ai lnebr,Kmel .Ln,KnaaSoe io .WtisadMi Aridor* Meir and Watkins C. Simon Shome, Kuntala Long, R. Kimberly and Klinkenberg, p125A David by regulated is that signals assembly site lipid exit ER of cascade A ARTICLE RESEARCH ß eevd1 uy21;Acpe 2Jnay2014 January 22 Accepted 2013; July 19 Received ([email protected]) correspondence for *Author 3500 Medicine, USA. of 15261, School PA Pittsburgh Pittsburgh, of Street, University Terrace Biology, Cell of Department 2005; al., Sar1, et of (Bielli activity GTP release the vesicle enhance by GTPase-dependent both constricted to is Sec31 residues leading neck catalytic by vesicle of site A position GTP-binding hydrolysis. the Sar1 of the residue hedral is arginine optimization within a catalytic that and a form of 2007) Sec23 to Provision al., by polymerizes 2008). al., et and et layer (Fath (Stagg cage inner (ACE1) forms the 1 The complex, onto 2003). element Sec13/31 recruited al., the coat et of ancestral Miller composed 1998; an is al., which sorting et 2001; layer, thus Kuehn al., outer 1998; et motifs, al., (Aridor exit vesicles et budded ER Aridor into sequences, incorporation for peptide cargo binding short multiple layer, for presents inner and sites coat lipids The ER. acidic membranes the binds deform from Sar1–Sec23/24, to to released and (ERES) vesicles and exit sites cytosolic buds for exit into destined ER These proteins at cargo 2001). membranes select protein ER Schekman, on Sec13/31 small assemble and and the proteins Sec23/24 of (Antonny cytosolic composed the is complexes and (COPII) II Sar1 complex GTPase protein coat The INTRODUCTION Sec23ip Phosphatidylinositol-4- phosphate, p125A, ERES, COPII, WORDS: KEY exit. nucleating ER for Sec16A, and ERES from association at COPII coat membrane of the segregation stabilize spatial to the used regulate was SAM– module the by DDHD recognition Lipid assembly. p125A- ERES for functional required mediated in were respectively, interface domains, hydrophobic SAM a and and DDHD cluster the basic A domain. DDHD recognition the lipid central by selective conserved and that A self-assembly promoted cells. proteins domain in SAM membranes transfer PI4P-rich phosphates to PI targeted phosphatidylinositol was DDHD and and C-terminal phospholipases PA a recognized in defined found we p125A, domain control to Within signals lipid assembly. used p125A p125A. ERES required that ER step to a prior at ERES exit p125A-COPII-coated from Sec16A segregated (ERES) that spatially as find sites is we exit blocks, temperature ER known using at activity, By undefined. assembly coat (officially remain functional control directing and to steps p125A sorting layers the both yet cargo and with mediate interact proteins COPII Sec16A SEC23IP) of biogenesis. layers outer vesicle and inner The ABSTRACT 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,16–78doi:10.1242/jcs.138784 1765–1778 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. nvitro in and BuetlPrye l,20;Fra ta. 08.I yeast, In 2008). export dynamic al., ER et the and Farhan where assembly 2006; ERES al., membranes supports et ER (Blumental-Perry PI4P of on PI4KIII generation (PI4P) 2008). III (PI4K) phosphate Sec16 al., mammalian 4-kinase of activities phosphoinositide et the and by mediated changes (Sec16A) to is activities load ERES cargo of in adaptation al., the et example, (Zanetti For 2011). demands are budding biosynthetic and reactions physiological sorting with couple minimal activities that vesicle in proteins interacting measured by and controlled 1998). COPII activities al., selection of et COPII Matsuoka set cargo 2001; However, minimal al., et The basic (Antonny 2010). activities recapitulates formation al., et proteins Long coat 2005; al., et Lee upr h ipaeeto e1AwiedrcigCOPII directing while exit. ER Sec16A promotes which to of ERES, and at PI4P displacement nucleation as such to the signals lipid prior is support decode Sec16 to ERES functions which p125A-COPII-coated p125A in exit. from cascade at molecular segregated assembly a spatially reverse COPII identify to now direct We signals to ERES. and lipid al., coupling uses et layer p125A 2010). Tani Sec16-inhibited that 2005; A1 al., al., hypothesize phospholipase et et We Shimoi PA-preferring (Ong 1999). 2000; of ER 2010). al., et family the (Mizoguchi al., from a enzymes Shimoi export to 2007; et cargo belongs al., and p125A et (Ong 2005) (Iinuma al., organization layers ERES et p125A for thus coat that required Sec23, demonstrate is two studies binds overexpression also and the it Knockdown be where linking membranes to to uniquely remains recruited exit is Sec16-mediated A and ER of 2010). support reversal Schwartz, to linkage and the defined. coat Whittle for of 2012; allows inhibition al., the that et inhibits mechanism Sato, and Sar1–Sec23/24, Kung (Yorimitsu C-terminal Sec23/24 onto binds 2012; The layer inhibition outer which Sar1. the to of Sec16p, recruitment of leading of layers, activity linkage outer domain GTPase functional and Sec31-enhanced the inner hinders of coat Sec16p the (Supek However, liposomes between lipids 2002). synthetic acidic al., on for recruitment et has substitutes COPII signals promoting Sec16p lipid in and observed. Sec16 between previously link been A Sec16–COPII their defined. ERES activities, be at budding to two enhances COPII ER. remain control these the generation, subunits, PI4P from which and interactions budding by COPII vesicle mechanisms COPII all The potentiates and with assembly interacts Sec16p erimn fmmainCPIfo yoo smaue on measured is cytosol ERES from the COPII functional when observed mammalian for 2008). is of dependency al., required recruitment similar et whether Farhan is examined 2006; ER We al., et the (Blumental-Perry organization at liposomes PI4P-enriched production to COPII PI4P with recruited is p125A RESULTS 15 ofcal nw sSC3P id e3 ncytosol in Sec31 binds SEC23IP) as known (officially p125A a eeae hshtdlnstl4- phosphatidylinositol generates a (Farhan 1765

Journal of Cell Science (Sar1 n eurdP4 fatos13 i.1) h Sar1-dependent The 1A). Fig. activation 1-3; Sar1 (fractions by PI4P to induced required was cytosol and Sec23) from by recruitment (measured COPII liposomes Effective gradients. sucrose iooe eeicbtdwt yoo ntepeec of presence the control in cytosol or (Sar1 with PI4P-containing active incubated constitutively membranes. were liposomes defined synthetic ARTICLE RESEARCH 1766 1 at tested 400 both PI4P. (LUV, (Sar1-GDP, vesicles requires inactive recruitment or p125A (Sar1-GTP) and Active COPII Sar1-dependent 1. Fig. 5 C 5 E 0 S 0 hlseo n 0 IPa 26 at PI4P 10% and cholesterol 10% PS, 10% PE, 35% PC, 35% rti)a RSadwsjxaoe oEGC(RI5)o i-og oprmns(P10.Arwed niaeEF-15 oaiigwt Sec31 5 with bars: localizing Scale EGFP-p125A (1:500). indicate gpp130 Arrowheads and (GPP130). (1:100) compartments ERGIC53 p1 cis-Golgi containing endogenous or membranes the (ERGIC53) with to probed observed ERGIC adjacent were (as to 1:100), A) hSec31a juxtaposed in with dilution (described p125A was predominantly (antibody reaction and colocalizes and recruitment p125A immunoprecipitation ERES COPII EGFP-tagged a by at expressed from cytosol Transiently protein) 1–3) 1 micros (E) liver (fractions ER and Sec31. liposomes rat to and ng PI4P-containing from cytosol Sec23 Floated 500 p125A-depleted (1:5000), depleted (D) ng, or p125A. was (50 control of (control) 5–8) from Recruit Sar1-GTP (lanes SNX9 (C) recruitment by presence labeled). Sec23 or recruited as Sar1-GTP-dependent p125A fractions was indicated. p125A. (floated as Sec23 (B) of blot, monitored. (1:500) western depletion hSec31a using by or verified (A) affected was dilution) not depletion 1:10,000 is (at membranes Sec23 ER against to antibodies with blot western by analyzed T39N a1GP a1poen,adioae yfotto in floatation by isolated and proteins, Sar1 Sar1-GDP) , m )cmoe f4%popaiycoie(C,3%popaiyehnlmn P) 0 hshtdleie(S n 0 hlseo or cholesterol 10% and (PS) phosphatidylserine 10% (PE), phosphatidylethanolamine 35% (PC), phosphatidylcholine 45% of composed M) H79G emdSr-T)o inactive or Sar1-GTP) termed , m ,50 g, m ˚ o oradfotdot urs rdet(ilie l,20) rcin were Fractions 2005). al., et (Bielli gradient sucrose a onto floated and hour 1 for C ia oue eeicbtdwt a ie yoo n ytei ag unilamellar large synthetic and cytosol liver rat with incubated were volume) final l m ,fnlvlm 60 volume final g, AB a1dpnetCPIrcutett lae iooe sdpneto PI4P. on dependent is liposomes floated to recruitment COPII Sar1-dependent (A,B) htteln ewe ne n ue otlyr ih be might layers coat outer and suggest inner they yet between recruitment, link coat in 1998) the PI4P yeast al., for that et role purified (Matsuoka a of membranes support PI4P- and synthetic recruitment to of the proteins COPII with monitoring fractions agreement in studies are Sec23-containing results onto previous The directed all) 1B). (Fig. was liposomes not (Sec31) containing cage (albeit COPII outer distinct the of recruitment m )i oedpnetmne nteasne(ae –)or 1–4) (lanes absence the in manner dose-dependent a in L) ora fCl cec 21)17 7517 doi:10.1242/jcs.138784 1765–1778 127, (2014) Science Cell of Journal m m. eto Sec23 of ment mswas omes o p125A for 25A

