Peetades eateto ahlg,Sho fMdcn,Uiest of USA. University 48109-0602, Medicine, MI of Arbor, School Ann Pathology, Michigan, of Department address: *Present USA. 90095, CA Angeles, 91343, CA Angeles, Los System, Care USA. Health Angeles Los Greater Administration laVagin Olga A stability toxin remarkable botulinum its by determines proteins protease cytoskeletal septin of Recruitment ARTICLE RESEARCH 3294 2014 May 22 Accepted 2013; November 18 Received ` of 3 inhibition Montal, in 2006; 1 Aoki, resulting and attachment (Dolly proteins, factor exocytosis sensitive neurotransmitter (SNARE) N-ethylmaleimide two) the soluble case, receptor single inside a the chains in therapies. (or, light one of their cleave cosmetic specifically deliver they and and where a cells, neurons disorders of to neurogenic bind treatment BoNTs of long-term for of variety success therapeutically wide the toxins and these severe botulism the using human both of underlies symptoms neuroparalytic which from neurons, release cholinergic transmitter peripheral inhibit (BoNTs) neurotoxins Botulinum INTRODUCTION stability, Protein Ubiquitylation protease, A Degradation, toxin Botulinum Septin, WORDS: KEY proteolytic LCA-related degradation. is activity. of neuroparalytic stabilization LCA presumably co-clustering long-lasting and increased the LCA–septin for by and dileucine-mediated crucial oligomerization septins the interaction with septin Therefore, LCA and clustering prevented of septin-2 and septins of impairment of silencing or and degradation the forchlorfenuron non-proteasomal LCA Similarly, L428A/L429A The and LCA. of LCA. proteasomal of co-clustering in accelerated presence accumulated decreased the which in clusters, mutation only septin-7, mutation clusters and plasma-membrane-associated the these septins-2 LCA in that with wild-type The colocalizing showed septins. concentrated with interactome LCA was of LCA spectrometry interaction Mass the the cells. prevented and neuronal of cultured of in analysis proteolytic effects expressed the stability LCA and studied the localization of the interactome, we the elucidate longevity, on both To mutation LCA L428A/L429A mice. for in shorten mechanism neurotoxin is cellular the LCA significantly of several a in effects in for mutation neuroparalytic to L428A/L429A results persists The (LCA) that neurons. known A motor release in type neurotransmitter months neurotoxin of botulinum blockade of the by chain 25 light protein synaptosomal-associated of cleavage Proteolytic ABSTRACT ail Lewis Ramilla uhrfrcrepnec ([email protected]) correspondence for Author h P-ee nttt,PsrwMs pcrmtyLbrtr,UL,Los UCLA, Laboratory, Spectrometry Mass Pasarow Institute, NPI-Semel The Veterans and UCLA Medicine, of School Physiology, of Department 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,39–38doi:10.1242/jcs.146324 3294–3308 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. 2 eateto ilgclSine,Alra n. rie A962 USA. 92612, CA Irvine, Inc., Allergan Sciences, Biological of Department 1, ` liaTokhtaeva Elmira , 2 ereSachs George , 1 ar Wheeler Larry , 1 atnE Garay E. Patton , 2 oe Aoki Roger , 2 uetSouda Puneet , tblt fLAi ernlcells. neuronal remarkable in the LCA for of crucial are stability we interactions Furthermore, these LCE. identified that with dileucine- demonstrate interact a have not in do we LCA but to manner, LCA, dependent bind versus that mutant LCA proteins L428A/L429A cytoskeleton wild-type membrane the the with and interacting LCE proteins prolongs 2011). of al., mutant, analysis et (Wang L428A/L429A mice its in proteolytically BoNT/E to dileucine-containing of not effects the neuroparalytic but finding to the LCA, LCE of by inactive of supported degradation intracellular is fusion the that dileucine-dependent interpretation that its suggest an both to Such results owing interactions. shorten The impeded mice 2011). is dramatically in LCA al., neurotoxin to the et of (Wang shown effects neuroparalytic been and proteolytic has 2004), lsammrn oaiaino C (Ferna LCA of localization membrane plasma gemn ihtedt bandpeiul nP1 cells in PC12 cells in SiMa previously obtained in active data is the GFP–LCA with that Fig. agreement material indicating (supplementary S1A), cells SiMa GFP–LCA-expressing product cleavage (Ferna LCA-catalyzed the with interact specifically that oTAi ernlclsaepol understood. poorly of are action cells long-lasting neuronal the applications. in govern therapeutic BoNT/A that its mechanisms for the extraordinary treatment advantageous However, The medical is for 1998). but challenge botulism, al., a of et presents (Eleopra BoNT/A clinical weeks of the longevity 4 on BoNT/E than of depending effects less months the last whereas 12 2006), to Aoki, and 3 (Dolly indication from last effects the therapeutic BoNT/A The but of unknown. 2014), BoNT are al., H protein et the type (Dover of The characteristics identified action. A recently their BoNTs, been of has of persistence serotype serotypes in Seven vary G, 2009). through Bouvet, and Popoff 2010; ta. 04.Adul uain 48/49,i dileucine- a in (E L428A/L429A, mutation, motif (Ferna double containing membrane A LCE plasma 2004). shorter-lived al., the the et with contrast, associate In the not of LCA. for does important leaflet the be inner of to the stability suggested with cellular been LCA has membrane of plasma (LCE) Association the BoNT/E 2010). of al., chain et by light (Tsai the followed is ubiquitylation than to degradation susceptible proteasome less is (LCA) BoNT/A a eetdb etr ltaayi nttlcl yae sa as SNAP-25 residues producing acid lysates SNAP-25 amino in C-terminal cell nine that the known total of is cleavage It catalyzes in S1A). LCA Fig. analysis material (supplementary protein blot kDa western 75 by cells detected SiMa non-differentiated cells was in neuronal expressed cultured transiently in LCA GFP–LCA of activity and Localization RESULTS ee ytecmaaietne assetoer (MS/MS) spectrometry mass tandem comparative the by Here, hnepesdi erbatm el,telgtcanof chain light the cells, neuroblastoma in expressed When 2 ´ n se Fernandez-Salas Ester and dzSlse l,21)dtce adblw2 D in kDa 25 below band a detected 2012) al., et ndez-Salas 3 aaBassilian Sara , 424 YL)i C htprilyimpairs partially that LCA in FYKLL) 1 uinP Whitelegge P. Julian , 197 Baie l,19) Antibodies 1993). al., et (Blasi 2, * ´ dzSlse al., et ndez-Salas ´ ndez-Salas 3 ,

Journal of Cell Science h lsammrn Fg B omfed4 i.1,zo field zoom 1C, and Fig. 4; cytoplasm 3; field the zoom field between 1B, distributed (Fig. zoom membrane or 1B, plasma either 4) the (Fig. field was zoom cytoplasm mutant 1C, the Fig. L428A/L429A in the When found cells, in S1E). predominantly LCA SiMa Fig. wild-type in material the expressed (supplementary of fractions detection exclusive membrane in fractions, almost cytosolic cells the to and membrane SH-SY5Y mutant, contrast both in fractionated (r)L428A/L429A detected was recombinant in protein the data, with these transfected with Along 2004). muto lae NP2 y05 n 2 the delivering and by or transfection 1 plasmid by 0.5, in either introduced cells, by LCA neuronal Therefore, S1B). the SNAP-25 Fig. in material cleaved increase (supplementary gradual of the from a amount in seen SNAP-25 differentiated as cleaved reagent manner to delivery dose-dependent protein delivered a using (rLCA) cells PC12 LCA recombinant In shown). addition, not (data SNAP-25 endogenous cleaved cells SH-SY5Y (Ferna ARTICLE RESEARCH rvosy h 48/49 uathdbe on obe (Ferna distributed to cells partially PC-12 found and in cytoplasm membrane been the the throughout had with mutant associated loosely L428A/L429A