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Original citation: Breeze, Emily, Dzimitrowicz, Natasha, Kriechbaumer, Verena, Brooks, Rhiannon, Botchway, Stanley W., Brady, Jacob P., Hawes, Chris, Dixon, Ann, Schnell, Jason R., Fricker, Mark D. and Frigerio, Lorenzo. (2016) A C-terminal amphipathic helix is necessary for the in-vivo tubule-shaping function of plant reticulon. Proceedings of the National Academy of Sciences of the United States of America, 113 (39). pp. 10902-10907. Permanent WRAP URL: http://wrap.warwick.ac.uk/81523 Copyright and reuse: The Warwick Research Archive Portal (WRAP) makes this work by researchers of the University of Warwick available open access under the following conditions. Copyright © and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners. To the extent reasonable and practicable the material made available in WRAP has been checked for eligibility before being made available. Copies of full items can be used for personal research or study, educational, or not-for-profit purposes without prior permission or charge. Provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way. Publisher’s statement: http://dx.doi.org/10.1073/pnas.1605434113 A note on versions: The version presented here may differ from the published version or, version of record, if you wish to cite this item you are advised to consult the publisher’s version. Please see the ‘permanent WRAP URL’ above for details on accessing the published version and note that access may require a subscription. For more information, please contact the WRAP Team at: wrap@warwick.ac.uk

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A C-terminal amphipathic helix is necessary for the in vivotubule-shapingfunctionofaplantreticulon

EmilyBreeze1,NatashaDzimitrowicz1,VerenaKriechbaumer4,RhiannonBrooks2,StanleyW.Botchway3,JacobP.Brady5,ChrisHawes4,AnnDixon2,JasonR.Schnell5,

MarkD.Fricker6andLorenzoFrigerio1,*

1SchoolofLifeSciencesand2DepartmentofChemistry,UniversityofWarwick,CoventryCV47AL,UnitedKingdom3CentralLaserFacility,ScienceandTechnologyFacilitiesCouncil(STFC)RutherfordAppletonLaboratory,ResearchComplexatHarwell,DidcotOX110QX,UnitedKingdom4PlantCellBiology,BiologicalandMedicalSciences,OxfordBrookesUniversity,OxfordOX30BP,UnitedKingdom5DepartmentofBiochemistry,UniversityofOxford,OxfordOX13QU,UnitedKingdom6DepartmentofPlantSciences,UniversityofOxford,OxfordOX13RB,UnitedKingdom*correspondingauthorRunningtitle:NovelstructuralfeaturesofaplantreticulonKeywords:Amphipathichelix/endoplasmicreticulum/reticulon/plant

Charactercount:

Classification:BiologicalSciences;CellBiologyAbstract

Reticulons(RTN)areaclassofendoplasmicreticulum(ER)membraneproteinsthat are capable ofmaintaining highmembrane curvature, thus helping shapetheERmembraneintotubules.ThemechanismofactionofRTNsishypothesisedtobeacombinationofwedging,resultingfromthetransmembranetopologyoftheir conserved reticulon homology domain, and scaffolding, arising from theabilityofRTNstoformlow-mobilityhomo-oligomerswithinthemembrane.WehavestudiedtheplantRTNisoformRTN13,whichhaspreviouslybeenshowntolocatetoERtubulesandtheedgesofERcisternaeandtoinduceconstrictionsinERtubuleswhenoverexpressed,andhaveidentifiedaregionintheC-terminuscontaining a putative amphipathic helix (APH). Herewe show that deletion of

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thisregionordisruptionofthehydrophobicfaceofthepredictedhelixabolishestheabilityofRTN13toinduceconstrictionsofERtubulesinvivo.Thesemutants,however,stillretaintheirabilitytointeractandformlow-mobilityoligomersintheERmembrane.Hence,ourevidenceindicatesthattheconservedAPHisakeystructuralfeatureforRTN13functioninvivoandweproposethatRTN,likeothermembranemorphogens,relyonAPHsfortheirfunction.SignificanceStatement

To our knowledge, this is the first invivo demonstration that reticulon (RTN)proteins,responsiblefortheshapingandmaintenanceofERmembranetubules,rely on a highly conserved C-terminal amphipathic helix (APH) for theirmorphogenic function. Previously it was thought that RTN could bend the ERmembranebothbyassumingawedge-like topologyandby formingoligomericarcs. We show here that deleting or mutating the APH region abolishes thefunction of a plant RTN, but does not affect its capacity to oligomerise. Thisindicates that proteins of the RTN family use an APH to affect membranecurvature – a mechanism that is shared by several other membrane-shapingproteinfamilies.

Introduction

Asthegatewaytothesecretorypathway,theendoplasmicreticulum(ER)isresponsibleforsecretoryproteintranslocation,foldingandqualitycontrol,andisthuscentraltothemaintenance of cellular homeostasis (1). In plant cells, the ER consists of the nuclearenvelope and a dynamic peripheral network of cisternae and, more predominantly,tubules extending throughout the cytoplasm and across cell boundaries throughplasmodesmata. Several proteins have been implicated in the shaping of the ERmembrane. In plants these include ROOT HAIR DEFECTIVE3 (RHD3), which isorthologous to mammalian atlastins and yeast Sey1p and is likely important for theformationofthree-wayjunctions(2,3),andtheproteinsofthereticulon(RTN)family.The RTNs are preferentially associated with ER tubules and the curved edges of

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cisternae. When overexpressed in planta, RTNs induce severe constrictions of ERtubulesandareabletoconvertERmembranesheetsintotubules(4-6).

ThemechanismbywhichRTNsgenerateand/orstabilizecurvatureofamembranehasbeen attributed to the reticulon homology domain (RHD)— a conserved domain of~200aminoacidscontainingtwolonghydrophobicregionsflankingahydrophilicloop.These two hydrophobic regions can each be further subdivided into twotransmembrane domains (TMDs), resulting in a ‘W’ – like topology. The RHD is alsofoundintheDP1(deletedinpolyposis)familyofproteinsthatincludesYop1pinyeastand human REEPs (receptor expression-enhancing proteins). The four hydrophobicTMDsof theplantRHDareproposed to formwedge-likehairpins in the lipidbilayer,which, in combination with the RHD-mediated oligomerisation of RTNs into low-mobility oligomers, appear to be sufficient to induce membrane curvature (5-9). Wehave previously shown that shortening of three or all four TMDs of the Arabidopsisreticulon RTNLB13 (Reticulon-like protein B13; henceforth referred to as RTN13)resultsintheproteinlosingitsabilitytoinducetubules,formoligomers,andlocalisetohigh-curvaturemembranes(5,6).Interestingly,Zureketal.(10)observedasimilarlossofmembrane-shapingfunctionandtubulepartitioningthroughextendingthelengthofTMDsofhumanRTN4.Theseresultsindicatethatthepreciselengthofthehydrophobichairpins is critical forRTN functionality, possibly by allowing the formation of homo-oligomers(7).

Recently,Bradyetal.(11)usedsolutionNMRoflipid-anddetergent-containingmicellesof recombinant Yop1p to gain structural insights in this class of ER tubule-shapingproteins.Theyidentifiedanovelstructuralmotif,anamphipathichelix(APH)inYop1p.ThisAPHis located in theC-terminalregionof theprotein,near theC-terminalendofthe fourth TMD, and appears to be highly conserved across all RTN family proteins.Notably,anisolatedpeptideoftheYop1pAPHwasseentointeractstronglywithanionicmembranes and, while the native Yop1p protein can induce tubule formation whenreconstitutedintopolarlipidsinvitro,atruncatedversionofYop1plackingtheAPHwasunable to do so (9, 11). C-terminal cytosolic APHs have also been identified andcharacterizedinothermembraneshapingproteins,includingatlastin,inwhichtheAPHwasshown to facilitate thedestabilizationof the lipidbilayer toaidmembrane fusionandthree-wayjunctionformation(12,13).

