Kuhlunga emaprotein endleleni yekukhipha kubalulekile ekugcineni kuhlukaniswa kwemaseli kanye ne homeostasis. Ngekwengeta ngekuhlunga lokulawulwa ngemagobolondo, indzima yema lipids ekuhlungini kwe kinesin enchubeni yekutfutsa secretory ngumbuto losisekelo lokhona sikhatsi lesidze longaphendvulwa. Lapha, senta 3D ngaso sonkhe sikhatsi imibalabala leminyenti lesetulu-sincumo sesikhatsi sangempela kufakazela ku-vivo kutsi emaprotheni lasanda kuhlanganiswa glycosylphosphatidylinositol-langahambi ngetincenye letindze kakhulu te-ceramide lipid ahlanganiswa futsi ahlukaniswe abe ngema-endoplasms lakhetsekile Net usembraneme isayithi yekuphuma, lokunguyo. Ngetulu kwaloko, sikhombisa kutsi ubude beketanga le-ceramide ku-endoplasmic reticulum ulwelwesi bubalulekile kulokukhetsa kwekuhlunga. Sifundvo setfu siniketa bufakazi bekucala lobucondzile be-vivo bekuhlukanisa imithwalo yemaprotheyini ngekuya ngebudze be-lipid chain ibe tindzawo tekutfumela ngaphandle letikhetsiwe endleleni ye-secretory.
Emaseli e-eukaryotic, emaprotheyini lahlanganiswa ku-endoplasmic reticulum (ER) abese ayahlukaniswa ngesikhatsi sekutfutsa ngendlela yekukhipha imfihlo kute ahanjiswe endzaweni yawo lefanele yemaseli (1). Ngaphandle kwekuhlunga lokulawulwa yijazi, bekucatshangelwa sikhatsi lesidze kutsi ema-lipids latsite angaphindze asebente njengetindzawo tekuphuma letikhetsiwe ngekuwahlanganisa abe tindzawo letitsite te-membrane kutsi emaprotheni lakhetsekile (2-5). Kepha, kusesekhona kushoda kwebufakazi lobucondzile be-vivo kufakazela lendlela lekhonakalako lesekelwe ku-lipid. Kute silungise lenkinga lesisekelo, safundza emvubelo kutsi emaprotheni labhalwe nge-glycosylphosphatidylinositol (GPI-APs) atfunyelwa njani ngekwehluka kusuka ku-ER. GPI-APs kukhona tinhlobonhlobo lipid-xhumene cell ebusweni amaprotheni ( 6, 7). I-GPI-AP yiphrotheni lefihlekile lenamatsele emacembe langaphandle elucwencweni lwe-plasma ngekusebentisa incenye ye-glycolipid (i-GPI anchor). Bamukela tikhonkwane te-GPI njengekuguculwa kwekugcina ngemuva kwekuhumusha ku-ER lumen (8). Ngemuva kwekunamatsela, i-GPI-AP idlula nge-Golgi apharathasi (5, 9) kusuka ku-ER kuya ku-plasma ulwelwesi. Kuba khona kwetikhonkwane te-GPI kwenta kutsi i-GPI-AP itfutfwe ngekwehlukana kumaprotheni lakhishwa nge-transmembrane (kufaka ekhatsi lamanye emaprotheni e-plasma membrane) eceleni kwendlela yekukhipha (5, 9, 10). Emaseli emvubelo, ema-GPI-AP ahlukaniswe nalamanye emaprotheni lafihlekile ku-reticulum ye-endoplasmic, bese apakishwa abe ngema-vesicles lahlukile lasongwe nge-coat protein complex II (COPII) (6, 7). Tintfo letincumako talenchubo yekuhlukanisa enchubeni yekutfumela ngaphandle kwe-ER aticaci, kepha kucatshangelwa kutsi lendlela ingadzinga ema-lipids, ikakhulukati kulungiswa kabusha kwesakhiwo sencenye ye-lipid ye-GPI anchor (5, 8). Emvubelo, kuvuselelwa kwemafutsa e-GPI kucala ngekushesha ngemuva kwekutsi i-GPI inamatsele, futsi etimeni letinyenti, kwenta kutsi i-ceramide ibophe ku-26-carbon lendze lenemafutsa esidi (C26:0) (11, 12). C26 ceramide yi ceramide leyinhloko lekhicitwa ngemangcamuzane emvubelo kuze kube manje. Ihlanganiswa ku-ER futsi iningi layo litfunyelwa ku-apharathasi ye-Golgi nge-COPII vesicles (13). I-ER kutfunyelwa ngaphandle kwe-GPI-AP kudzinga ngalokukhetsekile kuhlanganiswa kwe-ceramide lokuchubekako (14, 15), futsi ngekujika, kuguculwa kwe-ceramide ibe yi-inositol phosphate ceramide (IPC) ku-apharathasi ye-Golgi kuncike ekuhlanganisweni kwe-GPI yesikhonkwane (16). Tifundvo te-Biophysical letinelucwencwe lwekufakelwa tikhombise kutsi ema-ceramides e-acyl chain lamadze kakhulu angahlangana kute akhe tindzawo letihlelekile letinetintfo letihlukile letibonakalako (17, 18). Lemininingwane iholela ekucabangeni kutsi i-C26 ceramide ne-GPI-AP lene-C26 ceramide isebentisa timphahla tabo letibonakalako kute ihlangane ibe tifundza letihlelekile noma tifundza letisendzaweni ye-ER membrane lipid lengcolile. Ikakhulukati yakhiwe ngema-glycerolipids lamafishane nalangagcwali (C16: 1 na C18: 1) (19, 20). Letifundza titawukhetsa kugcila etindzaweni letitsite tekuphuma te-ER (ERES), lapho khona i-ceramide kanye ne-GPI-AP lesekelwe ku-ceramide ingahanjiswa ngekubambisana iye eGolgi ku-vesicle lefanako letinikele ye-COPII (5).
Kulesifundvo, sihlole ngalokucondzile lendlela lesekelwe kumafutsa ngekusebentisa i-super-resolution confocal yesikhatsi sangempela se-microscopy (SCLIM), lokuyindlela ye-microscopy lehamba embili lengabuka ngaso sonkhe sikhatsi emaprotheni labhalwe nge-fluorescent Lemibala lemitsatfu kanye ne-diges lesetulu ye-lipelu s (3D image) (21, 22).
Sicale sasebentisa tebuchwepheshe be-SCLIM kute sichaze kabanti kutsi i-GPI-AP levamile lenelicembu le-C26 ceramide yahlolwa njani kusuka kumaprotheni lakhishwa nge-transmembrane ngemuva kwekushiya i-ER ku-S. Kute sihlole kuhlukaniswa kwe-ER, sasebentisa luhlelo lwefutfo lolungabona ngco umtfwalo losanda kuhlanganiswa longena ku-ERES ku-vivo (7, 23). Njengemtfwalo, sakhetsa i-C26 ceramide-based GPI-AP Gas1 lebhalwe ngephrotheni leluhlata lekhanyako (GFP) kanye nephrotheni lefihlekile ye-transmembrane Mid2 lebhalwe ngephrotheni lekhanyako lesedvute ne-infrared (iRFP), kokubili lokuhlose ulwelwesi lwe-plasma (24-26). Kule sec31-1 mutant lebucayi ngekushisa, lemithwalo lemibili ivetwa ngaphansi kwemgcugcuteli we galactose-inducible kanye nemaka ye ERES lesungulako. Ngesikhatsi sekushisa lokukhulu (37°C), ngoba i-sec31-1 mutation ithinta umsebenti we-COPII coat component Sec31 kuvimbela kuhluma kwe-COPII kanye nekutfumela i-ER, umtfwalo losanda kuhlanganiswa uyabutsela e-ER (23). Ngemuva kwekupholisa kuze kube sezingeni leliphansi lekushisa (24°C), emaseli e-sec31-1 lagucukako alulama endzaweni yekufihla, futsi umtfwalo lomusha wekwentiwa lobutfwele wacala kutfunyelwa ngaphandle kusuka ku-ER. Kubonakala kwe CLIM kwakhombisa kutsi linyenti le Gas1-GFP lesanda kuhlanganiswa kanye ne Mid2-iRFP solo libutselelwe ku ER ye sec31-1 mutant cells ngemuva kwekufukamela ku 37 ° C bese likhululwa ku 24 ° C imizuzu lesihlanu (Umfanekiso 1). Njengobe iMid2-iRFP isabalaliswa kulo lonkhe lucwencwe lwe-ER, futsi i-Gas1-GFP igcila futsi ibutselwe endzaweni yelucwencwe lwe-ER lengachubeki, kusabalalisa kwabo kwehluke ngalokuphelele (Umfanekiso 1, A kuya ku-C kanye ne-Movie S1). Ngetulu kwaloko, njengobe kukhonjisiwe kuMfanekiso 1D, licembu leGas1-GFP alinayo iMid2-iRFP. Lemiphumela ikhombisa kutsi i-GPI-AP kanye nemaprotheni e-transmembrane ahlukaniswe abe tifundza letehlukene te-ER membrane ekucaleni. Leliqoqo le-Gas1-GFP lisedvute ne-ERES letsite lebhalwe ngephrotheni yejazi le-mCherry ye-COPII Sec13 (Umfanekiso 1, E na-F, kanye ne-movie S1) (23).
