Ukuhlunga amaprotheni endleleni yokukhipha amaprotheni kubalulekile ekugcineni ukwakheka kwamaseli kanye ne-homeostasis. Ngaphezu kokuhlunga okuqhutshwa yigobolondo, indima yama-lipid ekuhlungeni i-kinesin enkambisweni yokuthutha i-secretory ngumbuzo oyisisekelo osekuyisikhathi eside ungakaphendulwa. Lapha, senza isithombe sesikhathi sangempela se-3D ngesikhathi esisodwa semibala eminingi ukuze sifakazele in vivo ukuthi amaprotheni asanda kuhlanganiswa e-glycosylphosphatidylinositol-immobilized ane-ceramide lipid moieties ende kakhulu ahlanganiswa futhi ahlukaniswe kuma-endoplasm akhethekile. Indawo yokuphuma e-Net, ehlukile kuleyo esetshenziswa amaprotheni e-transmembrane. Ngaphezu kwalokho, sibonisa ukuthi ubude beketanga le-ceramide ku-endoplasmic reticulum membrane bubalulekile kulokhu kukhetha kokuhlunga. Ucwaningo lwethu lunikeza ubufakazi bokuqala obuqondile in vivo bokuhlukanisa imithwalo yamaprotheni ngokusekelwe kubude beketanga le-lipid ezindaweni zokuthumela ezikhethiwe endleleni yokukhipha amaprotheni.
Kumaseli e-eukaryotic, amaprotheni akhiwe ku-endoplasmic reticulum (ER) abe esehlelwa ngesikhathi sokuthuthwa ngendlela eyimfihlo ukuze athunyelwe endaweni yawo efanele yamaseli (1). Ngaphezu kokuhlunga okuhlanganiswe yi-coat, kwacatshangwa isikhathi eside ukuthi ama-lipid athile angasebenza njengezindawo zokuphuma ezikhethiwe ngokuwahlanganisa ezindaweni ezithile ze-membrane kunamaprotheni athile (2-5). Kodwa-ke, kusenokuntuleka kobufakazi obuqondile be-in vivo bokufakazela le ndlela engenzeka esekelwe kuma-lipid. Ukuze sixazulule le nkinga eyisisekelo, sifunde ngemvubelo ukuthi amaprotheni asekelwe kuma-glycosylphosphatidylinositol (GPI) (GPI-APs) athunyelwa kanjani ngokuhlukile kusuka ku-ER. Ama-GPI-AP ayizinhlobo ezahlukahlukene zamaprotheni angaphezulu kweseli axhunywe kuma-lipid (6, 7). I-GPI-AP iyiprotheni ekhishwe enamathiselwe emapheshaneni angaphandle e-plasma membrane nge-glycolipid moiety (GPI anchor). Ayamukela ama-GPI anchor njengokuguqulwa okulondoloziwe ngemuva kokuhumusha ku-ER lumen (8). Ngemva kokunamathiselwa, i-GPI-AP idlula ku-Golgi apparatus (5, 9) isuka ku-ER iye ku-plasma membrane. Ukuba khona kwama-GPI anchors kubangela ukuthi i-GPI-AP ithuthwe ngokwehlukana nama-protein akhishwe yi-transmembrane (kufaka phakathi amanye ama-protein e-plasma membrane) endleleni yokukhipha (5, 9, 10). Kuma-yeast cell, ama-GPI-AP ahlukaniswa namanye ama-protein akhishwe ku-endoplasmic reticulum, bese efakwa kuma-vesicles ahlukile ahlanganiswe yi-coat protein complex II (COPII) (6, 7). Izinto ezibangela le nqubo yokuhlela enkambisweni yokuthumela i-ER azicaci, kodwa kucatshangwa ukuthi le ndlela ingadinga ama-lipid, ikakhulukazi ukuvuselelwa kwesakhiwo kwengxenye yama-lipid ye-GPI anchor (5, 8). Ku-yeast, ukuvuselelwa kwama-lipid e-GPI kuqala ngokushesha ngemva kokuba i-GPI inamathele, futhi ezimweni eziningi, kubangela ukuthi i-ceramide ibophe ku-26-carbon long-chain saturated fatty acid (C26:0) (11, 12). I-C26 ceramide iyi-ceramide eyinhloko ekhiqizwa amaseli emvubelo kuze kube manje. Yenziwe ku-ER futhi iningi layo lithunyelwa ku-Golgi apparatus ngama-vesicle e-COPII (13). Ukuthunyelwa kwe-ER kwe-GPI-AP kudinga ngokukhethekile ukwenziwa kwe-ceramide okuqhubekayo (14, 15), futhi ukuguqulwa kwe-ceramide kube yi-inositol phosphate ceramide (IPC) ku-Golgi apparatus kuncike ekuhlanganisweni kwe-GPI anchor (16). Izifundo ze-biophysical ngama-membrane okwenziwa zibonise ukuthi ama-ceramide amade kakhulu e-acyl chain angahlangana ukuze akhe ama-domain ahlelekile anezakhiwo zomzimba ezihlukile (17, 18). Le datha iholela emcabangweni wokuthi i-C26 ceramide kanye ne-GPI-AP ene-C26 ceramide zisebenzisa izakhiwo zazo zomzimba ukuze zihlangane ezindaweni ezihlelekile noma ezifundeni endaweni ye-ER membrane lipid engcolile. Yakhiwe kakhulu ngama-glycerolipids amafushane futhi angagcwali (C16:1 kanye ne-C18:1) (19, 20). Lezi zifunda zizogxila ngokukhethekile ezindaweni ezithile zokuphuma ze-ER (ERES), lapho i-ceramide kanye ne-ceramide-based GPI-AP zingathuthwa ndawonye ziye eGolgi nge-vesicle efanayo ye-COPII (5).
Kulolu cwaningo, sihlole ngqo le ndlela esekwe ku-lipid ngokusebenzisa i-super-resolution confocal real-time imaging microscopy (SCLIM), okuyindlela ye-microscopy esezingeni eliphezulu engabona ngasikhathi sinye amaprotheni anelebula elikhanyayo. Izithombe ezinemibala emithathu kanye ne-three-dimensional (3D) zinesisombululo esiphezulu kakhulu kanye nesivinini kumaseli aphilayo (21, 22).
