Ukuhlela iiproteni kwindlela yokufihla kubalulekile ekugcineni ukwahlulwahlulwa kweeseli kunye ne-homeostasis. Ukongeza ekuhleleni okuqhutywa yigobolondo, indima yee-lipids ekuhleleni i-kinesin kwinkqubo yokuthutha i-secretory ngumbuzo osisiseko osele ukho ixesha elide ongekaphendulwa. Apha, senza umfanekiso we-3D ngaxeshanye we-multicolor high-resolution real-time ukuze sibonise kwi-vivo ukuba iiproteni ezisandula ukwenziwa ze-glycosylphosphatidylinositol-immobilized ezine-ceramide lipid moieties ezinde kakhulu ziqokelelwa kwaye zahlulwe zibe zii-endoplasms ezikhethekileyo. Indawo yokuphuma eNet, eyahlukileyo kuleyo isetyenziswa ziiproteni ze-transmembrane. Ukongeza, sibonisa ukuba ubude be-ceramide kwi-endoplasmic reticulum membrane bubalulekile kolu khetho lokucoca. Uphononongo lwethu lubonelela ngobungqina bokuqala obuthe ngqo kwi-vivo bokuhlukanisa imithwalo yeproteni ngokusekelwe kubude be-lipid chain kwiindawo zokuthumela ezikhethiweyo kwi-secretory pathway.
Kwiiseli ze-eukaryotic, iiproteni ezenziwe kwi-endoplasmic reticulum (ER) emva koko zihlelwa ngexesha lokuthuthwa ngendlela eyimfihlo ukuze zisiwe kwindawo yazo efanelekileyo yeseli (1). Ukongeza ekuhleleni okubangelwa yi-coat, bekucingelwa ixesha elide ukuba ii-lipids ezithile zinokusebenza njengeendawo zokuphuma ezikhethiweyo ngokuzihlanganisa kwiindawo ezithile ze-membrane kunye neeproteni ezithile (2-5). Nangona kunjalo, kusekho ukunqongophala kobungqina obuthe ngqo kwi-vivo bokungqina le ndlela inokwenzeka esekelwe kwi-lipid. Ukuze kusonjululwe le ngxaki isisiseko, sifunde kwimvubelo indlela iiproteni ezixhonyiweyo ze-glycosylphosphatidylinositol (GPI) (GPI-APs) ezithunyelwa ngayo ngokwahlukileyo kwi-ER. Ii-GPI-APs ziintlobo ngeentlobo zeeproteni zomphezulu weseli ezixhunyiwe kwi-lipid (6, 7). I-GPI-AP yiproteni ekhutshweyo enamathele kwiincwadana zangaphandle ze-plasma membrane nge-glycolipid moiety (GPI anchor). Ziyamkela ii-GPI anchors njengotshintsho oluhlala luhleli emva kokuguqulwa kwi-ER lumen (8). Emva kokuncamathiselwa, i-GPI-AP idlula kwisixhobo seGolgi (5, 9) ukusuka kwi-ER ukuya kwi-plasma membrane. Ubukho bee-GPI anchors bubangela ukuba i-GPI-AP ithuthwe ngokwahlukileyo kwiiproteni ezikhutshwe yi-transmembrane (kuquka nezinye iiproteni ze-plasma membrane) kwindlela ye-secretory (5, 9, 10). Kwiiseli ze-yeast, ii-GPI-APs zahlulwe kwezinye iiproteni ezikhutshwe kwi-endoplasmic reticulum, emva koko zipakishwe kwii-vesicles ezikhethekileyo ezigqunywe yi-coat protein complex II (COPII) (6, 7). Izinto ezibangela le nkqubo yokwahlulahlula kwinkqubo yokuthumela ngaphandle kwe-ER azicacanga, kodwa kucingelwa ukuba le ndlela inokufuna ii-lipids, ingakumbi ukuhlaziywa kwesakhiwo senxalenye ye-lipid ye-GPI anchor (5, 8). Kwi-yeast, ukuhlaziywa kwe-lipid ye-GPI kuqala kwangoko emva kokuba i-GPI inamathele, kwaye kwiimeko ezininzi, kubangela ukuba i-ceramide ibophe kwi-26-carbon long-chain fatty acid (C26:0) (11, 12). I-C26 ceramide yeyona ceramide iphambili eveliswa ziiseli zemvubelo ukuza kuthi ga ngoku. Yenziwe kwi-ER kwaye uninzi lwayo luthunyelwa kwisixhobo seGolgi nge-COPII vesicles (13). Ukuthunyelwa kwe-ER kwe-GPI-AP kufuna ngokukodwa ukwenziwa kwe-ceramide okuqhubekayo (14, 15), kwaye ke ngoko, ukuguqulwa kwe-ceramide ibe yi-inositol phosphate ceramide (IPC) kwisixhobo seGolgi kuxhomekeke kukwenziwa kwe-GPI anchor (16). Izifundo ze-Biophysical ngee-membranes zokwenziwa zibonise ukuba ii-ceramide ze-acyl chain ezinde kakhulu zinokudibana ukuze zenze ii-domains ezicwangcisiweyo ezineempawu zomzimba ezizodwa (17, 18). Olu lwazi lukhokelela kwingcamango yokuba i-C26 ceramide kunye ne-GPI-AP kunye ne-C26 ceramide zisebenzisa iimpawu zazo zomzimba ukudibanisa kwiindawo okanye imimandla ecwangcisiweyo kwindawo ye-ER membrane lipid engcolileyo. Iquka ikakhulu i-glycerolipids emfutshane nengagcwaliyo (C16:1 kunye ne-C18:1) (19, 20). Ezi ndawo ziza kugxila ngokukhethekileyo kwiindawo ezithile zokuphuma kwi-ER (ERES), apho i-ceramide kunye ne-ceramide-based GPI-AP zinokuthuthwa kunye ziye eGolgi kwi-COPII vesicle efanayo (5).
Kolu phononongo, sivavanye ngokuthe ngqo le ndlela isekelwe kwi-lipid ngokusebenzisa i-super-resolution confocal real-time imaging microscopy (SCLIM), eyinkqubo ye-microscopy yanamhlanje enokuqaphela ngaxeshanye iiproteni ezibhalwe nge-fluorescent. Imifanekiso enemibala emithathu kunye ne-three-dimensional (3D) inesisombululo esiphezulu kunye nesantya kwiiseli eziphilayo (21, 22).
Siqale sasebenzisa itekhnoloji ye-SCLIM ukuchaza ngakumbi indlela eqhelekileyo ye-GPI-AP eneqela le-C26 ceramide ehlolwe ngayo kwiiproteni ezikhutshwe yi-transmembrane emva kokuphuma kwi-ER kwi-S. cerevisiae. Ukuze sijonge udidi lwe-ER, sisebenzise inkqubo yemfuza enokuyibona ngokuthe ngqo impahla entsha ehlanganisiweyo ingena kwi-ERES in vivo (7, 23). Njengempahla, sikhethe i-C26 ceramide-based GPI-AP Gas1 ebhalwe ngeproteni eluhlaza okwesibhakabhaka (GFP) kunye neproteni ekhutshwe yi-transmembrane Mid2 ebhalwe ngeproteni ye-fluorescent ekufutshane ne-infrared (iRFP), zombini ezijolise kwi-plasma membrane (24–26). Kwi-mutant ebuthathaka kubushushu be-sec31-1, ezi mpahla zimbini zibonakaliswa phantsi kwe-promoter ye-galactose-inducible kunye ne-constitutive ERES marker. Kubushushu obugqithisileyo (37°C), kuba utshintsho lwe-sec31-1 luchaphazela umsebenzi we-COPII coat component Sec31 ukuthintela ukuhluma kwe-COPII kunye nokuthunyelwa kwe-ER, impahla entsha ehlanganisiweyo iqokelelana kwi-ER (23). Emva kokuba ipholile ukuya kutsho kubushushu obuphantsi (24°C), iiseli eziguqulweyo ze-sec31-1 zabuya kwindawo efihlakeleyo, kwaye umthwalo omtsha owenziweyo oqokelelweyo waqala ukuthunyelwa ngaphandle kwi-ER. Umboniso we-CLIM ubonise ukuba uninzi lwee-Gas1-GFP ezintsha kunye ne-Mid2-iRFP zisaqokelelene kwi-ER yeeseli eziguqulweyo ze-sec31-1 emva kokufakwa kwi-incubation kwi-37°C zaza zakhutshwa kwi-24°C imizuzu emi-5 (Umfanekiso 1). Ekubeni i-Mid2-iRFP isasazwa kwi-membrane yonke ye-ER, kwaye i-Gas1-GFP ixinene kwaye iqokelelwe kwindawo ye-membrane ye-ER engaqhubekiyo, usasazo lwazo lwahlukile ngokupheleleyo (Umfanekiso 1, A ukuya ku-C kunye ne-Movie S1). Ukongeza, njengoko kubonisiwe kuMfanekiso 1D, iqela le-Gas1-GFP alinayo i-Mid2-iRFP. Ezi ziphumo zibonisa ukuba iiproteni ze-GPI-AP kunye ne-transmembrane zahlulwe kwiindawo ezahlukeneyo ze-membrane ye-ER kwangethuba. Iqela leGas1-GFP likufutshane ne-ERES ethile ebhalwe nge-mCherry's COPII coat protein Sec13 (Umfanekiso 1, E kunye no-F, kunye ne-movie S1) (23).