Journal of Cell Science ellrlclzto fteslce GPtge oan in domains EGFP-tagged selected cells. HeLa the transfected transiently of localization cellular o ils niaigpo odn.I otatt t agtn to targeting for its to contrast 701–989) of In because folding. (residues poor produce indicating to yields, difficult domain low were fragments DDHD Shorter analysis. the encompassing OI ne ae e2/4t Rmmrns(i.1) The 1C). (Fig. that of membranes mirrored liposomes recruitment ER PI4P-containing Sar1-dependent to to p125A the of Sec23/24 recruitment affect layer not inner terminus did COPII by N 2010), cytosol al., its (Ong from Sec31 et p125A on co-deplete to of shown segments Immunodepletion previously immunoprecipitation, 2010). separate al., cytosol using et in Sec31 (Ong Sec23 with associates binds 2005), (Ong al., and export et ER Shimoi 2010; and al., assembly et ERES regulates p125A regulated. ARTICLE RESEARCH ta. 05,adi h 15-otiigPLA p125A-containing the Nir2 in (Litvak and where protein 2005), (PI)-transfer al., inositol N- (Nir1-3), et phosphatidyl the a in receptor as proteins, functions B domain-interacting degeneration retinal terminal in DDHD metal domains. found phosphoesterase putative are in a domains found form typically can site that binding (DDHD) residues conserved four a contains which sterile , terminus, a C and domain its DDHD using binding. role membranes lipid-signal the binds in examined domains p125A first p125A selected we of this, specificity test and to To signals assembly. lipid ERES selective regulate recognizes p125A that hypothesized We cells lipid in support to cooperate ERES domains recognition SAM of and progression DDHD The the control to Sar1–Sec23/ membranes of assembly. ER recruitment initial onto link following may 24 layers p125A coat layer-associated two outer Thus, the 1D). (Fig. Sec31 of ta. 00.I h otx fmn utdmi rtis SAM proteins, multidomain many Yamashita of 2005; context al., the et In Shimoi 2010). 2010; al., al., et et (Sato family protein eotr uha h Hdmi fFp1(exle l,2005). His al., et a PI4P (Weixel Fapp1 typical prepared of domain like TGN PH We the behaving and as shown) compartments, such reporters not 2A) Fig. (gpp130 membranes (TGN46, of cis-Golgi Golgi rims the (ERGIC53), decorated PI4P-rich EGFP–DDHD ERGIC bound with Membrane also 2A). and association (Fig. pool cytosolic robust a in with showed distributed contrast, colocalize was not strikingly, By of did but, al., S3). higher domain Sec31 Fig. DDHD enlargement et material EGFP-tagged isolated supplementary At (Mizoguchi the the and 6 structures to compartments. (Fig. trans-Golgi membrane 2000) led the ERES shown) EGFP-p125A or COPII-coated 1E) not levels, Fig. (ERGIC53, expression compartment localize (gpp130, (TGN46, not intermediate cis-Golgi did network Golgi At the but to 1E). to, 1E), (Fig. Fig. ER adjacent region were the cell perinuclear ERES with, the sites, the the in latter in by distributed these EGFP- clustered (marked normally 2005), ERES or that subunit) at periphery al., Sec31 COPII previously et layer with outer (Shimoi As localized p125A p125A assays. using tagged endogenous oligomerization sedimentation for and and recognition shown lipid overlays in lipid-blot domains the of 643–704) (residues SAM p125A domains of Bowie, DDHD role hetero- and and the (Qiao or analyzed shown structures We been functional homo- 2005). also larger have into to that domains polymerize SAM ability to of motifs forms their Several interaction through associate. protein-protein function common modulate are domains 8 eiusln rsde 7–8 np2A n contain and p125A) in 779–989 (residues long residues 180 nvitro in nvivo in n idn fP4-ihmembranes PI4P-rich of binding and 6 tge rgeto h terminus C the of fragment -tagged and a oi SM.DH oan are domains DDHD (SAM). motif nvitro in nvitro In . nvivo In eaaye h role the analyzed we , eeaie the examined we , 1 phospholipase aloto ouinuo Zn upon solution that structures al., of sheet et out (Nagaya oligomeric ERES generates fall at and assembly dimerizes COPII 2002), regulates that protein 34 ipopae[PI(3,4)P cellular our bisphosphate analysis. defined with and (3,4) phosphatidylinositol PA recognized agreement recognized weakly for highly and also (PS), in domain phosphatidylserine The and, 2A,C). exerted (643–989) (Fig. PI5P PI4P domain observations, domains PI3P, to combined DDHD included specificity the that and fragment Importantly, larger SAM broad a binding produced ineffective somewhat both this weak we Given with recognition, 2B). only (Fig. lipid exposures) lipids displayed acidic toward prolonged blot specificities domain lipid with using DDHD (observed measured the when cells, overlays in membranes PI4P-rich oooosSMdmi fday lcrlkns (DAGK) kinase glycerol the diacyl that of demonstrated domain have remained SAM studies homologous domain Structural 2F). SAM Furthermore, (Fig. EGFP-tagged shown). cytosolic (not expressed to binding lipid- transiently lipid using the domains no Analysis showed possibilities, domain. DDHD overlay SAM these blot GST-tagged evaluate assemble a To generated may we selective avidity. binds lipid-binding it domain optimize SAM Alternatively, the phospholipid that selective lipids. possible enhanced is domain It SAM recognition. the that suggest of results The inclusion PIs. polyphosphate acidic more recognize Mn Kih ta. 00.A bevdwt h A oanof domain SAM the with observed As 2010). DAGK al., dimerize, to oligomerization et SAM domain monitor to (Knight the test easy of an with ability us providing the thus on dependent is formation oansoe olpdrcgiin(i.2) n transiently and 2D), His (Fig. recognition DDHD the EGFP-tagged lipid expressed assays, no in showed with overlay tested domain When them blot PI-X). their replaced termed lipid and examined (851-EGEEE-855, group (851-KGRKR-855) acid a and on glutamic residues focused domains we basic domain we of these DDHD hypothesis, the Within in this functionality. test by mutations assisted To is generated assembly. and domain domain-mediated DDHD SAM the within resides recognition assembly and recognition SAM lipid Our the support by to domains used DDHD are and interactions hydrophobic and Charge rsneo Zn the in of precipitated effectively presence domain SAM non-tagged The GST. rmt eetv ii eonto n ellrmembrane cellular and recognition domains which DDHD lipid in binding. and model selective SAM a assembled promote (not suggest of cytosolic activities results the remained cooperative these between (701–778) Collectively, region domains shown). linker DDHD GFP- A and the 2005). SAM encompassing al., et fragment (Weixel tagged observed domains as PI4P-binding shown), yet other (not with Golgi disassembly Golgi PI4P-enriched caused binding. to occasionally membrane localized avidity-based also optimize EGFP–SAM–DDHD may p125A-SAM the which by that domain, assisted suggested analysis is overlay recognition blot lipid lipid DDHD-mediated The below). see 2I, (Fig. ouiiyo h oto S rti a lovral fetdby affected Zn variably of also addition was the protein GST control the of solubility fslto ne hs odtos(i.2) diino Ca of Addition 2H). (Fig. conditions out these fell under quantitatively solution which of protein, the of polymerization robust rsne ifs yolsi itiuin(i.2) This EGFP–SAM–DDHD 2E). (Fig. when observed distribution also cytoplasmic was diffuse localization a presented 2+ nvivo in a iia fet nSMslblt ntson.The shown). (not solubility SAM on effects minimal had d ora fCl cec 21)17 7517 doi:10.1242/jcs.138784 1765–1778 127, (2014) Science Cell of Journal diino Zn of addition , and 2+ 2+ hra ie uatrmie soluble remained mutant dimer a whereas , nvitro in etu lae h A oanfo the from domain SAM the cleaved thus We . 2+ nlss(i.2 ugs htlipid that suggest 2) (Fig. analyses oGTp2ASMdmi e to led domain GST-p125A-SAM to 2 ,ytfrtems atddnot did part most the for yet ], PI-X 2+ idn.Zn binding. otisGlilclzto and localization Golgi its lost 6 tge SAM–DDHD -tagged 2+ idcdsheet -induced 1767 2+ d PI-X PI-X ,a or

Journal of Cell Science EERHARTICLE RESEARCH blsigpoenasml Kih ta. 00 ioand Qiao DAGK 2010; 1768 the al., In et (Knight 2005). test assembly Bowie, To protein the for assembly. abolishing available protein isolation information structural supports in DAGK used shown), targeting we hypothesis, not cellular this or and recognition 2F (Fig. lipid display DDHD not the that recognition. lipid suggest for results required The is shown). domain (not analyzed was GST-SAM or GST-SAM to added was [right Golgi His coating against seen antibody HRP-conjugated be using can overlay 1 and blot (B) 10 lipid membranes bars: domains. on PI4P-enriched Scale probed SAM of was image). and Sec31- domain, middle periphery DDHD 53, between the the (ERGIC colocalization to ERGIC of of targets and consisting lack 1:500)] domain shows dilution DDHD (antibody (arrowhead The TGN46) ERES EGFP–DDHD). image, Sec31-containing p125A. localized from of and dissociated module ERES (779–989) SAM–DDHD stained domain the EGFP–DDHD by expressed recognition Transiently lipid Cooperative 2. Fig. S ople P rcin a niae) sn etiuainadaayi nCoasesandgl.()SMadSAM and SAM (I) gels. Coomassie-stained on analysis and centrifugation using indicated), (as fractions (P) pellet to (S) xedn h rgett oti h ptemSMdmi 6399 ofre ii eetvt omnpopoyae I,P,P n PI(3,4)P and PS PA, PIs, monophosphorylated to selectivity lipid conferred (643–989) domain SAM upstream the contain to fragment the extending 5 nuae ih045m fZn(AOc) of mM 0.455 with incubated A oan(4–0)(eeae nPye ihrdarwedidctn h oiino osre ecn 60 ihnahdohbcdmrinterfa dimer hydrophobic a within (690) leucine conserved a of Zn position of the addition indicating by arrowhead promoted red is with oligomerization Phyre) GST-SAM in (H) (generated (643–704) domain SAM elcn ai tec frsde nteDH oano h A–DDmdl ihguai cdrsde (850-KGRKR-854 residues acid glutamic EGFP–DDHD with whereas module (left), membranes SAM–DDHD Golgi the to of targeted is domain domain DDHD EGFP–DDHD the The (E) in recognition. residues lipid of abolished stretch basic a replacing m .