WehaveidentifiedsuchanAPHintheC-terminalregionofRTN13,oneoftheshortestRTNisoformsinArabidopsisandourmodelproteinfortheplantRTNfamily(4-6).Here

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weshowthatthispredictedAPH,whilenotrequiredforoligomerformation,isessentialforthemembrane-shapingfunctionofRTN13invivo.DeletionoftheAPHordisruptionof itshydrophobic face results ina lossof tubule-formingactivity invivo butdoesnotaffecttheabilityoftheproteintoformhomotypicinteractions,ortolocalizetotheER.ResultsandDiscussion

Arabidopsishas21predictedRTNproteins,ofwhichRTN13isoneofthesmallest(206aminoacids), consistingof theRHDplusshortN-andC-terminalextensions.Wehavepreviously shown that RTN13 is localized to the tubular cortical ER and, whenectopicallyexpressed,iscapableofinducingconstrictionsinERtubulesandtoconvertERsheets intotubules,asdemonstratedforother, longerRTNisoforms(4-6).Assuch,RTN13canbeconsideredtobetheminimalfunctionalplantRTNprotein,andisthusausefulmodelforstudyingtherelationshipbetweenthestructuralmotifsandtopologyofRTNs,andtheirERmorphogenicproperties.

ToinvestigateifanAPHakintotheoneidentifiedinYop1p(11)wasconservedinplantRTNs, analysisof theaminoacid sequenceofRTN13wasperformed.This identifiedaregion of high helical hydrophobic moment (residues E160-K175; Fig 1A-C) which,when plotted as a function of its hydrophobicity, localised to the region occupied bymembranesurface-seekingproteins(14)(Fig.1D).AswithYop1p,theAPHisC-terminalto the fourpredictedTMDs(Fig.1,A-B).Theamphipathiccharacterof thisregion isahighly conserved feature across many eukaryotic RTN family proteins (Fig. 1D and(11)).

Thesecondarystructureofasynthetic16aminoacidpeptidederivedfromthisputativeAPHsequence(residues160-175)wasinvestigatedinincreasingconcentrationsofthezwitterionic detergent n-dodecylphosphocholine (DPC) using circular dichroism (CD)spectroscopy.Thepeptidewasreadilysolubleinaqueousbufferbutadoptedarandomcoil secondary structure as evidenced by the single negative maximum in the CDspectrumjustbelow200nm(Fig2,dashedline).AdditionofincreasingconcentrationsofDPCfrom10-200mMleadtoasteadyreductioninthepeakcorrespondingtorandomcoil secondary structure, and the emergence of negative maxima centred atapproximately208 and222nm, characteristic ofα-helical secondary structure.ThesedataindicatethatthisregionofRTN13hasapropensitytoassociatewithDPCmicelles,used here as a rough model for phosphatidylcholine membranes, and that thisassociation initiatesα-helix formation, thus supporting the presence of amembrane-

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associatedAPH.

To establish if the presence of the predicted APH is required in vivo for the ERmembrane remodeling phenotype induced by overexpression of RTN13, we firstgeneratedC-terminal truncationsofRTN13, taggedat theN-terminuswithYFP(4),byinsertingastopcodonimmediatelypriortothepredictedAPH(Δ160-206;Fig.1E),andbyexcisingthesequenceencodingthepredictedAPH(ΔAPH;Fig.1E).

When co-expressed transiently inNicotianabenthamiana epidermal cellswith the ERluminalmarker,GFP-HDEL, allmutants retained their ability to localize to tubularER(Fig. 3). Overexpression of full length YFP-RTN13 caused the typical, markedconstriction of ER tubules (Fig 3A), as previously reported. Tubule constrictionsdisplace the luminalmarker to discrete, punctate regions of the ER network, therebyresulting in a significant reduction in the degree of colocalisation of GFP-HDEL withYFP-RTN13(Fig.3E).DeletionoftheAPHplusC-terminalregion(Δ160-206)andoftheregionencodingtheAPHalone(ΔAPH)abolishedthisphenotype(Fig.3B,3D)suchthatthe luminalmarker GFP-HDEL colocalisedwith the tagged RTN13 throughout the ERnetwork(Fig.3E),indicatingthatthesemutants,whilecorrectlylocatingtotheER,areincapableofinducingconstrictions.

TofurtherinvestigateiftheputativeAPHalonewassolelyresponsiblefortheprotein’stubule forming capacity, an isoleucine residue in the centre of the predictedhydrophobic face of the APHwasmutated to a lysine residue (I165K). ThismutationintroducesapositivechargewithinthehydrophobicregionoftheAPH,whichdecreasesthemagnitudeof thehydrophobicmomentbya third relative towild-type, effectivelyabolishingamphipathicity in thehelix (Fig.1C).TheER tubule constrictionphenotypewasnotobservedupontransientexpressionofthisconstruct(Fig.3C).Thesteady-stateproteinexpressionlevelsofΔAPHwerecomparabletofulllengthYFP-RTN13,whereasI165KandΔ160-206mutantproteinexpressionwasslightly lower(SupplementalFig.1A).InfiltrationwithserialdilutionsofAgrobacteriumtumefaciens(OD600)carryingfulllengthYFP-RTN13,whichresultedinprogressivelyloweramountsofdetectableprotein(SupplementalFig.1B),didnotaffecttheappearanceoftheERconstrictionphenotypeevenatthelowestagrobacterialtitre(SupplementalFig.1C).Thus,weconcludethatthisphenotypeisunlikelytobeafunctionofsubtledifferencesintheproteinconcentrationsachievedwithinourstandardagroinfiltrationprocedure.

Taken together, these data indicate that the presence of an intact C-terminal APH is

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necessaryforthemembrane-shapingactivityofRTN13invivo.

InadditiontothetubuleconstrictionphenotypeshowninFig.3,weattemptedtoassesstheeffectof full-lengthRTN13andof theAPHmutantsonthediameterofERtubules.WeimagedtheERlumen(labelledbyGFP-HDEL)inthepresenceofeitherfull-lengthormutated YFP-RTN13, using the Airyscan detector of a Zeiss LSM 880 confocalmicroscope,whichisabletoachievealateralresolutionofaround140nm.Wecollectedimagesfrom10expressingcellsperconstruct(representativeimagesareshowninFig.4A, left-hand panels), and subjected each image to network analysis. The tubular ERnetwork was automatically extracted to give a single-pixel-wide skeleton. The localimage intensity normal to the skeleton was integrated to provide an estimate of therelativeamountofGFP-HDELpresentforeverypointalongeachtubule.Eachintegratedintensityprofilewasthenscannedtodetectintensitypeaks,correspondingtobulgesinthe tubules, and intensity troughs, corresponding to constriction sites (Fig. 4A, right-hand panels). Although the tubules are at or below the resolution limit, the relativeintensitiescanbeusedasaproxy for thewidthof theER tubules,with theadditionalassumptionthatGFP-HDELisevenlydistributedintheERlumen.