Iiseli ze-sec31-1 ziveza ukukhutshwa okubangelwa yi-galactose, i-ceramide ende ye-acyl chain (C26) ye-ceramide ye-GPI-AP Gas1-GFP (GPI-AP, eluhlaza) kunye neprotheyini ye-transmembrane Mid2-iRFP (TMP, eluhlaza okwesibhakabhaka) kwaye le khowudi ye-Constructive ERES ene-Sec13-mCherry (ERES, magenta) yafakwa kwi-37°C kangangemizuzu engama-30, yafuduselwa kwi-24°C, yaza yathathwa yi-SCLIM emva kwemizuzu emi-5. (A ukuya ku-C) ibonisa umfanekiso odibeneyo okanye omnye we-2D wendiza (A), umfanekiso we-2D oqikelelweyo we-10 z-sections (B) okanye umfanekiso we-3D cell hemisphere wempahla kunye ne-ERES markers (C). I-scale bar 1μm (A kunye no-B). Iyunithi yesikali yi-0.551μm (C). I-Gas1-GFP yafunyanwa kwiindawo ze-ER okanye amaqela ahlukeneyo, ngelixa i-Mid2-iRFP yafunyanwa yaza yasasazwa kuyo yonke i-ER membrane (C). (D) Igrafu ibonisa ubunzulu be-fluorescence ye-Gas1-GFP kunye ne-Mid2-iRFP kwi-Gas1-GFP cluster ecaleni komgca wotolo omhlophe (ekhohlo). I-AU, iyunithi engaqhelekanga. (E kunye no-F) bamele umfanekiso we-3D odibanisa iimpahla kunye nophawu lwe-ERES. Ii-Gas1-GFP clusters zifunyenwe kufutshane ne-ERES ethile. Iyunithi yesikali yi-0.551μm. (F) Utolo oluqinileyo olumhlophe lubonisa iqela le-Gas1-GFP elinxulunyaniswa ne-ERES. Iiphaneli eziphakathi nasekunene zibonisa umfanekiso we-3D odibeneyo owandisiweyo kunye nombono ojikelezileyo weqela le-Gas1-GFP elikhethiweyo.
Ubudlelwane obusondeleyo phakathi kweqela leGas1-GFP kunye ne-ERES ethile bubonisa ukuba iGas1-GFP inokungena kwi-ERES ekhethiweyo, eyahlukileyo kukhetho olusetyenziswa yiMid2-iRFP ukushiya i-ER. Ukujongana nale meko, silinganise umlinganiselo we-ERES kwimpahla enye okanye ezimbini kuphela (Umfanekiso 2, A ukuya ku-C). Sifumanise ukuba uninzi lwe-ERES (70%) luqulathe uhlobo olunye kuphela lwempahla. Umfanekiso ongezantsi woMfanekiso 2C ubonisa imizekelo emibini eqhelekileyo ye-ERES eneGas1-GFP kuphela (Umfanekiso 1) okanye iMid2-iRFP kuphela (Umfanekiso 2). Ngokwahlukileyo koko, malunga ne-20% ye-ERES iqulathe imithwalo emibini edibana kwindawo enye. Kufunyenwe ukuba ezinye i-ERES (10%) ziqulathe iintlobo ezimbini zempahla, kodwa zazahlulwe kwiindawo ezahlukeneyo ngokucacileyo. Ke ngoko, olu hlalutyo lwezibalo lubonisa ukuba emva kokuba i-ER ithunyelwe ngaphandle, i-GPI-AP Gas1-GFP kunye nemithwalo ye-transmembrane Mid2-iRFP zahlulwe zaba yi-ERES ezahlukeneyo (Umfanekiso 2D). Olu buchule bokuhlela luhambelana kakhulu nohlalutyo lwangaphambili lwe-biochemical (6) kunye nokugqitywa kwesimo (7). Singabona nokuziphatha komthwalo ovalelwe ngaphakathi kwi-ERES (Umfanekiso 2E kunye neMovie S2). Umzobo 2E ubonisa ukuba yinxalenye encinci kuphela yeGas1-GFP (ipaneli 3) okanye iMid2-iRFP (ipaneli 4) engena kwi-ERES ukusuka kwelinye icala kwaye ivalelwe kwindawo ethe cwaka. Iphaneli 5 yoMfanekiso 2E ibonisa ukuba iGas1-GFP kunye neMid2-iRFP ngamanye amaxesha zifumaneka kwi-ERES efanayo, kodwa zingena kumacala ahlukeneyo kwaye zixinene kwiindawo ezahlukeneyo ezinokumela ii-vesicles ze-COPII ezahlukeneyo. Sikwaqinisekisile ukuba ukwahlulwa okubonwayo kunye nokuhlelwa kwe-C26 ceramide-based GPI-AP Gas1 njenge-ERES ekhethiweyo kucacile kuba enye i-transmembrane secretion cargo, i-GFP-tagged plasma membrane protein Axl2 (27), ebonisa ukuziphatha okufanayo neMid2-iRFP. (Umfanekiso S1 kunye neMovie S3). I-Axl2-GFP esandul’ ukwenziwa isasazwa nge-membrane ye-ER efana ne-Mid2-iRFP (Umfanekiso S1, A kunye no-B), kwaye ihambelana ne-Mid2-iRFP kwii-ERES ezininzi (Umfanekiso S1, B ukuya ku-D). Iiphaneli 1 kunye ne-2 zoMfanekiso 1. I-S1C ibonisa imizekelo emibini eqhelekileyo ye-ERES apho ii-transmembrane cargoes ezimbini zidibana khona. Kule meko, zombini iimpahla zingena kunye kwi-ERES (Umfanekiso S1E, iPhaneli 3 kunye neMovie S3).
Iiseli ze-sec31-1 eziveza ukukhutshwa kwe-galactose inducible, i-Gas1-GFP (GPI-AP, eluhlaza) kunye ne-Mid2-iRFP (TMP, luhlaza okwesibhakabhaka) kunye ne-constitutive ERES labels Sec13-mCherry (ERES, magenta) zibekwe kwi-37 Emva kokufukamela imizuzu engama-30 kwi-°C, tshintshela kwi-24 °C ukuze ukhulule ibhloko yokukhupha, kunye nomfanekiso nge-SCLIM emva kwemizuzu engama-20. (A ukuya ku-C) Imifanekiso emele i-2D projection (A; scale bar, 1μm) okanye imifanekiso ye-3D cell hemisphere (B kunye ne-C; scale unit, 0.456μm) yempahla kunye ne-10 z-sections eziphawulwe yi-ERES. Iphaneli esezantsi kwi-(B) kunye nephaneli kwi-(C) zibonisa imifanekiso ecutshungulweyo ukubonisa kuphela iimpahla ezikhoyo kwi-ERES (magenta) [Gas1-GFP (grey) kunye ne-Mid2-iRFP (blue light)]. (C) Utolo oluvulekileyo: I-ERES ithwala kuphela iqhekeza elinye lomthwalo (1 ukuya ku-4). Utolo olungwevu: I-ERES iqulethe umthwalo owahlulwe ngokwahlukeneyo (5). Utolo olumhlophe oluqinileyo: I-ERES equlethe umthwalo obekwe kwindawo enye. Ngezantsi: I-ERES enye ekhethiweyo iqulethe iGas1-GFP (1) okanye iMid2-iRFP (2) kuphela. Ibha yesikali, i-100 nm. (D) Ubungakanani befotomicrograph echazwe kwi-(C). Ipesenti ephakathi ye-ERES equlethe umthwalo omnye kuphela (iGas1-GFP okanye iMid2-iRFP), umthwalo owahlulwe ngokwahlukeneyo kunye nomthwalo ogqithanayo. Kwizilingo ezintathu ezizimeleyo, i-n=432 kwiiseli ezingama-54. Ibha yempazamo = SD. Uvavanyo lwe-t olungenamitya emibini. *** P = 0.0002. (E) Umfanekiso we-3D we-ERES ekhethiweyo yomthwalo owahlulwe ngokwahlukeneyo ophawulwe ngo-(C). I-Gas1-GFP (luhlaza) (3) okanye iMid2-iRFP (luhlaza okwesibhakabhaka) (4) ingena kwi-ERES (magenta) ukusuka kwelinye icala kwaye inqunyelwe kwindawo encinci ngaphakathi kwe-ERES. Ngamanye amaxesha, zombini ezi ntlobo zemithwalo zingena kwi-ERES (5) efanayo ukusuka kwicala elinye kwaye zivaleleke kwindawo ekwanti ngaphakathi kwe-ERES. Ibha yesikali, 100 nm.