()TeEF-A oantasetyepesdi eaclssosdfuectslcdsrbto.Saebr 5 bar: Scale distribution. cytosolic diffuse shows cells HeLa in expressed transiently domain EGFP-SAM The (F) m. efrhrhpteie htteSMdmi,wihdoes which domain, SAM the that hypothesized further We d SMdmi ogiemtgnssamdat aimed mutagenesis guide to domain -SAM 2 L690E n nlzda nH. in as analyzed and d SMdmi,hydrophobic domain, -SAM (10 m fec) sidctd lgmrzto a olwdb h rcptto ftepoen rmsupernatant from proteins the of precipitation the by followed was Oligomerization indicated. as each), of M m /lo uiidHstge 15 rget(0–8)cnann h DDdmi,btntteSAM the not but domain, DDHD the containing (701–989) fragment p125A His-tagged purified of g/ml 2+ n ssniiet h 60 uain ufrwt rwtotteadto f20 of addition the without or with Buffer mutation. L690E the to sensitive is and A h DDdmi agt IPrc og ebaei isolation. in membrane Golgi PI4P-rich targets domain DDHD The (A) rcptt nrsos oZn to response in precipitate nietewl-yepoen GST-SAM protein, residue analysis. wild-type for single (L690E) a the SAM-domain generate p125A could Unlike the we in fully thus replacement is p125A, interface in dimer conserved hydrophobic The oligomerization. order Fg G,weesitouto facagdrsdea this at domain Zn residue and the charged dimerization mediate a of both of residues prevented oligomerization introduction position leucine whereas for 2G), and (Fig. required valine dimerization between interactions 6 a :0 iuin.Tefamn on ekyt cdclpd.()A nB: in As (C) lipids. acidic to weakly bound fragment The dilution). 1:500 (at ora fCl cec 21)17 7517 doi:10.1242/jcs.138784 1765–1778 127, (2014) Science Cell of Journal m .(-)Slcielpdrcgiini eedn namodule a on dependent is recognition lipid Selective (B-F) m. m 2+ PI-X .()Tepeitdsrcueo p125A of structure predicted The (G) m. L690E Fg HI.Tu,i common in Thus, 2H,I). (Fig. otGlitreig(ih) cl bar: Scale (right). targeting Golgi lost oan 045m ah were each) mM (0.455 domains R GE,tre PI-X) termed EGEEE, (L690E) 2+ idcdhigh- -induced m 2 Zn(AOc) M D si C: in As (D) . i not did ce. 2

Journal of Cell Science rvosyrpre,EGC3as ctee ndefined in scattered as and, also strongly compartments ERGIC53 ERGIC53 ERGIC ERGIC, reported, at perinuclear previously traffic in cargo accumulated retrograde and rfi niiinb o eprtrsrvasa exclusive an reveals temperatures low by inhibition Traffic Similarly, domain. Zn the with precipitated of SAM robustly domain addition, assembly SAM untagged order the while high therefore and DAGK with ARTICLE RESEARCH RI n og akr nclsicbtda reduced at incubated were COPII, cells incubated When 15 Golgi. to cells the at and retrograde relation we ER in and the between anterograde binding, in traffic markers slow preferentially p125A-membrane p125A that Golgi temperatures of of and site localization ERGIC the the bud COPII define to analyzed adjacent To membranes Golgi or sites. ERGIC on at or PI4P including ERES, PIs, monophosphorylated recognize may ERES p125A at p125A of localization oano 15 sfunctional. SAM is the p125A within of interface assembly domain hydrophobic the that suggest ˚ ne odtosta retboytei anterograde biosynthetic arrest that conditions under C d hssnl on uainaoihddimerization abolished mutation point single this , (L690E) eandcmltl oul.Teresults The soluble. completely remained 2+ eesdwe eundt 37 to returned 1991) when Svensson, rapidly reversed was and distribution This (Saraste S2A,C). Fig. material cytoplasm (supplementary the throughout puncta hc ylsbtenE n RI.Smlrt endogenous to Similar ERGIC. and ER between ERGIC53, cycles protein membrane which the coat analyzed sites we COPII-p125A membranes, ERES enlarged and whether ERGIC determine To both p125A-COPII S2A–B). where Fig. material (supplementary p125A)]. sites clustered largely compartments perinuclear (endogenous Golgi from 7A,B segregated Fig. sites and 3A, [Fig. ie Fg A.Icbto fclsa 10 were at cells sites of Incubation individual 3A). these while (Fig. at concentrated sites reduced, effectively was COPII was cytosolic and enlarged Sec31) by (marked xlsvl attoe ihCPIa oh15 p125A both Importantly, at 3A–C). COPII (Fig. with puncta partitioned enlarged exclusively in COPII 2002). collecting layers coalesced, Helenius, further and ERES (Mezzacasa conditions, export these membrane Under ER ER in bulk functional the are with and continuity ERES maintain COPII-coated that in membranes cargo biosynthetic folded of accumulation 15 at Importantly, S2B). Fig. (supplementary material conditions these under unperturbed largely remained xrsigmF-15 81 or)wr ananda 37 at maintained were hours) (8–12 mRFP-p125A during expressing ERES inhibition. COPII-coated traffic with temperature-induced associated p125A 3. Fig. Sc1 n Sc4 atbd t110dlto)i el incubated cells in dilution) 10 1:100 endogenous at at of (antibody Colocalization hSec24c (C) and extensive ERES. hSec31a the to indicate p125A arrowheads of A, colocalization in images areas Enlarged boxed (B) of (hSec31A). ERES with localization for analyzed nuae t15 at incubated ora fCl cec 21)17 7517 doi:10.1242/jcs.138784 1765–1778 127, (2014) Science Cell of Journal ˚ ,a nA cl as 5 bars: Scale A. in as C, ˚ Cor10 ˚ ,a niae,fr4hus hnfxdand fixed then hours, 4 for indicated, as C, ˚ ntson.Glimorphology Golgi shown). (not C m m. ˚ h ubro ERES of number the C ˚ ed oteselective the to leads C A eaclstransiently cells HeLa (A) ˚ Cand10 ˚ 1769 ,or C, ˚ C

Journal of Cell Science aeilFg 2) sarsl,YPp8wsas retdat arrested also was 15 YFP-p58 result, at (supplementary a overexpression ERES to As enlarged due S2C). likely Fig. most material ER, the in up ot RSmmrns ii eonto eitdb the by ERES mediated supports domains DDHD recognition localization. and SAM Lipid and of activity COPII membranes. cooperative with ERES co-segregates exclusively coats analysis p125A the Overall, that shown). not suggests and 7A,B Fig. below, (see p125A ucata eepriual vdn t15 at evident particularly were that puncta 15 at region perinuclear the in accumulated ERGIC53) (rat YFP-p58 overexpressed protein, ARTICLE RESEARCH 1770 PI4KIII and Sec16A whereas membranes, synthetic on COPII during assembly lipids acidic of requirement the for substitutes Sec16p exit during ER Sec16A from segregate ERES COPII-p125A-containing ebae r otdwt 15 n OI ne these under and 10 (Mezzacasa COPII at ERES S2C and of Assembly Fig. 2002)]. p125A Helenius, material with [supplementary coated conditions are membranes ˚ n 10 and C ˚ Cor10 ˚ ,adas ndsic scattered distinct in also and C, ˚ odmntaeta ERES that demonstrate to C ˚ .YPp8as backed also YFP-p58 C. ˚ eurdfunctional required C a ifrn eairt h eiula lseigo e1Aunder Sec16A of clustering exhibited perinuclear p125A the endogenous to behavior and Surprisingly, different temperatures. mRFP-p125A and low cytosol Sec23, ERGIC53/p58- at between results Sec31, reduced Sec16A The with is 4C). of which Fig. distribution membranes, associated in dynamic inset a closely and support 4 (Fig. and membranes containing adjacent sites RI (15 ERGIC e1ApeetdEE oaiainaddfue cytosolic (10 ER diffused the at and traffic localization slowing Importantly, ERES distribution. presented Sec16A e3 n 15 ihsmlrrsls(i.4adFg A.At 7A). Fig. and of 4 localization (Fig. the results similar to with relation 37 p125A with and both Sec16A Sec31 of EGFP-tagged localization a well the as temperatureas antibody), specific analyzed the using (KIAA0310, using We Sec16A endogenous p125A above. and described Sec16A blocks Sec31, localized hypothesis, this explore we To stages regulation. Sec16 at follow assembly or COPII precede support that that may hypothesized p125A We 2002). by al., recognition et lipid Supek cargo 2008; with al., assembly et ERES (Farhan load of adjustment during required both are ˚ ,edgnu e1AadtasetyepesdEGFP- expressed transiently and Sec16A endogenous C, Sc1)adEF-e1Awsdtrie.(,)CnrlHeLa Control (B,C) determined. was EGFP-Sec16A and hSec31a) el B rclstasetyepesn RPp2A()were (C) mRFP-p125A 10 expressing at transiently incubated cells or (B) cells e1A(–2hus eemitie t37 at maintained were hours) low (8–12 at Sec16A Sec16A from segregates p125A temperatures. coated ERES 4. Fig. e1AadYPp8drn 10 endogenous during of YFP-p58 transiently localization and cells the Sec16A HeLa for shows analyzed C YFP-p58 in expressing was Inset mRFP-p125A indicated. and as hSec31a determined, dilution), 1:1000 at (antibody niaehe3 AB rmF-15 C ie,sgeae from segregated 5 sites, bar: (C) Scale mRFP-p125A Sec16A. or (A,B) hSec31 indicate 15 ora fCl cec 21)17 7517 doi:10.1242/jcs.138784 1765–1778 127, (2014) Science Cell of Journal ˚ Cor10 ˚ ) e orbs olcino e1Aa perinuclear at Sec16A of collection robust to led C), ˚ o or,adtelclzto fEE mre by (marked ERES of localization the and hours, 4 for C A eaclstasetyepesn EGFP- expressing transiently cells HeLa (A) ˚ n h oaiaino noeosSec16A endogenous of localization the and C m m. ˚ nuain.Arrowheads incubations. C ˚ ,o nuae at incubated or C, ˚ )o at or C)