sec31-1 emaseli aveta kukhishwa kwe-galactose, umjikeleto lomudze we-acyl (C26) ceramide GPI-AP Gas1-GFP (GPI-AP, luhlata) kanye nephrotheni ye-transmembrane Mid2-iRFP (TMP, luhlata sasibhakabhaka) futsi loku kulebula kwe-ERES Lokwakhako kwa-Sec13-mCherry, imizuzu, yasuswa yaya ku 24°C, futsi yatsatfwa ngetitfombe yi SCLIM ngemuva kwemizuzu lengu 5. (A kuya ku C) ukhombisa ummeleli lohlanganisiwe noma sitfombe sinye se-2D sendiza (A), sitfombe sekubonisa se-2D setincenye letingu-10 te-z (B) noma sitfombe se-3D cell hemisphere semitfwalo kanye netimphawu te-ERES (C). Ibha yesikali 1μm (A na B). Iyunithi yesikali ngu-0.551μm (C). Gas1-GFP kwatfolakala etifundzeni letihlukile te-ER noma emacembu, kantsi i-Mid2-iRFP yatfolakala futsi yasakatwa kulo lonkhe lulwelwesi lwe-ER (C). (D) Igrafu ikhombisa kucina kwe-fluorescence lokuhlobene kwe-Gas1-GFP ne-Mid2-iRFP ecenjini le-Gas1-GFP eceleni kwemugca wemcibisholo lomhlophe (ngasencele). AU, iyunithi leyehlukile. (E na F) bamele sitfombe se-3D lesihlanganisa timphahla kanye nemamaki e-ERES. Emacembu e-Gas1-GFP atfolakala edvute ne-ERES letsite. Iyunithi yesikali ngu 0.551μm. (F) Umcibisholo lomhlophe locinile uphawula licembu le-Gas1-GFP lelihlobene ne-ERES. Emapaneli lasemkhatsini nalangesekudla akhombisa sitfombe se-3D lesikhulisiwe lesihlanganisiwe kanye nekubuka lokujikeletako kwe-Gas1-GFP cluster lekhetsiwe.
Budlelwane lobusedvute bendzawo emkhatsini we-Gas1-GFP cluster kanye ne-ERES letsite bukhombisa kutsi i-Gas1-GFP ingangena ku-ERES lekhetsiwe, leyehlukile ekukhetseni lokusetjentiswa yi-Mid2-iRFP kushiya i-ER. Kulungisa loku kungenteka, salinganisa silinganiso se-ERES semphahla yinye noma letimbili kuphela (Umfanekiso 2, A kuya ku C). Sitfole kutsi ema-ERES lamanyenti (70%) aneluhlobo lunye kuphela lwemtfwalo. Umfanekiso longentasi weMfanekiso 2C ukhombisa tibonelo letimbili letijwayelekile te-ERES lene-Gas1-GFP kuphela (Umfanekiso 1) noma i-Mid2-iRFP kuphela (Umfanekiso 2). Ngalokwehlukile, cishe 20% we ERES ucuketse imithwalo lemibili lehlangana endzaweni yinye. Kutfolakele kutsi lamanye ema ERES (10%) abenetinhlobo letimbili temitfwalo, kepha abehlukaniswe etindzaweni letehluke ngalokucacile. Ngako-ke, lokuhlatiya kwetibalo kukhombisa kutsi ngemuva kwekutsi i-ER itfunyelwe ngaphandle, i-GPI-AP Gas1-GFP kanye nemtfwalo we-transmembrane Mid2-iRFP ihlukaniswe ibe yi-ERES lehlukene (Umfanekiso 2D). Loku kusebenta kahle kwekuhlunga kuhambisana kakhulu nekuhlatiya kwe biochemical kwaphambilini (6) kanye nekutimisela kwe morphological (7). Singaphindze futsi sibuke kutiphatsa kwemitfwalo levalelwe yodvwa lengena e-ERES (Umfanekiso 2E kanye ne-Movie S2). Umfanekiso 2E ukhombisa kutsi kuphela incenye lencane ye Gas1-GFP (iphaneli 3) noma iMid2-iRFP (iphaneli 4) ingena ku ERES kusuka ngakulolunye luhlangotsi futsi ivalelwe endzaweni lehlukile. Iphaneli 5 yeMfanekiso 2E ikhombisa kutsi iGas1-GFP ne Mid2-iRFP ngalesinye sikhatsi itfolakala ku ERES lefanako, kepha ingena kusuka emaceleni lehlukene futsi igcila etigodzini letehlukene letingamelela ema vesicles lehlukene e COPII. Siphindze sacinisekisa kutsi kuhlukaniswa lokubonwe kanye nekuhlukaniswa kwe-C26 ceramide-based GPI-AP Gas1 njenge-ERES lekhetsiwe icacile ngoba lomunye umtfwalo wekukhipha umtimba we-transmembrane, iphrotheni ye-plasma lefakwe emamaki Axl2 (27 ), lekhombisa kutiphatsa lokufanako ne-Mid2-iRFP. (Sitfombe S1 kanye neMuvi S3). I-Axl2-GFP lesanda kuhlanganiswa isabalaliswa ngelucwencwe lwe-ER njenge-Mid2-iRFP (Umfanekiso S1, A na B), futsi ihlanganiswa ne-Mid2-iRFP kuma-ERES lamanyenti (Umfanekiso S1, B kuya ku D). Emaphaneli 1 na 2 eMfanekiso 1. S1C ukhombisa tibonelo letimbili letijwayelekile te-ERES lapho khona imithwalo lemibili ye-transmembrane ihlangana khona. Kuleti timo, totimbili letimphahla tingena ku-ERES ndzawonye (Umfanekiso S1E, Iphaneli 3 kanye neMovie S3).
Emaseli e-sec31-1 laveta kukhishwa kwe-galactose, i-Gas1-GFP (GPI-AP, luhlata) kanye ne-Mid2-iRFP (TMP, luhlata sasibhakabhaka) kanye ne-ERES lesungulako lebhalwe nge-Sec13-mCherry (ERES, i-magenta) yabekwa ku-37 Ngemuva kwekufukamela imfihlo kuya ku-°C imizuzu lengu-30. ibhulokhi, kanye nesitfombe nge-SCLIM ngemuva kwemizuzu lengu-20. (A kuya ku C) Titfombe tekubonisa letimele 2D (A; ibha yesikali, 1μm) noma titfombe te-3D cell hemisphere (B na C; iyunithi yesikali, 0.456μm) yemitfwalo kanye netigaba letingu-10 z-letibhalwe yi-ERES. Iphaneli lephansi ku (B) kanye nephaneli ku (C) kukhombisa titfombe leticubunguliwe kukhombisa kuphela timphahla letikhona ku ERES (magenta) [Gas1-GFP (mphunga) kanye ne Mid2-iRFP (luhlata sasibhakabhaka lolukhanyako)]. (C) Umcibisholo lovulekile: I-ERES ithwala umtfwalo munye kuphela (1 kuya ku-4). Umcibisholo lompunga: I-ERES icuketse imithwalo lehlukanisiwe (5). Umcibisholo lomhlophe locinile: ERES lecuketse imithwalo lesendzaweni lefanako. Ngezansi: I-ERES yinye lekhetsiwe icuketse iGas1-GFP (1) noma iMid2-iRFP (2) kuphela. Ibha yesikali, 100 nm. (D) Kulinganisa kwe-photomicrograph lechazwe ku (C). Liphesenti lelisemkhatsini le-ERES lecuketse umtfwalo munye kuphela (Gas1-GFP noma Mid2-iRFP), umtfwalo lohlukanisiwe kanye nemtfwalo lohlanganako. Kulokuhlolwa lokutsatfu lokutimele, n = 432 kumaseli langu-54. Ibha yeliphutsa = SD. Kuhlolwa kwe-t lokungabhangqwa lokunemisila lemibili. *** P = 0,0002. (E) Umfanekiso we-3D we-ERES lekhetsiwe yemitfwalo levalelwe yodvwa lebhalwe nge (C). I-Gas1-GFP (leluhlata) (3) noma i-Mid2-iRFP (leluhlata sasibhakabhaka) (4) ingena e-ERES (leluhlata sasibhakabhaka) kusuka ngakulolunye luhlangotsi futsi ivinjelwe endzaweni lencane ngaphakatsi kwe-ERES. Ngalesinye sikhatsi, totimbili tinhlobo temitfwalo tingena ku-ERES lefanako (5) kusuka eluhlangotsini lunye futsi tivalelwa endzaweni leyehlukile ngaphakatsi kwe-ERES. Ibha yesikali, 100 nm.
