phenotype. complex a in ing both neuronal and cells result non-neuronal are more severe and UNC-119 deletion affects genome harbors only one UNC119 gene. gene. UNC119 Accordingly, one only harbors genome β γ In contrast with vertebrates, the the vertebrates, with In contrast , to dissociate and traffic to the inner seg inner the to traffic and dissociate to g Supplementary Fig. 5b ). ). Notably, ODR-3, GPA-13 and T unc-119 δ 8 β may extract T extract may α . . γ extraction from membranes and the C. elegans C. 32 t . T . 1/2 unc-119 α deletion in the worm results in in results worm the in deletion Supplementary Fig. 3 of 3–5 min for T for min 3–5 of β -GTP and T and -GTP 6 4 . These sensory neurons are are neurons sensory These . γ s (166 min) for transducin to is known to associate with with associate to known is

relies on its 11 pairs of of pairs 11 its on relies Fig. 7b Fig. nature nature null mutant phenotypes β γ α , whose transport to transport whose , α α and rod outer seg outer rod and

δ transducin to dock ). The rate constant ’s intrinsic GTPase , and T β deletion model deletion d α γ Supplementary Supplementary ). Proper locali Proper ). NEUR is essential for essential is -GTP -GTP complex, α Fig. 3 Fig. β 2

and a a and OSCI −4 5a 9 γ C. elegans C. . Thus, a Thus, . subunits Fig. per s) ), trans α α b share pep EN ).

Fig. Fig. 5 t b C 1/2 3 α 5 8 ), ), E ------, . .

© 2011 Nature America, Inc. All rights reserved. 7. 6. 5. 4. 3. 2. 1. reprints/index.html. http://www.nature.com/ at online available is information permissions and Reprints Published online at http://www.nature.com/natureneuroscience/. The authors declare no competing interests.financial mouse. H.Z., C.P.H.,R.C., L.T. and W.B. wrote the manuscript. generated transgenic membranes, transducin and GTPasedetermined activity. M.W.D. and E.M.J. the unbound human UNC119 structure. C.D.G.R.C., and W.B. isolated ROS the determined structure. S.V., Y.C., J.S., Y.J.H., G.T.M.R.X., and L.T. determined F.G.W. and C.P.H. generated human T UNC119/acylated for H.Z. generated pulldown/light-induced translocation results and is responsible Blindness Senior Investigator Award. of Utah from Research to Prevent Blindness.of Utah,W.B. and unrestrictedis a recipient grants of to a theResearch DepartmentsCore toFacility, Prevent of Ophthalmology by a Center at theGrant University of theHealth Foundation (U54 GM074958), Fighting by Blindnessthe University to the (E.M.J.),Universityof Utah Macromolecule by a grant fromCrystallography the Protein Structuregrant), EY10848Initiative (G.I.) of and the NS034307US National (E.M.J.), grantsInstitutes by EY08123the Howardof (W.B.), Hughes EY019298Medical Institute(W.B.),crystallization EY014800-039003 screening. (NationalThis work Eyewas InstitutesupportedSynchrotron bycore US LightNational Source Institutes and G. DeTittaof Health Weof Hauptman thank R. AbramowitzWoodward andResearch J. InstituteSchwanof forfor access to the X4A beamline at the National Note: Supplementary information is available on the (UNC119–lauroyl-GAGASAEEKH) codes. Accession online the of at­version paper in the http://www.nature.com/natureneuroscience/. available are references associated any and Methods M species. other of systems sensory the in transport protein membrane in involved mechanisms elucidating and mouse both in data our Thus, mammals. to amoeba flagellated unicellular from organisms living all virtually in distributed is proteins, G with along UNC119, ing protein as well component as in a G necessary protein trafficking. protein 2), both of to which localize mouse and human cilia pigmentosa (retinitis and RP2 ARL3 with of UNC119 association the and intraflagellar transport, an argument that is further strengthened by cilium complex ment tion of which affects light-driven translocation of T T with interaction for candidate alternate An nature nature COM AUTH Acknowledgmen