Journal of Cell Science n A n DDdul uatp125A mutant double DDHD and SAM and sebycnrle ii eonto srqie oregulate to required is recognition lipid controlled Assembly under ERES COPII-p125A-coated (10 delay from either and that conditions Sec16 enhanced ERES The shown). of scattered not and segregation 4 enlarged 3, (Figs at Sec16A from segregating assembling conditions, these ARTICLE RESEARCH eurdfrSMdmi sebyadDDHD-supported and (p125A mutated residues were assembly specific 2) (Fig. which SAM-domain recognition in lipid for p125A Tagged required ERES. COPII at direct to recognition assembly lipid uses p125A that hypothesized We ERES at organization COPII ulaino OI ySec16. initial the by follows COPII that of stage late nucleation a regulate may p125A that suggest h A L9E rDH oan(IX a o be PI-X the not Importantly, because L690E 2B-I). and may (Fig. phenotype recognition protein lipid (PI-X) endogenous retain dominant the may domain mutants with a assemble DDHD may generate in mutants or mutations to Individual (L690E) sufficient hypothesis. SAM this test the to generated were ˚ )o nbe(15 enable or C) ˚ )so ag exit cargo slow C) PI-X p125A , IX L690E PI-X, L690E ) , 3) eas hsmtn ean h blt oitrc with interact p125A to mutant analyzed oligomerization ability we SAM interface, a the both dimerization Fig. the retains using material mutant p125A supplementary endogenous this and Because shown S3A). (not protein wild-type obt RSadteprncerGlirgo Fg 5). (Fig. region Golgi p125A perinuclear type, wild the to contrast and marked in ERES Importantly, both targeting occasional to with distribution cytosolic diffuse augmented nlzdb etr lt Fg Eadntson.EGFP- shown). when EGFP-p125A not levels 5). and (Fig. similar 7E ERES (Fig. at to blots localized expressed western p125A were al., by et mutants (Ong COPII analyzed terminus both All N unperturbed with the 2010). p125A by maintained wild-type are and layers mutated of interactions a lal vdn.Frtems at vrxrsinof overexpression part, most component the cytosolic For diffused evident. EGFP-p125A a clearly However, was ERES. to targeted h IXmtn.Oeepeso fEGFP-p125A of to Overexpression similar mutant. ERES, with PI-X association the and labeling diffuse exhibited ii eonto blte eedsbe.EGFP-p125A disabled. were abilities recognition lipid uat(p125A mutant ullnt GPp2Awl ye IX 60 rtedul mutant double the or L690E PI-X, (p125A type, wild EGFP-p125A full-length the assembly. by ERES binding PI4P 5. Fig. rEGFP-p125A or oanfo ap ntebcbn fa 60 uat EGFP-p125A mutant. PH L690E the an of with backbone substituted the been in EGFP-p125A Fapp1 has from a domain domain of DDHD expression the the where shows chimera panel bottom The indicated. obemtn p125A mutant double uruddwt el xrsigp125A expressing cell cells non-transfected with a surrounded p125A indicate Fapp1-PH-containing Arrowheads the cells. in chimera-expressing ERES restored and partially targeting was Membrane assembly assembly. ERES disrupted completely ora fCl cec 21)17 7517 doi:10.1242/jcs.138784 1765–1778 127, (2014) Science Cell of Journal L690E,PI-X PI-X n RS(Sc1)wr nlzdi eaclsas cells HeLa in analyzed were (hSec31a) ERES and ) IX L690E PI-X, L690E h oaiaino rninl xrse 1–4hours) (12–14 expressed transiently of localization The i o edt lseig sosre ihthe with observed as clustering, to lead not did IX L690E PI-X, uat eoeprl yooi,weesthe whereas cytosolic, partly become mutants ,i hc ohtedmrzto and dimerization the both which in ), SAM–DDHD otmmrn oaiainadhad and localization membrane lost IX L690E PI-X, oueo 15 controls p125A of module L690E *.Saebr 10 bar: Scale (*). Fg HI n a and 2H,I) (Fig. PI-X L690E IX L690E PI-X, a also was e to led 1771 L690E m m. PI-X ,

Journal of Cell Science niio fEE seby e3 otEE oaiainin localization ERES lost Sec31 assembly. p125A ERES of inhibitor oaie ntectpam(i.5.Mroe,p125A Moreover, 5). (Fig. cytoplasm the in localized lhuhi i o fetSc6 oaiain(o hw) These shown). (not localization Sec16A affect not did it although EERHARTICLE RESEARCH loihbtdteasml fEE hnmaue t10 1772 at measured when ERES of assembly the ERES. inhibited also at assembly COPII regulates membrane selective p125A-membrane p125A p125A whether for Indeed, tested by thus required We binding is ERES. at module binding by mediated SAM–DDHD recognition S3A). lipid Fig. the selective high material that supplementary suggest very results and diffuse at These 5 is even exhibited (Fig. maintained module levels and was expression localization lipid-binding which ERES distribution, cooperative cytosolic no presumed showed the disabled, which in IX L690E PI-X, IX L690E PI-X, epesn el n eanddiffusely remained and cells -expressing eaeatasdmnn negative trans-dominant a became IX L690E PI-X, ˚ C, el n nlpdbo vras iia op125A to Similar overlays. blot lipid in membranes on PI4P-rich and recognized cells domain at from DDHD assembly COPII derived The regulate recognition ERES. to required lipid is activities cooperative SAM–DDHD that suggest results fSMdmi nciain(sn h 60 mutation, L690E the (using inactivation domain p125A ( SAM domain DDHD of the of deletion ofr eetv eonto fP4 n,a loosre ihthe with observed Fapp1-PH also as 2006). and, al., PI4P of et recognition selective (Blumental-Perry pleckstrin-homology confers Fapp1 the domain of of domain, the module domain role PI4P-binding replacing (PH) recognition fide the artificially bona lipid test a by with further selective assembly, to a ERES staining truncation directing as Sec31 this by SAM–DDHD used analyzed p125A We as 6A). assembly, (Fig. ERES inhibited and L690E, ora fCl cec 21)17 7517 doi:10.1242/jcs.138784 1765–1778 127, (2014) Science Cell of Journal ie arw) cl a:5 bar: Scale (arrows). sites mRFP-p125A cells HeLa mRFP-p125A, (B) expressing (arrows). transiently colocalized are hSec31a and mRFP-p125A hours) (12–14 Sec16A expressing regulates ERES. p125A from by displacement recognition Lipid 6. Fig. rnfce el) n mRFP-p125A and cells), transfected atbd iuin120]aedssebe ncells p125A in mRFP disassembled expressing are 1:200)] [hSec31a dilution ERES (antibody indicated. as localization, hSec31 h olcino GPSc6 nmRFP-p125A in EGFP-Sec16A and of (arrow), collection YFP-Sec23a the co- with the mRFP-p125A to of opposed assembly of as segregation mRFP-p125A the the from for Note EGFP-Sec16A analyzed proteins. and transfected fixed of were localization hours 24 for indicated, mRFP-p125A D DDHD e octslcdseso fteprotein the of dispersion cytosolic to led ) L690E, L690E, D D DDHD DD+Fapp1-PH + DDHD D L690E, GPSc6,YPSc3,as YFP-Sec23A, EGFP-Sec16A, , 7–8)i h background the in 778–989) m A eaclstransiently cells HeLa (A) m. D DDHD L690E, for analyzed were atrssmark (asterisks L690E, D DD+Fapp1-PH + DDHD D DDHD IX L690E PI-X, L690E, or D DDHD ,

Journal of Cell Science og Fg AadFg ) motnl,i otatt expression to contrast in Importantly, 5). the p125A Fig. of to and targeting 6A preferential (Fig. some Golgi with distribution, punctate and fedgnu e3 tbt eiula og daetstsand sites adjacent Golgi perinuclear both assembly at maintained Sec31 endogenous and of with colocalized chimera the assembly, EERHARTICLE RESEARCH ebae Wie ta. 05.We xrse ncls the cells, in expressed When 2005). (p125A al., chimera Golgi et PI4P-rich to (Weixel isolation in membranes targeted is domain, DDHD isolated 7. Fig. e etpg o legend. for page next See IX L690E PI-X, L690E, rp125A or D DD +Fapp1-PH DDHD, L690E, D DDHD ipae ohcytosolic both displayed ) hc irpe ERES disrupted which , oeo IPi 15-eitdasml fERES. of assembly p125A-mediated the in supports PI4P PI4P- assembly of the ERES a role restore replace with partially to and artificially ability domain module binding The recognition 5). Fig. lipid and SAM–DDHD 6A (Fig. ERES peripheral n osblt sta ii eonto srqie otarget to required is recognition lipid that assembly? ERES is control possibility p125A by One recognition ERES lipid at could residency How p125A controls recognition Lipid ora fCl cec 21)17 7517 doi:10.1242/jcs.138784 1765–1778 127, (2014) Science Cell of Journal 1773

Journal of Cell Science p125A aeilFg 1)o EGFP-p125A or S1D) Fig. material emaue.Hwvr iie soito ihEE was ERES with association p125A limited EGFP- when However, observed measured. be 0scnso h eoeypae eefte nosingle into fitted initial T were averaged The overall phases with 5). 2006) (Fig. recovery al., et mutants the (Forster diffused exponentials these of observed the seconds of for 20 population explanation an cytosolic providing S1E), Fig. eltdadrsudcl ouain ihEF-15radEGFP- and EGFP-p125Ar p125A- with control, populations in p125A cell morphologies rescued Golgi KD and of of distribution depleted efficiency fractional transfection The partial (F) to cells. due exposures prolonged required 15 gen nioydlto :00 n Sc1 rd tEE arrested ERES at (red) 10 hSec31a at and 1:1000) dilution ER- antibody steady-state (green, p125A for required is traffic. module to-Golgi SAM–DDHD Functional 7. Fig. ARTICLE RESEARCH 1774 seconds). (3.36 EGFP-p125A (T or seconds) EGFP-p125A (3.14 YFP-Sec23 EGFP-p125A for recovery observed faster exhibited were Importantly, both (supplementary dynamics. kinetics shown) slower EGFP-p125A kinetics (not slightly mRFP-p125A or whereas recovery similar or S1C) 2006), typical Fig. al., material exhibited et COPII shown) shown. (Forster or (not S1B) is Fig. material experiments CFP-Sec31 (supplementary YFP-Sec23 independent both by three marked time-point each using in for events YFP- measured collected ERES 27–35 mRFP- of expressing or average S1) An at constructs Fig. p125A. material with (supplementary COPII EGFP-p125A the transfected cells Sec23, and HeLa analyzed transiently (FRAP). we proteins photobleaching were after possibility, p125A recovery on this fluorescence proteins of test coat associated To dynamics its and membranes. regulate with p125A may terminus ERES of recognition N time lipid its residency Alternatively, of the Sec31. association through and p125A ERES Sec23 However, to ERES. targeted to proteins is Sec31 associated and p125A n rfi a retdb nuaina 10 at incubation by arrested mRFP-p125Ar was RNAi-resistant Control traffic with (B) and transfected conditions. were similar cells under endogen- p125A-depleted (red) of or hSec31a localization and the (green) shows Sec16A ous Inset S2). Table material supplementary xrsigEF-agdpoen 9–2 el e ru) Unpaired cells group). all per for cells Student’s determined (93–220 were proteins phenotypes collected EGFP-tagged Golgi were expressing and images Ten experiment condition. each (mean each from cells for of KD performed analysis and were Statistical control experiments (G) in shown. morphology Golgi is Golgi missing condition intact or treatment (green) each Golgi under shattered (purple) (pink), Golgi dispersed (blue), n sntson h xrsino GPp2A wl ye n EGFP- and type) (wild EGFP-p125Ar of area expression analyzed The the p125A below shown. ran not EGFP is cells. KD and and antibody control comparable in GFP the 1:10,000) shows (EGFP-p125Ar; dilution panel clones upper EGFP-p125A-resistant actin The of and shown. expression 