Okulandelayo, sivavanye ingcamango yokuba i-long acyl chain ceramide (C26) ekhoyo kwi-ER membrane iqhuba ukuqokelelwa kunye nokuhlelwa kweGas1 kwi-ERES ekhethiweyo. Ngenxa yesi sizathu, sisebenzise uhlobo lwe-yeast oluguquliweyo i-GhLag1, apho ii-endogenous ceramide synthases ezimbini i-Lag1 kunye ne-Lac1 zatshintshwa yi-GhLag1 (i-Lag1 homolog yekotoni), nto leyo eyabangela uhlobo lwe-yeast olune-cell membrane uhlobo lwe-Ceramide olufutshane kunohlobo lwe-wild (Umfanekiso 3A) (28). Uhlalutyo lwe-Mass spectrometry (MS) lubonise ukuba kwii-wild-type strains, i-95% ye-ceramide iyonke yi-long very (C26) chain ceramide, ngelixa kwi-GhLag1, i-85% ye-ceramide inde kakhulu (C18 kunye ne-C16). ), yi-2% kuphela ye-ceramide ende kakhulu (C26) chain ceramide. Nangona ii-ceramides ze-C18 kunye ne-C16 zezona ceramides ziphambili ezifunyenweyo kwi-membrane ye-GhLag1 ukuza kuthi ga ngoku, uhlalutyo lwe-MS luqinisekisile ukuba i-anchor ye-GPI ye-Gas1-GFP echazwe kwi-strain ye-GhLag1 iqulethe i-ceramide ye-C26, efana ne-lipids yohlobo lwendalo. Umgangatho uyafana (Umzobo 3A) (26). Ke ngoko, oku kuthetha ukuba i-enzyme yokulungisa i-ceramide i-Cwh43 ikhetha kakhulu i-ceramide ye-C26, njengoko kubonisiwe kuMfanekiso 26, ibandakanya ngokukhethekileyo i-anchor ye-GPI evela kwinani elincinci le-ceramide ye-C26 kwi-strain ye-GhLag1. S2 (29). Nangona kunjalo, i-membrane yeseli ye-GhLag1 ngokusisiseko iqulethe i-ceramide ye-C18-C16 kuphela, ngelixa i-Gas1-GFP isenayo i-ceramide ye-C26. Le nyaniso yenza olu hlobo lube sisixhobo esifanelekileyo sokusombulula ingxaki yobude be-acyl chain ye-membrane ceramide kwi-ER. Indima ecingelwayo yeklasi kunye nokwahlula. Emva koko, siqale safunda ubuchule be-C26 Gas1-GFP bokuqokelelana kwiikluster kwi-GhLag1 kunye ne-allele ye-mutant evakalelwa bubushushu ye-sec31-1 nge-microscopy ye-fluorescence eqhelekileyo, apho kukho kuphela uthotho olude (C18-C16) kwi-ER membrane Ceramide (Umzobo 3). Siqaphele ukuba kwi-sec31-1, uninzi lwe-Gas1-GFP lwalugxile kwiikluster, ngelixa i-Gas1-GFP kwi-sec31-1 GhLag1 ene-ceramide ER membrane ende (C18-C16) yayingadityaniswanga kwaye yasasazwa kwi-ER membrane yonke. Ukuchaneka, kuba i-C26 ceramide-based clustering inxulumene kakhulu ne-ERES ethile (Umfanekiso 1), emva koko sihlolisise ukuba le nkqubo inokubandakanya na umsebenzi we-ER export protein mechanism. I-GPI-AP isebenzisa inkqubo ekhethekileyo ye-COPII yokuthumela ngaphandle kwe-ER, elawulwa ngokukhutheleyo yi-Ted1's structure remodeling yenxalenye ye-glycan ye-GPI anchor (30, 31). I-recombinant GPI-glycan emva koko iqatshelwa yi-transmembrane cargo receptor p24 complex, ethi yona ikhethe i-Lst1, eyi-isoform ethile ye-COPII cargo binding subunit enkulu i-Sec24, eyenza i-GPI-AP-rich COPII Vesicles iyimfuneko (31-33). Ke ngoko, sakhe i-double mutant edibanise ukususwa kwezi proteins zi-single (i-p24 complex component i-Emp24, i-GPI-glycan remodeling enzyme i-Ted1 kunye ne-COPII subunit ethile i-Lst1) kunye ne-sec31-1 mutant strain, kwaye sazifunda. Ngaba kunokwenzeka ukwenza i-Gas1-cluster GFP (Umfanekiso 3). Siqaphele ukuba kwi-sec31-1emp24Δ kunye ne-sec31-1ted1Δ, i-Gas1-GFP ayihlanganiswanga kwaye isasazwa kuyo yonke i-ER membrane, njengoko kubonwe ngaphambili kwi-sec31-1 GhLag1, ngelixa kwi-sec31-1lst1Δ, i-Gas1-GFP Njenge-sec31-1. Ezi ziphumo zibonisa ukuba ukongeza ekubeni kukho i-C26 ceramide kwi-membrane ye-ER, ukuhlanganiswa kwe-Gas1-GFP kufuneka kudibane ne-p24 complex, kwaye akudingi ukufunyanwa kwe-Lst1 ethile. Emva koko, sihlolisise ukuba ubude be-chain ye-ceramide kwi-membrane ye-ER bunokulawula ukubopha kwe-Gas1-GFP kwi-p24. Nangona kunjalo, sifumanise ukuba ukubakho kwe-C18-C16 ceramide kwi-membrane akuchaphazeli ii-GPI-glycans ezakhiwe ngokutsha yi-p24 complex (Imifanekiso S3 kunye ne-S4, A kunye ne-B) okanye ukubopha kwi-GPI-AP kunye nokuthumela ngaphandle i-GPI-AP. Fumana i-subtype ye-COPII Lst1 (Umfanekiso S4C). Ke ngoko, ukuhlanganiswa kwe-C26 ceramide-dependent clustering akudingi ukusebenzisana kweproteni neendlela ezahlukeneyo ze-ER export protein, kodwa kuxhasa indlela eyahlukileyo yokwahlula eqhutywa bubude be-lipid. Emva koko, sihlalutye ukuba ubude be-ceramide acyl chain kwi-membrane ye-ER bubalulekile na ekuhleleni ngempumelelo kwe-Gas1-GFP njenge-ERES ekhethiweyo. Ekubeni iGas1 kuhlobo lweGhLag1 ene-short-chain ceramide ishiya i-ER ize ingene kwi-plasma membrane (Umfanekiso S5), sikholelwa ukuba ukuba ukuhluzwa kuqhutywa bubude be-ceramide acyl chain, iGas1 kuhlobo lweGhLag1 inokudluliselwa kwenye indawo kwaye inqunyulwe. Iimpahla ze-ERES ezine-membrane efanayo.
(A) I-membrane yeseli yeGhLag1 ikakhulu iqulethe ii-ceramides ezimfutshane ze-C18-C16, ngelixa i-anchor ye-GPI yeGas1-GFP isenayo i-C26 IPC efanayo neeseli ze-wild-type. Ngaphezulu: uhlalutyo lobude be-acyl chain ye-ceramide kwi-membrane yeseli ye-wild-type (Wt) kunye ne-GhLag1p strains nge-mass spectrometry (MS). Idatha imele ipesenti ye-ceramide iyonke. Umyinge wezilingo ezintathu ezizimeleyo. Ibha yempazamo = SD. Uvavanyo lwe-t olungenamitya emibini. **** P ＜0.0001. Iphaneli esezantsi: Uhlalutyo lwe-MS lobude be-acyl chain ye-IPC ekhoyo kwi-anchor ye-GPI yeGas1-GFP (GPI-IPC) echazwe kwi-wild-type kunye ne-GhLag1p strains. Idatha imele ipesenti yesignali iyonke ye-IPC. Umyinge wezilingo ezizimeleyo ezintlanu. Ibha yempazamo = SD. Uvavanyo lwe-t olungenamitya emibini. ns, alubalulekanga. P = 0.9134. (B) Ii-micrograph ze-Fluorescence ze-sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ kunye neeseli ze-sec31-1lst1Δ ezibonisa i-Gas1-GFP ebangelwa yi-galactose zifakwe kwi-37°C kangangemizuzu engama-30 zaza zadluliselwa kwi-Enza i-microscopy ye-fluorescence eqhelekileyo emva kwama-24°C. Utolo olumhlophe: Iqela le-ER Gas1-GFP. Utolo oluvulekileyo: I-Gas1-GFP engadityaniswanga isasazwa kwi-membrane ye-ER yonke, ibonisa i-ER uphawu lwe-nuclear ring staining. I-Scale bar, 5μm. (C) Ubungakanani be-photomicrograph echazwe kwi-(B). Ipesenti ephakathi yeeseli ezinesakhiwo se-Gas1-GFP. Kwiimvavanyo ezintathu ezizimeleyo, iiseli ze-n≥300. I-Error bar = SD. Uvavanyo lwe-t olungenamitya emibini. **** P ＜0.0001.