Siqale sasebenzisa ubuchwepheshe be-SCLIM ukuze sichaze kabanzi ukuthi i-GPI-AP evamile eneqembu le-C26 ceramide ihlolwe kanjani kumaprotheni akhishwe yi-transmembrane ngemuva kokuphuma ku-ER ku-S. cerevisiae. Ukuze sihlole ukuhlukaniswa kwe-ER, sisebenzise uhlelo lwezakhi zofuzo olungabona ngqo impahla esanda kuhlanganiswa ingena ku-ERES in vivo (7, 23). Njengempahla, sikhethe i-GPI-AP Gas1 esekelwe ku-C26 ceramide ebhalwe nge-protein fluorescent eluhlaza (GFP) kanye ne-protein ekhishwe yi-transmembrane Mid2 ebhalwe nge-protein fluorescent eseduze ne-infrared (iRFP), zombili ezihlose i-plasma membrane (24-26). Ku-mutant ezwela ukushisa kwe-sec31-1, le mpahla emibili ivezwa ngaphansi kwe-promoter ye-galactose-inducible kanye ne-constitutive ERES marker. Ekushiseni okukhulu (37°C), ngoba ukuguqulwa kwe-sec31-1 kuthinta umsebenzi we-COPII coat component Sec31 ukuvimbela ukuhluma kwe-COPII kanye nokuthunyelwa kwe-ER, impahla esanda kuhlanganiswa iqoqana ku-ER (23). Ngemva kokupholisa kufikela ezingeni lokushisa eliphansi (24°C), amaseli ashintshiwe e-sec31-1 aphinde atholakala endaweni yokukhipha, futhi impahla entsha yokwenziwa eqoqwe yaqala ukuthunyelwa isuka ku-ER. Ukuboniswa kwe-CLIM kubonise ukuthi iningi lama-Gas1-GFP amasha kanye ne-Mid2-iRFP asaqongelelwe ku-ER yamaseli ashintshiwe e-sec31-1 ngemva kokufakwa ku-37°C bese ekhishwa ku-24°C imizuzu emi-5 (Isithombe 1). Njengoba i-Mid2-iRFP isatshalaliswa kulo lonke ulwelwesi lwe-ER, kanti i-Gas1-GFP igxilile futhi iqoqwe endaweni yolwelwesi lwe-ER engaqhubeki, ukusatshalaliswa kwayo kuhlukile ngokuphelele (Isithombe 1, A kuya ku-C kanye ne-Movie S1). Ngaphezu kwalokho, njengoba kuboniswe ku-Figure 1D, iqembu le-Gas1-GFP alinayo i-Mid2-iRFP. Le miphumela ikhombisa ukuthi amaprotheni e-GPI-AP kanye ne-transmembrane ahlukaniswe ezindaweni ezahlukene zolwelwesi lwe-ER ekuqaleni. Iqoqo le-Gas1-GFP liseduze ne-ERES ethile ebhalwe nge-mCherry's COPII coat protein Sec13 (Isithombe 1, E no-F, kanye ne-movie S1) (23).
Amaseli e-sec31-1 aveza ukukhishwa okubangelwa yi-galactose, i-ceramide ende ye-acyl chain (C26) GPI-AP Gas1-GFP (GPI-AP, eluhlaza) kanye neprotheyini ye-transmembrane Mid2-iRFP (TMP, eluhlaza okwesibhakabhaka) kanye nale lebula ye-ERES Constructive ERES Sec13-mCherry (ERES, magenta) yafakwa ku-37°C imizuzu engama-30, yathuthelwa ku-24°C, futhi yathathwa yi-SCLIM ngemva kwemizuzu emi-5. (A kuya ku-C) ikhombisa isithombe esihlanganisiwe noma esisodwa se-2D sendiza (A), isithombe se-projection se-2D sezingxenye eziyi-10 z (B) noma isithombe se-hemisphere yeseli ye-3D sempahla kanye nama-ERES markers (C). Ibha yesikali 1μm (A no-B). Iyunithi yesikali ingu-0.551μm (C). I-Gas1-GFP itholakale ezifundeni noma emaqenjini e-ER ahlukene, kuyilapho i-Mid2-iRFP itholakale futhi yasatshalaliswa kulo lonke i-membrane ye-ER (C). (D) Igrafu ikhombisa ukuqina kokukhanya kwe-Gas1-GFP kanye ne-Mid2-iRFP eqenjini le-Gas1-GFP eceleni komugqa womcibisholo omhlophe (kwesobunxele). I-AU, iyunithi engaqondakali. (E kanye no-F) bamele isithombe se-3D esihlanganisa izimpahla kanye nophawu lwe-ERES. Amaqoqo e-Gas1-GFP atholakale eduze kwe-ERES ethile. Iyunithi yesikali ingu-0.551μm. (F) Umcibisholo omhlophe oqinile uphawula iqembu le-Gas1-GFP elihlotshaniswa ne-ERES. Amaphaneli aphakathi nakwesokudla abonisa isithombe se-3D esihlanganisiwe esikhulisiwe kanye nombono ojikeleziwe weqembu le-Gas1-GFP elikhethiwe.
Ubudlelwano obuseduze bendawo phakathi kweqembu le-Gas1-GFP kanye ne-ERES ethile bubonisa ukuthi i-Gas1-GFP ingangena ku-ERES ekhethiwe, okuhlukile ekukhetheni okusetshenziswa yi-Mid2-iRFP ukushiya i-ER. Ukuze sibhekane nalokhu kungenzeka, silinganise isilinganiso se-ERES sempahla eyodwa noma ezimbili kuphela (Isithombe 2, A kuya ku-C). Sithole ukuthi iningi le-ERES (70%) liqukethe uhlobo olulodwa lwempahla. Isithombe esingezansi se-Figure 2C sibonisa izibonelo ezimbili ezijwayelekile ze-ERES ene-Gas1-GFP kuphela (Isithombe 1) noma i-Mid2-iRFP kuphela (Isithombe 2). Ngokuphambene nalokho, cishe u-20% we-ERES uqukethe imithwalo emibili ehlangana endaweni efanayo. Kutholakale ukuthi amanye ama-ERES (10%) aqukethe izinhlobo ezimbili zemithwalo, kodwa ahlukaniswe ezindaweni ezihlukile ngokusobala. Ngakho-ke, lokhu kuhlaziywa kwezibalo kukhombisa ukuthi ngemva kokuba i-ER ithunyelwe kwamanye amazwe, i-GPI-AP Gas1-GFP kanye nemithwalo ye-transmembrane Mid2-iRFP zihlukaniswe ngama-ERES ahlukene (Isithombe 2D). Lokhu kusebenza kahle kokuhlunga kuhambisana kakhulu nokuhlaziywa kwamakhemikhali okwedlule (6) kanye nokunqunywa kwesimo (7). Singabona futhi ukuziphatha kwempahla evalelwe yodwa engena e-ERES (Isithombe 2E kanye ne-Movie S2). Isibalo 2E sibonisa ukuthi ingxenye encane kuphela ye-Gas1-GFP (iphaneli 3) noma i-Mid2-iRFP (iphaneli 4) engena kwi-ERES isuka kolunye uhlangothi futhi ivalelwe endaweni ehlukanisiwe. Iphaneli 5 yesithombe 2E ikhombisa ukuthi i-Gas1-GFP kanye ne-Mid2-iRFP ngezinye izikhathi zitholakala ku-ERES efanayo, kodwa zingena ezinhlangothini ezahlukene futhi zigxile ezifundeni ezihlukene ezingase zimele ama-vesicles e-COPII ahlukene. Siphinde saqinisekisa ukuthi ukuhlukaniswa okubonwayo kanye nokuhlukaniswa kwe-C26 ceramide-based GPI-AP Gas1 njenge-ERES ekhethiwe kuqondile ngoba enye i-transmembrane secretion cargo, i-GFP-taged plasma membrane protein Axl2 (27), ekhombisa ukuziphatha okufanayo ne-Mid2-iRFP. (Isithombe S1 kanye ne-Movie S3). I-Axl2-GFP esanda kuhlanganiswa isatshalaliswa nge-membrane ye-ER njenge-Mid2-iRFP (Isithombe S1, A kanye no-B), futhi ihambisana ne-Mid2-iRFP kuma-ERES amaningi (Isithombe S1, B kuya ku-D). Amaphaneli 1 kanye no-2 eSithombe 1. I-S1C ikhombisa izibonelo ezimbili ezijwayelekile ze-ERES lapho imithwalo emibili ye-transmembrane ihlangana khona. Kulezi zimo, zombili izimpahla zingena ndawonye ku-ERES (Isithombe S1E, Iphaneli 3 kanye ne-Movie S3).