et Our Our data provide evidence for UNC119 functioning as a lipid-bind C. C. elegans hn, . Kn, . Pi, . Le J NUC19 Nelra ooou of homologue Naegleria NgUNC-119, J. Lee, & S. Paik, S., Kang, S., Chung, L.C. Keller, their and H. Zhang, family Arf the of proteins G small The S. Munro, & A.K. Gillingham, centriolar the with interacts CEP290 J.G. Gleeson, & S.R. Krishnaswami, J., Kim, H. Jin, Rosenbaum,Witman,J.L.& G.B.Intraflagellar transport. UNC-119, localizes to the flagellar rootlet. flagellar the to localizes UNC-119, Cell Biol. Mol. WD40 domain protein POC1 is required for centriole duplication and length control. segments. (2007). 8857–8862 outer photoreceptor to subunits regulators. primary the to localization Rab8 cilium. for required is and PCM-1 component satellite cilia. to proteins membrane traffics (2002). 813–825 P h 3 O 7 E ods . The detection of UNC119 in the mouse photoreceptor sensory TI R R NEUR et al. et Hum. Mol. Genet. Mol. Hum. CON NG immunostaining and imaging. generatedR.C. ITC results. R.C., et al. et Annu. Rev. Cell Dev. Biol. Dev. Cell Rev. Annu. FI et al. et The conserved Bardet-Biedl syndrome proteins assemble a coat that coat a assemble proteins syndrome Bardet-Biedl conserved The TRIBUTI OSCI

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E I 17

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s c i e n c e

9 website.

, 1 5 .

104

3 - - - , ,

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167 281 582 279 100

60 27 21 41 39 s  , , , , , , , , , ,

© 2011 Nature America, Inc. All rights reserved. UNC119 E function using REFMAC models were rebuilt using O replacementusing PHASER using DENZO and SCALEPACK, respectively of the Stanford Synchrotron Radiation Lightsource.maintained at Data100 Kwere during integrated data collection. and Datascaled erol were (vol/vol), collected pH 5.0) andat cooled beamby plungingline intoBL9-2 liquid nitrogen. Crystals were buffer (40% PEG 5000, 0.1 M sodium acetate, 0.1 M potassium acetate, 12% glyc 40 1.8 and 5.0 acetate,potassiumpH peptidemixture and 3 dropswerecontaininggrownin Crystals 3 °C. 4at h 1 leastat prepared by mixing purified UNC119 and the T DTT.proteinsolutionwas mM The 5 andNaCl mM 100 Tris7.4, mM pH 10 royl-GAGASAEEKH). Purified UNC119 and the T UNC119 ( U ID: 3GQQ) have been deposited in the Protein Data Bank. MolProbity sented in COOT using PHENIX manually with built refined was model the of remainder The Solvent flattening and partial model building were performed using RESOLVE integrated and using scaled the HKL2000 package SynchrotronNational the Light at Source X4A using beamline an at ADSC Quantum-4dataset SAD CCD a detector. collect Data to were used and K 100 at maintained were crystals Single propane. liquid in frozen and oil paratone to andweretransferred weeks 3–4 after mm 0.1 0.05, 0.025, to grew crystals The °C. 4 at 5.0 pH acetate, sodium M 0.1 acetate, potassium M (wt/vol), 0.1 4000 PEG 40% containing solution with ratio 1:1 a in grown were determination °C 4 at oil paraffin under tallization homogeneity was verified by SDS-PAGE and MALDI-TOF mass spectrometry. in buffer containing 10 mM TrisHCl, 100 mM NaCl, 5 mM DTT, pH matography7.5. Protein (Qiagen) and gel filtration (Superdex 75, Amersham/GEEscherichia coli Healthcare) 14-15C expression vector (Novagen). Selenomethionyl protein was expressed in GenomicsConsortiumtarget proteinHR3066a).The clonedwasintopET the estab the pipeline production of protein part high-throughput a lished as out carried was 56–240) (residues protein UNC119 C ated using standard methods. were also examined in AWB,AWCandmarkers integrated stablyGFP above neuronsexpressing the and lin-15(n765ts) X kyIs104[str-1::GFP, lin-15(+)] X strains used in this work were Riverside)California,P.and Sengupta(Brandeis University). Stablyintegrated were obtained from Caenorhabditis Genetics Center, M. Maduro (University of elegans C. G.I. were returned to the dark for 3, 6 or 24 h. the mice were illuminated by 2,000 lx for 60 min. Followingadaptation, the light-adaptation,pupils of the mice micewere dilated with 1% tropicamine (wt/vol) and of Utah. To dark-adapt, we placed the mice in the dark forapproved 12 byh theor Institutionalmore. ForAnimal Carelight- and Use Committee of the University Animals. ONLINE MET nature nature (Amersham/GEvector pGEX-2T a ofsites BamHI/EcoRI the intoclonedand xpression and purification of recombinant bovine U bovine recombinant of purification and xpression rystallization of U of rystallization NC Crystals were mounted in a nylon loop, briefly immersed in cryoprotectionnylonloop,immersedinbriefly amounted were Crystalsin SHELXE/D Preliminary crystallization trials were performed using the microbatch crys µ 1 l paraffin oil. Crystals were harvested for data collection after 10–14 d. kyIs140[str-2::GFPlin-15(+)]+ 2 119/lauroyl- . Gnat1 cDNA was amplified by PCR from a bovine retina cDNA library library cDNA retina bovine a from PCR by amplified was cDNA Mice were maintained in a 12:12 h dark-light cycle. The animals’ care was Supplementary Table 1 NEUR Supplementary Table 1 strains. 4 7 −/− . The atomic coordinates and structure factors for UNC119 (PDB 4 BL21(DE3) Magic,+ and usingpurified Ni-NTA affinitychro 3 animals were obtained from J. Lem (Tufts University). was used to locate selenium sites and calculate initial phases. initial calculate and sites selenium locate to used was OSCI H ODS G Strains were maintained using standard methods. Strains methods. standard using maintained were Strains A NC unc-119(ed3) EN G µ 4 6 ASA l40% PEG 5000 (wt/vol), 0.1 M sodium acetate, 0.1 M 119. 5 Dt cleto ad eieet ttsis r pre are statistics refinement and collection Data . C 0 . Structures were checked using MolProbity E 4 4 8 oyIs[P(odr-10)::gfp] V 9 Expression and purification of truncated humantruncated ofpurification Expressionand EEK using PDB code 3GQQ as the search model. The and refined against a maximum likelihood target . The quality of the model was checked using cytl rwh n dt collection. data and growth crystal H ) was co-crystallized with a T µ mutants. Double mutant strains were gener for examining AWC neurons l 30% isopropanol (vol/vol) at 4 °C, under isopropanol°C, 30% (vol/vol)4 atl 4 1 . UNC119 crystals useful for structure structure for useful crystals UNC119 . Unc119 4 2 . Data were phased by molecular α −/− for examining AWB neurons peptide (1:1.1 molar ratio) for for examining AWA neurons, mice were from the colony of 4 2 α . peptide were dissolved in 4 0 (Northeast Structural Structural (Northeast NC 119 protein. 119 µ α l UNC119 T UNC119 l 3 peptide (lau 9 . The AWA,The. 4 5 and was was and 4 7 . The 3 4 α 9 4 ------. ,