Ngemuva kwaloko, savivinya umcabango wekutsi i-acyl chain ceramide lendze (C26) lekhona ku-ER membrane ishayela kuhlanganiswa lokutsite kanye nekuhlunga i-Gas1 ibe yi-ERES lekhetsiwe. Kute kwentiwe loku, sasebentisa luhlobo lwemvubelo loluguculwe lwe-GhLag1, lapho khona ema-synthases lamabili e-ceramide e-endogenous Lag1 kanye ne-Lac1 atsatfwa sikhundla yi-GhLag1 (i-homolog ye-Lag1 yekotini), lokuholele eluhlobo lwemvubelo loluneluhlobo lwe-Ceramide lolunelucwencwe lweseli lolufishane kuneluhlobo lwesiganga (Umfanekiso 3A) (2). Kuhlatiya kwe Mass spectrometry (MS) kwakhombisa kutsi etinhlotjeni tetinhlobo tesiganga, 95% we ceramide lephelele yindze kakhulu (C26) i-ceramide yemaketanga, kantsi ku GhLag1, 85% we ceramide yindze kakhulu (C18 na C16). ), 2% kuphela we-ceramide lomudze kakhulu (C26) we-ceramide yeketanga. Noma nje iC18 ne C16 ceramides yiyo ceramides leyinhloko letfolakele elucwencweni lwe GhLag1 kuze kube ngunyalo, kuhlatiya kwe MS kuphindze kwacinisekisa kutsi i-anchor ye GPI ye Gas1-GFP levetwe kuloluhlobo lwe GhLag1 icuketse iC26 ceramide, lefanana ne lipids yeluhlobo lwesiganga. Ikhwalithi iyafana (Umfanekiso 3A) (26). Ngakoke, loku kusho kutsi i-enzyme yekulungisa kabusha i-Cwh43 ikhetsa kakhulu i-C26 ceramide, njengoba kukhonjisiwe kuMfanekiso 26, ikhetsa kufaka sikhonkwane se-GPI kusuka esilinganisweni lesincane se-C26 ceramide ku-GhLag1 strain. S2 (29). Noma kunjalo, ulwelwesi lweseli le GhLag1 ngekuyinhloko lucuketse i-C18-C16 ceramide kuphela, kantsi i-Gas1-GFP isene-C26 ceramide. Leliciniso lenta loluhlobo lube lithulusi lelihle kakhulu lekusombulula ngalokukhetsekile inkinga yebudze be-acyl chain ye-membrane ceramide ku-ER. Indima hypothetical ekilasini kanye nekuhlunga. Ngemuva kwaloko, sacala safundza likhono le-C26 Gas1-GFP lekubutsela ndzawonye ngemacembu ku-GhLag1 nge-allele legucukako lebucayi yekushisa ye-sec31-1 ngekusebentisa sibonakhulu se-fluorescence levamile, lapho khona kuphela i-chain lendze (C18-C16) lekhona ku-ER ulwelwesi Ceramide (Fig. 3). Sibonile kutsi ku sec31-1, iningi le Gas1-GFP laligcizelelwa ngemacembu, kantsi i Gas1-GFP ku sec31-1 GhLag1 lene (C18-C16) lendze ceramide ER ulwelwesi ikakhulukati akazange ahlanganiswe futsi asakatwe ku Ku lonkhe ulwelwesi ER. Kuba ngco, ngoba C26 ceramide-based clustering kuhlobene kakhulu ERES ethize (Umfanekiso 1), saphenya ngemuva kwaloko kutsi ngabe lenchubo ingaphindze ifake umsebenti ER kutfumela emaprotheni indlela. GPI-AP usebentisa luhlelo lolukhetsekile lwe-COPII lwekutfumela ngaphandle kwe-ER, lolulawulwa ngenkhutsalo yi-Ted1′s yekulungisa kabusha kwesakhiwo se-glycan ye-GPI anchor (30, 31). I-GPI-glycan lehlanganisiwe ibese iyabonakala yi-transmembrane yemukela umtfwalo we-p24, lephindze ikhetse i-Lst1, lokuyi-isoform letsite ye-COPII lenkhulu yekubopha umtfwalo Sec24, leyakha i-GPI-AP-ecebile ye-COPII Vesicles iyadzingeka (331). Ngako-ke, sakha i-mutant lephindvwe kabili leyahlanganisa kucishwa kwalamaprotheyini langawodvwa (i-p24 ingxenye leyinkimbinkimbi Emp24, i-enzyme yekuvuselela i-GPI-glycan Ted1 kanye ne-subunit lekhetsekile ye-COPII Lst1) nge-sec31-1 legucukako, futsi safundza ngayo Kungenteka yini kutsi kwakhiwe i-GFP1-clu3). Sibonile kutsi ku-sec31-1emp24Δ kanye ne-sec31-1ted1Δ, i-Gas1-GFP ikakhulukati ayihlanganiswa futsi isabalaliswe kulo lonkhe lulwelwesi lwe-ER, njengobe bekubonwe ngaphambilini ku-sec31-1 GhLag1, kantsi ku-sec31-1lst1Δ, i-Gas1-GFP Njenge-sec3. Lemiphumela ikhombisa kutsi ngetulu kwekuba khona kwe C26 ceramide ku ER membrane, kuhlanganiswa kwe Gas1-GFP nako kudzinga kubopha ku p24 complex, futsi akudzingi kucashwa lokutsite kwe Lst1. Ngemuva kwaloko, sahlola kutsi kungenteka yini kutsi ubude beketanga le-ceramide ku-ER membrane lingalawula kuboshwa kwe-Gas1-GFP kuya ku-p24. Kepha, satfola kutsi kuba khona kwe C18-C16 ceramide kule membrane akuyitsintsi i GPI-glycans leyakhiwe kabusha yi p24 complex (Tibalo S3 na S4, A na B) noma kubopha ku GPI-AP kanye nekutfumela i GPI-AP. emandla. Butha COPII luhlobo loluncane Lst1 (Umfanekiso S4C). Ngakoke, kuhlanganiswa lokuncike ku C26 ceramide akudzingi kusebentisana kwemaprotein netindlela letehlukene tekutfumela emaprotein eER, kepha kusekela lenye indlela yekuhlunga lecondziswa budze be lipid. Ngemuva kwaloko, sahlatiya kutsi ngabe budze be-ceramide acyl chain ku-ER membrane bubalulekile yini ekuhlukaniseni ngemphumelelo kwe-Gas1-GFP njenge-ERES lekhetsiwe. Njengoba iGas1 esigabeni se GhLag1 lene ceramide lencane ishiya i ER bese ingena elucwencweni lwe plasma (Umfanekiso S5), sikholwa kutsi uma kuhlunga kucondziswa budze be ceramide acyl chain, iGas1 esigabeni se GhLag1 ingacondziswa kabusha futsi iwele. ERES timphahla nge ulwelwesi efanayo.