the Previously, membrane the at of distribution LCA clustered prevents mutation L428A/L429A The the of plane the in located membrane. structures be lateral ring-like might the membranes structures plasma the of rod-like the in the projections along of shapes sections Some rod-like horizontal 1A). and in (Fig. seen cells cell the the of of at plane shapes planes middle ring-like top had and clusters bottom distributed These the evenly 1A). being (Fig. membrane than the rather cells at membrane plasma in the PC12 LCE along and 2010). in LCA al., of et localization (Tsai and similar cells cells a neuronal claiming S1F) SiMa data recent in Fig. GFP–LCE (Ferna material of detection (supplementary with microscopy agreement in S1E), the confocal Fig. By in material containing (supplementary mostly cells process. protein SH-SY5Y strong this detected of fractionation fractionation was is after the fraction (rLCE) cytosolic association during LCE intact recombinant might this by contrast, plasma remain or LCA that to the protein–protein and that by enough to membrane interaction, suggest bind plasma protein–lipid can results the any it with the or associate that domain Therefore, suggests transmembrane membrane. a that Fig. possess sequence not material other does (supplementary rLCA LCA fractions of detection S1E). membrane in in resulted methods primarily different two by reagent) using non- cells PC12 In and Chariot differentiated S1C). into Fig. the 1A,C) introduced material (Fig. was rLCA (supplementary GFP–LCA cells addition, differentiated of SH-SY5Y distribution in both Similar found SiMa 1B). in were (Fig. membrane differentiated plasma that the at aggregates cells small or and clusters in SNAP-25 endogenous the tail. with C-terminal its cleaves interacts protein, mature ifrnitdS-YYclstasetdwt LA(Chariot rLCA showing with transfected data PC12 cells SH-SY5Y in previous differentiated (Ferna GFP–LCA with of localization agreement S1D). cells anti- membrane in Fig. plasma using are punctate material immunofluorescence results (supplementary These by antibodies detected polyhistidine as membrane sdtce ycnoa irsoy F–C accumulated GFP–LCA microscopy, confocal by detected As eakby C a cuuae npthso clusters or patches in accumulated was LCA Remarkably, ´ ´ dzSlse l,20) iial,GPLAepesdin expressed GFP–LCA Similarly, 2004). al., et ndez-Salas dzSlse l,20) hs eut otatwt the with contrast results These 2004). al., et ndez-Salas TM ´ dzSlse l,20) rcinto of Fractionation 2004). al., et ndez-Salas rti eieyraetwslclzdt the to localized was reagent delivery protein ´ dzSlse al., et ndez-Salas m frLCA of g TM eemn hte h cdcrsdei h E the in residue motif, acidic To dileucine the trafficking. whether acidic protein for determine putative SiMa important a be in to 1C) known reside (Fig. (D/E)xxxL(L/I), both L429 differentiated in and observed L428 and wild- cells. were the 1B) mutant of (Fig. L428A/L429A to distribution non-differentiated its in contrast and differences in the The LCA homogeneous, type. type wild and was Importantly, the even membrane of rather the that membrane. in but mutant clustered, found plasma L428A/L429A not was the mutant the of L428A/L429A distribution the at cells, of predominantly minority a In 3). ml grgt oaiaino C ttepam membrane. or plasma clustered the the at for LCA crucial of are L428 localization that that suggesting aggregate acids 6), amino small and the 5 are 1B, fields L429 mutant (Fig. zoom and 1C, membrane E424A Fig. plasma the 5; the field LCA, zoom along wild-type distribution the to mutated clustered Similar we had LCA, alanine. of to localization E424 and trafficking in involved 49 nLAdaaial hresbt rtoyi and proteolytic both neurotoxin L428A/ shortens the mutation of dramatically double effects the LCA neuroparalytic that in shown L429A have studies Recent in LCA of cells stability neuronal decreases mutation L428A/L429A The xrse nSM el,w oprdtesaiiyo h wild- the of stability the LCA compared of we stability cells, the SiMa affects mutation in this expressed whether test To 2011). eetdi el ogrta C H227Y/L428A/L429A cells. LCA SiMa in increases degradation than mutation LCA L428A/L429A of rate longer the growth the that that confirming cell cells cells 2B), was (Fig. of H227Y inhibit in LCA Importantly, enrichment LCA. might no detected and or of LCA growth levels that lower express preferential by suggesting in SNAP-25 2A,B), resulting respective and (Fig. of their LCA counterparts than cleavage L428A/L429A both time active its longer of a and catalytically mutants for LCA inactive persisted wild-type L428A/L429A the the Interestingly, to of mutant. those similar were not to mutants H227Y/L428A/L429A similar and levels look presumably initial were H227Y The of 1C), medium. expression Fig. low-serum they of cells in and incubation their 2C transfection, though to Fig. owing (compare to Even cells L428A/ differentiated the 1B,C). prior of (Fig. that the differentiated to mutant similar in the 3), in field found distributed L429A zoom predominantly or 2C, evenly 2) (Fig. field cytoplasm either zoom 2C, H227Y/ (Fig. was The membrane 1). field mutant zoom wild-type 2C, L428A/L429A the (Fig. aggregates membrane small to plasma or the similar clusters along mutant, in expressing accumulated H227Y predominantly cells The LCA, 2B). in (Fig. assay H227Y/L428A/ L429A and same H227Y mutants, LCA the inactive proteolytically performed we LCA of assay persistence, cell-based this with interfered SNAP-25 endogenous mutation the the that LCA. of suggesting of that stability than 2A), decreases slower (Fig. the was mutant in GFP–LCA LCA decrease L428A/L429A wild-type wild-type The the the similar. of of were amount mutant expression L428A/L429A of the levels and initial the The of after blotting. amount after the lysed and and were transfection, GFP–LCA h wells following 24 periods separate transfection. time after in indicated days medium grown 20 cell to cells was allowed up FBS) Transfected for This medium overgrowth cells. (0.5% without normal SiMa survival To non-differentiated proliferation, low-serum growth. of cell transfection cell by of prolonged rate replaced during the proteins decrease mutated and type oecuetepsiiiyta C-aaye laaeo the of cleavage LCA-catalyzed that possibility the exclude To ora fCl cec 21)17 2430 doi:10.1242/jcs.146324 3294–3308 127, (2014) Science Cell of Journal b atni ellstswr nlzdb western by analyzed were lysates cell in -actin nvivo in 424 YL oi is motif FYKLL Wn tal., et (Wang 3295

Journal of Cell Science EERHARTICLE RESEARCH 3296 ora fCl cec 21)17 2430 doi:10.1242/jcs.146324 3294–3308 127, (2014) Science Cell of Journal mgsaeidctdb numbers. ‘zoom’ by the indicated of are position images The the cells. show of to outlines used was staining F-actin (phalloidin) (C). cells SiMa and differentiated (B) non-differentiated mutation in E424A, both the the not to but owing L428A/L429A, LCA or of clustered distribution of aggregate loss show Confocal images LCA. microscopy of localization on consensus motif of dileucine-based Effect the (B,C) in cell. mutations the the of in section membrane middle plasma the mostly along are seen cell, structures the rod-like of the sections whereas top and in bottom seen the predominantly are The structures membrane. ring-like plasma the at small aggregates or and clusters (arrows) (arrowheads) ring-like rod-like in seen is GFP–LCA. GFP–LCA (WT) wild-type cell the SiMa expressing differentiated a z- of showing sectioning images microscopy membrane. Confocal plasma (A) the at LCA distribution of aggregate small or impairs clustered mutation L248A/L249A 1. Fig.