Ukuze sisombulule le ngxaki ngokuthe ngqo, senze umfanekiso we-SCLIM we-Gas1-GFP kunye ne-Mid2-iRFP kwi-GhLag1 kunye ne-allele ye-mutant evakalelwa bubushushu ye-sec31-1 (Umfanekiso 4 kunye ne-Movie S4). Emva kokuba i-ER igcinwe kuma-37°C kwaye kamva yakhululwa kuma-24°C, uninzi lwe-Gas1-GFP entsha eyenziweyo ayizange ihlanganiswe kwaye isasazwe kuyo yonke i-membrane ye-ER, njengoko kubonwe zii-microscopes eziqhelekileyo (Umfanekiso 4, A kunye no-B). Ukongeza, ipesenti enkulu ye-ERES (67%) ibandakanya iintlobo ezimbini zempahla ebekwe kuyo (Umfanekiso 4D). Iiphaneli 1 kunye ne-2 ze-Figure 4C zibonisa imizekelo emibini eqhelekileyo ye-ERES ene-Gas1-GFP edibeneyo kunye ne-Mid2-GFP. Ukongeza, zombini iimpahla zaqokelelwa kwi-ERES efanayo (Umfanekiso 4E, iphaneli 3 kunye ne-movie S4). Ke ngoko, iziphumo zethu zibonisa ukuba ubude be-ceramide acyl chain kwi-membrane ye-ER bubungqina obubalulekileyo bokuhlanganiswa kunye nokuhlelwa kwe-ER protein.
Iiseli zeSec31-1 GhLag1 eziveza ukukhutshwa kwe-galactose, iGas1-GFP (GPI-AP, eluhlaza) kunye neMid2-iRFP (TMP, luhlaza okwesibhakabhaka) kunye ne-Sec13-mCherry (ERES, magenta) ene-ERES ebhalwe yi-Sec13-mCherry (ERES, magenta) efakwe kwi-37°C. Qhubeka imizuzu engama-30, yehla uye kwi-24°C ukuze ukhuphe ukukhutshwa, kwaye umfanekiso nge-SCLIM emva kwemizuzu engama-20. (A ukuya ku-C) Imifanekiso emele i-2D projection (A; scale bar, 1μm) okanye imifanekiso ye-3D cell hemisphere (B kunye ne-C; scale unit, 0.45μm) ye-10 z-sections eziphawulwe yi-cargo kunye ne-ERES. Iphaneli esezantsi kwi-(B) kunye nephaneli kwi-(C) zibonisa imifanekiso ecutshungulweyo ukubonisa kuphela iimpahla ezikhoyo kwi-ERES (magenta) [Gas1-GFP (grey) kunye neMid2-iRFP (blue light)]. (C) Utolo olumhlophe oluzaliswe yi-ERES: ERES, iimpahla ziyadibana. Utolo oluvulekileyo: I-ERES iqulethe into enye kuphela. Iphaneli esezantsi: I-ERES ekhethiweyo inempahla ehambelanayo (1 kunye no-2) ephawulwe kwi (C). Ibha yesikali, yi-100 nm. (D) Ubungakanani befotomicrograph echazwe kwi (C). Kwiyunithi ze-sec31-1 kunye ne-sec31-1 GhLag1, kuqukwe umthwalo omnye kuphela (iGas1-GFP okanye iMid2-iRFP), kunye nepesenti ephakathi ye-ERES yempahla ehlukanisiweyo kunye nempahla ehambelanayo. Kwizilingo ezintathu ezizimeleyo, i-n = 432 kwiiseli ezingama-54 (sec31-1) kunye ne-n = 430 kwiiseli ezingama-47 (sec31-1 GhLag1). Ibha yempazamo = SD. Uvavanyo lwe-t olungenamitya emibini. *** P = 0.0002 (sec31-1) kunye ** P = 0.0031 (sec31-1 GhLag1). (E) Umfanekiso we-3D we-ERES ekhethiweyo enempahla ehambelanayo (3) ephawulwe kwi (C). I-Gas1-GFP (eluhlaza) kunye ne-Mid2-iRFP (eluhlaza okwesibhakabhaka) zisondela kwi-ERES (magenta) ukusuka kwicala elinye kwaye zihlala kwindawo enye enomda we-ERES. Ibha yesikali, yi-100 nm.
Olu phononongo lubonelela ngobungqina obuthe ngqo kwi-vivo bokuba imithwalo yeproteni esekwe kwi-lipid ihlulwe kwiindawo ezikhethiweyo zokuthumela ngaphandle kwindlela yokufihla, kwaye ityhila ukubaluleka kobude be-acyl chain ekukhetheni ukwahlulahlula. Sisebenzisa indlela enamandla nephambili ye-microscopy ebizwa ngokuba yi-SCLIM, sibonise i-Gas1-GFP entsha eyenziweyo (i-plasma membrane enkulu ye-GPI-AP ene-acyl chain ende kakhulu (C26) ceramide lipid portion) kwi-yeast) Iindawo eziqokelelwe kwi-disrect ERs zidibene ne-ERES ethile, ngelixa iiproteni ezikhutshwe kwi-transmembrane zisasazwa kulo lonke i-ER membrane (Umfanekiso 1). Ukongeza, ezi ntlobo zimbini zeempahla zingena kwi-ERES ezahlukeneyo ngokukhetha (Umfanekiso 2). Ubude be-acyl chain ye-ceramide yeselula kwi-membrane buncitshisiwe ukusuka kwi-C26 ukuya kwi-C18-C16, iqela le-Gas1-GFP liphazanyiswa kummandla we-ER ohlukeneyo, kwaye i-Gas1-GFP itshintshelwa kwenye indawo ukuze ishiye i-ER ne-transmembrane protein nge-ERES efanayo (Umfanekiso 3 kunye nomfanekiso 3). 4).
Nangona i-GPI-AP isebenzisa indlela ekhethekileyo yeproteni ukuphuma kwi-ER, sifumanise ukuba ukwahlulwahlulwa okuxhomekeke kwi-C26 ceramide akuxhomekanga kwiindlela ezahlukeneyo zeproteni ezinokukhokelela kwi-ERES specialization (Imifanekiso S4 kunye ne-S5). Endaweni yoko, iziphumo zethu zixhasa indlela eyahlukileyo yokwahlulahlula eqhutywa kukuhlanganiswa kweproteni esekelwe kwi-lipid kunye nokukhutshwa okulandelayo kwezinye izinto ezithwala imithwalo. Izinto esizibonileyo zibonisa ukuba ummandla weGas1-GFP okanye iqela elinxulunyaniswa ne-ERES ethile alinayo iproteni ekhutshwe yi-transmembrane Mid2-iRFP, nto leyo ebonisa ukuba iqela le-C26 ceramide-dependent GPI-AP liya kwenza kube lula ukungena kwabo kwi-ERES efanelekileyo, kwaye kwangaxeshanye, likhuphe i-transmembrane. Izinto ezikhutshwayo zingena kule ERES ithile (Imifanekiso 1 kunye ne-2). Ngokwahlukileyo koko, ukubakho kwe-C18-C16 ceramide kwi-ER membrane akubangeli ukuba i-GPI-AP yenze imimandla okanye amaqela, ngoko azikhupheli okanye zithathe indawo yeeproteni ezikhutshwe yi-transmembrane kwi-ERES efanayo (Imifanekiso 3 kunye ne-4). Ngoko ke, sicebisa ukuba i-C26 ceramide iqhube ukwahlukana kunye nokuhlelwa ngokulungiselela ukuhlanganiswa kweeproteni ezidityaniswe ne-ERES ethile.