(A) Ulwelwesi lweseli le-GhLag1 ikakhulukati lucuketse ema-ceramides lamafishane e-C18-C16, kantsi sikhonkwane se-GPI se-Gas1-GFP sisane-C26 IPC lefanako njengemaseli eluhlobo lwesiganga. Ngetulu: kuhlatiya budze be-acyl chain ye-ceramide elucwencweni lwemaseli eluhlobo lwesiganga (Wt) kanye netinhlobo te-GhLag1p nge-mass spectrometry (MS). Idatha imelela iphesenti ceramide lilonkhe. Silinganiso sekuhlola lokutsatfu lokutimele. Ibha yeliphutsa = SD. Kuhlolwa kwe-t lokungabhangqwa lokunemisila lemibili. **** P <0,0001. Iphaneli lengephansi: Kuhlatiya kwe-MS kwebudze be-acyl chain ye-IPC lekhona ku-Gas1-GFP (GPI-IPC) i-GPI anchor levetwe eluhlobo lwesiganga kanye netinhlobo te-GhLag1p. Idatha imelela iphesenti IPC lilonkhe lebantfu labange signal. Silinganiso sekuhlola lokusihlanu lokutimele. Ibha yeliphutsa = SD. Kuhlolwa kwe-t lokungabhangqwa lokunemisila lemibili. ns, akubalulekile. P = 0.9134. (B) Titfombe letincane tekukhanya te-sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ kanye ne-sec31-1lst1Δ emaseli laveta i-galactose-lekhutsatwa yi-Gas1-GFP yakhuliswa phansi ku-37 ° C kuze kube ngu-30°C. ngemuva kwa 24°C. Umcibisholo lomhlophe: ER Gas1-GFP iqoqo. Umcibisholo lovulekile: I-Gas1-GFP lengakahlanganiswa isabalaliswa kulo lonkhe lucwencwe lwe-ER, ikhombisa kugcoba kwendandatho yenuzi lenesici se-ER. Ibha yesikali, 5μm. (C) Kulinganisa kwe-photomicrograph lechazwe ku (B). Iphesenti lelisemkhatsini lemangcamuzane ngesakhiwo se-Gas1-GFP lene-punctate. Kulokuhlolwa lokutsatfu lokutimele, n≥300 amaseli. Ibha yeliphutsa = SD. Kuhlolwa kwe-t lokungabhangqwa lokunemisila lemibili. **** P <0,0001.
Kucatulula ngco lenkinga, senta SCLIM kubonakala Gas1-GFP kanye Mid2-iRFP ku GhLag1 nge sec31-1 lokushisa-kubucayi allele mutant (Umfanekiso 4 kanye Movie S4). Ngemuva kwekutsi i-ER igcinwe ku 37°C futsi ngemuva kwaloko ikhululwe ku 24°C, linyenti le Gas1-GFP lesanda kuhlanganiswa alizange lihlanganiswe futsi lisakateke kulo lonkhe lulwelwesi lwe ER, njengoba kubonwe ngetibonakhulu letivamile (Umfanekiso 4, A na B ). Ngetulu kwaloko, liphesenti lelikhulu le-ERES (67%) lifaka tinhlobo letimbili temtfwalo lohlangene kuyo (Umfanekiso 4D). Emaphaneli 1 na 2 eMfanekiso 4C akhombisa tibonelo letimbili letijwayelekile te-ERES ngekuhlangana kweGas1-GFP kanye neMid2-GFP. Ngetulu kwaloko, totimbili letimphahla tatsatfwa ku-ERES lefanako (Umfanekiso 4E, iphaneli 3 kanye ne-movie S4). Ngako-ke, imiphumela yetfu ikhombisa kutsi budze be-ceramide acyl chain ku-ER membrane kuyintfo lebalulekile yekuhlanganiswa kwemaprotheni e-ER kanye nekuhlukaniswa.
Sec31-1 Emaseli eGhLag1 laveta tintfo letikhishwako letibangwa yigalactose, iGas1-GFP (i-GPI-AP, leluhlata) kanye ne-Mid2-iRFP (i-TMP, leluhlata sasibhakabhaka) kanye ne-ERES-lebhalwe nge-ERES (ERES, i-magenta) lesungulako. Chubeka imizuzu lengemashumi lamatsatfu, wehlele ku 24°C kwentela kukhipha tintfo letikhishwako, bese utsatsa sitfombe nge SCLIM ngemuva kwemizuzu lengemashumi lamabili. (A kuya ku C) Titfombe tekubonisa letimele 2D (A; ibha yesikali, 1μm) noma titfombe te-3D cell hemisphere (B na C; iyunithi yesikali, 0.45μm) yetincenye letingu-10 z-letiphawulwe ngumtfwalo kanye ne-ERES. Iphaneli lephansi ku (B) kanye nephaneli ku (C) kukhombisa titfombe leticubunguliwe kukhombisa kuphela timphahla letikhona ku ERES (magenta) [Gas1-GFP (mphunga) kanye ne Mid2-iRFP (luhlata sasibhakabhaka lolukhanyako)]. (C) Umcibisholo logcwele lomhlophe: ERES, timphahla tiyahlangana. Umcibisholo lovulekile: I-ERES icuketse intfo yinye kuphela. Iphaneli lengephansi: I-ERES lekhetsiwe inetimphahla letihlangene (1 na 2) letibhalwe ku (C). Ibha yesikali, 100 nm. (D) Kulinganisa kwe-photomicrograph lechazwe ku (C). Kumayunithi e-sec31-1 kanye ne-sec31-1 GhLag1, kufakwe umtfwalo munye kuphela (Gas1-GFP noma Mid2-iRFP), kanye neliphesenti lelisemkhatsini le-ERES yemtfwalo lohlukanisiwe kanye nemtfwalo lohlanganako. Kulokuhlolwa lokutsatfu lokutimele, n = 432 kumaseli langu-54 (sec31-1) kanye n = 430 kumaseli langu-47 (sec31-1 GhLag1). Ibha yeliphutsa = SD. Kuhlolwa kwe-t lokungabhangqwa lokunemisila lemibili. *** P = 0,0002 (sigaba 31-1) kanye ** P = 0,0031 (sigaba 31-1 GhLag1). (E) Umfanekiso we-3D we-ERES lekhetsiwe lenemitfwalo lehlanganisiwe (3) lebhalwe ku (C). I-Gas1-GFP (luhlata) ne-Mid2-iRFP (leluhlata sasibhakabhaka) isondzela ku-ERES (leluhlata sasibhakabhaka) kusuka eceleni lokufanako bese ihlala endzaweni yinye levinjelwe yi-ERES. Ibha yesikali, 100 nm.
Lolu cwaningo luniketa bufakazi lobucondzile be-vivo kutsi imithwalo yemaprotein lesekelwe ku-lipid ihlukaniswe ibe tindzawo tekutfumela ngaphandle letikhetsiwe endleleni ye-secretory, futsi iveta kubaluleka kwebudze be-acyl chain yekukhetsa kuhlukanisa. Kusebentisa indlela lenemandla futsi lehamba embili yekubuka ngesibonakhulu lebitwa ngekutsi yi-SCLIM, sakhombisa i-Gas1-GFP lesanda kuhlanganiswa (ulwelwesi lwe-plasma lolukhulu lwe-GPI-AP lolune-acyl chain lendze kakhulu (C26) incenye ye-ceramide lipid) ku-yeast ) Letifundza letihlangene nge-transmbrame lekhetsekile lesakateke nge-ES, ulwelwesi lwe-ER (Umfanekiso 1). Ngetulu kwaloko, letinhlobo letimbili temphahla tingena ku-ERES lehlukene ngekukhetsa (Umfanekiso 2). Budze be-acyl chain ye-ceramide yemaseli kulelucwencwe buncishiswa kusuka ku-C26 kuya ku-C18-C16, licembu le-Gas1-GFP liyaphazamiseka libe sigodzi se-ER leyehlukile, bese i-Gas1-GFP iyabuyiselwa indlela kute ishiye i-ER ngephrotheni ye-transmembrane nge-ERES lefanako (Umfanekiso 3 kanye neMfanekiso3). 4).
Noma i-GPI-AP isebentisa indlela lekhetsekile yemaprotheni kuphuma ku-ER, satfola kutsi kuhlukaniswa lokuncike ku-C26 ceramide akunciki ekusebentisaneni kwemaprotheni lehlukile langaholela ekukhetsekeni kwe-ERES (Tibalo S4 na S5). Esikhundleni saloko, lokutfolwe ngitsi kusekela lenye indlela yekuhlukanisa lecondziswa kuhlanganiswa kwemaprotheyini lasekelwe ku-lipid kanye nekungafakwa lokulandzelako kwaletinye titfwalo. Lokubonwako kwetfu kukhombisa kutsi sifundza se-Gas1-GFP noma licembu lelihlobene ne-ERES letsite liswele iphrotheni lekhishwa yi-transmembrane Mid2-iRFP, lokukhombisa kutsi licembu le-GPI-AP lencike ku-C26 ceramide litawusita ekungeneni kwabo ku-ERES lefanele, futsi ngesikhatsi lesifanako, lingafaki le-transmembrane letsite I-ESU 2). Ngalokwehlukile, kubakhona kwe C18-C16 ceramides ku ER membrane akubangeli kutsi i GPI-AP yakhe tifundza noma emacembu, ngako ayikhiphi noma ifake esikhundleni semaprotheni lakhishwa nge transmembrane abe yi ERES lefanako (Tibalo 3 na 4). . Ngako-ke, siphakamisa kutsi i-C26 ceramide ishayela kuhlukaniswa kanye nekuhlukaniswa ngekutsi kwentiwe lula kuhlanganiswa kwemaprotheni lahlobene ne-ERES letsite.