WethusestimatedthewidthofthetubulesinunperturbedER(GFP-HDELalone)andinthe presence of full-length or mutated YFP-RTN13. Our analysis showed thatconstrictionscanberegularlydetectedeveninunperturbedERtubules(Fig.4A,bottompanel; Fig. 4B).However,weobserved that, in thepresenceof full-lengthYFP-RTN13,the mean tubule width and the width at the constriction sites were significantlynarrower than inunperturbedER (Fig.4B). In contrast, in thepresenceof theRTN13APHmutants, themeanwidthof theER tubuleswasnot significantlydifferent to thatobserved in the unperturbed network. These data indicate that ectopic expression ofRTN13 negatively affects thewidth of ER tubules and that this property requires thepresenceofanAPH.

Since the RTN proteins are known to form both hetero- and homo-oligomers, whichcreate immobile, arc-like scaffolds in the tubularERmembrane, it is possible that theremoval ormutation of the APH affects the ability of RTN13 to oligomerise. Previouswork indicated that altering the length of the TMDs within the RHD abolished RTNfunction, but this was concomitant with a loss of the protein’s capacity to homo-oligomerise(5).TotestwhetherourAPHmutantshadsimilarlylosttheabilitytoformlowmobilityhomo-oligomersand/ortointeractwithwild-typeRTN13,weusedsucrosesedimentation velocity gradients, fluorescence recovery after photobleaching (FRAP)

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analysis and Förster resonance energy transfer (FRET), measured directly byfluorescencelifetimeimaging(FLIM).

We isolated microsomes from agroinfiltrated N. benthamiana leaves, solubilized themembraneswith1%digitoninandresolvedtheextractsonacontinuous5-30%sucrosegradient,withcomparisonagainstknownmolecularweightmarkerproteins.FractionswereanalysedbySDS-PAGEandimmunoblottedwithantiGFPantiserum.Fig.5showsthat both full length YFP-RTN13 and the APH mutants form high molecular weightoligomericcomplexesthathavecomparablesedimentationproperties,peakingaroundamolecularmassof~200-250kD.This indicates that thesecomplexesmaycontain4-5monomers,which iscomparable to thatobserved foryeastandmammalianreticulons(7).

FRAP of a region of the tubular ER expressing YFP-RTN13 appears relatively slow incomparisontonon-membrane-shapingERmembraneproteins,asobservedpreviously(5, 7) (Supplemental Fig. 2). In comparison, our previously described mutant ∆TM4,wherethe4TMDsofRTN13wereeachshortenedtoalengthof17residues(5)showsmarkedlyhighermobility(SupplementalFig.2Band(4)),withaFRAPprofilewhichissignificantlydifferenttothatobservedinfulllengthRTN13(Chi-squaredtestfortrend;p-value0.001;SupplementalFig.2E). Incontrast,both theYFP-RTN13mutant lackingthe C-terminal region (∆160-206; Supplemental Fig. 2C), and the I165K mutant(SupplementalFig.2D),displayedrecoverykinetics thatarecomparablewith thoseoffull length (p-value0.181and0.245, respectively).Thesedata indicate thatmutationsaffecting the putative APH regions do not affect the protein’s capacity to form homo-oligomers.

WealsousedFRET-FLIManalysistotestwhethertheabovemutantswerestillabletointeractwith full lengthRTN13.YFP(donor)was fusedto themutantRTN13proteinsandmCherry (mCh; acceptor) to the full lengthRTN13 sequence.Donor and acceptorconstructswereco-infiltratedintoN.benthamianaepidermalcellsandFRET-FLIMusedtoassessprotein-proteininteractions.ThelifetimevaluesforeachRTN13pairisshownin Table 1. Supplemental Fig. 3 shows fluorescence lifetimes of 35S:YFP:RTN13expressedaloneorincombinationwith35S:mCh:RTN13,asrepresentativeexamplesoftheresultsobtainedfromFRET-FLIManalyses.

FRET-FLIMhasbeenusedextensively inplantatoconfirmprotein-proteininteractionsidentified through a range of other techniques (6, 15). A reduction in the lifetime of

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donorfluorescencebyaslittleas0.2nsinthepresenceofanacceptorfluorophorehasbeenshowntobeindicativeofadirectinteractionbetweentwoproteinssuchthattheyareincloseenoughproximity(1-10nm)forquenchingtooccur(15-17).Here,allofthecombinationstesteddisplayedatleasta0.35nsdecreaseinlifetimewhenexpressedasa donor/acceptor pair compared with expression of the donor alone (Table 1;SupplementalFig.3).Hence, removalof theAPHordisruptionof itshydrophobic facedoesnotaffecttheabilityoftheproteintohomo-oligomeriseortointeractwiththefulllengthRTN13isoform.ThisobservationisinagreementwithpreviousstudiesofRTN13and other RTN interactions, where a truncated version of RTN13 lacking the two C-terminalTMDswasstillshowntoformhomotypicinteractions(6).

While theroleofAPHs insculptingmembraneshasbeenwelldocumented forseveralclasses of proteins including Sar1, epsin, endophilin and ArfGAP1 ((18, 19) andreferences therein), the apparent absence of an APH in proteins of the RTN familysuggestedthat theirmembrane-sculptingactionarose fromacombinationofwedging,due to theirmembrane topology, and scaffolding, resulting from their ability to formimmobileoligomers(7,20).Indeed,alteringthelengthoftheTMD,bothbyshorteningand extension, resulted in a loss of function. Altering TMD length, however, alsoimpactedontheabilityoftheproteintooligomerise,makingitimpossibletoassesstherelativecontributionofthetwomechanismsdescribedabove.Ourdataindicatethatthenewlyidentified,conservedAPHattheCterminusofRTN13isnecessaryforitsabilitytoshapeERtubules invivo.Lossof theAPHormutations thatdisrupt itsamphipathicnature do not affect RTN13’s ability to form oligomers but abolish its capacity toconstrictERtubules.Previously,Shibataetal.(7)showedthatmutantsofScRTN1pwithdefects inoligomerizationwerestillableto induceERtubule formation. Inthe lightoftheresultspresentedhere,wenowsuggestthatthisislikelyduetotheC-terminalAPHregion remainingunaffected in thosemutants. In conclusion,while it is likely that theRHDalone issufficient toguaranteereticulonoligomerisation, itsmainrolemaybe toprovide an environment for the correct insertion and interaction of the crucialmembrane-shapingAPH.

MaterialsandMethods

In silico analysis of RTN13. A consensus prediction of the membrane topology ofAtRTN13wasperformedusingTOPCONS(21)withfurtherrefinementofthepositionof

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the TMDs based on sequence alignments with the human RTNs and experimentalevidence described previously (5, 11). The predicted topology of RTN13was plottedusingtheTEXtopopackageinLaTeX(22).Basedonthealignment,aputative16aminoacid APH in the region C-terminal to the TMD was identified in AtRTN13 using theHELIQUEST web server algorithm (23). The membrane-seeking properties of theAtRTN13APHandthecorrespondingregion in250sequencesobtainedfromaBLASTsearchagainstthefullAtRTN13sequencewereevaluatedasdescribedbyEisenbergetal.(24).