Ungayifezekisa njani le C26 ceramide-dependent clustering kwindawo ethile ye-ER? Ukuthambekela kwe-membrane ceramide ukwahlukana ecaleni kunokubangela ukuba i-GPI-AP kunye ne-C26 ceramide zenze ii-lipids ezincinci nezilandelelaniswe ngoko nangoko kwindawo engaqhelekanga ye-lipid ye-membrane ye-ER equlethe i-glycerolipids ezimfutshane nezingenamanzi. Ii-Quality clusters (17, 18). Ezi qela zincinci zexeshana zinokudityaniswa ngakumbi zibe zii-clusters ezinkulu nezizinzileyo emva kokubopha kwi-p24 complex (34). Ngokuhambelana noku, sibonise ukuba i-C26 Gas1-GFP kufuneka isebenzisane ne-p24 complex ukuze yenze ii-clusters ezinkulu ezibonakalayo (Umfanekiso 3). I-p24 complex yi-heterozygous oligomer eyenziwe ziiproteni ezine ezahlukeneyo ze-p24 transmembrane kwi-yeast (35), ebonelela nge-multivalent binding, enokukhokelela ekudibaniseni ii-clusters ezincinci ze-GPI-AP, ngaloo ndlela ivelise i-Stable cluster enkulu (34). Ukusebenzisana phakathi kwe-ectodomains yeprotein ye-GPI-APs kunokubangela ukuhlanganiswa kwazo, njengoko kubonisiwe ngexesha lokuthuthwa kwazo yiGolgi kwiiseli ze-epithelial ezi-mammalian polarized (36). Nangona kunjalo, xa i-C18-C16 ceramide ikhona kwi-ER membrane, xa i-p24 complex ibopha kwi-Gas1-GFP, amaqela amakhulu ahlukeneyo akayi kwakheka. Indlela esisiseko inokuxhomekeka kwiimpawu ezithile zomzimba nezekhemikhali ze-long acyl chain ceramide. Izifundo ze-Biophysical zee-artificial membranes zibonisa ukuba nangona zombini ii-acyl chain ceramide ezinde (C24) nezimfutshane (C18-C16) zinokubangela ukwahlukana kwesigaba, kuphela ii-long acyl chain ceramide (C24) ezinokukhuthaza i-Curvature ephezulu kunye ne-film bending ukuze ziphinde zenze ifilimu. Ngokubhekiselele kumacala omabini (17, 37, 38). Kuye kwaboniswa ukuba i-transmembrane helix ye-TMED2, i-homologue yomntu ye-Emp24, isebenzisana ngokukhetha ne-sphingomyelin esekwe kwi-ceramide ye-C18 kwi-cytoplasmic lobules (39). Sisebenzisa ii-molecular dynamics (MD) simulations, sifumanise ukuba zombini ii-ceramide ze-C18 kunye ne-C26 ziqokelelana zijikeleze ii-cytoplasmic lobules ze-Emp24 transmembrane helix, kwaye zinokhetho olufanayo (Umfanekiso S6). Kubalulekile ukuqaphela ukuba oku kubonisa ukuba i-transmembrane helix ye-Emp24 inokukhokelela ekusasazweni okungalinganiyo kwee-lipids kwi-membrane. Esi sisiphumo samva nje esisekelwe kwiiseli zezilwanyana ezincancisayo. Ii-MD simulations ezifanayo zibonisa nokubakho kwee-ether lipids (40). Ke ngoko, sicinga ukuba i-C26 ceramide kwi-lobules ezimbini ze-ER26 ityebile apha ekhaya. Xa i-GPI-AP kwi-luminal lobules ibopha ngokuthe ngqo kwi-multivalent p24 kwaye ukuqokelelwa kwe-C26 ceramide ejikeleze i-p24 kwi-cytoplasmic lobules, inokukhuthaza ukuhlanganiswa kweProtein kunye nokugoba kwe-membrane kwenziwa ngeminwe (41), nto leyo ebangela ukuba i-GPI-AP yahlulwe ibe ziingingqi ezahlukeneyo ezikufutshane ne-ERES, nto leyo ekwathanda iindawo ezigobileyo kakhulu ze-ER membrane (42). Iingxelo zangaphambili zixhase indlela ecetywayo (43, 44). Ukubopha kwe-oligolectins, ii-pathogens okanye ii-antibodies kwi-ceramide-based glycosphingolipids (GSL) kwi-plasma membrane kubangela ukuhlanganiswa kwe-GSL enkulu, kuphucula ukwahlukana kwesigaba kwaye kubangele ukuguqulwa kwe-membrane kunye nokufakwa ngaphakathi (44). Iwabuchi njl. (43) Kufunyenwe ukuba xa kukho imixokelelwane ye-acyl ende (C24) kodwa engekho mfutshane (C16), i-ligand ene-multivalent ebotshelelwe kwi-GSL lactosylceramide yabangela ukwakheka kwamaqela amakhulu kunye nokungena kwe-membrane, kwaye i-cytoplasm Lyn-mediated signal transduction kwiincwadana idityaniswe yimixokelelwane ye-acyl kwii-neutrophils ezidibeneyo.
Kwiiseli ze-epithelial ezi-mammalian polarized, ukuxinana kwenethiwekhi ye-anti-Golgi (TGN) ukuya kwinqanaba le-apical plasma membrane kulawula ukwahlukana kunye nokuhlelwa kwe-GPI-AP (10, 45). Olu kuhlanganiswa luqhutywa yi-GPI-AP oligomerization (36), kodwa kunokuxhomekeka kubude be-ceramide chain esibufumana kwi-yeast. Nangona i-GPI-AP ye-mammalian ine-ether lipid-based anchor, kwaye isakhiwo sayo seekhemikhali sahlukile kakhulu kwi-acyl chain ceramide ende kakhulu, uphando lwakutshanje lufumanise ukuba zombini ii-lipids zineempawu zomzimba kunye neekhemikhali ezifanayo ngokwendalo kunye nomsebenzi (40). Ke ngoko, inxalenye ye-ether lipid kwiiseli ze-mammalian inokufana ne-C26 ceramide kwi-yeast, kwaye indima yayo kukudibanisa ne-long-chain ceramide kwi-membrane ukukhuthaza ukuhlanganiswa kunye nokuhlelwa kwe-GPI-AP. Nangona oku kunokwenzeka kusafuneka kuvavanywe ngokuthe ngqo, iziphumo zangaphambili zixhasa ukuba ukuthuthwa kwe-long acyl chain ceramide ukuya emzimbeni weGolgi akwenziwa ziiproteni zokudlulisa i-cytoplasmic, kodwa kuxhomekeke ekuhlanganisweni kwee-anchors ze-GPI ezifana ne-yeast. Ke ngoko, indlela yokuguqula izinto ngokuqhubekayo ibonakala ikwazi ukuthutha ngokukhetha i-long acyl chain ceramide kunye ne-GPI-AP (13, 16, 20, 46, 47) kwi-vesicle efanayo yokuthutha.
Kwiinkqubo zeeseli ze-epithelial ze-yeast kunye ne-mammalian polarized, ukuhlanganiswa kwe-GPI-AP kunye nokwahlukana kwezinye iiproteni ze-plasma membrane konke kwenzeka ngaphambi kokuba kufike kumphezulu weseli. UPaladino et al. (48) bafumanise ukuba kwi-TGN yeeseli ze-epithelial ze-mammalian polarized, ukuhlanganiswa kwe-GPI-AP akufuneki kuphela ekuhleleni ii-GPI-APs kwi-apical plasma membrane, kodwa kulawula nokuhlelwa kwe-GPI-APs kunye nomsebenzi wayo webhayoloji. Umphezulu weseli. Kwi-yeast, olu phononongo lubonise ukuba iqela le-GPI-AP elixhomekeke kwi-C26 ceramide kwi-ER linokulawula ukuhlelwa kweqela kunye nomsebenzi osebenzayo we-GPI-AP kwi-plasma membrane (24, 49). Ngokuhambelana nale modeli, iiseli ze-GhLag1 zi-allergy kwi-GPI inhibitors okanye iziyobisi ezichaphazela ukuthembeka kodonga lweseli (28), kwaye imfuneko yeqela le-Gas1-GFP elisebenzayo (49) le-tip ceramide eqikelelweyo ekudibaneni kweeseli ze-yeast ibonisa i-G​​ Iziphumo ezinokwenzeka zomzimba zeeseli ze-hLag1. Impazamo ye-GPI-AP. Nangona kunjalo, ukuvavanya ngakumbi ukuba ulungelelwaniso olusebenzayo lomphezulu weseli lucwangcisiwe kwi-ER ngendlela yokuhlela ngokusekelwe kubude be-lipid kuya kuba ngumxholo wophando lwethu lwexesha elizayo.