Journal of Cell Science EERHARTICLE RESEARCH h eobnn ih hisdlvrdt h el yteprotein the by cells the Chariot containing to reagent PC12 delivered transfection chains differentiated light in recombinant confirmed the also was mutation h nraei erdto aeo C yteL428A/L429A the by LCA of rate degradation in increase The TM splmnaymtra i.S2). Fig. material (supplementary C A rH2Y() Student’s (B), H227Y or (A) LCA h essec flgtcan ndfeetae C2clswas cells PC12 SNAP-25 differentiated detecting in by chains determined light of persistence The hw.Fatn(hlodn tiigwsue oso h ulnso cells. of outlines the are show medium to mean low-serum used are a was Results in staining Non- days distribution. (phalloidin) clustered 3 F-actin of for shown. loss incubated residues a cells in alanine resulted SiMa to LCA differentiated L429 of and mutant L428 inactive of of the distribution in mutation aggregate additional or the clustered that the whereas showing change LCA, images not microscopy did Confocal mutation (C) H227Y LCA. the of span life the shortens GFP–LCA by the normalized of signal quantification Densitometry and immunoblotting. GFP–LCA low-serum of in by amount incubation determined cell the of and periods transfection, time following mutants indicated medium the H227Y/L428A/L428A after or lysed the H227Y were or the (B) LCA and LCA. (WT) (A) of wild-type mutant time the L428A/L428A life expressing the cells decreases Non-differentiated mutation (A,B) L428A/L429A The 2. Fig. om,oewudepc uainidcdices nthe in increase mutation-induced ubiquitylated a of formation expect L428A/L429A of would the rate one If the forms, S2). increased Fig. also of that mutation material to compared (supplementary as mutant L428A/L429A with LCE consistent the of are of results life these amount longer Thus, the 150%). the (by to degree but Similarly, greater lactacystin, a LCA, adding lactacystin. by of adding increased of by was rate LCE polyubiquitylated by forms inhibited the polyubiquitylated was 50% increases which of mutation by degradation the proteasomal increased that indicating were no lactacystin, has mutant the of inhibitor forms polyubiquitylated L428A/L429A the proteasome of amount a the contrast, In why effect. by explaining degraded that slightly proteasome, polyubiquitylated suggest only are results the of LCA These of 3A). amount forms (Fig. polyubiquitylated the LCA the wild-type change the of not exposure forms Cell did polyubiquitylation. to lactacystin only subject not to is that LCA the indicate also in results but absent of The were LCE but 3A). absence levels, (Fig. similar wild- cells at the the non-transfected LCE both wild-type In in and detected LCA amount). type were forms total ubiquitylated the inhibitor, of (detection GFP an a using antibody or forms) immunoblotting polyubiquitylated of by (detection analyzed antibody ubiquitin and antibody anti-GFP an using immunoprecipitated were proteins proteasomal expressed without positive The or lactacystin. a with the pre-incubated as were used by cells was SiMa been 2010), Transfected al., degraded control. has et (Tsai which and cells LCE, neuronal ubiquitylated in and pathway be control, negative to a reported as and/or proteasome used SiMa irreversible were ubiquitylation Non-transfected cells 1995). the al., intracellular et (Fenteany used lactacystin inhibitor to we degradation, LCA the proteasomal increases of mutation L428A/L429A susceptibility the whether determine To in cells proteases neuronal non-proteasomal by and proteolysis proteasomal LCA both accelerates mutation L428A/L429A The 48/49 uatwsdcesdb 0 fe 4days. L429 and 14 L428 after include that 40% residues C-terminal by 22 decreased the of of almost Removal was activity rLCE The mutant cell of of S2). persisted days L428A/L429A Fig. activity 14 after material the disappeared rLCA (supplementary and while days incubation 3 days, after wild-type 14 80% by for declined average of on unchanged activity proteolytic h 48L2 iecn sipratfrLAstability. LCA of for important activity is that proteolytic confirming dileucine S2), in L428/L429 Fig. the material decrease (supplementary dramatic time over more LCA a in resulted rSNAP-25 or ora fCl cec 21)17 2430 doi:10.1242/jcs.146324 3294–3308 127, (2014) Science Cell of Journal 180 6 ..( s.d. LEcevdSA-5 o 4dy.The days. 14 for SNAP-25) (LCE-cleaved b atnsga hwta h 48/49 mutation L428A/L429A the that show signal -actin n 5 ) * 3). P t , -test. .1cmae ihtewild-type the with compared 0.01 197 LAcevdSNAP-25) (LCA-cleaved b atnwas -actin 3297

Journal of Cell Science EERHARTICLE RESEARCH 3298 their of of rate rate the the not but increases formation. results forms, mutation The polyubiquitylated 3A). L428A/L429A of (Fig. degradation LCA the wild-type the that the for in suggest lower than was mutant polyubiquitylation even the of the level for Remarkably, the by lactacystin, decreased 3A). of (Fig. presence is LCA mutation of L428A/L429A forms the polyubiquitylated the lactacystin, of of of absence absence amount the the in in However, LCA inhibitor. of proteasomal forms polyubiquitylated of amount yooa rtae Fg B.Gvnta G12 niethe unlike MG-132, that Given of 3B). activity (Fig. the proteases 1991), hence and al., lysosomal acidification et which lysosomal bafilomycin, (Yoshimori preventing of V-type-ATPase thus presence lysosomal the the in inhibits observed also but minor was L428A/L429A A the mutant of 3B). amount the (Fig. in MG-132 increase significant statistically by L428A/ increased the of was proteasome amount mutant reversible the L429A contrast, the In by 3B). affected (Fig. not MG-132 inhibitor was lysates cell total in iial,teaon ftennuiutltdwl-yeLCA wild-type non-ubiquitylated the of amount the Similarly, ora fCl cec 21)17 2430 doi:10.1242/jcs.146324 3294–3308 127, (2014) Science Cell of Journal C,tehaycano h nioyused antibody the immunoprecipitation. of for chain heavy IgG the bands; HCh, unidentified ?, cells; NT, non-transfected immunoprecipitation; IP, analysis; blot Student’s western inhibitors), (no control with eut niae htbt G12adbafilomycin and MG-132 both that the indicates of results Quantification immunoblotting. by determined The and h. LCs 8 of for amount inhibitors degradation the in of or presence absence the in cycloheximide were with LCs incubated Cells GFP-tagged LCs. respective of the amount expressing total of the inhibitors on the degradation of LCA Effect wild-type (B) the forms. on polyubiquitylated effect no but L428A/L429A mutant, the and LCE of forms polyubiquitylated the of shows accumulation LCs. results lactacystin-induced various the the of of quantification amounts Densitometric total detect monoclonal to GFP antibody with re-probed and was stripped membrane The an antibody. with ubiquitin-specific detected were LCs Polyubiquitylated were antibody. LCs GFP GFP-tagged with lysed. immunoprecipitated lactacystin and without h or 16 with for measured. incubated were were (LCs) Cells chains (A) light the of effects on the inhibitors and proteases), calpain proteasomal, and of lysosomal, (inhibitor MG-132 V-type or lysosomal ATPase) of (inhibitor (proteasome bafilomycin lactacystin inhibitor), to exposed wild-type or were mutant, LCE L428A/L429A or LCA, wild- (WT) the type expressing cells degradation. SiMa intracellular Non-differentiated to the LCA increases of mutation sensitivity L428A/L429A The 3. Fig. eut r mean MG-132. are the inhibitor Results in degradation not the but of absence, presence, the of in amount GFP–LCA the mature decreases septin-2 that of indicates downregulation results the immunoblotting. of by Quantification determined was lysates and septin-2 cell MG- LCA, of of amount presence The or 132. with absence incubated the were in and cycloheximide Methods in and described Materials as the siRNA control or septin-2 with were treated Non- GFP–LCA degradation. expressing LCA cells of differentiated rate by the septin-2 increases of siRNA Downregulation not (C) but LCA. LCE, wild-type and the mutant L428A/L429A the protect 6 ..( s.d. b atni oa ellstswas lysates cell total in -actin n 5 ) * 3). P , .1compared 0.01 t ts.WB, -test. b atni total in -actin