1:2500) are panel, 1:10,000) western (middle panel, by p125A (lower efficiency of as knockdown expression (labeled p125A Endogenous recognizable of blots. not Analysis was (E) morphology purple). (vesiculated, Golgi missing, shattered cells, and some (pink) In color- dispersed green). and (blue), gpp130 shown intact analyzing are including by morphologies coded, cells Golgi HeLa observed in Typical 5 quantified localization. bar: was Scale morphology the morphology. Golgi and Golgi (D) populations shattered Golgi Golgi in intact in increase of change concomitant loss dramatic with the (arrowheads) Note GFP. morphology as for S2), Table visualized material and (supplementary indicated, dsRNAi directed GFP- p125A or expressing control stably treated with cells were HeLa (GalNAcT2-GFP) cell (C) N-acetylgalactosaminyltransferase-2 depleted (asterisk)]. tagged indicates cells arrowhead rescued shown, to are next images [merge analyzed was K 5 ˚ ncnrladp2Adpee el,a niae (dsRNAi, indicated as cells, p125A-depleted and control in C IX L690Er PI-X, PI-X L690E, .6scns hwdfse ieis eas EGFP- Because kinetics. faster showed seconds) 2.56 IX L690E PI-X, t ts a sdt etfrsgiiatdfeecsbtengroups. between differences significant for test to used was -test L690E sidctd h ecnaeo el ihete natGolgi intact either with cells of percentage The indicated. as , eeas eetdb h 15-pcfcatbd but antibody p125A-specific the by detected also were A eg mgssoigtelclzto fendogenous of localization the showing images Merge (A) i o sebea eie ie,i ol not could it sites, defined at assemble not did (T K 5 .8scns)adEGFP-p125A and seconds.) 2.98 IX L690E PI-X, PI-X ˚ .Telclzto fhSec31a of localization The C. a nlzda reduced at analyzed was splmnaymaterial (supplementary L690E 6 ...Three s.d.). (supplementary m m. K PI-X for i.SC oisS,2.Wl yeadp125A and type Wild material S1,S2). (supplementary Movies EGFP-Sec16A S3C; Fig. within engulfed were ftVVGfo h R(ue eouinSMimages, SIM resolution Analysis (super S3-S5). exit Movies ER to and leads S3B the that Fig. shift material from supplementary temperature tsVSV-G a containing upon virus evident of structures membranes were stomatitis these tsVSV-G ER (vesicular containing on by structures Venus-tsVSV-G 1999). Bud marked al., protein glycoprotein). et and ERES, were cargo Tani 2005; sites the of 6B al., Fig. et large Shimoi enlargement shown) The 2000; not (mRFP-p125A, al., EGFP-p125A, uniform et and (Mizoguchi S3A demonstrated marked Fig. experiments material a supplementary in previously to used overexpression as such hours, led of if Overexpression 16 ERES. examine from p125A to to displacement Sec16 hours expression to 12 24 lead displaces would the to from far, prolonged actively so p125A presented therefore p125A of We that time ERES. ERGIC hypothesized or from ERES Sec16A We Sec16A at 3,4). blocks from (Figs traffic segregated temperature-induced nucleation during ERES ERES in stage late p125A-Sec23-Sec31-coated a at functions p125A rsneo ii-idn ouei 15 otosthe PI4KIII of controls Depletion the localization. p125A Sec16A Therefore, control in layers. module COPII-ERES. from COPII lipid-binding Sec16A of a both displacement of with presence similarly interact (mRFP- inactivated was we module 2002), lipid-binding direct p125A to in al., the proteins binding p125A overexpressed which et PI4P whether analyzed therefore use binding. (Supek We COPII-membrane stabilizing might thus liposomes displacement, p125A Sec16A to that hypothesized binding COPII shown). (not analyzed was was large Sec16A localization endogenous by Similar to shown when as S3C). observed size, adjacent Fig. in nm material found (supplementary 1500 to SIM 500 was from varied Sec16A that sites rounded Occasionally, row). versus middle top 6B, Fig. EGFP-Sec16A-mRFP-p125A; with YFP-Sec23-mRFP- p125A (compare EGFP-Sec16A low lacking ERES large clearly at p125A-induced contrast, were and By expressed 3,4). Sec31 EGFP- (Figs When Sec16A, ERES at with whether sites. Sec23 colocalized analyzed these largely We mRFP-125A from levels, shown). YFP- displaced not co-expressed is by the and or Sec16A analyzed 6B Sec31 as Indeed, (Fig. endogenous COPII, Sec23 of of S3B). colocalization layers Fig. both the collected Endo-H material ERES remained enlarged (using it conditions, (supplementary inhibited Golgi somewhat expression was functional p125A cis/medial traffic although extreme that the showed these at under arrival digestion), tsVSV-G of eyhg xrsinlvl splmnaymtra i.S3A). Fig. p125A material (supplementary levels Overexpressed expression high very eetv nSc6dslcmn.I akdcnrs omRFP- to contrast p125A marked mRFP- In displacement. p125A, Sec16 in defective ie fetvl olce GPSc6 Fg B.Analysis 6B). p125A (Fig. that evident demonstrated EGFP-Sec16A these SIM occasionally collected Importantly, using S3A). now effectively Fig. were material sites supplementary sites 6B; enlarged (Fig. but cytosolic, 15 eiec tEE ebae orglt COPII regulate to membranes ERES that at controls assembly. suggest module residency SAM–DDHD results the p125A The by recognition localization. at lipid turnover selective stable enhanced prevented that suggesting ERES shown), (not temperatures h eut ugs htP4 o ako)cnrglt 15 to p125A regulate can of) lack (or PI4P that suggest results The in lipids acidic for requirements the negates Sec16p As IX L690E PI-X, ora fCl cec 21)17 7517 doi:10.1242/jcs.138784 1765–1778 127, (2014) Science Cell of Journal rdltd(p125A deleted or ) L690E, IX L690E PI-X, D DDHD eanddsesdee at even dispersed remained L690E, L690E, lormie largely remained also D DDHD D DDHD a idcdsites -induced L690E, skonto known is r also are ) D DDHD

Journal of Cell Science splmnaymtra i.S) hs eut upr model a support results increased 2008). These hSec31a S4). with al., Fig. material colocalization at were et (supplementary where clustered sites, (Farhan numbers perinuclear now demonstrated ERES PI4KIII which previously peripheral Importantly, as hSec31a, whereas reduced, of sites, would redistribution perinuclear depletion to such PI4KIII how led siRNA-mediated asked localization. Sec16A We affect assembly. ERES inhibit ARTICLE RESEARCH B hl nacn T yrlsst upr ae tp,icuigSr xhneb e3o e2,adSr-nue eil ekcntito and constriction retent neck coat vesicle drives Sar1-induced This and layers. Sec23, coat on between Bet3 linking by effective exchange for Sar1 allow including to steps, layers later outer support and to (C). inner hydrolysis fission COPII GTP from enhancing Sec16 while of (B) ERES. displacement at the cascade promotes budding p125A COPII The 8. Fig. the by evidenced further at incubated as cells p125A-depleted 2005) in ERES 10 al., of of disruption loss et to pronounced leads 7E) (Shimoi (Fig. RNAi ERES using p125A of Depletion of membrane- module SAM–DDHD binding displacement the of the contributions Functional regulate assembly. signals COPII during PI4P ERES which from Sec16A in 8) (Fig. ciiyi Rei,w elcdedgnu 15 ihEGFP- with p125A endogenous replaced we p125A p125A exit, to ER functional module in the lipid-binding activity examine SAM–DDHD of To and the disruption 2010). of the membrane al., contribution to et leading of (Ong ER, morphology the inhibition Golgi from kinetic secretion cargo causes soluble 7B). (Fig. depletion mRFP-p125A of p125A form the by RNAi-resistant restored an was of conditions expression these under assembly ERES inset). civda rvosyrpre Oge l,21)(i.7E). Golgi (Fig. intact 2010) the was cell in al., reduction dramatic p125A et mitotic to (Ong led endogenous p125A a reported of Depletion of previously of as depletion suggestive achieved detectable Effective a perhaps missing population. cells compartment, Golgi (4) or and (vesiculated) loosely Golgi region cell; shattered (2) the perinuclear completely throughout the region; (3) dispersed in nuclei; perinuclear located the Golgi the around (1) dispersed to 7D): or (Fig. Morphology localized packed and phenotypes 7D). Golgi distinct (Fig. 7C) four intact (Fig. with morphology heterogeneous localization gpp130 was using GalNAcT2-GFP was quantified morphology using Golgi activities. analyzed traffic steady-state overall for ˚ ,udrcniin hr e1Ai ergtd(i.7 and 7A (Fig. segregated is Sec16A where conditions under C, IX L690E PI-X, eaaye og opooya reporter a as morphology Golgi analyzed We . a elto e ocutrn fSc6 at Sec16A of clustering to led depletion AC olwn nta soito fCPIlyr ihSc6o Rmmrns() h idn fP4 by PI4P of binding the (A), membranes ER on Sec16 with layers COPII of association initial Following (A–C) a depletion niiino e1 ergto rmEE Fg , and 4,6 (Figs Golgi maintain functional to ERES required into to morphology. activities translated traffic and from steady-state also in S1) S3), defects segregation of Fig. Fig. material association Sec16 material supplementary reduced of (supplementary to morphological 5), ERES inhibition robust (Fig. lipid- with to assembly SAM–DDHD led p125A the ERES which of in p125A, activity in defects of the reduction module in a results binding defects these to Overall, that and 7F,G). suggest (Fig. morphology compartments Golgi Golgi shattered intact of restoration )t tblz ebaebnig(upeetr aeilFig. material (supplementary 1, binding (Figs membrane module recognition that stabilize lipid protein to a a uses p125A, 2) layers, which COPII in both cascade links a identified We activities ERES. budding vesicle at control mediating proteins in COPII-interacting sufficient are biogenesis, subunits core COPII Although DISCUSSION idn.Adfndfnto fSMdmisi protein is domains SAM of membrane function in involved defined is A domains, which p125A, DDHD of binding. and terminus C SAM the contains that suggested studies ERES Previous module lipid-binding at SAM–DDHD activity The and nucleation molecular a COPII 8). of signaling directs (Fig. progression al., the lipid that et control Nagaya 2003) as a cascade 2008; al., al., et such support et Pathre Farhan activities 2002; results 2006; al., regulatory The et which (Blumental-Perry 5,7). to (Figs in leading assembly S2–S4), model Fig. ERES Sec16A material from supplementary functional COPII and of segregation 3,4,6 spatial (Figs the promotes and S1) GPp2A(i.7-)rvre hs fet,namely EGFP- of effects, expression contrast, intact By p125A restoring 7F,G). these and (Fig. of morphology populations form Golgi reversed Golgi resistant shattered RNAi the 7E-G) an eliminating (Fig. of expression as EGFP-p125A The 7F,G). specific (Fig. was morphology cells effect shattered RNAi-treated control in with increase compared when concomitant a and population IX L690E PI-X, ora fCl cec 21)17 7517 doi:10.1242/jcs.138784 1765–1778 127, (2014) Science Cell of Journal a nfetv,laigt nyapartial a only to leading ineffective, was 1775 ion