Iintlobo zeSaccharomyces cerevisiae ezisetyenzisiweyo kulo msebenzi zidweliswe kwiTheyibhile S1. Iintlobo zeMMY1583 kunye neMMY1635 zeSCLIM zomfanekiso weeseli eziphilayo zakhiwe ngasemva kweW303. Ezi ntlobo ziveza iSec13-mCherry enethegi yeprotheyini ekhanyayo zakhiwe kusetyenziswa indlela esekelwe kwipolymerase chain reaction (PCR) ene-pFA6a plasmid njengetemplate (23). Uhlobo oluveza iMid2-iRFP olubhalwe ngeprotheyini ekhanyayo phantsi kolawulo lwe-GAL1 promoter lwakhiwe ngolu hlobo lulandelayo. Ukwandiswa kwePCR kolandelelwano lwe-iRFP-KanMx oluvela kwi-pKTiRFP-KAN vector (isipho se-E. O'Shea, inombolo ye-Addgene plasmid 64687; http://n2t.net/addgene: 64687; isazisi sezixhobo zophando (RRID): Addgene_64687) Kwaye lufakwe kwi-C-terminus ye-endogenous Mid2. Emva kokuba ulandelelwano lwe-genome ye-Mid2-iRFP lwandisiwe lwaza lwadityaniswa kwi-promoter ye-GAL1, lwadityaniswa kwindawo ye-Not I-Sac I ye-integration plasmid pRS306. I-plasmid pRGS7 eyaphumayo yahlengahlengiswa nge-Pst I ukuze idityaniswe kwindawo ye-URA3.
I-Gas1-GFP fusion gene ibonakaliswa phantsi kolawulo lwe-GAL1 promoter kwi-centromere (CEN) plasmid, eyakhiwe ngolu hlobo lulandelayo. Ulandelelwano lwe-Gas1-GFP lwandiswe yi-PCR kwi-pRS416-GAS1-GFP plasmid (24) (isipho sika-L. Popolo) kwaye yahlanganiswa kwindawo ye-Xma I–Xho I ye-CEN plasmid pBEVY-GL LEU2 (isipho sika-C). Miller; Addgene plasmid number 51225; http://n2t.net/addgene: 51225; RRID: Addgene_51225). I-plasmid ephumayo yabizwa ngokuba yi-pRGS6. I-Axl2-GFP fusion gene ikwabonakaliswa phantsi kolawulo lwe-GAL1 promoter ye-pBEVY-GL LEU2 vector, kwaye ukwakhiwa kwayo kulandelayo. Ulandelelwano lwe-Axl2-GFP lwandisiwe kwi-plasmid ye-pRS304-p2HSE-Axl2-GFP (23) yi-PCR, yaza yafakwa kwindawo ye-Bam HI-Pst I ye-vector ye-pBEVY-GL LEU2. I-plasmid ephumayo yabizwa ngokuba yi-pRGS12. Ulandelelwano lwee-oligonucleotides ezisetyenzisiweyo kolu phononongo ludweliswe kwiTheyibhile S2.
Olu hlobo longezelelwa nge-0.2% adenine kunye ne-2% glucose [YP-dextrose (YPD)], i-2% raffinose [YP-raffinose] rich yeast extract protein p (YP) medium (1 % Yeast extract kunye ne-2% protein ept). (YPR)] okanye i-2% galactose [YP-galactose (YPG)] njengomthombo wekhabhoni, okanye kwi-synthetic minimal medium (0.15% yeast nitrogen base kunye ne-0.5% ammonium sulfate) ukongeza ii-amino acids ezifanelekileyo kunye neziseko ezifunekayo kwisondlo, kwaye ziqulathe i-2% glucose (synthetic glucose minimal medium) okanye i-2% galactose (synthetic galactose minimal medium) njengomthombo wekhabhoni.
Kwimifanekiso yexesha langempela, iiseli eziguqulweyo ze-sec31-1 ezibuthathaka kubushushu eziveza ulwakhiwo phantsi kwe-GAL1 promoter zikhuliswe kwindawo ephakathi ye-YPR kwi-24°C ubusuku bonke ukuya kwisigaba esiphakathi. Emva kokungeniswa kwi-YPG kwi-24°C kangangeyure e-1, iiseli zafakwa kwi-SG kwi-37°C imizuzu engama-30, zaze zadluliselwa kwi-24°C ukuze zikhutshwe kwi-secretion block. I-Concanavalin A yasetyenziselwa ukulungisa iiseli kwi-slide yeglasi kwaye yathathwa yi-SCLIM. I-SCLIM yindibaniselwano ye-Olympus IX-71 inverted fluorescence microscope kunye ne-UPlanSApo 100×1.4 numerical aperture oil lens (Olympus), i-high-speed kunye ne-high-signal-to-noise ratio rotating disc confocal scanner (Yokogawa Electric), i-custom spectrometer, kunye ne-custom cooling. I-image intensifier yenkqubo (iHamamatsu Photonics) inokubonelela ngenkqubo yelensi yokukhulisa ngokukhulisa kokugqibela kwe-×266.7 kunye nekhamera yesixhobo esidibeneyo netshaja ephindaphinda ii-electron (iHamamatsu Photonics) (21). Ukufunyanwa komfanekiso kwenziwa yisoftware eyenzelwe wena (iYokogawa Electric). Kwimifanekiso ye-3D, sisebenzise i-piezoelectric actuator eyenzelwe wena ukushukumisa ilensi ejolise ngokuthe nkqo, kwaye saqokelela iindawo ezibonakalayo eziyi-100 nm ngokwahlukeneyo kwi-stack. Umfanekiso we-Z-stack uguqulwa ube yidatha ye-voxel ye-3D, kwaye umsebenzi we-theory point spread osetyenziselwa i-rotary disc confocal microscope usetyenziselwa ukucubungula i-deconvolution yi-Volocity software (PerkinElmer). Ngokusebenzisa isoftware yeVolocity ukuze ifikelele ngokuzenzekelayo kuhlalutyo lwendawo enye, i-ERES kuquka nempahla ilinganisiwe. Uhlalutyo lwe-line scan lwenziwe kusetyenziswa isoftware yeMetaMorph (iiMolecular Devices).
Sebenzisa isoftware yeGraphPad Prism ukuze ubone ukubaluleka kwezibalo. Kuvavanyo lwe-t lomfundi olunemisila emibini kunye novavanyo oluqhelekileyo lwendlela enye yohlalutyo lokungafani (ANOVA), umahluko phakathi kwamaqela uthathwa njengonempembelelo enkulu kwi-P <0.05 (*).
Kwi-fluorescence microscopy yeGas1-GFP, iiseli ze-log phase zakhuliswa ngobusuku bonke kwi-YPD zaze zaqokelelwa nge-centrifugation, zahlanjwa kabini nge-phosphate buffered saline, zaza zafakwa kwi-incubator ubuncinane imizuzu eli-15, zaza zaqhubeka phantsi kwe-microscope njengoko kuchaziwe ngaphambili. Jonga (24). I-Leica DMi8 microscope (HCX PL APO 1003/1.40 oil PH3 CS) ixhotyiswe ngelensi ejolise kuyo, isihluzi se-L5 (GFP), ikhamera yeHamamatsu kunye nesoftware ye-Application Suite X (LAS X) yasetyenziswa ekufumaneni.
Iisampulu zahluzwa nge-SDS sample buffer kwi-65°C kangangemizuzu eli-10, zaza zahlulwa nge-SDS-polyacrylamide gel electrophoresis (PAGE). Uhlalutyo lwe-immunoblotting, i-10 μl yesampulu yafakwa kumzila ngamnye. I-antibody ephambili: Sebenzisa i-rabbit polyclonal anti-Gas1 kwi-dilution ye-1:3000, i-rabbit polyclonal anti-Emp24 kwi-dilution ye-1:500, kunye ne-rabbit polyclonal anti-GFP (isipho esivela ku-H. Riezman) kwi-dilution ye-1:3000. I-antibody yegundane ye-monoclonal anti-Pgk1 yasetyenziswa kwi-dilution ye-1:5000 (isipho esivela ku-J. de la Cruz). I-antibody yesibini: I-Horseradish peroxidase (HRP) conjugated goat anti-rabbit immunoglobulin G (IgG) esetyenziswe kwi-dilution ye-1:3000 (Pierce). I-IgG yebhokhwe echasene ne-HRP edityaniswe ne-HRP isetyenzisiwe xa ixutywa yi-1:3000 (Pierce). Indawo yokusabela komzimba ibonwe ngendlela ye-chemiluminescence ye-SuperSignal West Pico reagent (Thermo Fisher Scientific).