Amaseli e-sec31-1 aveza ukukhishwa kwe-galactose okungafakwanga, i-Gas1-GFP (GPI-AP, eluhlaza) kanye ne-Mid2-iRFP (TMP, eluhlaza okwesibhakabhaka) kanye nelebula ye-ERES equkethe i-Sec13-mCherry (ERES, magenta) abekwe ku-37 Ngemva kokufukamela imizuzu engama-30 ku-°C, thuthela ku-24 °C ukuze ukhulule ibhulokhi yokukhishwa, kanye nesithombe nge-SCLIM ngemuva kwemizuzu engama-20. (A kuya ku-C) Izithombe ezimele ze-2D projection (A; scale bar, 1μm) noma izithombe ze-3D cell hemisphere (B kanye no-C; scale unit, 0.456μm) yempahla kanye nezingxenye eziyi-10 z ezimakwe yi-ERES. Iphaneli engezansi ku-(B) kanye nephaneli ku-(C) zibonisa izithombe ezicutshungulwe ukuze zibonise kuphela izimpahla ezikhona ku-ERES (magenta) [Gas1-GFP (grey) kanye ne-Mid2-iRFP (oluhlaza okwesibhakabhaka olukhanyayo)]. (C) Umcibisholo ovulekile: I-ERES ithwala ucezu olulodwa lomthwalo (1 kuya ku-4). Umcibisholo ompunga: I-ERES iqukethe umthwalo ohlukanisiwe (5). Umcibisholo omhlophe oqinile: I-ERES iqukethe umthwalo obekwe ndawonye. Ngezansi: I-ERES eyodwa ekhethiwe iqukethe i-Gas1-GFP (1) noma i-Mid2-iRFP (2) kuphela. Ibha yesikali, i-100 nm. (D) Ukulinganiswa kwesithombe se-photomicrograph esichazwe ku-(C). Iphesenti elimaphakathi le-ERES eliqukethe umthwalo owodwa kuphela (i-Gas1-GFP noma i-Mid2-iRFP), umthwalo ohlukanisiwe kanye nomthwalo ohambisanayo. Ezivivinyweni ezintathu ezizimele, i-n=432 kumaseli angu-54. Ibha yephutha = SD. Ukuhlolwa kwe-t okunemisila emibili okungabhangqiwe. *** P = 0.0002. (E) Isithombe se-3D se-ERES ekhethiwe yomthwalo ohlukanisiwe omakwe ngo-(C). I-Gas1-GFP (eluhlaza) (3) noma i-Mid2-iRFP (eluhlaza okwesibhakabhaka) (4) ingena ku-ERES (emagenta) ohlangothini olulodwa futhi ikhawulelwe endaweni encane ngaphakathi kwe-ERES. Ngezinye izikhathi, zombili izinhlobo zempahla zingena ku-ERES efanayo (5) zisuka ohlangothini olufanayo futhi zigcinwe endaweni ehlukanisiwe ngaphakathi kwe-ERES. Ibha yesikali, 100 nm.
Okulandelayo, sihlole umbono wokuthi i-long acyl chain ceramide (C26) ekhona ku-ER membrane iqhuba ukuhlanganiswa kanye nokuhlelwa kwe-Gas1 ku-ERES ekhethiwe. Ukuze sifeze lokhu, sisebenzise uhlobo lwemvubelo oluguquliwe i-GhLag1, lapho ama-endogenous ceramide synthases amabili i-Lag1 ne-Lac1 athathelwa indawo yi-GhLag1 (i-Lag1 homologue yekotini), okwaholela ohlotsheni lwemvubelo olune-cell membrane uhlobo lwe-Ceramide olufushane kunohlobo lwe-wild (Isithombe 3A) (28). Ukuhlaziywa kwe-Mass spectrometry (MS) kubonise ukuthi ezinhlotsheni ze-wild-type, ama-95% e-ceramide iyonke yi-ceramide ende kakhulu (C26), kanti ku-GhLag1, ama-85% e-ceramide yinde kakhulu (C18 kanye ne-C16). ), ama-2% kuphela e-ceramide yi-ceramide ende kakhulu (C26). Nakuba ama-ceramide e-C18 kanye ne-C16 kuyi-ceramide eyinhloko etholakale kulwelwesi lwe-GhLag1 kuze kube manje, ukuhlaziywa kwe-MS kuqinisekisile nokuthi i-anchor ye-GPI ye-Gas1-GFP evezwe kulwelwesi lwe-GhLag1 iqukethe i-ceramide ye-C26, efana nama-lipids e-wild-type. Ikhwalithi iyafana (Isithombe 3A) (26). Ngakho-ke, lokhu kusho ukuthi i-enzyme yokulungisa i-ceramide i-Cwh43 ikhetha kakhulu i-ceramide ye-C26, njengoba kuboniswe kuMfanekiso 26, ifaka ngokukhethekile i-anchor ye-GPI kusuka enanini elincane le-ceramide ye-C26 kulwelwesi lwe-GhLag1. S2 (29). Noma kunjalo, i-membrane yeseli ye-GhLag1 ngokuyisisekelo iqukethe i-ceramide ye-C18-C16 kuphela, kuyilapho i-Gas1-GFP isenayo i-ceramide ye-C26. Leli qiniso lenza lolu hlobo lube ithuluzi elifanele lokuxazulula inkinga yobude be-acyl chain ye-membrane ceramide ku-ER. Indima ecatshangelwayo yeklasi nokuhlunga. Ngemuva kwalokho, saqala safunda ikhono le-C26 Gas1-GFP lokuqongelela ngamaqoqo ku-GhLag1 nge-allele eguquguqukayo ezwela izinga lokushisa ye-sec31-1 nge-microscopy evamile ye-fluorescence, lapho kuphela uchungechunge olude (C18-C16) olukhona ku-ER membrane Ceramide (Isithombe 3). Sibone ukuthi ku-sec31-1, iningi le-Gas1-GFP laligxile kumaqoqo, kuyilapho i-Gas1-GFP ku-sec31-1 GhLag1 ene-ceramide ER membrane ende (C18-C16) yayingahlanganiswanga futhi yasatshalaliswa ku-ER membrane yonke. Ukuze kube sobala, ngoba i-C26 ceramide-based clustering ihlobene eduze ne-ERES ethile (Isithombe 1), ngokulandelayo siphenye ukuthi le nqubo ingabandakanya yini nomsebenzi we-ER export protein mechanism. I-GPI-AP isebenzisa uhlelo olukhethekile lwe-COPII lokuthumela ngaphandle kwe-ER, olulawulwa ngenkuthalo yi-Ted1's structural reformation yengxenye ye-glycan ye-GPI anchor (30, 31). I-recombinant GPI-glycan ibe isiqashelwa yi-transmembrane cargo receptor p24 complex, yona ekhetha ngokukhetha