Journal of Cell Science ciiymyb hrdi te ii rcsigezmsthat enzymes processing DAGK lipid In other transport. in vesicular This 2). in shared (Fig. function be analysis DDHD overlay may the blot by lipid activity by recognition suggested lipid as for domain, avidity the enhances domain etrdb iperpaeeto h DDdmi iha with domain partially DDHD was the S3), of replacement Fig. simple material by supplementary deleted restored mutant) or and (PI-X) (L690E mutated disabled 5,6 is (Figs is domain DDHD interface lipid-binding dimer (4) EGFP–DDHD the and SAM 2); (3) the is (Fig. which when membranes activity, 2005); organizing lost chimera Golgi and al., targeting PI4P-rich ERES resulting mediated et to p125A targeted (Shimoi the is ERES domain and at not module localizes were domains, the lipid-recognition replace domains DDHD can p125B–SAM–DDHD p125A these 2012). and al., of et SAM (Inoue defined contributions its individual using the to although PI4P localizes al., that et p125A recognizes shows Shimoi of member p125A 2012; Golgi, family al., full-length a 2). et p125B, (Fig. (1) (2) (Inoue be 2005); module: recognition might PS PIP this PI4P for for that specificity and target suggest evidence primary PA of a lines PIs, several However, monophosphorylated recognizes agtdt IPrc og ebae Fg ) uain nthe in Mutations 2). (Fig. (DDHD was domain membranes EGFP–DDHD Golgi isolation, and PI4P-rich in to cells targeted in lipid-recognition expressed When form domain. to ERES. at function hubs By processing may 2007). SAM-DAGK al., between SAM-p125A et interactions supports heterodimeric (Raaijmakers further analogy, Arap3 domain Ship2-SAM with The heterodimerization 2010). the recognition al., of membrane et for domain ERES (Nakatsu required SAM similarly clathrin-mediated be of regulates The may which endocytosis, 2002). inhibition Ship2, al., for 5-phosphatase domain et inositol PH required (Nagaya this undefined of is are specificities domains lipid-binding although PH assembly and SAM activities lipid-binding ute blse h agtn fEGFP–SAM–DDHD of targeting the abolished further hniswl-yevrin oevr eeino h DDHD the of deletion higher Moreover, yields at version. (p125A bacteria domain wild-type in produced its was than and cells in aggregate ntfrp2Ata euae OI seby(i.5). recognition. (Fig. lipid assembly the COPII in define regulates recognition that lipid cluster to p125A a for provides PI-X needed unit module SAM–DDHD basic are the studies the Together, structural of DDHD Future contributions the S3). carrying Fig. proteins to similarly ERES dispersed ls ooo,teSMdmi fDAGK of is its domain SAM with common SAM of in the that, activity showed homolog, to assembly we close First, used basic p125A. substrates. the is in and functional suggest which complexes findings assembled 2005), Our between Bowie, avidity and increase (Qiao oligomerization ARTICLE RESEARCH 1776 (p125A mutations the DDHD by destabilize recognition not lipid did abolished also module SAM–DDHD shown), not domain, eurdt upr RSognzto n ciiy(is5-7; (Figs activity S3). mutation and and S1 we organization Figs point is material Third, ERES p125A supplementary single within support 2). interface (Fig. to assembly a oligomerization required SAM the of abolished that site demonstrated introduction the and within conserved is 00,tep2ASMdmi lgmrzdwe on to bound when oligomerized domain Zn p125A-SAM the 2010), ii eonto otlkl eie ntep125A-DDHD the in resides likely most recognition Lipid nvitro In eas 15-A akdmmrn targeting membrane lacked p125A-SAM Because 2+ eod edmntae httebscasml interface assembly basic the that demonstrated we Second, . nlsssget htteSMDH module SAM–DDHD the that suggests analysis PI-X L690E, IX L690E PI-X, ,wihpeetdGlibnigi el (and cells in binding Golgi prevented which ), nvitro in D nvitro in DDHD is56adsplmnaymaterial supplementary and 5,6 Figs , PI-X blse ebaebnigand binding membrane abolished ) Fg ) hs uain probably mutations These 2). (Fig. efvramdli hc this which in model a favor we , eas h rti i not did protein the because d ouecontaining module a , d Kih tal., et (Knight nvivo in d PI-X PI-X and or h eut ugs htp2AcnueP4 olclz n regulate and ERES. localize at to activities PI4P use COPII can p125A that Collectively, suggest 2005). 2002; results al., et the al., (Litvak Nir-2 and et 2010) al., (Nakajima et Yamashita p125B (PI4P- supplementary including Golgi in and proteins, found rich)-targeted are 6 domains (Fig. DDHD (6) ERES S3,S4); Fig. material from Sec16A of displacement eeino h DDdmi np2Ao siRNA-mediated or p125A in domain DDHD PI4KIII the (5) 6); of Fig. deletion (Fapp1-PH, domain PI4P-binding well-characterized uehmtpclyi h iiiyo RbdstsadPI4P and sites 2006; bud al., et ER (Blumental-Perry sites of these at vicinity rather initiated but the is Golgi formation in the with homotypically fuse not yeast, fuse Unlike do membranes. vesicles Golgi COPII with mammalian vesicles COPII of fusion on Subsequent PI4P (Lorente-Rodrı 2000). Golgi of al., inactivation sequestration PIK1 prolonged et that to 2002). failed shown (Audhya Stt4 al., have and studies traffic PIK1 et kinases ER-to-Golgi (Supek PI4 is major affect membranes yeast dependency the synthetic such of Depletion However, on yeast activities. only in two PI4P observed first the the particular provided between in Sec16p, by link and abrogated is lipids which assembly, acidic COPII on dependency regulation ERES A in p125A of Role niiino ucinlculn ewe OI aesis layers COPII between determined. Sec16p be which coupling to by yet mechanism has functional a reversed thus, of COPII, of yeast; evolution in inhibition the COPII lacking in addition is of late which a segregation is p125A the how Sec16A. determined from regulates be to binding remains It p125A-membrane cascade. 6 signals assembly lipid the (Fig. employing control reactions thus to S3), these Fig. in material uses module supplementary and and stabilize binding 7A,B) to lipid (Fig. required SAM–DDHD Sec16A its is from p125A segregation layers. following ERES COPII dissociation facilitate both Sec16 to linking promoting mechanism budding, while a COPII PI4KIII provide with of may interacts progression layers, by the which COPII 6; the p125A, or of (Fig. S4). both S3) p125A Fig. slowed of material Fig. module (supplementary is lipid-binding material traffic the also supplementary of is yet inhibited is inactivation displacement is conversely active, and by Sec16A p125A of 4). displacement is overexpression (Fig. by enhanced low exit ERGIC enhanced At at arrested ER from subsequently 2009). This displaced when al., is et maintained it ERES. (Hughes exit, state ER p125A-controlled removed steady inhibit proper slightly that at establishes is temperatures sites it Sec16A bud as Indeed, layers. from coat its the yet between membranes from linkage ER segregated on with physically nucleation COPII the and is of cascade initiates budding Sec16 Sec16 a explain 8). with roles in (Fig. steps interactions to sequential supportive COPII represent each model model, PI4P-p125A and this working In inhibitory Sec16. plausible contradictory a seemingly component provide vital a is al., is results it et release, for linkage vesicle (Fromme dispensable enhances budding is only functional vesicle Sec16p of Whereas However, 2002; completion 2007). al., hinder 2012). the et for Supek Sato, interactions required 2012; al., and et Sec16p-COPII Sec31- Yorimitsu (Kung inhibiting activity thus 2008). GAP layers, stimulated COPII al., between linkage functional et (Farhan PI4KIII ER-localized the and 2008). al., et Farhan e1Ai eurdt ulaenwEE,adbt Sec16A both and ERES, new nucleate to required is Sec16A a ora fCl cec 21)17 7517 doi:10.1242/jcs.138784 1765–1778 127, (2014) Science Cell of Journal elto,wihrdcsP4 ees ohihbtthe inhibit both levels, PI4P reduces which depletion, ´ uzadBroe 01,ytihbto sexerted is inhibition yet 2011), Barlowe, and guez nvivo in a r eurdt ananteesites these maintain to required are oihbtE oGlitraffic Golgi to ER inhibit do nvitro in udn and budding a nvivo in nvitro in depletion Our . or