Indlela yekufeza loku kuhlanganiswa lokuncike ku-C26 ceramide kube yindzawo letsite ye-ER? Lokuthambekela kwe ceramide yelucwencwe kutsi ihlukane ngeseceleni kungenta kutsi iGPI-AP ne C26 ceramide yakhe emafutsa lamancane futsi lahlelwe ngekushesha esimeni semafutsa lesingakavami kakhulu selucwencwe lwe ER lolucuketse ema glycerolipids lamafishane nalangagcwali. Emacembu ekhwalithi (17, 18). Lamacembu lamancane esikhashana angaphindze ahlanganiswe abe ngemacembu lamakhulu, lacinile ngemuva kwekubopha ku-p24 complex (34). Kuhambisana naloku, sakhombisa kutsi i-C26 Gas1-GFP idinga kusebentisana ne-p24 complex kute yakhe emacembu lamakhulu labonakalako (Umfanekiso 3). Le p24 inkimbinkimbi yi-oligomer le-heterozygous leyakhiwe ngemaprotheni lamane lahlukene e-p24 transmembrane ku-yeast (35), leniketa kubopha lokunyenti, lokungaholela ekuhlanganisweni kwemacembu lamancane e-GPI-AP, ngaleyo ndlela kukhicite licembu lelikhulu le-Stable (34). Kusebentisana emkhatsini we-ectodomains yemaprotheyini e-GPI-APs kungaphindze kube neligalelo ekuhlanganisweni kwawo, njengobe kukhonjisiwe ngesikhatsi sekutfutsa kwabo kwe-Golgi kumaseli e-epithelial la-polarized etilwane letimunyisako (36). Kepha, uma i-C18-C16 ceramide ikhona elucwencweni lwe-ER, uma le-p24 leyinkimbinkimbi ibopha i-Gas1-GFP, emacembu lamakhulu lahlukene angeke akhiwe. Indlela lesembili ingancika etimphahleni letitsite tenyama kanye nemakhemikhali e-acyl chain ceramide lendze. Tifundvo te Biophysical tetintfo letiphilako tetintfo letifakelwako tikhombisa kutsi noma kokubili lokudze (C24) kanye nalokufishane (C18-C16) kwe acyl chain ceramides kungabangela kwehlukana kwesigaba, kuphela ema ceramides lamadze e acyl chain (C24) langakhutsata Kugoba lokusetulu kanye nekugoba kwefilimu kwentela kubumba kabusha lefilimu. Ngekusebentisana (17, 37, 38). Kukhonjisiwe kutsi i-helix ye-TMED2, i-homologue yebantfu ye-Emp24, ihlangana ngekukhetsa ne-sphingomyelin lesekelwe ku-C18 ceramide kuma-lobules e-cytoplasmic (39). Kusebentisa kulingisa kwemandla emamolekhyuli (MD), satfola kutsi kokubili i-C18 ne-C26 ceramides ibutselana eceleni kwema-lobules e-cytoplasmic e-Emp24 transmembrane helix, futsi inetintfo letifanako (Umfanekiso S6). Kuyaphawuleka kutsi loku kukhombisa kutsi i-helix ye-transmembrane ye-Emp24 ingaholela ekusabalalisweni kwe-asymmetric kwe-lipids elucwencweni. Lona ngumphumela wakamuva losekelwe kumaseli etilwane letimunyisako. Kulingisa lokufanako kwe-MD kuphindze kukhombise kuba khona kwe-ether lipids (40) . Ngakho-ke, sicabanga kutsi C26 ceramide ku lobules ezimbili ER26 ecebile endzaweni. Lapho i-GPI-AP kuma-lobules e-luminal ibopha ngco ku-p24 lenemandla kanye nekugcwala kwe-C26 ceramide lezungete i-p24 kuma-lobules e-cytoplasmic, kungakhutsata kuhlanganiswa kweProtein lokuhambisanako kanye nekugoba kwelucwencwe kukhicitwa ngeminwe (41), kwenta kutsi i-GPI-AP iphindze ihlukane ibe sifundza lesiphakeme se-adES. tifundza te-ER ulwelwesi (42). Imibiko yangaphambilini yasekela indlela lephakanyisiwe (43, 44). Kuboshwa lokunyenti kwema-oligolectins, emagciwane noma ema-antibodies kuma-glycosphingolipids lasekelwe ku-ceramide (GSL) elucwencweni lwe-plasma kuvusa kuhlanganiswa lokukhulu kwe-GSL, kutfutfukisa kwehlukaniswa kwesigaba futsi kubangela kugucuka kwelucwencwe kanye nekungena ngaphakathi (44). Iwabuchi njll (43) Kwatfolakala kutsi lapho kunemaketanga lamadze (C24) kepha hhayi lamafishane (C16) e-acyl, i-ligand ye-multivalent leboshelwe ku-GSL lactosylceramide yakhutsata kwakheka kwemacembu lamakhulu kanye nekuhlaselwa kwelucwencwe, futsi kuguculwa kwesiginali ye-cytoplasm Lyn-lelawulwa yi-neugytropyl kumacembe e-chagittropy yi-interna.
Emaseli e-epithelial la-polarized etilwaneni letimunyisako, kugcila kwenethiwekhi ye-anti-Golgi (TGN) kuya ezingeni le-plasma ye-apical kulawula kuhlukaniswa kanye nekuhlunga kwe-GPI-AP (10, 45). Loku kuhlanganiswa kucondziswa yi-GPI-AP oligomerization (36), kepha kungaphindze kuncike ebudeni bemaketanga e-ceramide lesitfola kumvubelo. Noma nje i-GPI-AP yetilwane letimunyisako ine-ether lipid-based anchor, futsi sakhiwo sayo semakhemikhali sehluke kakhulu ku-acyl chain ceramide lendze kakhulu, lucwaningo lwamuva lutfole kutsi bobabili ema-lipids anetimphawu letifanako tekutigucukela kwemvelo kanye netemakhemikhali Futsi umsebenti (40). Ngakoke, lencenye ye ether lipid kumaseli etilwane letimunyisako ingafana ne C26 ceramide ku yeast, futsi indzima yayo kutsi ihlangane ne ceramide lendze lesetulu kule membrane kwentela kukhutsata kuhlanganiswa kwe GPI-AP kanye nekuhlunga. Noma loku kungenteka kusadzinga kuhlolwa ngalokucondzile, lokutfolwe ngaphambilini kusekela kutsi kuhanjiswa kwe acyl chain ceramide lendze kuya emtimbeni we Golgi akukwentiwa ngemaprotein ekudlulisela e-cytoplasmic, kepha kuya ngekuhlanganiswa kwema-anchor e-GPI njengemvubelo. Ngako-ke, indlela yekutigucukela kwemvelo yekulondvolota ibonakala ikhona kukhetsa co-kutfutsa i-acyl chain ceramide lendze kakhulu kanye ne-GPI-AP (13, 16, 20, 46, 47) ku-vesicle lefanako yekutfutsa.
Emvubelo kanye netinhlelo temaseli e-epithelial e-polarized etilwaneni letimunyisako, kuhlanganiswa kwe-GPI-AP kanye nekuhlukaniswa nalamanye emaprotein e-plasma membrane konkhe kwenteka ngaphambi kwekutsi kufike etulu kwemaseli. Paladino kanye nalabanye. (48) watfola kutsi ku-TGN yemaseli e-epithelial la-polarized etilwaneni letincelisako, kuhlanganiswa kwe-GPI-AP akudzingeki kuphela ekuhlukaniseni lokukhetsiwe kwe-GPI-APs kuya ku-apical plasma membrane, kepha futsi kulawula inhlangano yekuhlanganiswa kwe-GPI-APs kanye nemsebenti wayo wetintfo letiphilako. Indzawo yeseli. Emvubelo, lolu cwaningo lwabonisa kutsi C26 ceramide-ancike GPI-AP iqoqo ku ER ungalawula inhlangano iqoqo kanye umsebenti ukusebenza GPI-AP ulwelwesi plasma (24, 49). Kuhambisana nale modeli, emaseli e-GhLag1 ayagula ku-GPI inhibitors noma imitsi letsintsa bucotfo belubondza lwemaseli (28), kanye nesidzingo semacembu lasebentako e-Gas1-GFP (49) e-tip ceramide lekhonjiswe ekuhlanganisweni kwemangcamuzane emvubelo kukhombisa G Imiphumela lekhonakalako ye-hLag1. Liphutsa le-GPI-AP. Kepha, kuhlolwa lokunyenti kutsi ngabe inhlangano lesebentako yemhlabatsi wemaseli ihlelelwe kusuka ku-ER ngendlela yekuhlunga lesekelwe ebudeni be-lipid kutawuba sihloko selucwaningo lwetfu lwesikhatsi lesitako.