PBS, resuspended in 400 resuspendedin PBS, removed by centrifugation at 4 °C for 10 min. The pellet was washed once with werecomponents soluble The inhibitors.protease and DTT mM 1 with PBS protein and TEV were removed by Ni 25 °C) in 2l of 500 mM NaCl, 50 mM Tris pH 7.4, and 1 mM DTT. Unprocessed was removed by incubation with TEV protease (~1 mg per and100 mg1 protein,mM DTT. 20 h atFractions were assayed bufferby andSDS-PAGE, eluted with 300 mM imidazole in 700pooled, mM NaCl, 50 mM Trisand pH 7.4,the 12× His-tag column(Amersham/GE Healthcare), washedcolumnwithten volumes lysisof (45min, 20,000 for1hat 4°C, followed by sonication. The lysate was clarified by centrifugation pH 7.4, 1 mM DTT) and protease inhibitors10 mg(PMSF, ml aprotinin, leupeptin, pepstatin)night at 19 °C. Cells were harvested by centrifugation, resuspended, and lysed in E. coli andverified by DNA sequencing. The construct was expressed in BL21 Codon+ to a 12× His-tag using a QuickChange site-directed mutagenesis kit (Stratagene) a pET151/D-TOPO vector (Invitrogen). The 6×amplified His-tag by inPCR thefrom vectora human was retina converted cDNA libraryE and directionally cloned into sion size of 30 kDa (Millipore). and reduced glutathione protocol was removed manufacturer’s by a Microcon the centrifuge filter to with an exclu according Healthcare) (Amersham/GE GSTrapcolumn a by followed column Healthcare) (Amersham/GE Histrapby a purified was protein The °C. 37 at h 5 for IPTG mM 0.1 by induced was recombinantprotein the of Expression Biolab). England (New ER2556 strain of the Healthcare). A His-tag (6 histidines) was placed immediately following the ATG followed by 30 min dark-adaptation. The retinas were homogenized in 200 homogenizedin dark-adaptation.wereretinas min The 30 followed by retinas were prepared from wild-type mice undergoing 20 min light-adaptation E obtained from C.K. Chen (Virginia Commonwealth University). body to rod T Biotechnology), 1:6,000 UW101 (antibody to GCAP1), 1:10,000 for UUTA (antithe primary antibodies were 1:10,000 for SC389 (antibody to rod T membrane.lulose Membranes described wereprocessedas T of detection(for 15% or Immunoblot. using antibody to T in 1× PBS buffer, pulled down with GST-UNC119 and identified by western blot assaysusingandGST-UNC119.GST Solubilizedtransducin reconstituted was fraction was removed by insolublecentrifugation. The The DTT). supernatantmM 0.1 EDTA,was mM used for1 pulldown7.4, TrisHClpH mM (10 (dark) homogenized in 1× PBS buffer containing 40 Utah Peptide Core facility. and GAGASAEEKH peptides, respectively. All peptides were synthesized by the bated with 10 Core Facility at the University of Utah. Spectrometry Mass the in out carried was LC-MS/MS by sequencing protein SDS-PAGE.12.5% byresolved The proteinsbuffer.were gel-loading The SDS were pulled down by glutathione beads and the bound proteins were eluted with lowed by overnight incubation at 4 °C. GST-UNC119 and its interacting proteinslysatewas mixed with 10–80 cocktail (Roche). The insoluble debris was removed by centrifugation. The Naretina mM 10 KCl, mM 2.7 NaCl, mM (137 buffer PBS ml 1 in sonication brief by assays. Pulldown Amersham/GE (SD200, filtrationHealthcare; gel and100 mM NaCl, DTT) 25 mM mM Tris 1 pH 7.4, 7.4, 1 pH mM Tris DTT) chromatography. mM 25 anionexchange (HiTrap Q FF, GE Life Sciences, 20–1,000 mM NaCl gradient in was collected in the flow through, concentrated, and purified to homogeneity by xpression and purification of recombinant human U xtraction of transducin from mouse retinal membrane by U o pldws ih ih- n dr-dpe rtns mue eia were retinas mouse retinas, dark-adapted and light- with pulldowns For Forthe competitive binding assay ( 2 HPO cells (Stratagene) in ZY autoinduction media for 6 h at 37 °C and then over UNC119 − 4 . 1.76 mM KH mM 1.76 . 1 lysozyme in lysis buffer (20 mM imidazole, 700 mM NaCl, 50 mM Tris µ α Proteins were separated by 12.5% (for detection of T cDNA. The construct was transformed into the g GST-UNC119 in the presence of 150 ) and 1:5,000 for GN2 (antibody to rod T g )and soluble hUNC119 protein was bound to aNi For protein sequencing, a bovine retina was homogenized was retina bovine a sequencing, protein For α (UUTA). 2 PO µ α l PBS, and aliquoted in four microcentrifugetubes.four in aliquotedand PBS, l and T and 4 µ pH 7.4) with 1 mM DTT and proteaseand inhibitor DTT mM 1 with 7.4) pH gof purified GST-UNC119 or 40 γ 2+ ) SDS-PAGEnitrocel) a transferredto and sepharose chromatography, cleaved protein Fig.3 a µ ),400 M GTP (light) or hypotonic buffer µ µ lof retina lysate was incu NC g lauroyl-GAGASAEEKH γ ). UUTA and GN2 were 119. doi:10.1038/nn.2835 2 4