Journal of Cell Science yooa n yooi rtae.Smlry C a protected was LCE Similarly, both proteases. specifically of cytosolic which involvement and bafilomycin, the lysosomal suggests by degradation, than lysosomal MG-132 inhibits by of mutant protection greater the The proteases. non-proteasomal the are by mutant in the degraded that, of suggest forms non-ubiquitylated results the inhibitors, the and of 1997), absence (Lee al., et proteases Tawa cysteine also 1998; non-lysosomal Goldberg, but proteasomal, and only lysosomal not some inhibits lactacystin, specific more ARTICLE RESEARCH NR rtisN DND ND ND a complex protein Adaptor proteins SNARE Gene cytoskeleton Septin GFP–LCE or GFP–LCA wild- the expressing number) cells (accession of Protein lysates total DOC GFP-LCE 0.5% wild-type and NP40 or 1% GFP-LCA from L428A/L429A antibody or anti-GFP type by immunoprecipitated Proteins 1. Table and the NP-40 1% either from co- mutant of L428A/L429A proteins its constructs or the LCA GFP-fused wild-type of with analysis nano-liquid identify immunoprecipitated comparative To (nLC)-MS/MS a LCA. performed of chromatography we stability partners, the the binding to determine these not LCA-interacting but might the with type, that as wild considered proteins protease the the be can to this degradation. mutant, bind L428A/L429A intracellular of increases that from proteins interaction it the Therefore, protect the mutation that for residues proteins leucine particular important L428A/L429A two be these degradation, might the to LCA of susceptibility that the Given through LCA motif with both dileucine interact longevity-essential that by proteins of LCA Identification non-modified and LCA proteases. the non- cytosolic ubiquitylated by and of lysosomal induces the proteolysis degradation of mutation to accelerates degradation L428A/L429A LCA proteasomal The than rapid non-ubiquitylated susceptible proteases. the more proteasomal addition, 2010), degradation, is In al., resistant LCE proteolysis. highly proteasomal et are proteasomal LCA (Tsai of undergo to data forms polyubiquitylated published rapidly the whereas previously LCE with consistent of polyubiquitylated the by forms However, degraded polyubiquitylation. intracellular also is LCE proteases. cytosolic and non-ubiquitylated lysosomal 3B), (Fig. that lactacystin by not suggesting but bafilomycin, and MG-132 both by rti cr n h ecnaeo eunecvrg rmoeo h he xeiet r hw.N,ntdetermined. not ND, shown. are experiments val three The the otherwise. specified of unless one shown, from are coverage immunoprecipitates GFP sequence in of detected percentage not the and and experiments different score three protein in detected proteins the Only eetdi n ftetreexperiments. three the of one in Detected etn5ioom1(P9449 ET 3(0 DND ND ND ND ND ND ND ND ND ND ND (4) 48 (2) ND 54 (10) 33 (16) 50 (8) 83 (36) 505 (33) 352 SEPT5 (26) SEPT3 (13) 321 78 SEPT6 SEPT2 subunit complex AP-1 SEPT9 SEPT11 SEPT7 (NP_9945439) 1 isoform Septin-5 (NP_9837294) A isoform Septin-3 (NP_119626208) CRA_a isoform Septin-11, (NP_055944) B isoform Septin-6 (NP_16256489) c isoform Septin-9 (NP_148352329) 2 isoform Septin-7 (NP_4758158) Septin-2 gonorrhoeae] [Neisseria protein fluorescent Green chain Light E type neurotoxin Botulinum [Clostridium chain light A type Neurotoxin Cotiimbtlnm (C7EWM8_CLOBO) botulinum] [Clostridium nsmay h eut hwta ohLAadLEundergo LCE and LCA both that show results the summary, In (ACJ06700) (AAO21363) botulinum] b (NP_663782) 1 a oTA 79(5 66(60) 1636 (65) 1739 BoNT/A1 oTE17 (64) 1077 (45) 551 (33) 664 (33) 662 gfp BoNT/E PB D7 3 ND (3) 74 ND AP1B1 cr %o coverage) of (% Score GFP–LCA Wild-type h rti cr n etd oeaewr infcnl oe in wild-type the lower in significantly those to were compared as coverage immunoprecipitates peptide mutant the and both were LCA However, score mutant. septin-9 its protein and and wild-type the LCA Septin-7 wild-type with L429. the both and the LCA with L428 detected of by in mediated interaction is the minor exclusively septins that be and suggesting detected to septin-3 immunoprecipitates, appear were septin-11, coverage, septin-6, septin-5 were and Septin-2, septins, which partners. score major other septin-5, interacting lower as and Several with septin-3 1). them detected (Table septin-11, implicating LCA septin-6, namely of for septin-9, found partners and were interacting coverage septin-7 sequence of of values Mascot septin-2, percentage the highest of and The by amount scores immunoprecipitate. the the the calculated to approximation, in proportional coverage present rough as protein considered sequence a be can with As and the software LCA 1). that score (Table GFP of showed protein proteins software interaction in septin Mascot the detected by several impairs not data was mutation the were L428A/L429A of that analysis interactors. analysis GFP LCA a proteins The specific with as nLC-MS/MS the considered As were co-immunoprecipitated only immunoprecipitates cells parallel construct. proteins so SiMa the alone, respective a on of a performed control, lysates with (DOC) negative transfected deoxycholate transiently sodium 0.5% rti aiyta nldsa es 4hgl eae GTP- related highly various in 14 roles least important play at that as proteins includes cytoskeletal a identified to binding that belong were Septins family interaction. confirming protein septins LCA–septin nLC-MS/MS, the of GFP the by specificity and GFP–LCE the of BoNT/E for cells both none partners be SiMa for interacting However, can scores of as Mascot 1). efficiency high lysates (Table high the a cell GFP– for that with inferred the shows immunoprecipitated protocol was from same LCE the antibody using immunoprecipitated GFP–LCE proteins. GFP proteins expressing these the the with of LCA by analysis of mutation nLC-MS/MS interaction the The the that suggesting impaired 1), severely (Table immunoprecipitates LCA ora fCl cec 21)17 2430 doi:10.1242/jcs.146324 3294–3308 127, (2014) Science Cell of Journal cr %o coverage) of (% Score GFP–L428A/L429A cr %o coverage) of (% Score GFP–LCE Wild-type e fthe of ues 3299

Journal of Cell Science ugs htspismgtpoetLAfo intracellular from LCA prevented also protect results 3), the might and 1), (Table septins degradation. 2 septins (Figs that with mutation, plasma LCA stability suggest L248A/L429A of specific interaction LCA the the the to that decreased cytoskeleton LCA Given which membrane of regions. these attachment for membrane stable LCA role of adaptor in no a interaction and proteins provide suggests stable LCA the septins data between contrast, with The interaction By complexes. specific 2011). proteins previously a be Barbieri, was to for as and unlikely evidence LCA is (Chen of hence, tethering suggested and, membrane transient for and/or responsible weak is proteins with subunits these of interaction specific S1), LCA. detected a of Table also lack were material the subunits (supplementary indicating both immunoprecipitates but from GFP 1), immunoprecipitate in (Table L428A/L429A lysates cell in in total found experiment was subunit single one a and GFP–LCA S1), extracts in Table membrane material detected DOC (supplementary any was 0.2% subunits or from detect the AP-3 isolated of immunoprecipitates not AP-2, One (AP-1, 1). did complexes (Table AP-4) also protein adaptor complexes cells from subunits SiMa lysates protein cell GFP–LCA-expressing total adaptor from of isolated with immunoprecipitates GFP–LCA of interaction LCA (Ferna in motif Tbe1 nsieo h atta C lae NP2 in SNAP-25 cleaves determine GFP–LCA LCA with To that co-immunoprecipitate S1A,B). proteins fact Fig. SNARE nLC-MS/MS the material whether (supplementary of by cells spite SiMa immunoprecipitates in GFP–LCA 1) (Table in and Barral, detected Mostowy and Saarikangas 2012; 2010; al., Russell, et and 2011). Nakahira 2012; (Hall Cossart, processes cellular ARTICLE RESEARCH 3300 non- LCA both of colocalization cells, Fig. demonstrated SNAP-25 SiMa material have with studies (supplementary in Earlier differentiated S3B). LCA and its with differentiated showed colocalization SNAP-25 endogenous partial total of Immunofluorescence in proteins SNARE cells with SiMa LCA of colocalization Partial acidic putative a in L429 and L428 Fig. E DOC of material motif, 0.2% (supplementary location dileucine in analysis The detected blot was S3A). western GFP–LCA by with extracts SNAP-25, but the of syntaxin-1, with of not Consistent co-immunoprecipitation amount. results, protein nLC-MS/MS GFP–LCA when the of low to very abundance is compared the immunoprecipitates detected GFP–LCA that in indicate proteins GFP– the these coverage and in were sequence score exclusively protein of the found percentage of SNAP-25, values were The them, immunoprecipitates. 