GenerationofRTN13constructs.A complete list of all primersused in this study isgiven in Supplemental Table 1. All PCRs were performed using high fidelity DNApolymerase(Accuzyme,Bioline).ThecodingregionofRTN13wasclonedandfusedtoeYFPaspreviouslydescribed (4), andused to generate aYFP:RTN13Gateway®Entryclone in pDONRZeo (ThermoFisher Scientific). A point mutation in the entry clone(I165K)wascreated throughsitedirectedmutagenesisPCRwithoverlapping forwardand reverse primers containing the appropriate sequence mismatch, followed bydigestionofthemethylatedtemplatewith10UDpnI (NewEnglandBiolabs) for1hat37oC, and transformation into DH5α chemically competent E. coli cells. A mutatedversion of RTN13 inwhich the APH alonewas excised (∆APH)was similarly createdusing overlapping primers homologous to the sequence before and after the desireddeletion, in a two stepprocess (deletion of E160-L169, followedbydeletion ofG170-K175).ThenativeormutatedYFP:RTN13inpDONRZeowassubsequentlyusedasaPCRtemplate to create an ampliconwithXbaIandSacI recognition sites,whichwere thendigested(XbaIandSacI,NewEnglandBiolabs),gelpurified(QIAquickGelExtractionkit,Qiagen) and ligated (T4 DNA ligase, ThermoFisher Scientific) into the correspondingsitesinpVKH18-EN6expressionvector(25).AC-terminaltruncationofRTN13inwhicha stop codonwas inserted afterE159 (RTN13∆160-206)was similarly generated. ForFRET-FLIManalysis,amCherry:RTN13fusionwascreatedbyfusionPCRinwhichPCRfragments(10-50ngeach)ofmCherrywitha3’overhangtoRTN13,andRTN13witha5’overhangtomCherry,weremixedandPCRamplified.

Transientexpressionanalysis.AllRTN13constructsweretransformedbyheat-shockinto Agrobacterium tumefaciens strain GV3101 and were transiently expressed inNicotiana benthamiana leaf epidermal cells by agroinfiltration at an OD600 of 0.5, aspreviously described (26). In addition, serial dilutions of Agrobacterium tumefaciens

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carrying the 35S:YFP:RTN13 construct were made from OD600 0.5-0.05 foragroinfiltration.

Proteinresolutionanddetection

TotalproteinswereisolatedfrominfiltratedN.benthamianaleavesbyhomogenizinginaproteinextractionbuffer(0.2MNaCl,1mMEDTA,2%2-mercaptoethanol,0.2%TritonX-100, 0.1M Tris-HCl pH 7.8, complete protease inhibitor cocktail tablet [RocheDiagnostics Ltd]). For sucrose gradient analysis, ER microsomes were isolated frominfiltrated leaves as described (27). Microsomal pellets were resuspended in 100µlTKMGlysisbuffer(50mMTris-HCl,pH7.0,150mMKCl,2mMMgCl2,10%glycerol,1mMEDTA,1mMPMSF,1mM4-(2-aminoethyl)benzenesulfonylfluoridehydrochloride)containing 1%digitonin. Solubilized lysatewas centrifuged for 10min at 12,000 g toseparate out any remaining leaf debris. Microsomes were loaded onto continuous 5-30%(w/v)sucrosegradientsandultracentrifugedat166,000gfor4h30’at25oCinaBeckmanSW55Tirotor.TwentygradientfractionswerecollectedandsubjectedtoSDS-PAGEon15%acrylamidegels.500µgeachofferritin(440kDa),aldolase(158kDa)andovalbumin (44 kDa) (GE Healthcare) were used as molecular weight standards, andstainedwith Coomassie Brilliant Blue R-250 following electrophoresis. ProteinsweretransferredontoPVDFmembranesandsubjectedtoimmunoblottingwithanti-GFP-HRPantisera (Miltenyi Biotec Ltd.). Immunoblots were developed by enhancedchemiluminescence(ECL;Promega)usinga ImageQuantLAS4000 imagingsystem(GEHealthcare).

Confocalmicroscopy.Freshlyexcisedleafsamplesweremountedinwaterandimagedon a LeicaTCS SP5 confocalmicroscopewith a 63x oil immersionobjective lens. GFPand eYFPwere excited at 488nm and514nm, respectively. GFPwas detected in the494-513nmrangeandeYFPinthe524-554range.SimultaneousdetectionofGFPandYFP was performed by combining the settings indicated above using the sequentialscanning facility of the microscope, as instructed by the manufacturer. For FRAPanalysis,sampleswereimagedwithaZeissLSM880equippedwitha63xoilimmersionobjective.YFPwasexcitedat514nmanddetectedinthe520to560nmrange.ForERnetworkanalysis,sampleswereimagedusinga100X/1.46NAoil immersionobjectiveonaZeissLSM880equippedwithanAiryscandetector. Imageswereacquiredusingadefinedregionofinterestwithanaverageof4with2048x2048pixelsofimagesizeand8bit imagedepth, takingcare thateverypartofeach imageremained fullywithin thedynamicrangeofpixelintensity.

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Colocalisationanalysis.ColocalisationanalysiswascompletedusingtheImageJ-basedopensourceprocessingpackageFijiv1.49r(28).Firstamedianfilter(radius=0.5pixels)wasusedtoreducesaltandpeppernoiseandarollingballfilter(radius=20pixels)wasusedtoreducethebackgroundnoiseineachimage.TheRGBimagewasthensplitinto8-bitredandgreenchannelimages,andanROIforeachimagewasselected.Saturationlevelwassetat0.1%tonormalisetheimages.Minimumpixelintensitywassetto0foreachchannel.TomeasurethePearson’sCoefficienttheCoLoc_2pluginwasused(pointspreadfunction=3pxandCostesiterations=50).Aminimumof8images,correspondingto8differentcells,wasusedforeachRTNconstruct.ER network analysis.ER cisternae were automatically segmented from single plane(x,y)imagescollectedwiththeAiryScandetectorbyanimageopeningsteptoremoveallfeatures smaller than maximum tubule diameter (estimated as the full-width half-maximum (FWHM) from amanual transect). This was followed by an active-contoursteptoshrinkthesegmentedregionbacktomatchtheintensityprofileofthecisternae.Toautomatically extract the tubularERnetwork, imageswerebackgroundsubtractedthenrescaledusingcontrast-limitedadaptivehistogramequalisation(CLAHE).TheERtubuleswereenhancedusingaseriesoforientedlogGaborfilters,typicallyappliedover5 spatial scales and six orientations to calculate the local phase-congruency (29-31).Whilstthephase-congruencyfilterscharacteriseanumberofrelevantimageproperties,the local weighted mean phase angle (‘Feature type’) provided the most robustsubsequent segmentation. The tubular networkwas segmented from the feature typeimage using hysteresis thresholding and binary thinning to give a single-pixel wideskeleton. To ensure closely appressed regionswere notmergedduring the hysteresisthreshold,alllocalintensityminimawereidentifiedusinganh-minimumtransformandsettozero.Thewidthof the tubuleswasestimatedby severaldifferentmethods.Toestimate thetubuleFWHM,thepeakheightwasestimatedfromtheintensity,sampledforeachpixelin the skeleton; the distance was estimated from the distance transform of the pixelskeleton; and the50% threshold estimated fromwhere thepixel intensity falls belowhalf thepeak, assuminga localbackgroundof zeroafterbackgroundsubtraction.ThisreturnstheFWHMoftheobjectconvolvedwiththepoint-spreadfunction.Alternatively,a granulometry approach was used whereby the intensity image was subjected to aseries of image openings (erosion followed by dilation) that successively removedstructuresasthesizeoftheopeningkernelexceedstheunderlyingobject.Thisresultsin