i-Lst1, okuyi-isoform ethile ye-subunit enkulu ye-COPII cargo binding Sec24, okwenza i-GPI-AP-rich COPII Vesicles iyadingeka (31-33). Ngakho-ke, sakhe i-mutant ephindwe kabili ehlanganise ukususwa kwalawa maprotheni angawodwa (i-p24 complex component Emp24, i-GPI-glycan remodeling enzyme Ted1 kanye ne-COPII subunit Lst1 ethile) nohlobo lwe-sec31-1 mutant, futhi sazifunda. Kungenzeka yini ukwakha i-Gas1-cluster GFP (Isithombe 3). Sibone ukuthi ku-sec31-1emp24Δ kanye ne-sec31-1ted1Δ, i-Gas1-GFP ayihlanganisiwe futhi isatshalaliswa kulo lonke ulwelwesi lwe-ER, njengoba kubonwe ngaphambilini ku-sec31-1 GhLag1, kuyilapho ku-sec31-1lst1Δ, i-Gas1-GFP Njenge-sec31-1. Le miphumela ikhombisa ukuthi ngaphezu kokuba khona kwe-C26 ceramide ku-membrane ye-ER, ukuhlanganiswa kwe-Gas1-GFP nakho kudinga ukubopha ku-complex ye-p24, futhi akudingi ukuqashwa kwe-Lst1 ethile. Ngemuva kwalokho, sihlole ukuthi kungenzeka yini ukuthi ubude beketanga le-ceramide ku-membrane ye-ER bungalawula ukubopha kwe-Gas1-GFP ku-p24. Kodwa-ke, sithole ukuthi ukuba khona kwe-C18-C16 ceramide ku-membrane akuthinti ama-GPI-glycans akhiwe kabusha yi-complex ye-p24 (Izibalo S3 kanye ne-S4, A kanye ne-B) noma ukubopha ku-GPI-AP kanye nokuthekelisa ikhono le-GPI-AP. Thola i-subtype ye-COPII Lst1 (Isithombe S4C). Ngakho-ke, ukuhlanganiswa okuxhomeke ku-C26 ceramide akudingi ukusebenzisana kwamaprotheni nezindlela ezahlukene zokuthumela amaprotheni e-ER, kodwa kusekela indlela ehlukile yokuhlunga eqhutshwa ubude bamafutha. Ngemuva kwalokho, sihlaziye ukuthi ubude beketanga le-ceramide acyl ku-membrane ye-ER bubalulekile yini ekuhlukanisweni okusebenzayo kwe-Gas1-GFP njenge-ERES ekhethiwe. Njengoba i-Gas1 kuhlobo lwe-GhLag1 ene-ceramide emfushane iphuma ku-ER bese ingena kulwelwesi lwe-plasma (Isithombe S5), sikholelwa ukuthi uma ukuhlunga kuqhutshwa ubude be-ceramide acyl chain, i-Gas1 kuhlobo lwe-GhLag1 ingaqondiswa kabusha futhi ihlanganiswe. Izimpahla ze-ERES ezinelwelwesi elifanayo.
(A) I-membrane yeseli ye-GhLag1 ikakhulukazi iqukethe ama-ceramide amafushane e-C18-C16, kuyilapho i-anchor ye-GPI ye-Gas1-GFP isenayo i-C26 IPC efanayo namaseli e-wild-type. Ngenhla: ukuhlaziywa kobude be-acyl chain ye-ceramide ku-membrane yeseli yezinhlobo ze-wild-type (Wt) kanye ne-GhLag1p nge-mass spectrometry (MS). Idatha imele iphesenti le-ceramide iyonke. Isilinganiso sezilingo ezintathu ezizimele. Ibha yephutha = SD. Ukuhlolwa kwe-t okunemisila emibili engabhangqiwe. **** P <0.0001. Iphaneli engezansi: Ukuhlaziywa kwe-MS kobude be-acyl chain ye-IPC ekhona ku-anchor ye-GPI ye-Gas1-GFP (GPI-IPC) evezwe kuzinhlobo ze-wild-type kanye ne-GhLag1p. Idatha imele iphesenti lesignali iyonke ye-IPC. Isilinganiso sezilingo ezinhlanu ezizimele. Ibha yephutha = SD. Ukuhlolwa kwe-t okunemisila emibili engabhangqiwe. ns, akubalulekile. P = 0.9134. (B) Ama-micrograph e-Fluorescence e-sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ kanye namaseli e-sec31-1lst1Δ aveza i-Gas1-GFP ebangelwa yi-galactose afakwe ku-37°C imizuzu engama-30 futhi adluliselwa phansi ukuze enze i-microscopy ejwayelekile ye-fluorescence ngemva kwama-24°C. Umcibisholo omhlophe: Iqoqo le-ER Gas1-GFP. Umcibisholo ovulekile: I-Gas1-GFP engahlanganisiwe isatshalaliswa kulo lonke ulwelwesi lwe-ER, ikhombisa i-ER nuclear ring staining. Ibha yesikali, 5μm. (C) Ukulinganiswa kwe-photomicrograph echazwe ku-(B). Iphesenti elimaphakathi lamaseli anesakhiwo se-Gas1-GFP esinama-punctate. Ezivivinyweni ezintathu ezizimele, amaseli angu-n≥300. Ibha yephutha = SD. Ukuhlolwa kwe-t okunemisila emibili okungabhangqiwe. **** P ＜0.0001.
Ukuze sixazulule le nkinga ngqo, senze ukuboniswa kwe-SCLIM kwe-Gas1-GFP kanye ne-Mid2-iRFP ku-GhLag1 nge-allele ye-mutant ezwela ukushisa kwe-sec31-1 (Isithombe 4 kanye ne-Movie S4). Ngemva kokuthi i-ER igcinwe ku-37°C futhi kamuva yakhululwa ku-24°C, iningi le-Gas1-GFP esanda kuhlanganiswa alizange lihlanganiswe futhi lisatshalaliswe kulo lonke ulwelwesi lwe-ER, njengoba kubonwe ngama-microscope avamile (Isithombe 4, A kanye no-B). Ngaphezu kwalokho, iphesenti elikhulu le-ERES (67%) lifaka izinhlobo ezimbili zempahla ehlanganisiwe kuyo (Isithombe 4D). Amaphaneli 1 kanye no-2 eSithombe 4C abonisa izibonelo ezimbili ezijwayelekile ze-ERES ezine-Gas1-GFP ehambisanayo kanye ne-Mid2-GFP. Ngaphezu kwalokho, zombili izimpahla zaqoqwa ku-ERES efanayo (Isithombe 4E, iphaneli 3 kanye ne-movie S4). Ngakho-ke, imiphumela yethu ikhombisa ukuthi ubude be-ceramide acyl chain kulwelwesi lwe-ER luyisici esibalulekile sokuqoqwa kanye nokuhlukaniswa kwamaprotheni e-ER.