Unc119 . The dilutionsThe of. E. coli NC α µ α and GCAP1) gof GST fol 2+ , Santa Cruz 119. expression cDNA was sepharose Mouse µ ------l

© 2011 Nature America, Inc. All rights reserved. tated using a Typhoon Trio (GE Health Sciences) and Image Quant software. (Brinkmann). The PEI plates were washed in 0.12 M LiCl (30 min) and quanti aliquots were withdrawn at 0, 5, 10, 15 and 20 min and spotted on PEI cellulose UNC119(2.27 5 mM MgCl (4.5 ducin 4 pmoles (0.17 bovine retinas as previously described G using the MicroCal Origin Software. fordataexperimentalanalysiswerebufferblankssubtractedintofrom the the hUNC119 samples at 180-s intervals. Data obtained from the peptide injections MicroCal ITC-200 MicroCalorimeter at 25 °C. The peptide was injected into the above-describedthe peptidesin buffer.the ITCmeasurements were done ona and hUNC119 concentrations PeptidewereDTT. varied mM as a 1 result and of theNaCl varying solubilitymM of100 7.4), (pH Tris mM 25 in suspended were samples protein UNC119 humanrecombinant and GAGASAEEKH, or IT conjugated secondary antibodies were diluted 1:300. T rod to (antibodyUUTA described as formed sections. retina of Immunocytochemistry cells were harvested 48 h after transfection. Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. The site-directedmutagenesis kit(Stratagene). Hek293 werecells transfected using mutantG2Agenerated gene.Thewasconstruct byeGFP replace the vectorto pEGFP-N2 of sites NotI and XhoI the into cloned and library cDNA bovine vitro In pellets were subjected to western blot using antibody to T 100 supernatantresuspendedtheand removed.wasin pelleted was waspellet The tion) were added, respectively. After overnight incubation at 4 °C, the membrane18 In these four tubes, 9 doi:10.1038/nn.2835 TPase assay.TPase C µ . µ g GST-UNC119, and 18 The transducinlauroyl-,peptidesTheN-terminal myristoyl-GAGASAEEKH l PBS. 10 PBS. l expression of T ofexpression µ 2 g), 61 g), , 1 mM DTT and 0.1 mM EDTA. Some assays included 133 pmoles Transducin and depleted ROS membranes were purified from purified weremembranes ROS Transducindepleted and µ µ µ g) rhodopsin (in depleted ROS membranes), 54 pmoles trans g) orpmolesg)130 ofAssaysBSA. were incubated at2 °C, 35 l of the supernatant from each tubes and their correspondingtheirsupernatant andtubes the eachof from l µ M GTP spiked with spiked GTP M 5 µ . Dilutions for the primary antibodies were 1:1,000 for 1:1,000 were antibodies primary the for Dilutions . g GST, 9 a and T and α µ ) and 1:1,000 for G8 (antibody to GRK1). FITC- GRK1). to (antibody G8 for 1:1,000 and ) g GST-UNC119 and 40 µ a g GST and 40 ( G 2 2A). 7 .The GTPase assay mix (20 γ