16, of LCA not syntaxin two only and However, syntaxin-1a but S1). Table material (supplementary proteins, SNARE- and specific proteins interacting as SNARE them Several proteins. confirming LCA-interacting S1), but and extracts Table membrane immunoprecipitates, material DOC septin-11 0.2% (supplementary GFP–LCA in immunoprecipitates, septin-9, GFP in in septin-7, not detected cell were septin-2, total in from septin-5 1), found (Table that isolated to immunoprecipitates Similar lysates extracts. nLC-MS/ GFP–LCA membrane DOC an of 0.2% performed analysis we conditions, MS extracting milder under h eut niaeta soito fGPLAwt SNARE with GFP–LCA of association that indicate results The upiigy NP2 rohrSAEpoen eenot were proteins SNARE other or SNAP-25 Surprisingly, ´ dzSlse l,20) oee,nCM/Sanalysis nLC-MS/MS However, 2004). al., et ndez-Salas 197 nnuoa C2cls(Ferna cells PC12 neuronal in 424 YL,sget h novmn fthis of involvement the suggests FYKLL, ´ dzSlse al., et ndez-Salas hsefc a atal rvne yM-3,idctn that indicating degradation. MG-132, of from by 3C). LCA amount prevented (Fig. protects The 53% partially septin-2 incubation. by was siRNA 8-h effect septin-2 this This by during mature the decreased for also the GFP–LCA was cycloheximide in at and GFP–LCA with specifically after before or incubated look h presence were 2 To h samples 24 h. the by 8 all in detected for At GFP–LCA, MG-132 as incubated of 3C). 50%, (Fig. were absence by cells septin-2, analysis expression transfection, septin-2 targeting blot resulted transfection, in GFP–LCA western (siRNAs) decrease to prior a RNAs h in 24 and of interfering 48 mixture performed the small with cells SiMa two of transfections degradation consecutive LCA Two increases septin-2 of Downregulation etn2a h lsammrn fSM el,cnitn with consistent cells, SiMa of of membrane intensity plasma fluorescence the mean at that Statistical the septin-2 demonstrated BoNT/ increased profiles S4). exposure intensity in Fig. BoNT/A fluorescence septin-2 the material of performed of (supplementary analysis and clustering cells overnight show A-exposed BoNT/A low- images nM and 10 high- magnification representative or binding, The 1 immunofluorescence. receptor septin-2 to following cells occurs cells SiMa it plasma as the neuronal translocation to and delivered the internalization, be (Ferna will of LCA neurons the membrane to settings, motor sensitive these In as primary 2012). be to differentiated as demonstrated used previously BoNT/A we membrane. we BoNT/A, that LCA to cells plasma SiMa exposure of cell the presence neuronal after the structures. at oligomeric that septin LCA of suggest assembly the of cells results of induces mutant- involvement non-transfected the clustering the the in with Furthermore, in consistent that in that are to septins results so proteins The similar membrane, 5B,C). cytoskeletal was (Fig. the cells these and at expressing septin-2 of regions both LCA-positive of distribution accumulation LCA in Importantly, the 1). of septin-7 (Table decreased and impairment mutation mutation septin-2 this the the by both 5A) with septins (Fig. with with consistent interaction immunoblotting LCA 5B,C), (Fig. by of septin-7 detected colocalization of L428A/L429A as decreased co-immunoprecipitation and septin-2 The with decreased cytoskeleton. re- LCA a septin dramatically in results the mutation presence non-transfected LCA- of LCA of that in membrane organization suggesting plasma regions 4A,B), plasma the (Fig. membrane to the cells other or at to cells regions expressing more compared much LCA-positive as displayed in membrane 4C). septin-7 (Fig. accumulation and cells intense LCA-expressing septin-2 of both sections Remarkably, high-resolution basal from 4A,B). the seen (Fig. of as membrane images shapes ring-like plasma have the LCA clusters along with These colocalized distributed precisely clusters were in cells septin-7 SiMa and in septin-7 septin-2 and Both septin-2 with LCA of Colocalization itiuino F–C nrsrce ein tteplasma the at regions S3B,C). restricted Fig. clustered non- material in evenly to (supplementary membrane opposed GFP–LCA are both as syntaxin-1 of membrane and in distribution plasma (supplementary the SNAP-25 cells along Both found distributed S3C). SiMa Fig. was differentiated material syntaxin-1 and differentiated with colocalization 04.Tedfeecsaepoal u otefc htthe SNAP-25 that fact and the both recognizes to SNAP-25 cells SiMa due of intact probably immunostaining for are used antibody differences The 2004). odtriewehrLAidcdspi lseigoccurs clustering septin LCA-induced whether determine To ora fCl cec 21)17 2430 doi:10.1242/jcs.146324 3294–3308 127, (2014) Science Cell of Journal 197 iial,ol partial only Similarly, . nvivo in ´ dzSlse al., et ndez-Salas eexposed We .

Journal of Cell Science EERHARTICLE RESEARCH 08.Teefcso C nmtss elmgainadcell and migration cell mitosis, on FCF FCF al., 2004). of et and Hu of effects al., 2010; al., formation The yeast et et the 2008). (DeMay Iwase induced structures in septin and 2008; large assembly abnormally both al., septin et normal dynamics, impaired septin (Hu affect cells forchlorfenuron and to mammalian found oligomerization, organization recently been septin assembly, has of compound we LCA, This inhibitor of (FCF). stability for an important are used septins whether and assess localization To on assembly LCA septin of of stability inhibitor an of Effect the at BoNT/A. with septin-2 treatment of upon dose- the clustering membrane a with in plasma in membrane consistent the manner, clusters in intensity dependent the to fluorescence of high frequency with septin-2 the sites increased also of exposure BoNT/A recruitment membrane. LCA-induced the itiuino C ttepam ebae(i.6B), of (Fig. L428A/L429A distribution the clustered membrane for homogeneous seen plasma pattern decreased and increased the the more-even resembled and transfection, which a at LCA, LCA LCA in LCA after resulting of of h 14 distribution localization cells the cytosolic to added fnndfeetae iaclswt 100 with Incubation cells 6A). SiMa (Fig. of elongated non-differentiated presence linear more of the became elongated in cells addition, of inhibitor, In the formation arrowheads). punctuated the 6A, disrupted and (Fig. induced inhibitor filaments The ring-like and arrows). structures the 6A, these (Fig. cells, evident control are structures of in section 100 performed with bottom immunofluorescence incubated septin-2 cells the the from 2011). evident al., et is septin-2 Sidhaye of 2008; depletion al., by et (Hu induced siRNA those by to similar were adhesion h feto h niio nspi raiaini iacells SiMa in organization septin on inhibitor the of effect The ora fCl cec 21)17 2430 doi:10.1242/jcs.146324 3294–3308 127, (2014) Science Cell of Journal etn2i h iglk structures. ring-like the in and septin-2 LCA of colocalization LCA- showing non-differentiated cells of transfected sections basal the images of High-magnification (C) images. merged the ofiin (mean coefficient septins (mean with The colocalization regions. of boxed coefficient the of show images Insets magnified (arrowheads). membrane cells plasma non-transfected the in at more that much to is compared (arrows) intense membrane regions plasma LCA-positive the in at septin-7 and septin-2 of both Immunofluorescence cells. SiMa plasma and differentiated the non-differentiated at in clusters both in membrane (B) septin-7 and (A) to aggregates. small septin-7 or and clusters septin-2 membrane-associated recruits LCA 4. Fig. 6 s.d., m C o Fg A.I the In 6A). (Fig. h 7 for FCF M n septin-2 with colocalized is LCA (A,B) 5 2 olwdb h overlap the by followed 12) 6 s.d., n 5 m 2 sidctdbelow indicated is 12) C,wihwas which FCF, M 3301