Tinhlobo te Saccharomyces cerevisiae letisetjentiswe kulomsebenti tibalwe ku Lithebula S1. Le MMY1583 kanye ne MMY1635 tinhlobo te SCLIM tekutsatsa titfombe temaseli laphilako takhiwa ngemuva kwe W303. Letinhlobo letiveta i-Sec13-mCherry lenethegi yemaprotheyini lakhanyako takhiwe kusetjentiswa indlela lesekelwe ku-polymerase (PCR) nge-pFA6a plasmid njengesifanekiso (23). Loluhlobo loluveta iMid2-iRFP lebhalwe ngeprotheyini lekhanyako ngaphansi kwekulawula kwemgcugcuteli we GAL1 lwakhiwa ngalendlela lelandzelako. Kukhuliswa kwe-PCR kwekulandzelana kwe-iRFP-KanMx kusuka ku-pKTiRFP-KAN vector (sipho sa-E. O'Shea, inombolo ye-plasmid ye-Addgene 64687; Ngemuva kwekulandzelana kwe-genome ye-Mid2-iRFP kwakhuliswa futsi kwakhicitwa kumgcugcuteli we-GAL1, kwahlanganiswa endzaweni ye-Not I-Sac I ye-plasmid pRS306 yekuhlanganiswa. I-plasmid pRGS7 lephumelelako yahlanganiswa ne-Pst I kute ihlanganiswe ne-URA3 locus.
Lelijini lekuhlanganiswa kwe Gas1-GFP livetwa ngaphansi kwekulawula kwemgcugcuteli we GAL1 ku plasmid ye centromere (CEN), leyakhiwe ngalendlela lelandzelako. Lokulandzelana kwe-Gas1-GFP kwakhuliswa yi-PCR kusuka ku-plasmid ye-pRS416-GAS1-GFP (24) (sipho sa-L. Umlimi; Inombolo ye-plasmid ye-adjene 51225; 51225; RRID: Sihlanganiso_51225). Le plasmid lephumelelako yabitwa ngekutsi yi- pRGS6. I-Axl2-GFP fusion gene iphindze ivezwe ngaphansi kwekulawula kwe-GAL1 umgcugcuteli we-pBEVY-GL LEU2 vector, futsi kwakhiwa kwayo kunjalo. Lokulandzelana kwe-Axl2-GFP kwakhuliswa kusuka ku-plasmid ye-pRS304-p2HSE-Axl2-GFP (23) nge-PCR, futsi kwakhicitwa endzaweni ye-Bam HI-Pst I ye-pBEVY-GL LEU2 vector. Le plasmid lephumelelako yabitwa ngekutsi yi-pRGS12. Kulandzelana kwe-oligonucleotides lesetjentiswe kulolu cwaningo kubhalwe kuThebula S2.
Loluhlobo lwangetwa nge 0.2% we adenine kanye na 2% we glucose [YP-dextrose (YPD)], 2% we raffinose [YP-raffinose] lecebile yemvubelo lekhishwa ngemaprotein p (YP) lesemkhatsini (1 % Imvubelo lekhishwako kanye na 2% wemaprotein ept). (YPR)] noma 2% we-galactose [YP-galactose (YPG)] njengemtfombo wekhabhoni, noma ngendlela lencane yekwentiwa (0.15% wemvubelo ye-nitrogen base kanye na-0.5% we-ammonium sulfate) kufaka ema-amino esidi lafanele kanye netisekelo letidzingekako tekudla , Futsi lecuketse 2% we-gactose lencane ye-glucose ( (i-galactose yekwentiwa lencane lesemkhatsini) njengemtfombo wekhabhoni.
Kute kutfolakale titfombe ngesikhatsi sangempela, emaseli lagucukako labucayi ngekushisa e-sec31-1 laveta kwakha ngaphansi kwemgcugcuteli we-GAL1 akhuliswa ku-YPR medium ku-24°C ebusuku kuya esigabeni se-log lesemkhatsini. Ngemuva kwekufakwa ku YPG ku 24°C lihora linye, lamaseli afutfunyelwa ku SG ku 37°C imizuzu lengemashumi lamatsatfu, bese adluliselwa ku 24°C kutsi akhululwe kusuka ku secretion block. I-Concanavalin A yasetjentiswa kulungisa lamaseli esilayidi sengilazi futsi yatsatfwa ngetitfombe yi-SCLIM. I-SCLIM yinhlanganisela ye-Olympus IX-71 sibonakhulu sekukhanya lokugucukile kanye ne-UPlanSApo 100×1.4 lens yemafutsa ekuvula ngetinombolo (i-Olympus), sivinini lesikhulu kanye nesilinganiso lesiphakeme sesiginali-kuya-emsindvweni lejikeletako ye-disc spectrometer ye-confocal scanner (Yokogawa Electric) (I-Photonics ye-Hamamatsu) inganiketa luhlelo lwe-lens lekhulisako ngekukhulisa kwekugcina lokungu-×266.7 kanye nekhamera yetintfo letihlanganiswe nekushaja lephindzaphindza ema-electron (I-Photonics ye-Hamamatsu) (21). Kutfolakala kwetitfombe kwentiwa ngesofthiwe yesiko (Yokogawa Gezi). Kute titfombe te-3D, sasebentisa i-actuator ye-piezoelectric leyentiwe ngekwemkhuba kute idvudvute lens lenhloso ngco, futsi sabutsela ndzawonye tincenye letibonakalako letingu-100 nm ngaphandle kwemfumba. Lesitfombe se-Z-stack siguculwa sibe yidatha ye-voxel ye-3D, futsi umsebenti wekusakateka kwemaphuzu etifundvo losetjentiselwa sibonakhulu se-confocal ye-disc lejikeletako usetjentiselwa kucubungula ku-deconvolution ngesoftware ye-Volocity (PerkinElmer). Ngekusebentisa isoftware yeVolocity kutsi ivele umkhawulo wekuhlatiya indzawo yekuhlanganyela, i-ERES kufaka ekhatsi imithwalo yakalwa. Kuhlatiya kwe-line scan kwentiwa kusetjentiswa isoftware ye-MetaMorph (Tintfo Temamolekhyuli).
Sebentisa isoftware ye-GraphPad Prism kute utfole kubaluleka kwetibalo. Ku-t-test yeMfundzi lenemisila lemibili kanye nekuhlatiya lokujwayelekile kwendlela yinye yekuhluka (ANOVA), umehluko emkhatsini wemacembu utsatfwa njengalonemtselela lomkhulu ku-P <0.05 (*).
Ku-fluorescence microscopy ye-Gas1-GFP, emaseli esigaba se-log akhuliswa busuku bonkhe e-YPD futsi ahlanganiswa nge-centrifugation, agezwa kabili nge-phosphate buffered saline, bese afutfunyelwa e-ice lokungenani imizuzu lengu-15, bese ayachubeka ngaphansi kwesibonakhulu njengoba kuchaziwe ngaphambilini (Check24). Sibonakhulu se-Leica DMi8 (i-HCX PL APO 1003/1.40 oyili PH3 CS) lesifakwe lensi lenemgomo, luhlu lwe-L5 (GFP), ikhamera ye-Hamamatsu kanye nesofthiwe ye-Application Suite X (LAS X) yasetjentiswa kutfola. .
Lamasampula aguculwa nge SDS isampula buffer ku 65°C imizuzu lelishumi, bese ahlukaniswa nge SDS-polyacrylamide gel electrophoresis (LIKHASI). Kuhlatiya kwe-immunoblotting, 10 μl wesampula walayishwa ngelayini. I-antibody leyinhloko: Sebentisa i-anti-Gas1 ye-polyclonal yanogwaja ngesikhatsi sekuhlanjululwa nga-1:3000, i-anti-Emp24 ye-polyclonal yanogwaja ngekuncibilika nga-1:500, kanye ne-anti-GFP ye-polyclonal yanogwaja (sipho lesivela ku H. Riezman) ngekuncibilika kwa-1:3000. Leligundvwane lelilwa ne Pgk1 lasetjentiswa ngekuhlanjululwa kwa 1:5000 (sipho lesivela ku J. de la Cruz). I-IgG yesibili: I-Horseradish Peroxidase (HRP) lehlanganisiwe yembuti lelwa nenogwaja (IgG) lesetjentiswa ngekuncibilikisa nga 1:3000 (Pierce). IgG yembuti lehlanganiswe ne HRP lelwa neligundvwane yasetjentiswa ngesikhatsi sekuhlanjululwa kwa 1:3000 (Pierce). Indzawo yekuphendvula kwemasotja emtimba yabonwa ngendlela yekukhanya kwemakhemikhali ye-SuperSignal West Pico reagent (i-Thermo Fisher Scientific).