-P Gnat1 Immunocytochemistry was per was Immunocytochemistry 32 -GTP, 20 mM Tris HCl pH 7.5, pH TrisHCl-GTP, mM 20 µ M GTP (final concentration), cDNA was amplified from a from amplified cDNAwas µ M GTP (final concentra α (UUTA). µ l) contained µ - - - - l

44. 43. 42. 41. 40. 39. product from the fusion PCR was microinjected into the germline. was fused to the 5 from amplified of sequence promoter 2.7-kb A PCR. by amplified was sequence The in which the GFP cDNA was fused in frame to the 5 PCR from pPD104.53 (provided by A. Fire, Stanford) and clonedinjection wasgenerated intoby fusion Briefly, PCR. GFPthegene wasamplified P#MM016, by C gifts from G. Jansen (Center for Biomedical Genetics). Olympus confocal microscope. Polyclonal ODR-3 and GPA-13 antibodies were stained following standard methods. The images were acquired using an FV1000 of Immunocytochemistry 50. 49. 48. 47. 46. 45. ell-specific rescue of rescue ell-specific

ewlie, .. OV ad EOV: uoae srcue ouin n density and solution structure automated RESOLVE: SHELXD. and SOLVE T.C.Terwilliger, with solution Substructure in G.M. collected Sheldrick, data & diffraction T.R. Schneider, X-ray of Processing D. Minor, & Z. Otwinowski, for system automated An D.M. Blow, & D.L. P.D.,Maeder, Stewart, N.E., Chayen, chemotaxis T.B.Acton, Reprogramming C.I. Bargmann, & B.E. Kimmel, E.R., Troemel, Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular of Refinement E.J. Dodson, & A.A. Vagin, G.N., Murshudov, T.A.,Jones, J.Y.,Zou, S.W.Cowan, building for methods Improved M. Kjeldgaard, & A.J. McCoy, I.W.Davis, P.D.Adams, graphics. molecular for tools model-building Coot: K. Cowtan, & P.Emsley, Crystallogr. D Biol. Crystallogr. Biol. D Crystallogr. mode. oscillation (1990). micro-batch protein crystallization and screening. Consortium. Genomics Structural (1997). 161–169 240–255 (1997). 240–255 structuresby the maximum-likelihood method. A Crystallogr. Acta models. these in errors of location the and maps density electron in models protein (2007). 658–674 acids. nucleic and structuredetermination. Crystallogr. Biol. D Crystallogr. modification. responses: sensory neurons define olfactory preferences in preferences olfactory define neurons sensory responses: gfp - unc119 et al. et et al. et t al. et fusion gene including the flanking transcription termination transcription flanking the including gene fusion Methods Enzymol. Methods C. elegans C. t al. et ′ end of the MolProbity: all-atom contacts and structure validation for proteins for validation structure and contacts all-atom MolProbity: Robotic cloning and Protein Production Platform of the Northeast the of Platform Production Protein and cloning Robotic Methods Enzymol. Methods

HNX bidn nw otae o atmtd crystallographic automated for software new building PHENIX: 47 Nucleic Acids Res. Acids Nucleic hsr rsalgahc software. crystallographic Phaser C. elegansC.unc-119 , 110–119 (1991). 110–119 , ActaCrystallogr. Biol.Crystallogr.D genomic DNA. The 3 The DNA.genomic C. elegans.C. gfp-unc119

58 60

374 Methods Enzymol. Methods , 1772–1779 (2002). 1772–1779 , , 2126–2132 (2004). 2126–2132 ,

, 22–37 (2003). 22–37 , 276

Animalswereandpermeabilized, fixed 35 fusion gene by nested-PCR. The PCR , 307–326 (1997). 307–326 , , W375–383 (2007). W375–383 ,

mutant. ActaCrystallogr. D Biol. Crystallogr. J. Appl. Crystallogr. ′ end of the the of end nature nature

′ TheDNA for used micro 394 of unc-119 . pl Crystallogr. Appl. J. , 210–243 (2005). 210–243 ,

58 , 1948–1954, (2002). . elegans C. NEUR gpa-13 genomic DNA.