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anintermediate(x,y,s)image,wheresincreaseswiththesizeofthedisk-shapedkernel.Theintensityofeachpixelinitiallydecreasesslowlywithsasthekernelsamplesmoreoftheobject,butthenreducesdramaticallyoncetheboundaryoftheobjectisreached,and the kernel only samples the background. The transition point for any pixel isdetermined from the maximum (negative) gradient of the granulometry curve.However, thisapproachconstrains theradius to integerpixelsvalues,andalsosuffersfromdigital approximationof smallkernels toa truedisk shapedkernel.Thus, ratherthanextract a specific size threshold, the integrated intensityunder thegranulometrycurve was calculated to provide a more nuanced interrogation of the local imageintensity profile. The integrated intensity cannot be directly related to the physicalwidth without additional assumptions about the relationship between fluorescenceintensityandsampledvolume.Nevertheless,thisapproachdoeshelpwithestimationofrelative tubulewidths, even if they are sub-resolution objects, provided it is assumedthatthefluorescentprobeisevenlydistributedthroughouttheER,andtheERiswithinthe sampling volume of the confocal defined by the point-spread-function (psf). Theaverageintensityfromthecisternalregionswastakenasameasureofafully-filledpsfwould yield when sampling sheets and tubes, and used to normalise the integratedintensityvaluesbetweenimages.Thepixelskeletonwasconvertedtoagraphrepresentationwithnodesatthejunctions,cisternaeandfreeends,connectedbyedgesalongeachtubule.Bulgesalongeachtubulewereestimatedfrompeaksintheintegratedintensityprofilethatwereatleast10%ofthemaximum intensity and also 5% greater than the signal on either side. Likewise,constrictions were determined using the same peak-finding process on the invertedintensityprofile.AlltheanalysisroutineswereimplementedinMATLAB(TheMathworks,Nantick,MA)andareavailableinastandalonepackageoraMATLABappfromwww.markfricker.org.FRAPanalysis.Forphotobleaching,thetubularERwasmagnifiedusinga10×zoomforcleartubuledetection.Nodrugswereusedandstationaryregionsofthenetworkweretargeted forbleaching. Imageswereacquiredevery0.41s.Threeprebleaching frameswere acquired and a region of interest 5 µm in diameter bleached at 100% laserintensityfor1frame.Afterphotobleaching,46framesweretakenat0.41sintervalsfora total of 21 s. Mean fluorescence intensity within the bleached area was measuredduring the recoveryphaseofFRAPexperimentsusingZeissZENsoftware.AnalysisoffluorescencerecoverywasperformedasdescribedbyShibataetal.(2008).Briefly,thefluorescence intensity of three regions of interest was measured: the photobleached

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region (PR), a regionoutsideof the tubularERnetworkprovidingoverallbackgroundfluorescence (BR),anda regionwithin theERnetwork thatwasnotphotobleached tocorrect for overall observational photobleaching and fluorescence variation (CR). Therelativefluorescenceintensity(I)foreachindividualFRAPexperimentwasbackgroundcorrectedandnormalisedusingthefollowingequation:I=[(PRt-BRt)/(PRt0-BRt0)*100]x[(100-CRt/CRt0)/100]+1where t0 values were averaged over the three pre-bleach scans. Normalised data foreachsetofFRAPexperimentswereplottedandaChi-square test for trendperformedusing GraphPad Prism 6.0 to compare the recovery curves of eachmutant to that ofwild-type.FRET-FLIManalysis.Data acquisition for FRET-FLIMwasperformed as describedbySparkesetal.(2010).Datawereanalyzedbyobtainingexcitedstatelifetimevaluesofaregionof intereston thenucleus, and calculationsweremadeusing theSPCImage5.4analysissoftware(BeckerandHickl).ThedistributionoflifetimevalueswithintheROIwere generated and displayed as a curve. Only values that had a Chi-squared scorebetween0.9and1.4wereused.Themedianlifetimevalueandminimumandmaximumvaluesforaquarterofthemedianlifetimevaluesfromthecurveweretakentogeneratethe range of lifetimes per sample. At least three nuclei per RTN combination wereanalyzed, and the average of the ranges taken. Results are from two independentexperiments.

PeptideSynthesisandPurification.ApeptidecorrespondingtoRTN13residues160-175 (EYGDQIQKHLGSLKDK) was synthesized at Insight Biotechnology Limited(Wembley, UK) using F-moc chemistry. The peptide was purified by reversed-phasehigh performance liquid chromatography (HPLC) using a linear acetonitrile gradientfrom30-100% containing 0.1% trifluoroacetic acid (TFA) on a Phenomenex C4 semi-preparative column. The purity of pooled peptide fractions was confirmed byelectrospray ionization time-of-flight mass spectroscopy (ESI-TOF-MS microTOF,Bruker)beforesubsequentlyophilization.Circular Dichroism (CD). CD spectra were measured using a Jasco J-1500spectropolarimeter equippedwith aPeltier thermally controlled cuvetteholder (JascoUK,GreatDunmow,UK)and1.0mmpath-lengthquartzcuvettes(Starna,OptiglassLtd,Hainault,UK).All spectrawere recorded from190 to300nmusinga2.0nmspectralbandwidth,0.1nmstepresolution,100nmmin-1scanningspeed,and1sresponsetime.

14

Thepeptidewaspreparedin25mMsodiumphosphatebuffer(pH7.0)inthepresenceandabsenceofthezwitterionicdetergentn-dodecylphosphocholine(DPC,AvantiPolarLipids, USA) at increasing concentrations ranging from 10-200 mM, maintaining aconstantpeptideconcentrationof94µM.CDspectrashownwerecollectedat37°C,andwereaveragedfromfourindividualspectraaftersubtractionoftheCDspectrumofthebuffer or buffer/detergent solution (as appropriate). CD data were fitted usingDichoweb(Contin/trainingset7)(32-34)toestimatethesecondarystructurecontent.

Acknowledgements

Thisworkwas fundedby theBBSRCERA-CAPSgrant ‘PERASPERA’ toLFandCH.WearegratefultotheBBSRCforaDTPstudentshiptoND.WethankPhilipYoungforhelpwithFRAPanalysis.

Authorcontributions

EBgenerated theconstructs,performed themajorityof theexperimentsandco-wrotethe manuscript; ND performed colocalization analysis; VK performed Airyscan imageacquisition;RBandADperformedCDanalysis;SBassisted in theproductionofFRET-FLIMdata; JBandJScarriedoutsequencealignmentsandAPHpredictions;CHhelpedwithdataanalysisandmanuscriptpreparation;MDFdevelopedtheERanalysistoolandperformed the analysis. LF conceived the study, performed some of the FRET-FLIMexperimentsandco-wrotethemanuscript.

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260.