Njengobe kuchaziwe ku (31), kuhlolwa kwemvelo kwe-immunoprecipitation kwentiwa ku-ER ingcenye lecebile. Ngamafuphi, geza emaseli emvubelo nge-TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride kanye nekuhlanganiswa kwekuvimbela i-protease) ku-600 nm ( OD600) ku-100 kabili. Yaphulwa ngebuhlalu bengilazi, kwase kutsi imfucumfucu yemaseli kanye nebuhlalu bengilazi kususwa nge centrifugation. Le supernatant yabese iyahlanganiswa ku 17,000 g imizuzu lelishumi nesihlanu ku 4°C. Le pellet yaphindze yamiswa ku TNE bese i-digitalis saponin yengetwa kuze kube ngu 1%. Lokumiswa kwafutfunyelwa lihora linye ngekujikeleta ku 4°C, bese letintfo letingancibiliki tasuswa ngekutsi tifakwe ku centrifugation ku 13,000 g ku 4°C imizuzu lengemashumi lasitfupha. Kute kuvikeleke kwe Gas1-GFP, cala ngekufukamela lesampula ngebuhlalu be agarose lobungenalutfo (ChromoTek) ku 4°C lihora linye, bese ufukamela nge GFP-Trap_A (ChromoTek) ku 4°C emahora lamatsatfu. Lobuhlalu lobuvulekile bagezwa tikhatsi letisihlanu nge TNE lecuketse 0.2% digoxigenin, bahlanjululwa nge buffer yesampula ye SDS, bahlukaniswe ku SDS-PAGE, futsi bahlatiya nge immunoblotting.
Njengobe kuchaziwe ku (31), kuncuma kwekuhlanganisa lokuphambukile kwentiwa ku-ER fraction lecebile. Kafishane, ingcenye ye-ER lececeshiwe yakhuliswa nge-0.5 mM dithiobis (i-succinimidyl propionate) (i-Pierce, i-Thermo Fisher yesayensi, i-Rockford, i-IL, eMelika; 20 ° C, imizuzu lengu-20). Lokuphendvula kwekuhlanganiswa kwacinywa ngekwengeta glycine (50 mM lokugcina lokuhlushwa, imizuzu lesihlanu, 20 ° C).
Njengobe kuchaziwe ngaphambilini (50), kuhlatiya kwe-MS kwe-ceramide ku-wild-type kanye ne-GhLag1 tinhlobo kwentiwa. Ngamafuphi, emaseli akhuliswa abe sigaba se exponential (3 kuya ku 4 OD600 emayunithi/ml) ku YPD ku 30°C, futsi emaseli langu 25×107 avunwa. Kusebenta kwawo kwemtimba kucinywa nge-tricchloroacetic esidi. Sebentisa sincibilikisi sekukhipha [i-ethanol, emanti, i-ether, i-pyridine kanye ne-4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] kanye ne-1.2 nmol yelizinga langekhatsi le-C17 ceramide (860517, i-Avanti polar lipid). Sebentisa i-reagent ye-monomethylamine [i-methanol, emanti, i-n-butanol kanye nesisombululo se-methylamine (4:3:1:5 v/v)] kwenta i-alkaline lencane yekukhicita emanti, bese usebentisa i-n-butanol legcwele emanti kwenta luswayi. Ekugcineni, loku lokukhishiwe kwaphindze kwafakwa ngendlela lenhle yekuncibilikisa [i-chloroform/methanol/emanti (2:7:1) + 5 mM ammonium acetate] kwase kujovwa ku-spectrometer yebuningi. Kugadza kwekuphendvula lokunyenti (MRM) kwentiwa kute kukhonjwe futsi kulinganiswe emamolekhyuli e-sphingolipid. I-TSQ Vantage ye-Termo Fisher Scientific ifakwe umtfombo we-ion ye-nanoflow yerobhothi i-Nanomate HD (I-Advion Biosciences, e-Ithaca, e-NY) yekuhlatiya emafutsa. Amandla ekushayisana alungiselelwe sigaba ngasinye se-ceramide. Idatha ye-MS yatfolakala ngendlela lenhle. Kukuphindzaphindza ngamunye kwetilwane, luphawu lwe-lipid lusemkhatsini wetilinganiso letintsatfu letitimele.
Njengobe kuchaziwe ku (31), emaseli (800 × 107) laveta iGas1-GFP abekwa ngaphansi kwekuvikela kwemvelo. Le Gas1-GFP lehlanjululwe yahlukaniswa yi SDS-PAGE futsi yadluliselwa ku ulwelwesi lwe polyvinylidene fluoride (PVDF). Le protein yabonakala ngekutsi kugcotjwe i-PVDF nge-amide lemnyama. Lelibhande le Gas1-GFP lajutjwa kusuka ku PVDF futsi lagezwa tikhatsi letingu 5 nge methanol futsi kanye ngemanti lasesigabeni se chromatography-MS (LC-MS). Ngekufukamela umgca welucwencwe nge-500μl 0.3 M NaOAc (pH 4.0), sivikelo kanye ne-500μl yekuhlanganiswa kwe-sodium nitrite 1 M lesandza kuncibilikiswa ku-37°C emahora lamatsatfu, lencenye ye-lipid ikhululwa kusuka ku-Gas1-GFP kanye ne-phocosa ino-inophamine le-lysed (51). Ngemuva kwaloko, lomgca we membrane wagezwa tikhatsi letine ngemanti elizinga le LC-MS, womiswa ngesikhatsi sekushisa ekamelweni, bese ugcinwa emoyeni we nitrogen ku -80°C kwaze kwaba ngulapho sekuhlatiya. Njengekulawula, isampula lengenalutfo ye-PVDF membrane yasetjentiswa ekuhlolweni ngamunye. I-lipid lekhishwe ku-Gas1-GFP yabese ihlaziywa yi-MS njengoba kuchaziwe (50). Ngamafuphi, imichilo ye-PVDF lecuketse i-GPI-lipid yaphindze yamiswa ku-75μl yesikhunta lesibi [i-chloroform / i-methanol (1: 2) + 5 mM ammonium acetate] futsi yadlula ku-ionization ye-electrospray (ESI)-MRM / MS Kuhlatiya kwetinhlobo te-sphingolipid (TSQ Vantage). Kulesi simo, idatha MS kwatholakala imodi ion embi.
Njengobe kushiwo ngaphambili, incenye ye-lipid ye-anchor ye-GPI yahlukaniswa ne-[3H] -inositol-lebhalwe ku-GPI-AP (16). Emafutsa ahlukaniswe ngekusebentisa luhlelo lwekuncibilikisa (55:45:10 chloroform-methanol-0.25% KCl) futsi abonakala ngekusebentisa iFLA-7000 (Fujifilm).