Njengoko kuchaziwe kwi (31), uvavanyo lwendalo lwe-immunoprecipitation lwenziwe kwi-ER fraction etyebisiweyo. Ngamafutshane, hlamba ii-yeast cells nge-TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride kunye nomxube we-protease inhibitor) kwi-600 nm (OD600) kwi-100 optical density kabini. Yaphulwa ngee-glass beads, emva koko ii-debris zeseli kunye nee-glass beads zasuswa nge-centrifugation. I-supernatant emva koko yafakwa kwi-centrifuge kwi-17,000 g imizuzu eli-15 kwi-4°C. I-pellet yaphinda yaxhonywa kwi-TNE kwaye i-digitalis saponin yongezwa kuxinzelelo lokugqibela lwe-1%. I-suspension yafakwa kwi-incubation iyure e-1 ngokujikeleza kwi-4°C, kwaye emva koko izinto ezinganyibilikiyo zasuswa nge-centrifugation kwi-13,000 g kwi-4°C imizuzu engama-60. Kwi-Gas1-GFP immunoprecipitation, qala ngokufunxa isampuli ngee-empty agarose beads (ChromoTek) kwi-4°C kangangeyure e-1, uze emva koko ufunxe nge-GFP-Trap_A (ChromoTek) kwi-4°C kangangeeyure ezi-3. Ii-immunoprecipitated beads zihlanjwe izihlandlo ezihlanu nge-TNE equlethe i-0.2% digoxigenin, zahlulwe nge-SDS sample buffer, zahlulwe kwi-SDS-PAGE, zaza zahlalutywa nge-immunoblotting.
Njengoko kuchaziwe kwi-(31), ukuqinisekiswa kokudibanisa kwenziwa kwi-fraction ye-ER etyetyisiweyo. Ngamafutshane, i-fraction ye-ER etyetyisiweyo yafakwa kwi-0.5 mM dithiobis(succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, USA; 20°C, 20 min). Impendulo yokudibanisa yacinywa ngokongeza i-glycine (50 mM final concentration, 5 minutes, 20°C).
Njengoko kuchaziwe ngaphambili (50), uhlalutyo lwe-MS lwe-ceramide kwiintlobo ze-wild-type kunye ne-GhLag1 lwenziwe. Ngamafutshane, iiseli zikhule zaya kwi-exponential phase (3 ukuya kwi-4 OD600 units/ml) kwi-YPD kwi-30°C, kwaye iiseli ezingama-25×107 zavunwa. I-metabolism yazo iyacinywa nge-trichloroacetic acid. Sebenzisa i-extraction solvent [ethanol, water, ether, pyridine kunye ne-4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] kunye ne-1.2 nmol yomgangatho wangaphakathi we-C17 ceramide (860517, Avanti polar lipid)). Sebenzisa i-monomethylamine reagent [methanol, water, n-butanol kunye ne-methylamine solution (4:3:1:5 v/v)] ukwenza i-alkaline hydrolysis encinci ye-extract, uze usebenzise i-water-saturated n-butanol ukususa ityuwa. Ekugqibeleni, isicatshulwa saphinda saxutywa kwi-solvent ye-positive mode [chloroform/methanol/water (2:7:1) + 5 mM ammonium acetate] saza safakwa kwi-mass spectrometer. Ukubeka iliso kwi-Multi-reaction (MRM) kwenziwa ukuze kuchongwe kwaye kulinganiswe iimolekyuli ze-sphingolipid. I-TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) ixhotyiswe nge-robotic nanoflow ion source Nanomate HD (Advion Biosciences, Ithaca, NY) yohlalutyo lwe-lipid. Amandla okungqubana alungiselelwe udidi ngalunye lwe-ceramide. Idatha ye-MS ifunyenwe kwi-positive mode. Kwi-replication nganye yebhayoloji, isignali ye-lipid yi-median yemilinganiselo emithathu ezimeleyo.
Njengoko kuchaziwe kwi-(31), iiseli (800×107) eziveza iGas1-GFP zafakwa kwi-immunoprecipitation yendalo. IGas1-GFP ecociweyo yahlulwa yi-SDS-PAGE yaza yadluliselwa kwi-membrane ye-polyvinylidene fluoride (PVDF). Iproteni yabonwa ngokufaka i-PVDF i-amide black. Ibhendi yeGas1-GFP yanqunyulwa kwi-PVDF yaza yahlanjwa izihlandlo ezi-5 nge-methanol kwaye kanye ngamanzi e-liquid chromatography-MS (LC-MS). Ngokufaka i-membrane strip nge-500μl 0.3 M NaOAc (pH 4.0), i-buffer kunye ne-500μl esandul’ ukunyibilikiswa yi-1 M sodium nitrite mixture kwi-37°C kangangeeyure ezi-3, i-lipid fraction ikhutshwa kwi-Gas1-GFP kwaye i-lysed. Ukukhululwa kwe-inosine phosphate ceramide phakathi kwe-glucosamine kunye ne-inositol (51). Emva koko, umcu we-membrane wahlanjwa izihlandlo ezine ngamanzi e-LC-MS grade, womiswa kubushushu begumbi, waza wagcinwa kwindawo ene-nitrogen kwi--80°C de kube luhlalutyo. Njengolawulo, kwasetyenziswa isampulu engenanto ye-PVDF membrane kwisilingo ngasinye. I-lipid ekhutshwe kwi-Gas1-GFP yahlaziywa yi-MS njengoko kuchaziwe (50). Ngamafutshane, imicu ye-PVDF equlethe i-GPI-lipid yaphinda yaxhonywa kwi-75μl negative mold solvent [chloroform/methanol (1:2) + 5 mM ammonium acetate] yaza yadlula kwi-electrospray ionization (ESI)-MRM/MS Analysis of sphingolipid species (TSQ Vantage). Kule meko, idatha ye-MS ifunyenwe kwi-negative ion mode.
Njengoko bekutshiwo ngaphambili, inxalenye ye-lipid ye-anchor ye-GPI yahlulwe kwi-[3H]-inositol-labeled GPI-AP (16). Ii-lipid zahlulwe nge-chromatography enomgangatho omncinci kusetyenziswa inkqubo ye-solvent (55:45:10 chloroform-methanol-0.25% KCl) kwaye zabonwa kusetyenziswa i-FLA-7000 (Fujifilm).
Iiseli eziveza iGas1-GFP (600×107) zihlanjwe kabini nge-TNE buffer ene-TNE buffer, zaza zaphulwa ngeeglasi beads, zaza zafakwa kwi-centrifuge ukususa ukungcola kweeseli kunye neeglasi beads. I-supernatant emva koko yafakwa kwi-centrifuge kwi-17,000 g iyure e-1 kwi-4°C. I-pellet yahlanjwa kwi-TNE yaza yafakwa kwi-1 U PI-PLC (Invitrogen) kwi-TNE equlethe i-0.2% digitalis saponin iyure e-1 kwi-37°C. Emva konyango lwe-enzyme, i-membrane yasuswa nge-centrifuge kwi-17,000 g kwi-4°C iyure e-1. Ukuze i-immunoprecipitate Gas1-GFP, i-supernatant yafakwa kwi-GFP-Trap_A (ChromoTek) kwi-4°C ubusuku bonke. I-Gas1-GFP ecociweyo eyahlulwe yi-SDS-PAGE yadaywa nge-Coomassie brilliant blue. Ibhendi yokudaya yeGas1-GFP yanqunyulwa kwimpunga ejikeleze umjelo wamanzi, emva koko emva kokuxubana ne-iodoacetamide kunye nokunciphisa nge-dithiothreitol, kwenziwa ukugaya kwi-gel nge-trypsin. I-Extract kunye ne-tryptic peptides kunye ne-peptides zomile nge-GPI-glycans. I-peptide eyomileyo yanyibilikiswa kwi-20 μl yamanzi. Faka inxalenye (8μl) kwi-LC. Ikholamu ye-octadecylsilane (ODS) (Develosil 300ODS-HG-5; ububanzi bangaphakathi obuyi-150 mm×1.0 mm; Nomura Chemical, Aichi Prefecture, eJapan) yasetyenziswa ukwahlula ii-peptides phantsi kweemeko ezithile ze-gradient. Isigaba esihambayo yi-solvent A (0.08% formic acid) kunye ne-solvent B (0.15% formic acid kwi-80% acetonitrile). Inkqubo ye-Accela HPLC (iThermo Fisher Scientific, eBoston, eMassachusetts) yasetyenziswa ukususa ikholam nge-solvent A kwimizuzu engama-55 ngesantya sokuhamba se-50 μl min-1 imizuzu emi-5, kwaye emva koko uxinzelelo lwe-solvent B lwandiswe ukuya kwi-40%., eMelika). I-eluate yangeniswa rhoqo kumthombo we-ESI ion, kwaye ii-tryptic peptides kunye nee-peptides ezine-GPI-glycans zahlalutywa yi-LTQ Orbitrap XL (i-hybrid linear ion trap-orbitrap mass spectrometer; iThermo Fisher Scientific). Kwi-MS setup, i-voltage yomthombo we-capillary yayimiselwe kwi-4.5 kV, kwaye ubushushu be-transfer capillary bugcinwe kwi-300°C. I-capillary voltage kunye ne-tube lens voltage zazimiselwe kwi-15 V kunye ne-50 V, ngokwahlukeneyo. Idatha ye-MS ifunyenwe kwimo ye-ion e-positive (isisombululo se-60,000; ukuchaneka kobunzima be-10 parts per million) kuluhlu lobunzima be-300/m/z mass/charge ratio (m/z) 3000. Idatha ye-MS/MS ifunyanwa nge-ion trap kwi-LTQ Orbitrap XL [iinombolo zokuqala ezi-3 apho idatha ixhomekeke khona, i-collision induced dissociation (CID)].