Amaseli e-Sec31-1 GhLag1 aveza ukukhishwa okubangelwa yi-galactose, i-Gas1-GFP (GPI-AP, eluhlaza) kanye ne-Mid2-iRFP (TMP, eluhlaza okwesibhakabhaka) kanye ne-Sec13-mCherry (ERES, magenta) ene-ERES ebhalwe nge-Sec13-mCherry (ERES, magenta) efakwe ilebula le-ERES. Qhubeka imizuzu engama-30, wehle uye ku-24°C ukuze ukhiphe ukukhishwa, bese uthatha isithombe nge-SCLIM ngemva kwemizuzu engama-20. (A kuya ku-C) Izithombe ezimele ze-2D projection (A; scale bar, 1μm) noma izithombe ze-3D cell hemisphere (B no-C; scale unit, 0.45μm) zezingxenye ezingu-10 z eziphawulwe yi-cargo kanye ne-ERES. Iphaneli engezansi ku-(B) kanye nephaneli ku-(C) zibonisa izithombe ezicutshungulwe ukuze kuboniswe kuphela izimpahla ezikhona ku-ERES (magenta) [Gas1-GFP (grey) kanye ne-Mid2-iRFP (oluhlaza okwesibhakabhaka olukhanyayo)]. (C) Umcibisholo ogcwele omhlophe: ERES, izimpahla ziyahlangana. Umcibisholo ovulekile: I-ERES iqukethe into eyodwa kuphela. Iphaneli engezansi: I-ERES ekhethiwe inezimpahla ezihambisanayo (1 no-2) ezimakwe ku-(C). Ibha yesikali, 100 nm. (D) Ukulinganiswa kwe-photomicrograph echazwe ku-(C). Kumayunithi e-GhLag1 e-sec31-1 kanye ne-sec31-1, kufakiwe umthwalo owodwa kuphela (i-Gas1-GFP noma i-Mid2-iRFP), kanye nephesenti elimaphakathi le-ERES yempahla ehlukanisiwe kanye nempahla ehambisanayo. Kuzivivinyo ezintathu ezizimele, i-n = 432 kumaseli angu-54 (sec31-1) kanye ne-n = 430 kumaseli angu-47 (sec31-1 GhLag1). Ibha yephutha = SD. Ukuhlolwa kwe-t okungenayo imigqa emibili. *** P = 0.0002 (sec31-1) kanye ne-** P = 0.0031 (sec31-1 GhLag1). (E) Isithombe se-3D se-ERES ekhethiwe enempahla ehambisanayo (3) esimakwe ku-(C). I-Gas1-GFP (eluhlaza okotshani) kanye ne-Mid2-iRFP (eluhlaza okwesibhakabhaka) zisondela ku-ERES (magenta) ohlangothini olufanayo futhi zihlala endaweni efanayo evinjelwe yi-ERES. Ibha yesikali, 100 nm.
Lolu cwaningo lunikeza ubufakazi obuqondile be-in vivo bokuthi imithwalo yamaprotheni asekelwe ku-lipid ihlukaniswa ngezindawo zokuthumela ezikhethiwe endleleni yokukhipha, futhi yembula ukubaluleka kobude be-acyl chain ekukhetheni ukuhlukaniswa. Sisebenzisa inqubo enamandla nesezingeni eliphezulu ye-microscopy ebizwa ngokuthi i-SCLIM, sibonise i-Gas1-GFP esanda kuhlanganiswa (i-plasma membrane enkulu i-GPI-AP ene-acyl chain ende kakhulu (C26) ceramide lipid portion) kumvubelo ) Izifunda eziqoqwe kuma-ER ahlukene zihlotshaniswa nama-ERES athile, kuyilapho amaprotheni akhishwe yi-transmembrane esatshalaliswa kulo lonke i-ER membrane (Isithombe 1). Ngaphezu kwalokho, lezi zinhlobo ezimbili zezimpahla zingena kuma-ERES ahlukene ngokukhetha (Isithombe 2). Ubude be-acyl chain ye-ceramide yeselula ku-membrane buncishisiwe kusuka ku-C26 kuya ku-C18-C16, i-Gas1-GFP cluster iphazanyiswa esifundeni se-ER esihlukile, futhi i-Gas1-GFP iqondiswa kabusha ukuze ishiye i-ER ne-transmembrane protein nge-ERES efanayo (Isithombe 3 kanye nesithombe 3). 4).
Nakuba i-GPI-AP isebenzisa indlela ekhethekile yeprotheyini ukuphuma ku-ER, sithole ukuthi ukuhlukaniswa okuxhomeke ku-C26 ceramide akuncikile ekusebenzisaneni okuhlukile kweprotheyini okungaholela ekukhetheni kwe-ERES (Izithombe S4 kanye ne-S5). Esikhundleni salokho, okutholakele kwethu kusekela indlela ehlukile yokuhlukanisa eqhutshwa ukuhlanganiswa kwamaprotheni asekelwe ku-lipid kanye nokukhishwa okulandelayo kwezinye izinto ezithwalayo. Okubonwe yithi kubonisa ukuthi isifunda noma iqembu le-Gas1-GFP elihlotshaniswa ne-ERES ethile alinalo iphrotheyini ekhishwe yi-transmembrane Mid2-iRFP, okubonisa ukuthi iqembu le-GPI-AP elixhomeke ku-C26 ceramide lizokwenza kube lula ukungena kwabo ku-ERES efanele, futhi ngesikhathi esifanayo, likhiphe i-transmembrane. Ukukhishwa kungena kule ERES ethile (Izithombe 1 kanye no-2). Ngokuphambene nalokho, ukuba khona kwama-ceramide e-C18-C16 ku-ER membrane akubangeli i-GPI-AP ukuthi yakhe izifunda noma amaqoqo, ngakho-ke awakhiphi noma awathathi indawo yamaprotheni akhishwe yi-transmembrane ku-ERES efanayo (Izithombe 3 kanye no-4). Ngakho-ke, siphakamisa ukuthi i-C26 ceramide iqhube ukuhlukaniswa nokuhlukaniswa ngokusiza ukuhlanganiswa kwamaprotheni axhunywe ku-ERES ethile.