Journal of Cell Science EERHARTICLE RESEARCH uigcl nuainwt rwtotFF etr blot Western FCF. without h 8 or for 3302 and with before cells incubation h 2 degradation, cell for on cycloheximide specifically during with FCF substantial incubated To of LCA. were a effect of FCF degradation caused the that increases determine suggesting also or 6C), biosynthesis (Fig. FCF inhibits content either to cellular LCA Exposure in decrease 5). (Fig. mutant h neato ewe C n etn eperformed we septin, and LCA between To preventing interaction 6D). by (Fig. degradation LCA the LCA decreases of FCF whether degradation MG-132, of evaluate FCF-induced inhibitor, rate LCA degradation the the degradation in of prevented the decrease Addition increases LCA. significant mature FCF a the that showed indicating lysates amounts, cell of analysis ora fCl cec 21)17 2430 doi:10.1242/jcs.146324 3294–3308 127, (2014) Science Cell of Journal A F–C-48/49 mutation. GFP–LCA-L428A/L429A AA, LL/ immunoprecipitation; IP, NT, cells; GFP–LCA; non-transfected wild-type WT, images. merged the ofiin (mean coefficient septins with (mean colocalization of regions. coefficient LCA-positive The in septin-7 both and of septin-2 accumulation decreased in also both The mutation (C) cells. septin-7 differentiated and and (B) non-differentiated septin-2 with LCA of colocalization decreases mutation The L428A/L429A (B,C) co- septin-2. that with LCA immunoprecipitates of amount the decreases L428A/ the by impaired mutation. L429A is septins co-clustering with and LCA interaction of the Both 5. Fig. 6 s.d., n 5 2 olwdb h overlap the by followed 12) 6 A h 48/49 mutation L428A/L429A The (A) s.d., n 5 2 sidctdbelow indicated is 12)

Journal of Cell Science EERHARTICLE RESEARCH h etn2imnpeiiae Fg E.Ipraty the Importantly, 6E). (Fig. and lysates immunoprecipitates cell total significantly septin-2 both total in but in GFP–LCA the fractions, of septin-2 amount of immunoprecipitated the amount decreased or the change 100 lysates not cell without did FCF GFP–LCA-expressing or to with exposure from incubated septin-2 cells of immunoprecipitation m C o 4h The h. 24 for FCF M h rp) h eut niaeta irpino septin of disruption LCA on between ‘IP/Lysate’ that interaction 6E, the indicate (Fig. impairs lysates results FCF cell by The total oligomerization to graph). the normalized in the of was amount amount the that in than its GFP–LCA decrease greater dramatic a co-immunoprecipitated was from the seen fraction as lysates, immunoprecipitated cell in the in decrease ora fCl cec 21)17 2430 doi:10.1242/jcs.146324 3294–3308 127, (2014) Science Cell of Journal oto n niios,Student’s inhibitors), (no control neato ewe etn2adLA Septin-2 LCA. and the the septin-2 of impairs between presence, FCF interaction the (E) in MG-132. not inhibitor GFP– but degradation mature absence, of the amount in the LCA decreases FCF of to exposure cells that indicates results the of immunoblotting. Quantification by determined and was LCA lysates of amount 100 The of MG-132. presence the the in in or cycloheximide absence with incubated were GFP– LCA expressing of cells degradation Non-differentiated induces GFP–LCA. FCF of by Disruption organization GFP (D) septin in cells. decrease FCF-exposed substantial in a fluorescence show images cells microscopy these for confocal FCF of without magnification or Low with h. incubated 7 were later GFP–LCA h with 14 transfected and were density plated similar cells with SiMa plasma Non-differentiated the (C) along membrane. LCA even/ of relatively distribution and homogeneous LCA of localization cytoplasmic 100 to exposed cells SiMa non- GFP-LCA-expressing of differentiated images microscopy High-resolution/high- confocal (B) magnification cells. SiMa filaments in elongated (arrowheads) of and formation (arrows) the 2 induces septin of structures punctuated increase degradation. FCF LCA by and organization septin-2 septin of of disruption downregulation Both 6. Fig. irpieefc nspi--C neato.Results mean interaction. are septin-2-LCA its on indicating effect lysates, disruptive cell in than dramatically the more in fractions GFP–LCA immunoprecipitated of septin-2 amount that the shows decreases results FCF the of lysates. quantification cell Densitometry total and fraction GFP–LCA in immunoprecipitated and immunoblotting septin-2 by both determined of was amount the for and FCF h, without 24 or with cells GFP–LCA-expressing incubation of after performed was immunoprecipitation 6 ..( s.d. m n C hwn increased showing FCF M 5 A C irpstern-ieand ring-like the disrupts FCF (A) ) * 3); P , .1cmae with compared 0.01 t -test. b m atni oa cell total in -actin C r20 or FCF M 3303 m M

Journal of Cell Science ttepam ebaeadtermral tblt fLAin LCA of stability remarkable the cells. and distribution neuronal aggregate membrane or plasma clustered the the from the at both LCA for for required is protects septins required which degradation. septins, are LCA with dynamics accelerates LCA degradation. of septin turn, interaction in normal which, Therefore, septins, and ARTICLE RESEARCH swa n rnin n ec sulkl ob epnil for responsible LCA be 3304 to unlikely the material is SNAP-25 hence of and with supplementary transient GFP–LCA analysis 2011). and 1; of weak (Table association global is Barbieri, that cells indicate a S1) and SiMa Table from (Chen in results the interactome neurons with our LCA in and of However, LCA association membrane of stable interaction for plasma the responsible that is suggested SNAP-25 have studies Recent partners LCA-interacting preferred proteins SNARE are than rather proteins cytoskeleton Membrane dileucine the with no interacted LIMP-II. directly However, of septin-7 LI- motif that 2008). so a possible provided, al., was is in binding it LIMP-II septin–AP-3 et for to evidence (Baust binds experimental by that manner mediated complex was motif-dependent that protein septin-7 this suggested and adaptor in authors LIMP-II AP-3 The residue, between LIMP-II. glutamine interaction in 2008). the mutated the al., been the particularly et had abolished motif (Baust residues, septin-7 ERAPLI, other 2 and SCARB2), No a LIMP-II protein as in between membrane known interaction LI/AA lysosome of also with mutation of (LIMP-II, crucial the interaction motif are Interestingly, that direct dileucine-containing residues interaction. acid in these amino this other involved whether for identify are to elucidate and to residues septins suggesting required leucine the 5), are septin-binding clearly Fig. two new studies a impairs 1; results of Further (Table part motif. maybe our mutation septins dileucine L428/L429 with contrast, the L428A/L429A that LCA By of the LCA. interaction that of stability or shown) 1B,C) demonstrate (Fig. not localization E424A of is the The any (data 1). change which with not (Table did complexes LCA 2009), protein mutation of adaptor Traub, interaction of subunits 2012; dileucine-specific the find analysis al., nLC-MS/MS not Our et did trafficking. Sitaram intracellular Mattera for 2003; 2011; important al., et many al., Janvier or 2003; of AP-2 et Traub, AP-1, and complexes domains protein (Bonifacino of LCA. adaptor AP-3 cytoplasmic interaction the the with of mediate the proteins to these C-terminus known in are the proteins, present transmembrane near motifs, motif Such results consensus the These E)xxxL(L/I) localization 1). through cells. subcellular Table neuronal for in interaction 5; LCA essential of is 3, persistence LCA–septin LCA and in 2, the (Figs motif of dileucine stability cells that the decreases SiMa demonstrate and membrane, in plasma distribution LCA the aggregate between at or LCA clustered interaction the of the prevents septins, abolishes and LCA mutation L428A/L429A the BoNT/A mutation PC12 in L428A/L429A membrane (Ferna plasma the the cells with that LCA of shown association decreases have studies motif dileucine Previous LCA of properties New DISCUSSION 48adL2 r oae nadluiecnann (D/ dileucine-containing a in located are L429 and L428 with LCA of interaction dileucine-mediated the conclusion, In nvivo in ´ dzSlse l,20)adsotn h fet of effects the shortens and 2004) al., et ndez-Salas Wn ta. 01.Hr edmntaethat demonstrate we Here 2011). al., et (Wang eiigseii ebaecmatet n o localizing for