23 OSCI gpa-13 promoter , 297–302 . Cell EN was

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40 91 53 C E - , , ,

1

SUPPLEMENTAL MATERIAL

UNC119 is required for G protein trafficking in sensory neurons

Houbin Zhang, Ryan N. Constantine, Sergey Vorobiev, Yang Chen, Jayaraman Seetharaman, Yuanpeng Janet Huang, Rong Xiao, Gaetano T. Montelione, Cecilia D. Gerstner, M. Wayne Davis, George Inana, Frank G. Whitby, Erik M. Jorgensen, Christopher P. Hill, Liang Tong, and Wolfgang Baehr

Inventory

Supplemental Table 1 UNC119 Crystallographic Data and Refinement Statistics

Supplemental Table 2 Comparison of the RMSD values for each of the six UNC119 chains and their associated ligands.

Supplemental Figures

Figure S1 Comparison of UNC119, PrBP/δ, and RhoGDI structures Fig. S1A. Greek key motif of the β-sandwich fold common to the three structures Fig. S1B. Ribbon representation of the geranylgeranyl binding protein RhoGDI (1DOA) and the prenyl binding protein PrBP/δ (1KSH) Fig. S1C. Sequence alignment of human UNC119, PrBP/δ, and RhoGDI Fig. S2 Structural alignment of UNC119 with PrBP/δ and RhoGDI Fig. SA,B. Structural alignment of UNC119 with PrBP/δ and RhoGDI Fig. S2C. Electron density surrounding the lauroyl-T peptide Fig. S3. Overlap of the UNC119 carbon-α coordinates and associated ligands Fig. S4 A–J. ODR-3 and GPA-13 trafficking defects in mutant C. elegans olfactory neurons

Nature Neuroscience: doi:10.1038/nn.2835 2

Table S1: UNC119 crystallographic data and refinement statistics

Data Crystal HR3066aa Au10pe

Space Group P212121 P212121 Unit Cell Dimensions a=77.89, b= 79.56, c=189.72 a=78.55, b=79.71 , c=189.59 Resolution (Å) 50.0 – 1.95 30.0 – 2.00 Resolution (Å) (high-resolution shell) (2.02 – 1.95) (2.07 – 2.00) # Reflections measured 1,169,802 1,252,665 # Unique reflections 166,290a 82,342 Redundancy 7.0 15.2 Completeness (%) 99.6 (100) 100 (100) 26 (2.9) 11 (2.5) Mosaicity (o) 0.37 1.1 Rsymb 0.088 (0.537) 0.116 (0.701)

Refinement Resolution (Å) 40.51 – 1.95 29.37 – 1.99 Resolution (Å) – (high-resolution shell) (1.97 – 1.95) (2.04 – 1.99) # Reflections used for refinement 165,606a 78,089 # Reflections in Rfree set (%) 8,332 (5.0) 4,139 (5.3) Rc 0.191 (0.247) 0.200 (0.226) Rfreed 0.214 (0.251) 0.250 (0.276) RMSD: bonds (Å) / angles (°) 0.005 / 1.3 0.012 / 1.304 (Å2): Tα peptide residues/ # atoms N/A 41 / 268 (Å2): UNC119 only / # atoms 33 / 8,265 29 / 8,299 (Å2): water molecules / # water 41 / 803 39 / 793 / most favored (%) 99 97

Values in parenthesis refer to data in the high resolution shell. a Friedel pairs were used in phasing and refining HR3066a. b Rsym = |I-|/I where I is the intensity of an individual measurement and is the corresponding mean value. c R = ||Fo|-|Fc||/|Fo|, where |Fo| is the observed and |Fc| the calculated structure factor amplitude. d Rfree is the same as R calculated with a randomly selected test set of 5% (HR3066aa) or 5.3% (Au10pe) reflections that were never used in refinement calculations.

Nature Neuroscience: doi:10.1038/nn.2835 3

Table S2. Comparison of the RMSD values for each of the six UNC119 chains and their

associated ligands.

Structures were overlapped on the UNC119 protein carbon-α atoms only. Ligand atoms were

not included in the overlap calculation. RMSD values are given for the overlap of all protein

atoms in each chain, all atoms related to residue 501 (glycine and the attached lauroyl group),

and the carbon-α trace for the peptide attached to the lauroyl group in each ligand.