Ungakufeza kanjani lokhu kuhlangana kwe-C26 ceramide endaweni ethile ye-ER? Ukuthambekela kwe-membrane ceramide ukuhlukana eceleni kungabangela i-GPI-AP kanye ne-C26 ceramide ukuthi zakhe ama-lipid amancane futhi ahlelwe ngokushesha endaweni engajwayelekile ye-lipid ye-membrane ye-ER equkethe ama-glycerolipid amafushane futhi angagcwali. Amaqoqo ekhwalithi (17, 18). Lawa maqoqo amancane esikhashana angahlanganiswa kakhulu abe amaqoqo amakhulu, azinzile ngemva kokubopha ku-p24 complex (34). Ngokuvumelana nalokhu, sibonise ukuthi i-C26 Gas1-GFP idinga ukusebenzisana ne-p24 complex ukuze yakhe amaqoqo amakhulu abonakalayo (Isithombe 3). I-p24 complex iyi-oligomer ye-heterozygous eyakhiwe ngamaprotheni amane ahlukene e-p24 transmembrane ku-yeast (35), enikeza ukubopha okuningi, okungaholela ekuhlanganiseni kwamaqoqo amancane e-GPI-AP, ngaleyo ndlela kukhiqize i-Stable cluster enkulu (34). Ukusebenzisana phakathi kwama-ectodomains e-protein e-GPI-APs nakho kungaba nomthelela ekuhlanganisweni kwawo, njengoba kuboniswe ngesikhathi sokuthuthwa kwawo yi-Golgi kumaseli e-epithelial aphethwe yi-mammalian polarized (36). Kodwa-ke, lapho i-ceramide ye-C18-C16 ikhona ku-membrane ye-ER, lapho i-p24 complex ibopha ku-Gas1-GFP, amaqoqo amakhulu ahlukene ngeke akheke. Indlela eyisisekelo ingancika ezicini ezithile zomzimba nezamakhemikhali ze-ceramide ende ye-acyl chain. Izifundo ze-biophysical ze-membranes zokwenziwa zibonisa ukuthi yize zombili i-ceramide ende (C24) nemfushane (C18-C16) ye-acyl chain zingabangela ukuhlukaniswa kwesigaba, i-ceramide ende ye-acyl chain (C24) kuphela engakhuthaza i-Curvature ephezulu kanye nokugoba kwefilimu ukuze kuphinde kuklanywe ifilimu. Ngokubhekisela komunye nomunye (17, 37, 38). Kuye kwaboniswa ukuthi i-transmembrane helix ye-TMED2, i-homologue yomuntu ye-Emp24, isebenzisana ngokukhetha ne-sphingomyelin esekelwe ku-C18 ceramide kuma-lobule e-cytoplasmic (39). Sisebenzisa ukulingisa kwe-molecular dynamics (MD), sithole ukuthi ama-ceramide e-C18 kanye ne-C26 aqoqana eduze kwama-lobule e-cytoplasmic e-Emp24 transmembrane helix, futhi anezintandokazi ezifanayo (Isithombe S6). Kubalulekile ukuqaphela ukuthi lokhu kubonisa ukuthi i-transmembrane helix ye-Emp24 ingaholela ekusakazweni okungalingani kwama-lipid ku-membrane. Lona umphumela wakamuva osekelwe kumaseli ezilwane ezincelisayo. Ukulingisa okufanayo kwe-MD kukhombisa nokuba khona kwama-lipid e-ether (40). Ngakho-ke, sicabanga ukuthi i-C26 ceramide kuma-lobule amabili e-ER26 icebile endaweni. Uma i-GPI-AP kuma-lobule e-luminal ibopha ngqo ku-p24 ene-multivalent kanye nokuqongelela kwe-ceramide ye-C26 ezungeze i-p24 kuma-lobule e-cytoplasmic, ingakhuthaza ukuhlanganiswa kwe-Protein okuhambisanayo kanye nokugoba kwe-membrane kukhiqizwa ngeminwe (41), okubangela ukuthi i-GPI-AP ihlukane ibe yizifunda ezihlukene eduze kwe-ERES, okuthanda nezifunda ezigobile kakhulu ze-membrane ye-ER (42). Imibiko yangaphambilini isekele indlela ehlongozwayo (43, 44). Ukubopha kwe-oligolectins ene-multivalent, ama-pathogens noma ama-antibodies kuma-glycosphingolipids asekelwe ku-ceramide (GSL) ku-membrane ye-plasma kubangela ukuhlanganiswa okukhulu kwe-GSL, kuthuthukisa ukuhlukaniswa kwesigaba futhi kubangele ukuguqulwa kwe-membrane kanye nokufakwa ngaphakathi (44). Iwabuchi njll. (43) Kutholakale ukuthi lapho kukhona amaketanga e-acyl amade (C24) kodwa angemafushane (C16), i-ligand ene-multivalent eboshelwe ku-GSL lactosylceramide yabangela ukwakheka kwamaqoqo amakhulu kanye nokungena kwe-membrane, kanti i-cytoplasm Lyn-mediated signal transduction kumapheshana ihlukaniswa ngamaketanga e-acyl kuma-neutrophils ahlangene.
Kumaseli e-epithelial ahlukaniswe ngama-mammalian, ukuhlushwa kwenethiwekhi ye-anti-Golgi (TGN) kuya ezingeni le-apical plasma membrane kulawula ukuhlukaniswa nokuhlelwa kwe-GPI-AP (10, 45). Lokhu kuhlanganiswa kuqhutshwa yi-GPI-AP oligomerization (36), kodwa kungancika futhi kubude be-ceramide chain esibuthola ku-yeast. Nakuba i-GPI-AP yama-mammalian ine-ether lipid-based anchor, futhi isakhiwo sayo samakhemikhali sihluke kakhulu ku-acyl chain ceramide ende kakhulu, ucwaningo lwamuva luthole ukuthi womabili ama-lipid anezakhiwo zomzimba kanye namakhemikhali ezifanayo ngokuziphendukela kwemvelo kanye nomsebenzi (40). Ngakho-ke, ingxenye ye-ether lipid kumaseli ama-mammalian ingase ifane ne-C26 ceramide ku-yeast, futhi indima yayo ukuhlanganisa ne-ceramide ende ku-membrane ukukhuthaza ukuhlanganiswa nokuhlelwa kwe-GPI-AP. Nakuba lokhu kungenzeka kusadingeka kuhlolwe ngqo, okutholakele ngaphambilini kusekela ukuthi ukuthuthwa kwe-long acyl chain ceramide emzimbeni we-Golgi akwenziwa ngamaprotheni okudlulisa i-cytoplasmic, kodwa kuncike ekuhlanganisweni kwama-GPI anchors njengemvubelo. Ngakho-ke, indlela yokuguquka kwemvelo ibonakala ikwazi ukuthutha ngokukhetha i-acyl chain ceramide ende kakhulu kanye ne-GPI-AP (13, 16, 20, 46, 47) ku-vesicle efanayo yokuthutha.