important for barriers and diffusion compartments as membrane act specific they defining proteins, binding other by addition, to In 2010). Krummel, intracellular and the (Gilden and cytoskeleton membrane plasma septins tubulins, the between and linkers actins as with serve interacting Stamenkovic also 2012; By 2010). Cossart, Yu, and and Mostowy membrane 2002; al., plasma et membrane (Bretscher the are with not 4,5-bisphosphate phosphatidylinositol with Septins interaction associate their but through 1). that LCA, (Table proteins wild-type mutant cytoskeletal the L428A/L429A of the partners of were interacting seven which proteins, septins, membrane GTP-binding as related known detected plasma highly 14 of both Of family a LCA. for form of the important stabilization of is and proteins tethering family cytoskeletal protein membrane is with septin LCA LCA the on of interaction proteins and SNARE data 2011) of particularly al., effect These et stabilizing (Yu proteins, transient. doubtful. a complexes and that SNARE SNARE and that of mean weak nature suggest other with dynamic is results highly with syntaxin, the associates addition, with interaction constantly In interaction, LCA. the SNAP-25 LCA–SNAP-25 from dissociates the the that with of consistent implying is nature conclusion This enzyme–substrate LCA. of stabilization the Fg )adwe nrdcdit el yepsr oBoNT/A to exposure by cells S4). into transfection Fig. introduced material by (supplementary when cells and in 4) overexpressed to (Fig. when septins recruits both LCA membrane, Moreover, the 4). and (Fig. membrane septin-2 plasma 2003; the LCA, with associated Kinoshita, structures ring-like Accordingly, in colocalized 2012; found 2012). are septin-7 Russell, Cossart, and and membrane-associated Hall Mostowy (Gilden form 2010; filaments to Krummel, and rings known and as are such Septins structures, hetero-oligomeric 2008). Cossart, al., Spiliotis and 2011; and Barral, et Mostowy and (Gilden Saarikangas 2010; 2012; al., sites Russell, et Nakahira 2012; and intracellular Hall specific 2010; Krummel, at proteins interacting C,ulk C,i nestv obflmcnadMG-132 and why bafilomycin LCA to explaining protect insensitive degradation, that is 3B). (Fig. septins cytosolic LCE, with unlike and interacts LCA, non-ubiquitylated lysosomal LCE, Similarly, LCA from not of but degradation. forms a polyubiquitylated LCA, but to proteasomal the LCA, due protect with is interact and from 3A) that LCE, septins (Fig. with the LCE of not of effect that indirect to or direct study compared LCA our of as forms in lactacystin polyubiquitylated presented of to sensitivity results of The lack this that LCA. of suggest stability to lower compared the as for responsible protein part, in least at probably, of zones restricted in membrane. structures plasma and ring-like the septins recruit LCA–septin actively induce to LCA appears to as LCA oligomerization well Importantly, septin 6). as normal (Fig. inhibiting septins, by with impaired LCA are co- stability of Similarly, 3A,B). interaction more Fig. and the becomes 2A,B; clustering LCA (Fig. at and degradation 5), to structures (Fig. susceptible plasma abolished hetero-oligomeric is the membrane the mutation, LCA–septin plasma When L428A/L429A septins. of the at by with formation prevented LCA clusters is of interaction interaction LCA-containing this the of requires membrane formation degradation The from the LCA at protects clusters membrane LCA/septins of formation Dileucine-mediated ycnrs,terslspeetdhr niaeta stable a that indicate here presented results the contrast, By h ako neato fLEwt etn Tbe1 is 1) (Table septins with LCE of interaction of lack The ora fCl cec 21)17 2430 doi:10.1242/jcs.146324 3294–3308 127, (2014) Science Cell of Journal

Journal of Cell Science bet omSAEcmlxs u hs opee r not are complexes these but complexes, SNARE form to able EERHARTICLE RESEARCH Iaae l,20;Knsiae l,20;Tage l,21;Xue 2011; al., et Tsang 2000; al., in et Kinoshita studies septins 2007; several al., in of et demonstrated (Ihara presence been has The SNAP-25, membranes cleaves presynaptic neurotransmission. and inhibiting septins with thus interacts it where membrane Fg C.LActlzdcevg ftenn -emnlamino C-terminal SNAP-25 nine generates the residues of acid cleavage zone LCA-catalyzed active the 7C). in (Fig. complexes event SNARE fusion vesicle–membrane from a release biosynthetic during after a formed diffusion is by lateral either SNAP-25 by membrane or free the the route of The outside portion membrane. this septins presynaptic to motif recruit delivered the this possibly of through dileucine could zone septins active L428/L429 LCA to 7C). binding the (Fig. LCA-bound LCA motif likely the conceal providing that a 7B), not shows with (Fig. 1XTG) 2W2D) does ID ID (PDB SNAP-25 (PDB SNAP-25 to LCA bound full-length LCA an truncated of vicinity a structures the crystal of known in of complexes superposition Montecucco, Importantly, SNARE SNAP-25 zone. of active free and formation cleaves the and (Pantano to to prior binds neurotoxins likely botulinum LCA by Therefore, cleaved 2014). the be cannot of complex fact SNARE any the a in with proteins consistent and SNARE LCA is that bind This cannot simultaneously. SNAP-25 proteins so SNARE 7A), drastically (Fig. (Breidenbach differs 2004) SNAP-25 SNAP-25 Brunger, 2002) LCA-bound and of the al., conformation of et conformation The (Chen the 2010). from complex al., SNARE et a Yang within 2004; al., et nmtrnuos C rbbyacmltsa h presynaptic the at accumulates probably LCA neurons, motor In 197 h lae SNAP-25 cleaved The . 197 is hrae l,21;Xee l,20) h pcfcsignaling to remain specific septins The by 2004). LCA al., identified. of 2012; al., be et stabilization et in Xue form Brand involved 2008; 2013; pathways to al., al., et et Baust known 2008; signaling Sharma al., are multiple et regulate (Amin Septins and events recruitment proteins. a protein for their other and scaffolds to owing with LCA syntaxin-1A of also interaction degradation is actin, attenuate It septins 2011). (e.g. that al., possible et cortical analyzed Hagiwara other transporter; were some glutamate-aspartate GFP- than that turnover Interestingly, to slower proteins binding proteins. exhibit physical LCA. cytoskeletal septins its to tagged stable from binding syntaxin-1, result relatively of for might the domain LCA available with of SNARE domain Stabilization single interact C-terminal al., a et the might of to Ito leaving N-terminal bound LCA 1999; the is al., that which SNAP-25 et in suggest Beites heterodimers, 2005; results SNAP-25–syntaxin-1 al., (Amin These et fusion Beites membrane 2009). 2008; inhibit to al., and et syntaxin-1 with interact 6) and LCA. 3 of effects 2, probably, neuroparalytic and, newly (Figs the proteolytic both degradation of be longevity remarkable intracellular the that will ensures the septins from in with as LCA LCA persists LCA, of protects LCA molecules association as by The long membrane. cleaved presynaptic as molecules SNAP-25 be SNAP-25 synthesized will As they SNARE-complex-bound 2014). dissociate, Montecucco, and (Pantano full-length exocytosis for competent nnuos etn2 etn5adspi- aebe on to found been have septin-8 and septin-5 septin-2, neurons, In ora fCl cec 21)17 2430 doi:10.1242/jcs.146324 3294–3308 127, (2014) Science Cell of Journal xctss eutn nabokg fsnpi eil fusion vesicle synaptic of blockage for a competent in not resulting SNAP-25 are exocytosis, LCA, that by complexes residues SNARE of acid forms cleavage amino the C-terminal After nine SNARE fusion. the from vesicle released synaptic or after synthesized complexes monomers newly SNAP-25 either cleaves are it the where that in zone, active membrane an presynaptic of the vicinity to septin LCA with tethers interaction filaments model dileucine-mediated putative the A that (C) activity. showing proteolytic LCA is the septins for with permissive the LCA that of indicating interaction L429 SNAP-25, dileucine-mediated and bound L428 by of ID hindrance spheres) (PDB no (purple LCA shows lines) full-length purple of a thin domain with 2W2D, core 1XTG) C-terminal ID the (PDB to SNAP-25 truncated bound a surface) of (gray structures LCA high-resolution of Superposition (B) LCA-catalyzed SNAP-25. with SNARE-complex-bound compatible of not cleavage is within complex other (orange), SNARE with synaptobrevin the and and domain (yellow) N-terminal syntaxin its SNAREs, C-terminal with the SNAP-25 of of interaction domain tight two these The the in complexes. SNAP-25 in protein of differences domain significant C-terminal bound the showing of SNAP-25 1XTG) conformation and ID 1KIL) (PDB ID LCA (PDB to SNAP-25 complex of SNARE structures a resolution within high of Superposition and (A) SNAP-25 neurotransmission. of of pool inhibition in free prolonged results the membrane of presynaptic of cleavage the persistent stabilization at that septins showing by model LCA postulated A 7. Fig. rbby aayi fet fLAi oo neurons. motor in LCA and, of proteolytic effects of paralytic longevity probably, remarkable an the providing for degradation, explanation septins intracellular with from LCA LCA of protects Association release. neurotransmitter and 3305 197