Chains Overlapped RMSD for all protein atoms RMSD for residue 501a RMSD of Cα traceb B on A 0.373 0.252 0.190 (501-507) C on A 0.459 0.315 0.180 (501-504) D on A 0.320 0.284 0.182 (501-504) E on A 0.449 0.370 0.323 (501-503) F on A 0.458 0.635 0.424 (501-507)

aRMSD values calculated using all atoms on residue 501 with residue 501 defined as glycine

plus the attached lauroyl group.

bNumbers in parentheses refer to residues on the ligand from which the RMSD values were

calculated.

Nature Neuroscience: doi:10.1038/nn.2835 6745 89123

Figure S1A The Greek Key Motif in UNC119, RhoGDI and PrBP/δ (related to Fig. 1). Greek key motif of the β-sandwich fold common to the three structures. Blue arrows depict the strands that form one β- sheet, while gray arrows depict strands of the second β-sheet.

Nature Neuroscience: doi:10.1038/nn.2835 PrBP/δ N 1KSH

RhoGDI 1DOA β 6

C N C Figure S1B Ribbon representation of the geranylgeranyl binding protein RhoGDI (1DOA) and the prenyl binding protein PrBP/δ (1KSH). RhoGDI is shown with Cys-geranylgeranyl of CDC42 present in its binding pocket. The presumed entrance to the hydrophobic pocket of PrBP/δ is indicated with an arrow. Note that entrances to the hydrophobic pocket in UNC119 (Fig. 4A ,B) and RhoGDI are located at the opposite edge of the β- sandwich fold. Figure created with PyMOL (www.pymol.org).

Nature Neuroscience: doi:10.1038/nn.2835

β1 β2

hUNC119Unc119 60 IGPEDVLGLQRITGDYLCSPEENIYKIDFVRFKIRDMDSGTVLFEIKKPPVSERLPINRRD PrBP/Pde6dδ 1 MSAKDERAREILRG------FKLNWMN--LRDAETGKILWQG-----TEDLSVPGVE RhoGDIRhoGDI 41 LDKDDESLRK------YKEALLGRVAADPNVPNVVVTGLTLVCSSAPGPLELD β3 α1 β4 β5 β6

hUNC119Unc119 121 LDPNAGRFVRYQFTPAFLRLRQVGA--TVEFTVGDKPVNNFRMIERHYFRNQLLKSFDFH PrBP/Pde6dδ 45 ------HEARVP--KKILKCKAVSR--ELNFSS-TEQMEKFRLEQKVYFKGQCLEEWFFE RhoGDIRhoGDI 88 LTGDLESFKK----QSFVLKEGVEYRIKISFRVNREIVSGMKYIQHTYRKGVKIDKTDYM

β7 α2 β8 β9

hUNC119Unc119 179 FGFCIPSSKNTCEHIYDFPPLSEELISEMIRHPYETQSDSFYFVDDRLVMHNKADYSYSGTP PrBP/Pde6dδ 94 FGFVIPNSTNTWQSLIEAAPESQMMPASVLTG--NVIIETKFFDDDLLVSTSRVRLFYV RhoGDIRhoGDI 144 VGSYGPR-----AEEYEFLTPVEEAPKGMLARG-SYSIKSRFTDDDKTDHLSWEWNLTIKKDWKD

Figure S1C Sequence Alignment of Human UNC119, PrBP/δ, and RhoGDI. Only C-terminal residues of UNC119 (60-240))( and RhoGDI (41- 204) are shown. β-strands are depicted as large red arrows for UNC119 and are underlined in red for PrBP/δ and RhoGDI. Identical residues are highlighted blue and similar residues gray.

Nature Neuroscience: doi:10.1038/nn.2835 RhoGDI ABPrBP/δ UNC119 UNC119

Figure S2A,B Structural Alignment of UNC119 with PrBP/δ and RhoGDI (related to Figure 4). (A) UNC119 (red) (PDB 3RBQ) aligned with PrBP/δ (blue). The Tα peptide is shown in dark gray and the acyl chain in green. Figure created with PyMOL (www.pymol.org). (B) UNC119 (red) aligned with RhoGDI (cyan). The Tα peptide is shown in dark gray and the acyl chain in green. The geranylgeranyl chain of the GTPase CDC42 bound to RhoGDI is shown in light gray. Figures created with PyMOL (www.pymol.org).

Nature Neuroscience: doi:10.1038/nn.2835 Fig. S2C Zhang et al.