Ezinhlelweni zamaseli e-epithelial e-yeast kanye ne-mammalian polarized, ukuhlanganiswa kanye nokuhlukaniswa kwe-GPI-AP kwamanye amaprotheni e-plasma membrane konke kwenzeka ngaphambi kokufika ebusweni beseli. UPaladino et al. (48) bathole ukuthi ku-TGN yamaseli e-epithelial e-mammalian polarized, ukuhlanganiswa kwe-GPI-AP akudingeki nje kuphela ekuhlukaniseni ama-GPI-AP ku-apical plasma membrane, kodwa futhi kulawula ukuhlelwa kokuhlanganiswa kwama-GPI-AP kanye nomsebenzi wayo webhayoloji. Ubuso beseli. Ku-yeast, lolu cwaningo lubonise ukuthi i-C26 ceramide-dependent GPI-AP cluster ku-ER ingalawula ukuhlelwa kweqembu kanye nomsebenzi osebenzayo we-GPI-AP ku-plasma membrane (24, 49). Ngokuhambisana nale modeli, amaseli e-GhLag1 azwela i-GPI inhibitors noma izidakamizwa ezithinta ubuqotho bezindonga zeseli (28), kanye nesidingo samaqoqo e-Gas1-GFP asebenzayo (49) e-tip ceramide aboniswe ekuhlanganeni kwamaseli e-yeast sibonisa i-G​​ Imiphumela engaba khona yomzimba yamaseli e-hLag1. Iphutha le-GPI-AP. Kodwa-ke, ukuhlola okwengeziwe ukuthi ngabe ukuhlelwa kokusebenza kobuso beseli kuhlelwe kusukela ku-ER ngendlela yokuhlunga ngokusekelwe kubude be-lipid kuzoba yisihloko socwaningo lwethu lwesikhathi esizayo.
Izinhlobo ze-Saccharomyces cerevisiae ezisetshenziswe kulo msebenzi zibhalwe kuThebula S1. Izinhlobo ze-MMY1583 kanye ne-MMY1635 ze-SCLIM zokuthwebula izithombe zamaseli aphilayo zakhiwe ngemuva kwe-W303. Lezi zinhlobo eziveza i-Sec13-mCherry enethegi yephrotheni ekhanyayo zakhiwe kusetshenziswa indlela esekelwe ku-polymerase chain reaction (PCR) ene-pFA6a plasmid njengethempulethi (23). Uhlobo oluveza i-Mid2-iRFP olubhalwe ngephrotheni ekhanyayo ngaphansi kokulawulwa kwephromotha ye-GAL1 lwakhiwe kanje. Ukwandiswa kwe-PCR kochungechunge lwe-iRFP-KanMx oluvela ku-vector ye-pKTiRFP-KAN (isipho sika-E. O'Shea, inombolo ye-Addgene plasmid 64687; http://n2t.net/addgene: 64687; isihlonzi semithombo yocwaningo (RRID): Addgene_64687) Futhi kufakwe ku-C-terminus ye-endogenous Mid2. Ngemva kokuthi uchungechunge lwe-genome lwe-Mid2-iRFP lukhuliswe futhi lwahlanganiswa kuphromotha ye-GAL1, lwahlanganiswa kusayithi le-Not I-Sac I le-plasmid pRS306 yokuhlanganiswa. I-plasmid pRGS7 eyalandela yahlukaniswa nge-Pst I ukuze ihlanganiswe ku-URA3 locus.
I-Gas1-GFP fusion gene ivezwa ngaphansi kokulawulwa kwe-GAL1 promoter ku-centromere (CEN) plasmid, eyakhiwe kanje. Uchungechunge lwe-Gas1-GFP lwandiswa yi-PCR kusuka ku-pRS416-GAS1-GFP plasmid (24) (isipho sika-L. Popolo) futhi lwahlanganiswa endaweni ye-Xma I–Xho I ye-CEN plasmid pBEVY-GL LEU2 (isipho sika-C). Miller; Inombolo ye-Addgene plasmid 51225; http://n2t.net/addgene: 51225; RRID: Addgene_51225). I-plasmid ephumela yaqanjwa ngokuthi i-pRGS6. I-Axl2-GFP fusion gene ivezwa ngaphansi kokulawulwa kwe-GAL1 promoter ye-pBEVY-GL LEU2 vector, futhi ukwakhiwa kwayo kungokulandelayo. Uchungechunge lwe-Axl2-GFP lwandiswe kusukela ku-pRS304-p2HSE-Axl2-GFP plasmid (23) yi-PCR, futhi lwahlanganiswa endaweni ye-Bam HI-Pst I ye-vector ye-pBEVY-GL LEU2. I-plasmid ephumela yaqanjwa ngokuthi i-pRGS12. Uchungechunge lwama-oligonucleotide asetshenziswe kulolu cwaningo lubhalwe kuThebula S2.
Lolu hlobo lwengezwe nge-adenine engu-0.2% kanye ne-glucose engu-2% [YP-dextrose (YPD)], i-raffinose [YP-raffinose] rich yeast extract protein p (YP) medium (1% Yeast extract kanye ne-2% protein ept). (YPR)] noma i-2% galactose [YP-galactose (YPG)] njengomthombo wekhabhoni, noma endaweni encane yokwenziwa (0.15% yeast nitrogen base kanye ne-0.5% ammonium sulfate) ukuze kufakwe ama-amino acid afanele kanye nezisekelo ezidingekayo ukuze kondliwe, Futhi equkethe i-2% glucose (synthetic glucose minimal medium) noma i-2% galactose (synthetic galactose minimal medium) njengomthombo wekhabhoni.
Ukuze kuthathwe izithombe ngesikhathi sangempela, amaseli aguquliwe e-sec31-1 azwela izinga lokushisa aveza ukwakheka ngaphansi kwe-promoter ye-GAL1 akhuliswe endaweni ephakathi ye-YPR ku-24°C ubusuku bonke kuya esigabeni esiphakathi nendawo. Ngemva kokungeniswa ku-YPG ku-24°C ihora eli-1, amaseli afakwa ku-SG ku-37°C imizuzu engama-30, abese edluliselwa ku-24°C ukuze akhishwe ku-secretion block. I-Concanavalin A yasetshenziswa ukulungisa amaseli ku-slide yengilazi futhi yathathwa isithombe yi-SCLIM. I-SCLIM iyinhlanganisela ye-Olympus IX-71 inverted fluorescence microscope kanye ne-UPlanSApo 100×1.4 numeral aperture oil lens (Olympus), i-rotation disc confocal scanner ejikelezayo ngesivinini esikhulu kanye ne-high-signal-to-noise ratio (Yokogawa Electric), i-custom spectrometer, kanye nokupholisa ngokwezifiso. I-image antensifier yesistimu (i-Hamamatsu Photonics) inganikeza uhlelo lwe-magnifying lens ngokukhulisa kokugcina okungu-×266.7 kanye nekhamera yedivayisi ehlanganisiwe yokushaja ephindaphinda ama-electron (i-Hamamatsu Photonics) (21). Ukutholwa kwesithombe kwenziwa yisoftware yangokwezifiso (i-Yokogawa Electric). Ezithombeni ze-3D, sisebenzise i-piezoelectric actuator eyenziwe ngokwezifiso ukuze sidlidlize ilensi eqondiwe ngokuqondile, futhi saqoqa izingxenye ze-optical eziyi-100 nm ezihlukene esitaki. Isithombe se-Z-stack siguqulwa sibe idatha ye-voxel ye-3D, futhi umsebenzi wokusabalala kwamaphuzu omqondo osetshenziselwa i-rotation disc confocal microscope usetshenziselwa ukucubungula i-deconvolution yi-Volocity software (PerkinElmer). Ngokusebenzisa isofthiwe yeVolocity ukuze kubekwe umkhawulo ngokuzenzakalelayo wokuhlaziywa kwendawo ehlangene, i-ERES kufaka phakathi impahla yalinganiswa. Ukuhlaziywa kokuskena kwemigqa kwenziwa kusetshenziswa isofthiwe ye-MetaMorph (Amadivayisi e-Molecular).