Emaseli laveta iGas1-GFP (600×107) agezwa kabili nge TNE buffer nge TNE buffer, bese aphulwa ngebuhlalu bengilazi, bese aya centrifuged kususa imfucumfucu yemaseli kanye nebuhlalu bengilazi. Le supernatant yabese iyahlanganiswa ku 17,000 g lihora linye ku 4°C. Le pellet yagezwa ku TNE yase iyafutfunyelwa nge 1 U PI-PLC (Invitrogen) ku TNE lecuketse 0.2% we digitalis saponin lihora linye ku 37°C. Ngemuva kwekwelashwa kwe enzyme, le membrane yasuswa ngekutsi ihlanganiswe ku 17,000 g ku 4°C lihora linye. Kute kuvikeleke iGas1-GFP, lendzawo lesetulu yafutfunyelwa nge GFP-Trap_A (ChromoTek) ku 4°C busuku bonkhe. Le Gas1-GFP lehlanjululwe lehlukaniswe yi SDS-PAGE yayigcotjwe nge Coomassie loluhlata sasibhakabhaka lolukhanyako. Lelibhande lekufaka emabala le Gas1-GFP lancunywa kusuka kulomphunga lozungete umsele wemanti, kwase ngemuva kwekutsi kuhlanganiswe nge iodoacetamide kanye nekunciphisa nge dithiothreitol, kugaya nge trypsin kwentiwa. Khipha bese womisa ema-peptides e-tryptic kanye nema-peptides lane-GPI-glycans. Le peptide leyomile yancibilikiswa ku 20 μl wemanti. Jova incenye (8μl) ku-LC. Ikholomu ye octadecylsilane (ODS) (iDevelosil 300ODS-HG-5; bubanti bangekhatsi 150 mm×1.0 mm; iNomura Chemical, Sifundza sase Aichi, eJapan) yasetjentiswa kwehlukanisa ema peptides ngaphansi kwetimo letitsite te gradient. Sigaba lesihambako sincibilikisa A (0.08% esidi yefomiki) kanye nesincibilikisi B (0.15% esidi yefomiki ku 80% we acetonitrile). Luhlelo lwe Accela HPLC (Thermo Fisher Scientific, Boston, Massachusetts) lwasetjentiswa kukhipha lekholomu ngesincibilikisi A kungakapheli imizuzu lengemashumi lasihlanu nesihlanu ngesilinganiso sekugeleta se 50 μl min-1 imizuzu lesihlanu, kwase kutsi lokucuketfwe kwesincibilikisi B kwakhuliswa kwaba ngu 40%. , eMelika). Le eluate yachubeka yetfulwa emtfonjeni we-ion ye-ESI, futsi ema-peptides e-tryptic kanye nema-peptides lane-GPI-glycans ahlaziywa yi-LTQ Orbitrap XL (i-spectrometer ye-ion lehlanganisiwe lehambisanako; i-Thermo Fisher Scientific). Kusetha kwe MS, ivoltheji yemtfombo we capillary yabekwa ku 4.5 kV, futsi lizinga lekushisa le capillary yekudlulisa lagcinwa ku 300°C. I voltage capillary kanye tube lens voltage kwabekwa 15 V kanye 50 V, ngokulandelana. Idatha ye-MS itfolakele ngendlela ye-ion lenhle (sincumo se-60,000; kuciniseka kwebukhulu betincenye letingu-10 ngesigidzi) esigabeni sebukhulu be-300/m/z bukhulu/silinganiso sekushaja (m/z) 3000. kushayisana lokubangelwa kuhlangana (CID)].
MD kulingisa kwentiwa kusetjentiswa GROMACS (52) isofthiwe kanye MARTINI 2 emandla insimu (53-55). I-CHARMM GUI Makhi Welucwencwe (56, 57) yabese isetjentiswa kwakha lucwencwe lolubili lolucuketse i-dioleoylphosphatidylcholine (i-DOPC) kanye ne-Cer C18 noma i-DOPC ne-Cer C26. Le topology kanye nekuhlanganiswa kwe Cer C26 kutsatfwa ku DXCE ngekususa lobuhlalu lobungetiwe kusuka emsileni we sphingosine. Sebentisa inchubo lechazwe lapha ngentasi kulinganisa luhlaka loluphindvwe kabili bese ulugijima, bese usebentisa ema-coordinates ekugcina eluhlelo kwakha luhlelo lolucuketse i-Emp24. Lendzawo ye-transmembrane yemvubelo Emp24 (tinsalela 173 kuya ku-193) yakhiwa njenge-α-helix kusetjentiswa isakhiwo se-molecule yethuluzi le-MD (VMD) lebonakalako (58). Ngemuva kwaloko, ngemuva kwekususa ema lipids lahlanganisiwe, le protein yakhiwa ngetinhlavu letimbi futsi yafakwa ku bilayer kusetjentiswa i CHARMM GUI. Luhlelo lwekugcina lucuketse 1202 DOPC kanye 302 Cer C26 noma 1197 DOPC kanye 295 Cer C18 kanye ne Emp24. Ionize uhlelo kuya lokuhlushwa 0.150M. Tiphindvwe letine letitimele tentiwa ngekwakheka lokubili kwe-bilayer.
Le lipid bilayer ilinganiswa ngekusebentisa inchubo ye CHARMM GUI, lefaka ekhatsi kunciphisa bese kulinganisa tinyatselo letingu 405,000, lapho khona kuvinjelwa kwesikhundla kuncishiswa kancane kancane futsi kucedvwe, bese sinyatselo sesikhatsi siyakhuliswa kusuka ku 0.005 ps kuya ku 0.02 ps. Ngemuva kwekulingana, ikhicita 6 μs ngesinyatselo sesikhatsi se 0.02 ps. Ngemuva kwekufaka Emp24, sebentisa inchubo lefanako ye-CHARMM GUI kunciphisa nekulinganisa luhlelo, bese ugijima 8 s ekukhiciteni.
Kutotonkhe tinhlelo, ngesikhatsi sekulinganisa, umfutfo ulawulwa yi Barendsen (59), futsi ngesikhatsi sekukhicita, umfutfo ulawulwa yi Parrinello-Rahman (60). Kuto tonkhe timo, umfutfo lomlinganiselo ngu 1 bar futsi kusetjentiswa luhlelo lwekuhlanganisa umfutfo we semi-isotropic. Enchubeni yekulinganisa kanye nekukhicita, i-thermostat (61) lenekulinganisa kabusha ngesivinini isetjentiswa kuhlanganisa lizinga lekushisa le protein, lipid kanye netinhlayiya te solvent ngekulandzelana. Ngesikhatsi sonkhe sekusebenta, lokushisa lokuhlosiwe ngu-310K. Lokusebentisana lokungabophi kubalwa ngekukhicita luhlu lwekubhaca kusetjentiswa luhlelo lwe Verlet ngekubeketela kwe 0.005 buffer. Lethemu le Coulomb libalwa kusetjentiswa insimu yekuphendvula kanye nebanga lekusika le 1.1 nm. Lelibito la Vander Waals lisebentisa luhlelo lwekusika lolunebanga lekusika le 1.1 nm, bese luhlelo lwekusika lwa Verlet lusetjentiselwa kuhamba lokungenteka (62).
Kusebentisa iVMD, budze begagasi lobujutjiwe emkhatsini webuhlalu be DOPC phosphate noma buhlalu be ceramide AM1 kanye ne protein ngu 0.7 nm, bese linani lemafutsa lasebentisana ne protein liyabalwa. Ngekuya kwefomula lelandzelako, bala sici sekucitsa-kucebisa (DE) njengase (63): sici se-DE = (linani lemafutsa laphelele kuphrotheni 0.7) kuphrotheni 0.7 (linani leCer kumafutsa laphelele)
Inani lelibikiwe litfolakala njengesilinganiso, futsi emabha ephutsa ngemakhophi lamane latimele e-SE. Kubaluleka kwetibalo kwesici se-DE kubalwa ngekuhlolwa kwe-t [(silinganiso se-DE-sici-1) / SE]. Bala inani le-P kusuka ekusabalalisweni kwemsila munye.
Lithuluzi le GROMACS lasetjentiswa kubala imephu ye 2D lateral density yeluhlelo lolucuketse i Emp24 ngaphakatsi kwe 250 ns yekugcina yekulandzelela. Kute kutfolakale imephu yekucebisa/yekucitsa ye ceramide, imephu yekucina kwe Cer ihlukaniswe ngesamba semephu ye Cer ne DOPC, bese ihlukaniswa ngekugcwala kwe Cer emtimbeni. Kusetjentiswa sikali sebalave lelifanako lembala.
Kute utfole tintfo letengetiwe talesihloko, sicela ubuke http://advances.sciencemag.org/cgi.
Lena yindzatjana levulekile lesakatwa ngaphasi kwemigomo ye-Creative Commons Attribution-Non-Commercial License, levumela kusetjentiswa, kusabalaliswa kanye nekukhicita kabusha nganoma nguyiphi indlela, uma nje kusetjentiswa kwekugcina akusiko kwenzuzo yekutsengisa futsi umcondvo kutsi umsebenti wekucala ulungile. Kudlilisela.
Caphela: Sicela kuphela kutsi unikete likheli lakho le-imeyili kute umuntfu lomncoma kulelikhasi ati kutsi ufuna ayibone le-imeyili nekutsi akusiyo i-spam. Angeke sitsatse noma ngumaphi emakheli e-imeyili.
Lombuto usetjentiselwa kuhlola kutsi usivakashi yini kanye nekuvikela kuhanjiswa kwe-spam lokuzenzakalelayo.
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 Lorgihoz -Linero), Waga, Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
3D high-resolution real-time imifanekiso kuveta kubaluleka ceramide chain ubude for emaprotheni kuhlunga etindzaweni letikhetsiwe lokukhipha.
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 Lorgihoz -Linero), Waga, Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
3D high-resolution real-time imifanekiso kuveta kubaluleka ceramide chain ubude for emaprotheni kuhlunga etindzaweni letikhetsiwe lokukhipha.
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