Journal of Cell Science yoo eepeae sdsrbdpeiul Rw ta. 96.His 1996). al., et (Rowe previously described as prepared were liver cytosol rat and microsomes ER NRK-derived with concentrations. protocols, DNA manufacturers’ optimized to according reagents, Sciences) (Life 2000 S1. Table p125A-Fapp1-PH material supplementary and in domains detailed is p125A chimera selected mutants, p125A expressing constructs a p125A, mRFP-tagged and Life where EGFP- of pEGFP-C1 construction and The modified frame. Pharmacy a of into University ligated unique (Tokyo using Japan) Tani, Tokyo, Science, Katsuko kindly by pFlag-CMW-6c-p125A, Lippincot- from provided Jennifer excised by was p125A provided (NIH). kindly Schwartz was YFP-p58 Spain). Barcelona, Pierce. from Pittsburgh, were University, antibodies HRP- Invitrogen. secondary Mellon Science, from conjugated were were (Carnegie Life antibodies secondary antibodies Linstedt Alexa-conjugated and Golgi-specific PA). Adam Mouse Pharmacy All by used. of were used. provided University Japan) provided (Tokyo Tokyo, (Sec16L) were Hachioji, Tani KIAA03100 anti Katsuko Roche) rabbit by and p125A (040905270001, anti HRP- monoclonal and anti-His Abcam) (ab3416-250, conjugated antibodies anti-GST HRP-conjugated a67,Acm,GP(360 nirgn,PI4KIII Invitrogen), (332600, Sigma-Aldrich), GFP F1804, BD Abcam), (M2, (ab6276, (612350, Flag anti-Sec31A Laboratories), Sciences), Transduction Life Enzo ERGIC53 ALX-804-602, Polysciences), (24240, (G1/93, GFP Abgent), AP114511b, Lab; Bethyl EERHARTICLE RESEARCH xrsinwr olce rmtreidpneteprmns(30 experiments independent three from EGFP collected for positive S2. were fields individual Table 10 expression material morphology, supplementary Golgi of in analysis described For is p125A of Knockdown analysis knockdown-replacement protocol. p125A purification GST bulk standard a Science) using Life cleaved Healthcare thrombin (GE 4B and GS-Sepharose on purified were proteins incubation standard a and under International) Source (37 (Cellgro) (Serum environment (FBS) penicillin-streptomycin serum bovine 5% fetal modified with 10% Dulbecco’s supplemented to Fisher-Scientific) in up (HyClone sub-confluence (DMEM) at media maintained Eagle’s were cells HeLa METHODS AND MATERIALS 0m rsHl(H80,10m al MET,1m PMSF HEPES mM mM [50 1 buffer EDTA, HNE mM with 1 washes three NaCl, (pH with by mM followed times 100 OG, 2% three 8.0), Ni-NTA- and (pH washed on Tris-HCl were mM loaded beads 50 were Protein-bound (Qiagen). proteins (MP Agarose Solubilized by Sarcosine n-Octyl- USA). supernatants homogenized 2% Biotechnology, and N-Lauroyl further with centrifugation supplemented were by 10% removed were that were 1995). lysates debris Cell to sonication. al., added was et Biomedicals) (Aridor previously .) on rtiswr ltdwt N ufrcnann 0 mM SAM 500 and containing SAM GST-tagged buffer 1993). HNE Neel, with and (pH eluted (Frangioni imidazole imidazole were mM 25 proteins with Bound supplemented 7.4). buffer HNE with washes three agdSr 7GadT9 rtiswr uiida described His as 1995). purified Balch, and were Rowe proteins 1995; T39N al., et and (Aridor H79G previously Sar1 tagged PG(. M o or t37 at hours 4 for with mM) induced (0.1 was (Invitrogen) IPTG 21DE3 BL in expression SAM–DDHD and ujce opyilgclregula steps physiological identified to subjected The (Supek 2002). lipids acidic al., lack et yet Sec16p of COPII contain functionality in that liposomes documented for on the defects COPII to support similar presenting animals, sufficient these relatively mainly in provide activities are 2011), may syndrome mice Sec16 al., Waardenburg p125A-KO spermiogenesis. et However, of (Arimitsu 2010). development unaffected al., the et in (McGary gene in migration causative cell and crest 7 S3) (Fig. neural defects Fig. traffic material general to supplementary leads p125A of overexpression rnfcinwscridotuigEfcee(ign rLipofectamine or (Qiagen) Effectene using out carried was Transfection (GRC, Malhotra Vivek by provided kindly was EGFP-Sec16A h hsooia oeo 15 sukon ct elto or depletion Acute unknown. is p125A of role physiological The 5 .) 0 MNC,1m MgCl mM 1 NaCl, mM 300 7.4), ˚ ,5 CO 5% C, 2 .Atbde gis 15 (MSTP053, p125A against Antibodies ). Xenopus ˚ in hc a o edefined. be now can which tion, n el eelsda described as lysed were cells and C 15 elto nefrswith interferes depletion p125A . Hin nEE sebyaeprobably are assembly ERES in b IIand dIII Dguoyaoie(G (Gold (OG) -D-glucopyranoside 2 . MET,2 G and OG] 2% EGTA, mM 0.5 , andwaspredictedtobea Hin Sma IIst a de in- added was site dIII etito ie and sites restriction I a Cl Signaling), (Cell 6 -DDHD b -Actin L690E 6 - efre sn irsf xe 2010. Homoscedastic Excel Microsoft Corporation). using performed visually (Microsoft were Student’s 2010 field analyzed two-tailed Excel the were Data in Windows expression. cells p125A using All and morphology condition). Golgi each for scored for total in images 10 nietimnfursec a are u sdsrbdpreviously described as out carried was immunofluorescence Indirect Immunofluorescence same the in hours. 2 for 3% incubated with 1 and supplemented with EMD) supplemented Tween-20) V, buffer 1% Fraction 8.0), [Tris- (BSA, buffer pH BSA TBS-Tween in (TBS, hour saline 1 for buffered blocked were (Echelon) Strips PIP blot-overlay Lipid Scientific) (Fisher 15 7.4) in at (pH incubated incubated were HEPES were cells mM the hours 20 and 14–16 with for supplemented FP-proteins media expressing cells HeLa analysis Temperature-block R1K987t ...Dpstdi M o ees fe 2months. 12 after release (NIH) for Health PMC of in Institutes Deposited National M.A.]. the to by [R01DK092807 supported was study The Funding authors. manuscript all the from wrote comments and and project editing various the input, interpreted directed with and and performed conceived designed, M.A. M.A. experiments. and S.C.W. K.S., K.R.L., D.K., contributions Author interests. financial competing no declare authors The interests Competing reagents. Adam Bethesda, valuable Spain), (NIH, for Lippincott-Schwartz Barcelona, MD) Jennifer (GRC, and PA), Malhotra E. Pittsburgh, Vivek William (CMU, CA), Linstedt Japan), Jolla, University, (Tokyo La COPII Tagaya (TSRI, Mitsuo Balch measure and Tani Katsuko to thank We used 2003). al., and Acknowledgements et (Pathre levels, previously HSP70 described as and microsomes verified to concentration, Sec23 recruitment protein The 2005). similar for RLC. al., adjusted for from et immunoprecipitation were by (Bielli supernatants depleted resulting Sar1 previously were and described SNX9 (RLC) or as p125A cytosol performed liver were rat proteins using assays microsomes ER recruitment or LUV-COPII LUVs to recruitment Coat GST-SAM or GST-SAM Zn Olympus) Fluoview (IX-81 Olympus microscope an inverted on an 60 acquired using and were system Images confocal 1995). 1000 al., et (Aridor ie n rcse o analysis. for processed and fixed im(evleSinii) codn omnfcues protocols. manufacturers’ to X-Ray according CL Scientific), HyBlot and (Denville Scientific) film (Thermo Substrate Duration Extended (6 ufrcnann 0m rsHl(H75 n 0 MNC,and NaCl, mM 100 using (Invitrogen). visualized and Kit were 7.5) Proteins Staining above. SilverQuest (pH as determined Tris-HCl was mM polymerization 50 containing buffer esuigCoaseBu tiig o hobncevdproteins, thrombin-cleaved For staining. Blue SAM SAM, Coomassie SDS-PAGE using on visualized were gels fractions 18,000 pellet and at Supernatant centrifugation (Sorvall). by estimated was ori B-we upeetdwt %BAadHRP-conjugated and BSA His 3% anti with supplemented murine TBS-Tween in hour 1 (6 washed were f20 of V0AWV 20.31 OypsCroain n Adobe and Corporation) (Olympus 10.0.1). Version: Photoshop 02.00.03.10 (Adobe CS3 Photoshop V. FV10-ASW 6 2+ m i. nTSTenadvsaie sn ueSga etDura West SuperSignal using visualized and TBS-Tween in min.) 5 n5 MTi-C (pH Tris-HCl mM 50 in M ae oyeiainassay polymerization based m 6 Zn(OAc) M ora fCl cec 21)17 7517 doi:10.1242/jcs.138784 1765–1778 127, (2014) Science Cell of Journal A14 LPNojcie mgswr rcse using processed were Images objective. PLAPON 1.42 NA L690E 6 6 iueicbtos nTSTenadicbtdfor incubated and TBS-Tween in incubations) minute 5 rGTatbde.Sbeunl,srp eewashed were strips Subsequently, antibodies. GST or n Zn(OAc) and 2 ntesm ufrwsaddadpolymerization and added was buffer same the in t tsso h ecnaeo natGliwere Golgi intact of percentage the on -tests L690E m /lo S rHis or GST of g/ml 2 eedltdt ia ocnrto of concentration final a to diluted were 5 bt t045m)wr nuae in incubated were mM) 0.455 at (both .) 0 MNC.A qa volume equal An NaCl. mM 100 7.5), ˚ Cor10 ˚ o or.Smlswere Samples hours. 4 for C g sn oldmicrofuge cooled a using 6 agdpoen.Strips proteins. tagged 1777

Journal of Cell Science og .R,Ymmt,Y,Bkr .L,Wtis .C,Cye .B,Conway, B., C. Coyne, C., S. Watkins, L., A. S. Baker, Y., Lev, Yamamoto, R., and K. Long, H. Sabanay, S., Ramachandran, N., Dahan, V., R. Litvak, Schekman, and M. Ravazzola, E., Futai, S., Hamamoto, L., Orci, C., M. Lee, ose,R,Wis . imran . enu,E . eism,F., Verissimo, G., E. Reynaud, J. T., Goldberg, Zimmermann, and M., X. Weiss, Bi, P. R., H. D., Forster, Hauri, J. and Mancias, J. S., R. Fath, Kaufman, K., Tani, M., Weiss, A. H., O. Weisz, Farhan, C., S. Watkins, M., K. Weixel, J., C. Haney, A., Blumental-Perry, Aridor, and I. S. Bannykh, C., S. Watkins, G., Gabreski, J., D. C. Haney, S. A., Bielli, Emr, and M. Foti, A., K., Audhya, Sekimizu, H., Hamamoto, K., Nakao, T., Baba, T., Kogure, N., Arimitsu, Lippincott- H., T. Roberts, J., Weissman, S., Bannykh, N., K. E. Fish, W. M., Balch, Aridor, and C. Nuoffer, E. S., Bannykh, W. J., Balch, Weissman, and M., Aridor, T. Rowe, I., S. Bannykh, M., R. Schekman, Aridor, and L. Orci, S., Hamamoto, D., Madden, B., Antonny, ug .F,Pgn,S,Fti . ’ragl,J . uhnn . Dittmar, R., Buchanan, G., J. D’Arcangelo, E., R. Futai, S., Schekman, Pagant, F., and L. Kung, M. J. Herrmann, J., M. Kuehn, N., Arimitsu, M., Aridor, B., M. O’Brien, K., Nakamoto, A., Shiga, T., Iinuma, non,B n cemn R. Schekman, and B. Antonny, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.138784/-/DC1 online available material Supplementary material Supplementary ARTICLE RESEARCH 1778 C., Noakes, J., Mantell, J., K. Palmer, W., K., Schmidt, Eyaid, A., Budnik, M., H., Al-Balwi, Hughes, S., Hamamoto, M., Ravazzola, C., J. G. Fromme, B. Neel, and V. J. Frangioni, ngt .J,Juet .K,Pokwk,M . rpt . igr,M., Gingery, J., Kropat, L., M. Plotkowski, K., M. Joubert, J., M. Knight, M. Tagaya, T., Watanabe, A., Takemori, R., Ohtsuki, S., Sato, T., Baba, H., Inoue, n Rexport. ER M. and Aridor, and F. J. protein function. Nir2 secretory the by Golgi apparatus for Golgi