Journal of Cell Science CAGCATATATAAGTTCATGTGC-3 ta. 02.TercmiatHstge C rC) t L428A/ residues its C-terminal 22 (rLCA), the were LCA without variant His-tagged cells (Ferna (LCA truncated recombinant transfection its indicated, to The mutant, prior L429A 2012). Where days al., 3 CA). least et at Carlsbad, for reagent differentiated transfection Technologies, 2000 Lipofectamine (Life using transfection transient by CAATT-3 GCACATGAACTTATATATGCTGGACATAGATTATATGGAATAG- eedfeetae nRM ei ih2m GlutaMAX mM 2 with media RPMI in differentiated were ta. 04.Tepoen eedlvrdt -a ifrnitdPC12 differentiated (Ferna 7-day to previously delivered Chariot were the described using proteins cells (ATCC) The as 2004). al., purified et and synthesized 5 wild-type template: the a with as respectively, used mutant, were L428A/L429A primers the and following plasmid the mutants, L429A AAACAATCCAG-3 uaeei sn h ukhnemtgnsskt(Agilent the template: a kit mutant, as E424A plasmid 3 wild-type mutagenesis the the For with 5 used USA). QuikChange were TX, primers following Creek, the previously Cedar site-directed described Technologies, by using constructed were as N- mutagenesis GFP–LCA the of constructed mutants at L428A/L429A GFP were the with (Ferna LCE (GFP–LCE) wild-type the terminus and (GFP–LCA) terminus (Carlsbad, Technologies Life from otherwise. specified were unless reagents CA) culture tissue All WI). 1 supplement, AEIL N METHODS AND MATERIALS ARTICLE RESEARCH HS5 el eepae vrih,dfeetae o as and 3306 days, 3 for (20 differentiated rLCE or overnight, rLCA plated with rLCE transfected and were rLCA cells with transfected SH-SY5Y cells SH-SY5Y of Fractionation mM 2 with medium RPMI in grown were GlutaMAX VA) cells pheochromocytoma Manassas, rat PC12 acids. (ATCC, serum Germany; amino bovine non-essential fetal 10% mM Braunschweig, with 0.1 1640 RPMI and (DSMZ, in grown were 1999) cells al., et Marini SiMa cells neuronal neuroblastoma in LCE Human and LCA of Expression o naeeto C nospi-otiigmembrane- from LCA septin-containing protects into which degradation. intracellular LCA complexes, required cytoskeleton of is associated septins engagement with LCA for the of that interaction indicates proteins dileucine-mediated LCA-interacting of analysis proteomic otnDw,U)wr rw nmnmmesnilmdu/1 with medium/F12 essential HPA, minimum GlutaMAX (ECACC, in mM grown cells 2 were neuroblastoma UK) Down, human Porton SH-SY5Y serum. equine .%CO 5.0% oi,M) iaadS-YYclswr ananda 37 at maintained were pyruvate, cells SH-SY5Y sodium and mM 100 SiMa 1 MO). HEPES, and Louis, mM penicillin cell 10 three U/ml all by 100 for supplemented medium growth were The lines acids. amino non-essential mM 0.1 albd A S)acrigt auatrrspooo.Bify the Briefly, protocol. 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Beites, ta. 02) muolt eeqatfe ydnioer sn Zeiss 3.2. using version densitometry software, by 510 quantified LSM were Immunoblots 2012b). al., et eut n rt h aucit ...... n R.L and J.P.W., S.B., P.S., P.E.G., E.T., O.V., interpreted manuscript; and the analyzed wrote experiments, designed and project, results the led E.F.-S. and O.V. contributions Author Inc. hold Allergan and for Inc. consultant Allergan a of is employees G.S. are stock. E.F.-S. Allergan and R.A L.W., R.L, P.E.G, interests Competing legends. of figure number the and in significance specified Statistical are Excel). experiments Microsoft and software Student’s 4 using performed was analysis Statistical analysis Statistical 2011) S1. al., Table et material Tokhtaeva supplementary 2009; in al., and et (Doolittle previously described slice nLC-MS/ as by gel analyzed MS each and in USA) WI, contained Madison, Proteins (Promega, lane pieces. trypsinized the were each 12 into Then to sliced SDS-PAGE. and reducing Immunoprecipitated excised, cross-linked 4–12% was 2011). by was separated al., USA) were et proteins CA, (Tokhtaeva against View, beads antibody polyclonal protein-A–agarose Mountain rabbit the (Clontech, lysates, cell GFP to adding to prior exception nLC-MS/MS, the that, with by above described LCA as performed with was immunoprecipitation co-immunoprecipitated proteins analyze To proteins immunoprecipitated of nLC-MS/MS ets .L,Xe . osr .adTibe .S. W. Trimble, and R. Bowser, H., Xie, L., C. Beites, Krause, C., Thiele, L., Bourel, I., Parshyna, C., Czupalla, M., T., Anitei, T., Guszczynski, Baust, J., Kanungo, S., Kesavapany, L., Y. Zheng, D., N. Amin, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.146324/-/DC1 online available material Supplementary O.V. to material Inc. Supplementary Allergan, from grant a by supported was work This manuscript. the of Funding writing and results the of discussion study, contributed R.A. the and of L.W. conception G.S., J.P.W., to data; analyzed and experiments executed xrclua inlrgltdkns mtgnatvtdpoenkns 6 A. kinase septin Kotlyarov, morphology. protein regulates dendrite complex and and (mitogen-activated signaling function M. 5 3 kinase Gaestel, protein kinase [MAPK6])-MAPK-activated S., signal-regulated Meloche, extracellular S., Britsch, factors. H. Wong, the stabilize to forchlorfenuron molecule small the cytoskeleton. septin for requirements Cellular complex. J. Rizo, and SNAP-25. membrane-bound plasma with chain cortex. cell the at integrators A. serotype neurotoxin botulinum lysosomes. and endosomes to proteins ouiu toxins. botulinum SNAP-25. protein synaptic the cleaves selectively Su Drl1cmee ihapaSA o idn oteSAEcomplex. exocytosis. SNARE inhibits and the syntaxin to binds binding for alpha-SNAP J. with Biochem. competes CDCrel-1 proteins. membrane B. lysosomal Hoflack, targeting and E. C. (hCDCrel- H. SEPT5 Pant, septin and exocytosis. human P. modulates of Grant, 1) phosphorylation W., 5 Albers, kinase P., Cyclin-dependent Rudrabhatla, K., R. Sihag, hf .C,Nean .adJh,R. Jahn, and H. Niemann, C., T. dhof, ¨ ora fCl cec 21)17 2430 doi:10.1242/jcs.146324 3294–3308 127, (2014) Science Cell of Journal .LpdRes. Lipid J. Neuron 20) eai iaemtrto:aprilpoem finteracting of proteome partial a maturation: lipase Hepatic (2009). 385 20) he-iesoa tutr ftecomplexin/SNARE the of structure Three-dimensional (2002). 347-353. , u.J Neurol. J. 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Journal of Cell Science ioht,A,Nd,M n ioht,M. Y., Kinoshita, B. and Kim, M. A., Noda, J. A., Martina, Kinoshita, R., J. Rose, M., Boehm, Y., Kato, A. K., Toh-e, Janvier, and T. Oguchi, S., Okada, M., Iwase, K., Mizutani, I., Iwamoto, K., Sudo, N., Usuda, R., Morishita, K., Atsuzawa, R., H., Ito, Hikawa, A., Kitano, A., Tanigaki, A., T. Hagiwara, N., E. Yamasaki, Spiliotis, M., Ihara, and J. W. Nelson, Q., Hu, E. S. Russell, and A. P. Hall, e,D .adGlbr,A L. A. Goldberg, and H. D. Lee, M. Kinoshita, etay . tnar,R . ae .S,Co,S,Cry .J and J. E. Corey, S., Choi, Ferna S., W. Lane, F., R. Standaert, G., Fenteany, S. S. Arnon, and G. Xie, K., K. Hill, R., J. Barash, N., Dover, ARTICLE RESEARCH 3308 and H. Kimura, A., Kusumi, N., Morone, R., Hikawa, Y., Tanaka, A., Hagiwara, F. M. Krummel, and J. Gilden, Ferna otl M. Montal, lor,R,Tgoi . osto . eGads .adMneuc,C. Montecucco, and D. Grandis, De O., Rossetto, V., Tugnoli, R., Eleopra, atn,S n otcco C. Montecucco, and A., S. T. 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