Figure S2C Electron density surrounding the Lauroyl-T peptide. A stereoview showing a simulated annealing Fo-Fc omit map contoured at 2.5 sigma (red mesh). Phases were calculated following deletion of the ligand, application of random shifts (0.1 A), and refinement of the resulting model. UNC119 residues that comprise the walls of the cavity are shown with yellow carbon atoms, the acyl chain is colored green, and residues that comprise the remainder of the T peptide are colored gray.

Nature Neuroscience: doi:10.1038/nn.2835 Figure S3 Overlap of the UNC119 carbon-α coordinates and associated ligands. The UNC119 carbon-α coordinates for each of the six molecules in the asymmetric unit were overlapped and are shown in red. In each of the molecules the lauroyl-GAGASAEEKH ligand is bound in very similar fashion. The acyygl group of the li gand is shown in green and the ordered peptide residues of the ligand are colored gray. Figure created with PyMOL (www.pymol.org).

Nature Neuroscience: doi:10.1038/nn.2835 WT unc-119 ODR-3 ODR-3

A B

GPA-13 GPA-13

CD10 µm

Figure S4 A–D ODR-3 and GPA-13 trafficking defects in mutant C. elegans olfactory neurons. (A–D), localization of ODR-3 (A, B) and GPA- 13 (C,D) in wild-type (A,C) and unc-119 mutant (B,D) C. elegans olfactory neurons. In w ild-tOtype neurons, ODRDR-3dGPA3 and GPA-13 (green ) tra ffic norma lly to the olfactory cilia. In mutant neurons, the G protein subunits are mislocalized (B, ODR-3) or exhibit decreased expression (D, GPA-13).

Nature Neuroscience: doi:10.1038/nn.2835 WT unc-119 AWA

E F AWB

GH AWC

I J 10 µm

Figure S4 E–J ODR-3 and GPA-13 trafficking defects in mutant C. elegans olfactory neurons. E–J, structural Integrity of AWA, AWB and AWC Cilia in unc-119 C. elegans and restoration of ODR-3 and GPA-13 Localization. GFP was specifically expressed in AWA neurons (E,F), AWB neurons (()G,H), and AWC neurons ()(I,J) of wild-type ( left column) and unc- 119 mutant (right column) C. elegans worms. Reporter constructs were odr-10::gfp for AWA, str-1::GFP for AWB, and str-2::GFP for AWC. The structural integrity of these neurons is not compromised in the mutant (F,H , J) w hen compared to WT (E ,G .I) . Pl ease not e th at t ransgene ky ls 140 in the unc-119 mutant background only expressed GFP in one of the two AWC neurons (Fig. S4I,J), while some of the transgenic worms expressed GFP in both AWC neurons (unpublished data). Similar phenomena have been reported for other mutants (Troemel et al., Cell 99, 387-398 (1999).

Nature Neuroscience: doi:10.1038/nn.2835 Fig. S5 Light-induced translocation of transducin and return to the outer segment. (A) Schematic depiction of transducin translocation. Under intense light, TαGTP and Tβγ translocate separately to the inner segment and following GTP hydrolysis, associate with inner segment membranes as a heterotrimer TαGDPβγ. Following GDP/GTP exchange, which is very slow in the absence of rhodopsin, TαGTP is eluted from the membrane by the acyl-binding protein UNC119. Tβγ elutes and associates with a prenyl binding protein, either PrBP/δ or phosducin37. Both T α/UNC119 and Tβγ/PrBP diffuse freely and re - associate with a destination membrane after GTP hydrolysis. The destination membrane could be the cell membrane at the distal inner segment where IFT cargo is assembled or one of many outer segment disks. Nature Neuroscience: doi:10.1038/nn.2835 Fig. S5 Light-induced translocation of transducin and return to the outer segment. (B) Kinetic analysis of return to the OS. In the absence of a GEF (rhodopsin), solubilization of Tα governed by GDP/GTP exchange is slow (k= 10-4/sec)38, and is most likely the rate limiting step for the return of transducin. Likewise, the hydrolysis of TαGTP is slow (k=0.025 sec-1) in the absence of GAP, but not rate-limiting. GDP/GTP exchange maintains a rate constant of 0.0001/sec, resulting in only 0.01% of Tα solubilized per second 38, or one-third of Tα/hour, which explains the slow return to the outer segment.

Nature Neuroscience: doi:10.1038/nn.2835 6

Reference List

1. Hanzal-Bayer,M., Renault,L., Roversi,P., Wittinghofer,A., & Hillig,R.C. The complex of Arl2-GTP and PDE delta: from structure to function. EMBO J. 21, 2095-2106 (2002). 2. Troemel,E.R., Sagasti,A., & Bargmann,C.I. Lateral signaling mediated by axon contact and calcium entry regulates asymmetric odorant receptor expression in C. elegans. Cell 99, 387-398 (1999).

Nature Neuroscience: doi:10.1038/nn.2835