FigureLegends

Figure1. RTN13 possesses a putative, conserved C-terminal APH. (A)Schematic topology model of RTN13 in the ER membrane (total length, 206 aminoacids).Thepositionsof the fourTMDsof theRHD(predictedusing theTOPCONSwebserver (21)) are indicated alongside the APH. Residues are coloured by hydropathywhereby red is acidic, blue is basic, yellow is polar uncharged, and green ishydrophobic/nonpolar. Selected amino acids referred to in the text are indicated (B)Protein sequence alignment of the RHD and C-terminal region ofArabidopsisthalianaRTN13, Homo sapiens RTN1-4 and Saccharomyces cerevisiae Yop1p. Residues arecolouredasin(A)withtheexceptionofpolarunchargedaminoacids,colouredorange.Numbering is for RTN13 amino acid positions (C) Helicalwheel plot of the identifiedregionofhighestpredictedhydrophobicmomentofRTN13C-terminaltotheRHDandof the mutated form RTN13 I165K. The direction (line) and size (square) of thehydrophobic moment (µ) of the helices are depicted in pink (µ=0.28 [WT] and 0.18[I165K])(D)PlotofhydrophobicmomentagainsthydrophobicityfortheAPHofRTN13and the same region from250 sequencesobtainedbyaBLAST searchagainst the fullRTN13 sequence. The position of RTN13 is indicated (red star). The majority of thesequenceslieintheregionoftheplotexpectedformembrane-seekinghelices(24).(E)Schematic representation of the constructs used in this study (not to scale). TM,transmembranedomain;APH,putativeamphipathichelix;CT,C-terminalregion.KKSE,ERretentionmotifFigure 2. RTN13 residues 160-175 formanα-helix in the presence of detergent

micelles.CDspectraoftheputativeAPHpeptide(EYGDQIQKHLGSLKDK,residues160-175) dissolved in 25 mM sodium phosphate buffer (pH 7.0, dashed line) as well asincreasing concentrations of buffer-solubilized micelles. The peptide attained amaximumhelicalcontentof31%in200mMDPC(asestimatedbyfittingofthedataasdescribed in Materials andMethods). CD data fitting summary: Helix: 30.6%, Strand:21.3%,Turns:19.6%,disordered:27.8%

18

Figure3. Deletion of theAPHor disruption of its hydrophobic face impairs

RTN13 function. Nicotiana benthamiana epidermal cells were co-infiltrated withAgrobacterium tumefaciens carrying plasmids encoding either full length, mutant ortruncated forms of 35S:YFP:RTN13, together with the soluble ERmarker, GFP-HDEL.Representative confocal images of leaves transfected with (A) 35S:YFP:RTN13 (B)35S:YFP:RTN13Δ160-206 (C) 35S:YFP:RTN13 I165K (D) 35S:YFP:RTN13 ΔAPH. Scalebar=5µm. (E) AveragePearson’s correlation coefficients (PCCs) of cells coexpressingGFP-HDELandfulllength,truncatedormutatedformsof35S:YFP:RTN13,whereaPCCof +1 indicates perfect colocalisation, 0 indicates no colocalisation and -1 indicatesnegativecolocalisation.Errorbarsindicatethestandarderrorofthemean;n=8-9cells.Asterisks indicate a significantdifferencebetween full lengthRTN13and the relevantAPHmutant(Student’st-test;p<0.001).Figure 4. The APH is necessary for the action of RTN13 on the diameter of ERtubules.

(A) Nicotiana benthamiana epidermal cells were co-infiltrated with Agrobacteriumtumefaciens carrying plasmids encoding the indicated constructs. Cells were imagedwithaZeissAiryscandetector(left-handpanels)andsubjectedtoERnetworkanalysis(right-hand panels). (B) comparison of average ER tubule width and constrictionsresulting fromtheexpressionof full lengthYFP-RTN13or theAPHmutants.Asterisksindicatesignificantdifferences(Student’st-test;p<0.001).Figure5. RemovalormutationoftheAPHdoesnotaffectthecapacityofthe

resultingRTN13proteintoformoligomerswithintheERmembrane.

Microsomalmembranes isolated frominfiltratedN.benthamiana leavesexpressingtheindicated constructs were solubilized in 1% digitonin and separated on sucrosesedimentation velocity gradients. Gradient fractions were resolved by SDS-PAGE andgels subjected to immunoblottingwith anti-GFP antiserum.Molecularweightmarkers(kDa) are indicated at the top and on the left. The immunoblot for Δ160-206 isoverexposedduetothelowerproteinlevelsofthismutant(SupplementalFig.1A).

Table1. Fluorescence lifetimes of RTN13 pairs in FRET-FLIM analysis. Fulllength (35S:mCh:RTN13; acceptor) and mutated or truncated forms of RTN13(35S:YFP:RTN13∆160-206 or I165K; donor) were infiltrated alone or in combination

19

intoN.benthamiana leaf epidermal cells and protein-protein interactions assessed byFRET-FLIM analysis. For each measurement, a region of low mobility ER continuouswith the nuclear envelope was selected and the fluorescence lifetime of the donorfluorophore measured. Table shows the range of FLIM lifetimes, mean and standarddeviation (n=3-5 cells) and the p-values from Student’s t-test comparisons ofdonor+acceptor compared to donor alone,which indicate thatmutated and truncatedformsofRTN13arestillabletooligomerise.SupplementalFigure1.(A)ProteinexpressionofYFP-RTN13andAPHmutants.Equalamounts of protein from total protein extracts of agroinfiltratedN.benthamiana leafsectors were subjected to SDS-PAGE and immunoblot with anti GFP antiserum. CBB,Coomassie brilliant blue staining of the post-ECL blot. Numbers at right indicatemolecularweightmarkerinkD.(B)As in A, butwith extracts from leaves agroinfiltratedwith the indicated OD600 ofagrobacteriacarryingplasmidencodingYFP-RTN13.(C)Nicotianabenthamianaepidermalcellswereco-infiltratedwiththe indicatedOD600ofAgrobacteriumtumefaciens carrying plasmids encoding full length 35S:YFP:RTN13 ,togetherwiththesolubleERmarker,GFP-HDEL.Supplemental Figure2.Nicotianabenthamiana epidermal cellswere infiltratedwithAgrobacterium tumefaciens carrying plasmids encoding either full length, mutated ortruncated forms of 35S:YFP:RTN13 and subjected to FRAP analysis. (A-D)Representativepre-bleachingand0,10and20sframesfromtypicalFRAPexperiments.The circled areas represent regions undergoing photobleaching. Scale bar = 5µm (A)35S:YFP:RTN13 (B) 35S:YFP:RTN13ΔTM4 (C) 35S:YFP:RTN13Δ160-206 (D)35S:YFP:RTN13I165K(E)Fluorescenceintensitiesnormalizedtopre-bleachingvaluesplottedovertime.Errorbarsindicatethestandarderror;n=9-14cells.TheresultsofaChi-squaredtestfortrend(df=1)comparingtheFRAPprofileofeachmutanttothatofwild-typeareshown(**p<0.005).Supplemental Figure 3. FRET-FLIM analysis. Fluorescence lifetime data of35S:YFP:RTN13 (donor) expressed alone or in combination with 35S:mCh:RTN13(acceptor), as representative examples of the results obtained from FRET-FLIManalyses.(A)35S:YFP:RTN13alone(B)35S:YFP:RTN13+35S:mCh:RTN13.(i) A region of interest (ROI) on the ER continuous with the nuclear envelope wasselected based on the lifetime decay curves of points within the region having a�2

20

valuebetween0.9and1.4(iv).Pseudocolouredlifetimemaps(ii)depictthelifetimeofpointswithintheROI,andthedistributionoflifetimesacrosstheentireimageisshownin(iii).SupplementalTable1.OligonucleotideprimersusedforthegenerationoftheRTN13constructsinthisstudy.