Ukulinganisa kwe-MD kwenziwe kusetyenziswa isoftware ye-GROMACS (52) kunye ne-MARTINI 2 force field (53-55). I-CHARMM GUI Membrane Builder (56, 57) yaze yasetyenziswa ukwakha i-bilayer equlethe i-dioleoylphosphatidylcholine (DOPC) kunye ne-Cer C18 okanye i-DOPC kunye ne-Cer C26. I-topology kunye ne-coordinates ze-Cer C26 zithathwe kwi-DXCE ngokususa ii-beads ezongezelelweyo kumsila we-sphingosine. Sebenzisa inkqubo echazwe ngezantsi ukulinganisela umaleko ophindwe kabini kwaye uwusebenzise, ​​emva koko sebenzisa ii-coordinates zokugqibela zenkqubo ukwakha inkqubo equlethe i-Emp24. Indawo ye-transmembrane ye-yeast Emp24 (iintsalela eziyi-173 ukuya kwi-193) yakhiwa njenge-α-helix kusetyenziswa isakhiwo se-molecule yesixhobo se-visual MD (VMD) (58). Emva koko, emva kokususa ii-lipids ezigqunyiweyo, iproteni yaxutywa kakhulu yaza yafakwa kwi-bilayer kusetyenziswa i-CHARMM GUI. Inkqubo yokugqibela iqulethe i-1202 DOPC kunye ne-302 Cer C26 okanye i-1197 DOPC kunye ne-295 Cer C18 kunye ne-Emp24. Faka i-ion kwinkqubo kwi-concentration ye-0.150M. Kwenziwe iikopi ezine ezizimeleyo kwimixholo emibini ye-bilayer.
I-lipid bilayer ilinganiswe kusetyenziswa inkqubo ye-CHARMM GUI, equka ukunciphisa nokulinganisela amanyathelo angama-405,000, apho imida yendawo incitshiswa kancinci kancinci kwaye isuswe, kwaye inyathelo lexesha liyandiswa ukusuka kwi-0.005 ps ukuya kwi-0.02 ps. Emva kokulingana, ivelisa ii-µs ezi-6 ngenyathelo lexesha le-0.02 ps. Emva kokufaka i-Emp24, sebenzisa inkqubo efanayo ye-CHARMM GUI ukunciphisa nokulinganisela inkqubo, uze usebenze imizuzwana esi-8 kwimveliso.
Kuzo zonke iinkqubo, ngexesha lenkqubo yokulinganisela, uxinzelelo lulawulwa yi-barostat yeBerendsen (59), kwaye ngexesha lenkqubo yemveliso, uxinzelelo lulawulwa yi-barostat yeParrinello-Rahman (60). Kuzo zonke iimeko, uxinzelelo oluqhelekileyo yi-1 bar kwaye kusetyenziswa iskimu sokudibanisa uxinzelelo lwe-semi-isotropic. Kwinkqubo yokulinganisela kunye nemveliso, i-thermostat (61) ene-speed recalibration isetyenziselwa ukudibanisa ubushushu beprotheni, i-lipid kunye nee-solvent particles ngokulandelelana. Ngexesha lonke lokusebenza, ubushushu obujoliswe kuyo yi-310K. Intsebenziswano engeyiyo i-bonding ibalwa ngokuvelisa uluhlu lokubhangqa kusetyenziswa iskimu seVerlet esine-0.005 buffer tolerance. Igama leCoulomb libalwa kusetyenziswa intsimi yempendulo kunye nomgama onqunyiweyo we-1.1 nm. Igama leVander Waals lisebenzisa iskimu sokusika esinomgama onqunyiweyo we-1.1 nm, kwaye iskimu sokusika seVerlet sisetyenziselwa ukutyibilika okunokwenzeka (62).
Ukusebenzisa i-VMD, ubude bomda phakathi kwee-DOPC phosphate beads okanye ii-ceramide AM1 beads kunye neproteni yi-0.7 nm, kwaye inani lee-lipids ezisebenzisana neproteni liyabalwa. Ngokwefomula elandelayo, bala i-depletion-enrichment (DE) factor njengakwi-(63): i-DE factor = (isixa see-lipids ezipheleleyo kwiproteni 0.7) kwiproteni 0.7 (isixa se-Cer kwi-lipids ezipheleleyo)
Ixabiso elichaziweyo lifunyanwa njengomndilili, kwaye iibha zempazamo ziikopi ezine ezizimeleyo ze-SE. Ukubaluleka kwezibalo ze-DE factor kubalwa ngovavanyo lwe-t [(averageDE-factor-1)/SE]. Bala ixabiso le-P ukusuka kulwabiwo olunemsila omnye.
Isixhobo se-GROMACS sisetyenzisiwe ukubala imephu yoxinano lwe-2D ecaleni kwenkqubo equlethe i-Emp24 ngaphakathi kwe-250 ns yokugqibela yomkhondo. Ukuze kufunyanwe imephu yokutyebisa/yokuncitshiswa kwe-ceramide, imephu yoxinano lwe-Cer yahlulwe ngenani lemephu ye-Cer kunye ne-DOPC, ize yahlulwe ngenani le-Cer emzimbeni. Kusetyenziswa isikali semephu yombala ofanayo.
Ukuze ufumane ezinye izinto ezongezelelweyo kweli nqaku, nceda ujonge ku-http://advances.sciencemag.org/cgi/content/full/6/50/eaba8237/DC1
Eli linqaku elivulelekileyo elisasazwa phantsi kwemigaqo yeLayisensi yeCreative Commons Attribution-Non-Commercial, evumela ukusetyenziswa, ukusasazwa kunye nokuveliswa kwakhona kuyo nayiphi na indlela, lo gama nje ukusetyenziswa kokugqibela kungekuko ukuzuza kwezorhwebo kwaye ingqikelelo kukuba umsebenzi wokuqala uchanekile. Isalathiso.
Qaphela: Sikucela kuphela ukuba unikeze idilesi yakho ye-imeyile ukuze umntu omcebisayo kwiphepha azi ukuba ufuna abone i-imeyile kwaye ayisiyo spam. Asiyi kubhala naziphi na iidilesi ze-imeyile.
Lo mbuzo usetyenziselwa ukuvavanya ukuba ungumtyeleli na kwaye uthintele ukuthunyelwa kwe-spam ngokuzenzekelayo.
Sofia Rodriguez-Gallardo, Kazuo Kurokawa, Susana Sabido-Bozo, Alejandro Cortez · Gomez (Alejandro Cortes-Gomez), Atsuko Ikeda (Atsuko Ikeda), Valeria Zoni (Valeria Zoni), Auxiliadora Aguilera-Romero, Ana Maria Perez -Lineope Lopero, Ana Maria Perez Waga (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Imifanekiso ye-3D enesisombululo esiphezulu ngexesha langempela ibonisa ukubaluleka kobude betsheyini ye-ceramide ekuhleleni iiproteni kwiindawo ezikhethiweyo zemveliso.
Sofia Rodriguez-Gallardo, Kazuo Kurokawa, Susana Sabido-Bozo, Alejandro Cortez · Gomez (Alejandro Cortes-Gomez), Atsuko Ikeda (Atsuko Ikeda), Valeria Zoni (Valeria Zoni), Auxiliadora Aguilera-Romero, Ana Maria Perez -Lineope Lopero, Ana Maria Perez Waga (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Imifanekiso ye-3D enesisombululo esiphezulu ngexesha langempela ibonisa ukubaluleka kobude betsheyini ye-ceramide ekuhleleni iiproteni kwiindawo ezikhethiweyo zemveliso.
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