critical the in is level diacylglycerol the of Maintenance the vesicle. completes COPII and curvature a membrane of initiates fission helix N-terminal Sar1p (2005). unvro OI uuisa igeE xtsites. exit ER single at R. subunits Pepperkok, COPII of and turnover J. D. cage. Stephens, COPII the in proteins coat of organization in increases chronic and acute to load. sites cargo exit reticulum endoplasmic of Adaptation export. ER regulates sites M. Aridor, and fission. vesicle COPII control to 171 deformation membrane promotes and M. growth, cell secretion, in dynamics. Pik1p, membrane and organelle Stt4p 4-kinases, phosphatidylinositol al. et spermiogenesis. for M. required Tagaya, 585 is R., (Sec23ip) Taguchi, protein H., Sec23-interacting Nakanishi, A., Yamamoto, assembly. site export E. reticulum Biol. W. endoplasmic Cell with J. Balch, selection cargo and biosynthetic ER. J. the from Schwartz, export during machinery budding Biol. COPII Cell the J. by to selection reticulum Cargo endoplasmic in coats vesicle COPI transport. and Golgi COPII between coupling analogues. stable and GTP with coat 531-537. COPII the of Dynamics e2padSc6 oprt orglt h T yl fteCPIcoat. COPII the al. of et cycle L. GTP E. the Snapp, regulate S., to Hamamoto, J. cooperate R., EMBO Sec16p Rothstein, and J., R. Sec24p Reid, C., vesicles. J. transport ER-derived into sorting 391 protein direct interactions 9676. reticulum. endoplasmic the K. from Tani, traffic membrane and M. Tagaya, fhmnE-xtsts eurmnsfrtelclsto fSc6t transitional to Sec16 of localisation the for al. ER. requirements et P. sites: Watson, ER-exit P., human Verkade, of A., D. Carter, A., Johnson, assembly. coat COPII into insight provides L. disease Cell Orci, craniofacial and a R. of Schekman, P., basis Cosson, A., S. Boyadjiev, proteins. fusion (pGEX) S-transferase Biochem. glutathione active enzymatically coach. COPII omto yteSMdmi fdaygyeo kinase diacylglycerol of domain U. SAM J. the Bowie, by and formation S. S. Merchant, F., Sakane, KIAA0725p. A1 phospholipase K. Tani, and 17639. 20) euaino a1N2triu yGPbnigadhydrolysis and binding GTP by terminus NH2 Sar1 of Regulation (2005). 919-924. , 2171-2176. , 187-190. , .Cl Sci. Cell J. 13 623-634. , 31 210 MOJ. EMBO 152 141 1014-1027. , 21) oe fSMadDH oan nmmainintracellular mammalian in domains DDHD and SAM of Roles (2012). 179-187. , ur pn elBiol. Cell Opin. Curr. .Cl Biol. Cell J. 213-230. , 61-70. , 122 .Cl Biol. Cell J. 20) hshtdlnstl4popaefraina Rexit ER at formation 4-phosphate Phosphatidylinositol (2006). 2924-2934. , 21) a1asml euae ebaeconstriction membrane regulates assembly Sar1 (2010). 27 2043-2054. , e.Cell Dev. Cell 190 20) amla e1/20pasarl in role a plays Sec16/p250 Mammalian (2007). 131 ici.Bohs Acta Biophys. Biochim. 122 20) Repr:pbi rnprainb the by transportation public export: ER (2001). 115-128. , 875-893. , o.Bo.Cell Biol. Mol. 605-617. , 13 19) ouiiainadprfcto of purification and Solubilization (1993). 11 20) h a1GPs coordinates GTPase Sar1 The (2001). a.Cl Biol. Cell Nat. 438-443. , 671-682. , 20) ertr ag euae the regulates cargo Secretory (2006). 20) itntrlsfrteyeast the for roles Distinct (2000). 21) icbnigdie sheet drives binding Zinc (2010). 11 Cell 2673-2689. , ur Biol. Curr. .Bo.Chem. Biol. J. 7 129 d 225-234. , 1823 . Biochemistry 20) tutr and Structure (2007). 1325-1336. , 19) COPII-cargo (1998). 20) Organisation (2009). 930-939. , 20) h genetic The (2007). 19) Sequential (1995). 16 a.Cl Biol. Cell Nat. 173-179. , 21) p125/ (2011). 282 .Cl Biol. Cell J. ESLett. FEBS 49 17632- , (2005). (1998). (2001). (2008). (2012). 9667- , Nature Anal. Dev. 3 , aet,G,Phj,K . tdr . hm .adShka,R. Schekman, and S. Shim, S., Studer, B., K. Pahuja, G., Zanetti, K. Sato, and T. Yorimitsu, T. Sugiura, and S. Oka, N., Suzuki, H., Koga, T., Kumazawa, A., Yamashita, iouh,T,Nkjm,K,Htuaa . aaaa . ar,H P., H. Hauri, M., Nagahama, K., Hatsuzawa, K., Nakajima, T., L. Mizoguchi, Orci, S., Hamamoto, C., M. Lee, N., P. Malkus, H., T. Beilharz, A., E. Miller, A. Helenius, and A. Mezzacasa, Lorente-Rodrı n,Y . ag .L,Lo .S n og W. Hong, and S. L. B., Loo, F. L., Gertler, B. J., Tang, S., Domin, Y. R., Ong, Zoncu, L., Lucast, M., R. Perera, F., M., Nakatsu, Nagahama, H., Arai, J., Aoki, T., Mizoguchi, H., Sonoda, K., Nakajima, H. Kanoh, and J. Y. Jia, I., Wada, H., Nagaya, ai . iouh,T,Iaas,A,Htuaa .adTgy,M. Tagaya, and K. Hatsuzawa, A., Iwamatsu, T., Mizoguchi, R. K., Schekman, Tani, and L. Orci, S., Hamamoto, T., D. Madden, F., Supek, Gu A., Razvi, P., LaPointe, M., M., S. Aridor, Stagg, G., Gabreski, S., Uesaki, K., Nakamoto, I., Ezawa, W., Shimoi, U. J. Bowie, and F. Qiao, htl,J .adShat,T U. T. Schwartz, and R. J. A. Whittle, O. Weisz, and M. Aridor, C., S. Watkins, A., Blumental-Perry, M., K. Weixel, cay .L,Pr,T . od,J . h,H . alnfr,J .and B. J. Wallingford, J., H. Cha, O., J. S., Woods, J., Hamamoto, T. Park, Y., L., S. K. McGary, Bednarek, M., Amherdt, L., Orci, K., Matsuoka, ao . nu,H,Kgr,T,Tgy,M n ai K. Tani, and M. Tagaya, T., Kogure, H., Inoue, S., Sato, K. Svensson, and J. Saraste, E. W. Balch, and C. Nuoffer, H., Plutner, M., J. McCaffery, M., Aridor, T., Rowe, Z., Zhang, J., Koning, de H., Rehmann, L., Deneubourg, H., J. Raaijmakers, S., Alber, J., C. Haney, A., Bielli, A., Blumental-Perry, K., Shome, P., Pathre, oe .adBlh .E. W. Balch, and T. Rowe, n h euaino rti otn nmammals. in sorting protein of regulation the and sites. exit ER at formation vesicle 2942. COPII in Sec16 the of in acid D/phosphatidic 1 phospholipase DDHD1. containing of of domain activation DDHD involvement by Possible lysophosphatidylinositol (DDHD1): of Generation (2010). 347-361. oe amla e2pitrcigprotein. Sec23p-interacting Commun. mammalian novel K. Tani, and M. vesicles. Tagaya, transport into proteins membrane diverse of Cell capture ensure Sec24p R. Schekman, glycoprotein. and VSV tsO45 of secretion the in control quality for 6549. ome .adD ail,P. dynamics. pit Camilli, clathrin-coated De endocytic regulates and D. Toomre, p125. protein, 11329-11335. Sec23p-interacting mammalian K. a to Tani, homology and M. Tagaya, domains. PH and SAM its 302-316. via traffic ER-to-Golgi suppresses 15i oe amla e2pitrcigpoenwt tutrlsimilarity structural with protein proteins. Sec23p-interacting phospholipid-modifying mammalian to novel a is p125 vesicles. transport bud to proteins COPII Biol. of action the potentiates Sec16p organization. their K. in Tani, involved and and Chem. sites Biol. M. exit J. reticulum Tagaya, endoplasmic M., in localized Nagahama, A., Yamamoto, lmn,atmlt o sebyo h OI eil coat. vesicle COPII the of assembly for template a element, regulated 4-kinases. 4-phosphate phosphatidylinositol phosphatidylinositol by of populations Golgi Distinct (2005). oestruhotooosphenotypes. orthologous through models liposomes. M. defined E. Marcotte, chemically and proteins coat T. Cell purified Yeung, with and reconstituted R. Schekman, compartments. Golgi with vesicles COPII 216-229. of fusion in PI(4)P ftemmainSc3Sc1CPIsbope ofcltt ER-Golgi facilitate to subcomplex COPII Sec13/Sec31 mammalian transport. the of n ac,W E. assembly. W. Balch, and the to apparatus Golgi the cells. mammalian from in trafficking membrane plasma membrane regulates KIAA0725p transport. Golgi reticulum to ER endoplasmic in operating mammalian from derived COPI. recruit vesicles microsomes COPII (1996). domain-dependent SAM a L. in J. SHIP2 phosphatase manner. Bos, PI(3,4,5)P3 and the with C. interacts Erneux, S., ER Krugmann, and assembly COPII support to required is Sar1p GTPase export. small the M. Aridor, by and D G. Romero, C., S. Watkins, ehd Enzymol. Methods 114 93 158 263-275. , ora fCl cec 21)17 7517 doi:10.1242/jcs.138784 1765–1778 127, (2014) Science Cell of Journal MOJ. EMBO 497-509. , el Signal. Cell. 1029-1038. , .Cl Biol. Cell J. 279 Cell ge,A n alw,C. Barlowe, and A. ´guez, 144-149. , 280 134 21) ytmtcdsoeyo oovoshmndisease human nonobvious of discovery Systematic (2010). 22 10141-10148. , 474-484. , 20) tutrlbssfrcrorglto fCPIcoat COPII of regulation cargo for basis Structural (2008). 257 4059-4069. , 20) utpecrobnigstso h OI subunit COPII the on sites binding cargo Multiple (2003). 19 ici.Bohs Acta Biophys. Biochim. 190 49-53. , 1249-1257. , 20) h ayfcso SAM. of faces many The (2005). 19) xrsinadprfcto fmmainSarl. mammalian of purification and Expression (1995). 20) eemnto ffntoa ein fp2,a p125, of regions functional of Determination (2000). 331-345. , .Cl Biol. Cell J. 21) nihsit tutrladrgltr roles regulatory and structural into Insights (2012). 20) oe hshlps 1wt sequence with A1 phospholipase novel A (2002). 19) itiuino h nemdaeelements intermediate the of Distribution (1991). 20) h rniinlE eie boundary a defines ER transitional The (2002). .Bo.Chem. Biol. J. 21) tutr fteSc3Sc6edge Sec13-Sec16 the of Structure (2010). 21) h nstl5popaaeSHIP2 5-phosphatase inositol The (2010). .Cl Sci. Cell J. .Bo.Chem. Biol. J. 19) OI-otdvsceformation vesicle COPII-coated (1998). 135 kn . otr .S,Crahr B. Carragher, S., C. Potter, C., rkan, ¨ 21) eurmn o Golgi-localized for Requirement (2011). ESLett. FEBS rc al cd c.USA Sci. Acad. Natl. Proc. 895-911. , 20) ciaino phospholipase of Activation (2003). 20) icllcrlkns delta kinase Diacylglycerol (2002). 20) h IKefco Arap3 effector PI3K The (2007). 1801 100 280 .Cl Biol. Cell J. 21) 15 xssa part as exists p125A (2010). a.Cl Biol. Cell Nat. 415-430. , 274 711-720. , ice.Bohs Res. Biophys. Biochem. 584 10501-10508. , o.Bo.Cell Biol. Mol. 20505-20512. , 4389-4395. , 21) Golgi-localized (2010). c.STKE Sci. .Bo.Chem. Biol. J. Traffic o.Bo.Cell Biol. Mol. o.Bo.Cell Biol. Mol. .Cl Biol. Cell J. 190 20) 15is p125 (2005). 14 21) COPII (2011). 307-315. , 20-28. , 3 107 2005 833-849. , 23 2930- , (1999). (2002). 6544- , .Cell J. re7. , 190 277 13 22 , , , ,

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