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CLUSTAL O(1.2.1) multiple sequence alignment HsRTN4 -------------SVVDLLYWRDIK KTGVVFG-ASLFLLLSLTV- FS IVSVTAYIALALLSVTISFRI HsRTN1A -------------KAIDLLYWRDIK QTGIVFG-SFLLLLFSLTQ- FS VVSVVAYLALAALSATISFRI HsRTN2A -------------KVADLLYWKDTR TSGVVFT-GLMVSLLCLLH- FS IVSVAAHLALLLLCGTISLRV HsRTN3-A1 -------------SVHDLIFWRDVK KTGFVFG-TTLIMLLSLAA- FS VISVVSYLILALLSVTISFRI AtRTNLB13 MANDVTKDPTPKSDIVEDIYLWRRK KLAFSTLLVSTSTWILLSF- YG FTTITIVSWIGIAVVSMIFLW ScYop1p -------------ILQQLENKTNLP KSYLVAGLGFAYLLLIFINV GG VGEILSNFAGFVLPAYLSLVA HsRTN4 YKGVIQAIQKSDEGHPFRAYLESEVAISEELVQKYSNSALGHVNCTIKELRRLFLVDDLVD HsRTN1A YKSVLQAVQKTDEGHPFKAYLELEITLSQEQIQKYTDCLQFYVNSTLKELRRLFLVQDLVD HsRTN2A YRKVLQAVHRGDGANPFQAYLDVDLTLTREQTERLSHQITSRVVSAATQLRHFFLVEDLVD HsRTN3-A1 YKSVIQAVQKSEEGHPFKAYLDVDITLSSEAFHNYMNAAMVHINRALKLIIRLFLVEDLVD AtRTNLB13 GSLLRLLSKVEPELSG--------LEVSEEFVVETVRSCRMLMEEMVRWMFRVGAESEWFV ScYop1p LKTPT---------------------------------------------------STDDT HsRTN4 SLKFAVLMWVFTYVGALF NG LTLLILALISLF-SVPVIY E------ RHQAQIDHYLGLAN--- KNVKDAMAKIQAKIPGLKRKAE HsRTN1A SLKFAVLMWLLTYVGALF NG LTLLLMAVVSMF-TLPVVY V------ KHQAQIDQYLGLV-R-- THINAVVAKIQAKIPGAKRHAE HsRTN2A SLKLALLFYILTFVGAIF NG LTLLILGVIGLF-TIPLLY R------ QHQAQIDQYVGLVT--- NQLSHIKAKIRAKIPGTGALASAAAAVSGSKAKAE HsRTN3-A1 SLKLAVFMWLMTYVGAVF NG ITLLILAELLIF-SVPIVY E------ KYKTQIDHYVGIA-R-- DQTKSIVEKIQAKLPGIAKKKAE AtRTNLB13 FARTVLGFWILSRIGNLL DF HTCLFIGLVMGL-TVPKLW E------ EYGDQIQKHLGSL-KDK SKGAYNTTHEKILEMKNKLHHGTEEKVKKSE ScYop1p QLLTYWIVFSFLSVIEFW SK AILYLIPFYWFLKTVFLIY IALPQTG GARMIYQKIVAPL-TDR YILRDVSKTEKDEIRASVNEASKATGASVH HsRTN4 SLKFAVLMWVFTYVGALF NG LTLLILALISLF-SVPVIY E------ RHQAQIDHYLGLAN--- KNVKDAMAKIQAKIPGLKRKAE HsRTN1A SLKFAVLMWLLTYVGALF NG LTLLLMAVVSMF-TLPVVY V------ KHQAQIDQYLGLV-R-- THINAVVAKIQAKIPGAKRHAE HsRTN2A SLKLALLFYILTFVGAIF NG LTLLILGVIGLF-TIPLLY R------ QHQAQIDQYVGLVT--- NQLSHIKAKIRAKIPGTGALASAAAAVSGSKAKAE HsRTN3-A1 SLKLAVFMWLMTYVGAVF NG ITLLILAELLIF-SVPIVY E------ KYKTQIDHYVGIA-R-- DQTKSIVEKIQAKLPGIAKKKAE AtRTNLB13 FARTVLGFWILSRIGNLL DF HTCLFIGLVMGL-TVPKLW E------ EYGDQIQKHLGSL-KDK SKGAYNTTHEKILEMKNKLHHGTEEKVKKSE ScYop1p QLLTYWIVFSFLSVIEFW SK AILYLIPFYWFLKTVFLIY IALPQTG GARMIYQKIVAPL-TDR YILRDVSKTEKDEIRASVNEASKATGASVH

TM3 TM4 APH

130 140 150 160 170 180 190 200

CLUSTAL O(1.2.1) multiple sequence alignment HsRTN4 -------------SVVDLLYWRDIK KTGVVFG-ASLFLLLSLTV- FS IVSVTAYIALALLSVTISFRI HsRTN1A -------------KAIDLLYWRDIK QTGIVFG-SFLLLLFSLTQ- FS VVSVVAYLALAALSATISFRI HsRTN2A -------------KVADLLYWKDTR TSGVVFT-GLMVSLLCLLH- FS IVSVAAHLALLLLCGTISLRV HsRTN3-A1 -------------SVHDLIFWRDVK KTGFVFG-TTLIMLLSLAA- FS VISVVSYLILALLSVTISFRI AtRTNLB13 MANDVTKDPTPKSDIVEDIYLWRRK KLAFSTLLVSTSTWILLSF- YG FTTITIVSWIGIAVVSMIFLW ScYop1p -------------ILQQLENKTNLP KSYLVAGLGFAYLLLIFINV GG VGEILSNFAGFVLPAYLSLVA HsRTN4 YKGVIQAIQKSDEGHPFRAYLESEVAISEELVQKYSNSALGHVNCTIKELRRLFLVDDLVD HsRTN1A YKSVLQAVQKTDEGHPFKAYLELEITLSQEQIQKYTDCLQFYVNSTLKELRRLFLVQDLVD HsRTN2A YRKVLQAVHRGDGANPFQAYLDVDLTLTREQTERLSHQITSRVVSAATQLRHFFLVEDLVD HsRTN3-A1 YKSVIQAVQKSEEGHPFKAYLDVDITLSSEAFHNYMNAAMVHINRALKLIIRLFLVEDLVD AtRTNLB13 GSLLRLLSKVEPELSG--------LEVSEEFVVETVRSCRMLMEEMVRWMFRVGAESEWFV ScYop1p LKTPT---------------------------------------------------STDDT HsRTN4 SLKFAVLMWVFTYVGALF NG LTLLILALISLF-SVPVIY E------ RHQAQIDHYLGLAN--- KNVKDAMAKIQAKIPGLKRKAE HsRTN1A SLKFAVLMWLLTYVGALF NG LTLLLMAVVSMF-TLPVVY V------ KHQAQIDQYLGLV-R-- THINAVVAKIQAKIPGAKRHAE HsRTN2A SLKLALLFYILTFVGAIF NG LTLLILGVIGLF-TIPLLY R------ QHQAQIDQYVGLVT--- NQLSHIKAKIRAKIPGTGALASAAAAVSGSKAKAE HsRTN3-A1 SLKLAVFMWLMTYVGAVF NG ITLLILAELLIF-SVPIVY E------ KYKTQIDHYVGIA-R-- DQTKSIVEKIQAKLPGIAKKKAE AtRTNLB13 FARTVLGFWILSRIGNLL DF HTCLFIGLVMGL-TVPKLW E------ EYGDQIQKHLGSL-KDK SKGAYNTTHEKILEMKNKLHHGTEEKVKKSE ScYop1p QLLTYWIVFSFLSVIEFW SK AILYLIPFYWFLKTVFLIY IALPQTG GARMIYQKIVAPL-TDR YILRDVSKTEKDEIRASVNEASKATGASVH HsRTN4 SLKFAVLMWVFTYVGALF NG LTLLILALISLF-SVPVIY E------ RHQAQIDHYLGLAN--- KNVKDAMAKIQAKIPGLKRKAE HsRTN1A SLKFAVLMWLLTYVGALF NG LTLLLMAVVSMF-TLPVVY V------ KHQAQIDQYLGLV-R-- THINAVVAKIQAKIPGAKRHAE HsRTN2A SLKLALLFYILTFVGAIF NG LTLLILGVIGLF-TIPLLY R------ QHQAQIDQYVGLVT--- NQLSHIKAKIRAKIPGTGALASAAAAVSGSKAKAE HsRTN3-A1 SLKLAVFMWLMTYVGAVF NG ITLLILAELLIF-SVPIVY E------ KYKTQIDHYVGIA-R-- DQTKSIVEKIQAKLPGIAKKKAE AtRTN13 FARTVLGFWILSRIGNLL DF HTCLFIGLVMGL-TVPKLW E------ EYGDQIQKHLGSL-KDK SKGAYNTTHEKILEMKNKLHHGTEEKVKKSE ScYop1p QLLTYWIVFSFLSVIEFW SK AILYLIPFYWFLKTVFLIY IALPQTG GARMIYQKIVAPL-TDR YILRDVSKTEKDEIRASVNEASKATGASVH

No.

of s

eque

nces

∆APH TM2

TM1

TM3

TM4YFP CT KKSE

Figure 2

Peptide: EYGDQIQKHLGSLKDK

YFP-RTN13GFP-HDEL Merge

Full length

I165K

A

B Δ160-206

C

Figure 3

E

ΔAPH

YFP-RTN13GFP-HDEL Merge

D

0.000.100.200.300.400.500.600.700.800.901.00

Pearson'sRvalue

*** *** ***

Full

leng

thI1

65K

∆160

-206

∆APH

GFP-

HDEL

Figure 4

A

B

0

0.05

0.1

0.15

0.2

0.25

GFP-HDEL Fulllength I165K ∆160-206 ∆APH

Meanwidth(microns)

constrictions

average

***

***

Donor Acceptor Range of FLIM Lifetimes (ns)

Mean FLIM Lifetime (ns)

Std Dev (ns)

P value (two-tailed Student's t-test)

YFP:RTN13 - 2.85-3.03 2.96 0.090.0002

YFP:RTN13 mCh:RTN13 2.42-2.61 2.50 0.07

YFP:RTN13∆160-206 - 3.0-3.1 3.03 0.060.0238

YFP:RTN13∆160-206 mCh:RTN13 2.5-2.8 2.68 0.16

YFP:RTN13 I165K - 2.7-3.0 2.82 0.130.0049

YFP:RTN13 I165K mCh:RTN13 2.40-2.56 2.46 0.07

Table 1

440kD158kD44kD

∆160-206

YFP-RTN13

I165K

∆APH

Figure 5

46kD -

46kD -

46kD -

46kD -

OD600 0.5

OD600 0.1

OD600 0.2

OD600 0.05

YFP-RTN13GFP-HDEL Merge

0.5 0.2 0.1 0.05

Supplemental Figure 1

OD600:

CBB

Anti GFP

A B

C

- 46- 30- 23

- 58

- 46- 30- 23

- 58

CBB

Anti GFP

- 46- 30- 23

- 58

- 46- 30- 23

- 58

Full

leng

th

10 s0 spre-bleach

A

20 s

∆TM

4

20 s10 s0 spre-bleach

B

∆160

-206

pre-bleach

C

0 s 10 s 20 s

I165

K

pre-bleach

D

0 s 10 s 20 s

E

Supplemental Figure 2

10.0

20.0

30.0

40.0

50.0

60.0

70.0

0 2 4 6 8 10 12 14 16 18 20 22

Norm

aliz

ed R

elat

ive

Fluo

resc

ent I

nten

sity

(%)

Time (s)

full length∆TM4I165K∆160-206

Chi-square testfor trend χ2 P value

Full length vs I165K 1.351 0.2452Full length vs ∆160-206 1.794 0.1805Full length vs ∆TM4 10.49 0.0012 **

Supplemental Figure 3

2.52.0 3.0 ns

2.52.0 3.0 ns

A

B

i ii

iii

i ii iii

iv

iv

SupplementalTable1. Oligonucleotide primersusedforthegenerationoftheRTN13constructsinthisstudy.

Purpose Primer Name Sequence

attB PCR product for YFP:RTN13YFP:RTN13 LP (Gateway) AAAAAAGCAGGCTTCATGGTGAGCAAGGGCGAGGAGCTG

YFP:RTN13 RP (Gateway) CAAGAAAGCTGGGTCCTACTCTGATTTTTTCACTTTCTCTTCTGTACC

installing full length attB sitesattB1 adaptor GGGGACAAGTTTGTACAAAAAAGCAGGCT

attB2 adaptor GGGGACCACTTTGTACAAGAAAGCTGGGT

XbaI:YFP:RTN13:SacIYFP:RTN13 LP (RE cloning) CAGATATCTAGAATGGTGAGCAAGGGCGAGGAGCTG

YFP:RTN13 RP (RE cloning) CAGATAGAGCTCCTACTCTGATTTTTTCACTTTCTCTTCTGTACC

Sequencing insertspVKH18-En6 LB CCTTCGCAAGACCCTTCCTC

pVKH18-En6 RB CATGCAAGACCGGCAACAG

SDM of YFP:RTN13 pDONRZeo to generate YFP:RTN13 I165K

YFP:RTN13 I165K LP GGAGATCAAAAGCAGAAGCATTTGGGTAGCC

YFP:RTN13 I165K RP GTACTCTTCCCACAACTTTGGAACCGTCAACC

XbaI:YFP:RTN13∆APH:SacI (insertion of STOP codon after residue E159)

YFP:RTN13 LP (RE cloning) CAGATATCTAGAATGGTGAGCAAGGGCGAGGAGCTGYFP:RTN13∆APH+C RP (RE cloning)

CAGATAGAGCTCCTATTCCCACAACTTTGGAACCGTCAACC

Deletion of APH (E160-L169)YFP:RTN13∆APH LP1 CGGTTCCAAAGTTGTGGGAAGGTAGCCTTAAAGATAAATCGAAGG

G

YFP:RTN13∆APH RP1 CCCTTCGATTTATCTTTAAGGCTACCTTCCCACAACTTTGGAACCG

Deletion of APH (G170-K175)YFP:RTN13∆APH LP2 CGGTTCCAAAGTTGTGGGAATCGAAGGGAGCGT

YFP:RTN13∆APH RP2 ACGCTCCCTTCGATTCCCACAACTTTGGAACCG

mCh:RTN13 Fusion PCR. mCherry fragmentmCherry LP (RE cloning) CAGATATCTAGAATGGTGAGCAAGGGCGAGGA

mCherry:RTN13 RP (RE cloning) TGGATCTTTGGTCACGTCGTTGGCCTTGTACAGCTCGTCCATGC

mCh:RTN13 Fusion PCR. RTN13 fragmentmCherry:RTN13 LP (RE cloning) GCATGGACGAGCTGTACAAGGCCAACGACGTGACCAAAGATCCA

YFP:RTN13 RP (RE cloning) CAGATAGAGCTCCTACTCTGATTTTTTCACTTTCTCTTCTGTACC