Sebenzisa isofthiwe ye-GraphPad Prism ukuze unqume ukubaluleka kwezibalo. Ekuhlolweni kwe-t komfundi okunemisila emibili kanye nokuhlolwa okuvamile kwe-one-way of variance (ANOVA), umehluko phakathi kwamaqembu ubhekwa njengonethonya elikhulu ku-P <0.05 (*).
Ukuze kwenziwe i-fluorescence microscopy ye-Gas1-GFP, amaseli e-log phase akhuliswa ubusuku bonke ku-YPD futhi aqoqwa nge-centrifugation, ahlanzwe kabili nge-phosphate buffered saline, bese efakwa eqhweni okungenani imizuzu eyi-15, bese eqhubeka ngaphansi kwe-microscope njengoba kuchaziwe ngaphambilini Hlola (24). I-Leica DMi8 microscope (HCX PL APO 1003/1.40 oil PH3 CS) ifakwe ilensi eqondile, isihlungi se-L5 (GFP), ikhamera ye-Hamamatsu kanye nesofthiwe ye-Application Suite X (LAS X) yasetshenziswa ukuthola.
Amasampula ahlungwe nge-SDS sample buffer ku-65°C imizuzu eyi-10, abese ehlukaniswa yi-SDS-polyacrylamide gel electrophoresis (PAGE). Ukuze kuhlaziywe i-immunoblotting, kulayishwe i-10 μl yesampula ngomzila ngamunye. I-antibody eyinhloko: Sebenzisa i-rabbit polyclonal anti-Gas1 ekuxubeni okungu-1:3000, i-rabbit polyclonal anti-Emp24 ekuxubeni okungu-1:500, kanye ne-rabbit polyclonal anti-GFP (isipho esivela ku-H. Riezman) ekuxubeni okungu-1:3000. I-antibody yegundane ye-monoclonal anti-Pgk1 yasetshenziswa ekuxubeni okungu-1:5000 (isipho esivela ku-J. de la Cruz). I-antibody yesibili: I-Horseradish peroxidase (HRP) conjugated goat anti-rabbit immunoglobulin G (IgG) esetshenziswe ekuxubeni okungu-1:3000 (Pierce). I-IgG yembuzi ehlanganiswe ne-HRP elwa negundane yasetshenziswa ekuncibilikisweni okungu-1:3000 (Pierce). Indawo yokusabela komzimba yabonwa ngendlela ye-chemiluminescence ye-SuperSignal West Pico reagent (Thermo Fisher Scientific).
Njengoba kuchaziwe ku-(31), kwenziwa ukuhlolwa kwemvelo kwe-immunoprecipitation engxenyeni ye-ER ecebile. Ngamafuphi, geza amaseli emvubelo nge-TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride kanye nengxube ye-protease inhibitor) ku-600 nm (OD600) ku-100 optical density kabili. Yaphulwa ngama-glass beads, kwase kususwa ama-debris esisele kanye nama-glass beads nge-centrifugation. I-supernatant yabe isifakwa ku-centrifuge ku-17,000 g imizuzu eyi-15 ku-4°C. I-pellet yaphinde yamiswa ku-TNE kwathi i-digitalis saponin yengezwa ekugxilweni kokugcina okungu-1%. Ukumiswa kwafakwa i-incubation ihora eli-1 ngokujikeleza ku-4°C, kwabe sekususwa izingxenye ezinganyibiliki nge-centrifugation ku-13,000 g ku-4°C imizuzu engama-60. Ukuze uthole i-Gas1-GFP immunoprecipitation, qala ngokufaka isampula kusengaphambili ngama-agarose beads angenalutho (ChromoTek) ku-4°C ihora eli-1, bese ufaka i-GFP-Trap_A (ChromoTek) ku-4°C amahora ama-3. Ama-immunoprecipitated beads ahlanzwe izikhathi ezinhlanu nge-TNE equkethe i-0.2% digoxigenin, asuswa nge-SDS sample buffer, ahlukaniswe ku-SDS-PAGE, futhi ahlaziywa nge-immunoblotting.
Njengoba kuchaziwe ku-(31), ukunqunywa kokuxhumanisa kwenziwa engxenyeni ye-ER ecebile. Kafushane, ingxenye ye-ER ecebile yafakwa i-0.5 mM dithiobis(succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, USA; 20°C, 20 min). Ukusabela kokuxhumanisa kwaqedwa ngokungeza i-glycine (ukuhlushwa kokugcina okungu-50 mM, imizuzu emi-5, 20°C).
Njengoba kuchaziwe ngaphambilini (50), kwenziwa ukuhlaziywa kwe-MS kwe-ceramide ezinhlotsheni ze-wild-type kanye ne-GhLag1. Ngamafuphi, amaseli akhuliswe abe yisigaba se-exponential (amayunithi angu-3 kuya kwangu-4 e-OD600/ml) ku-YPD ku-30°C, kwathi amaseli angu-25×107 avunwa. I-metabolism yawo iqedwa nge-trichloroacetic acid. Sebenzisa i-extraction solvent [i-ethanol, amanzi, i-ether, i-pyridine kanye ne-4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] kanye ne-1.2 nmol yekhwalithi yangaphakathi ejwayelekile ye-C17 ceramide (860517, i-Avanti polar lipid)). Sebenzisa i-monomethylamine reagent [i-methanol, amanzi, i-n-butanol kanye nesisombululo se-methylamine (4:3:1:5 v/v)] ukwenza i-alkaline hydrolysis encane ye-extract, bese usebenzisa i-n-butanol egcwele amanzi ukuze ususe usawoti. Ekugcineni, lesi siqeshana saphinde samiswa ku-solvent yemodi enhle [i-chloroform/methanol/water (2:7:1) + 5 mM ammonium acetate] futhi safakwa ku-mass spectrometer. Ukuqapha kwe-Multi-reaction (MRM) kwenziwa ukuze kutholakale futhi kulinganiswe ama-molecule e-sphingolipid. I-TSQ Vantage tertiary quadrupole mass spectrometer (i-Thermo Fisher Scientific) ifakwe umthombo we-robotic nanoflow ion i-Nanomate HD (i-Advion Biosciences, i-Ithaca, NY) wokuhlaziywa kwe-lipid. Amandla okungqubuzana alungiselelwe isigaba ngasinye se-ceramide. Idatha ye-MS itholwe kumodi enhle. Kokuphindwa ngakunye kwezinto eziphilayo, isignali ye-lipid iyisilinganiso sezilinganiso ezintathu ezizimele.
Njengoba kuchaziwe ku-(31), amaseli (800×107) aveza i-Gas1-GFP afakwa ngaphansi kwe-immunoprecipitation yemvelo. I-Gas1-GFP ehlanziwe yahlukaniswa yi-SDS-PAGE yabe isidluliselwa kulwelwesi lwe-polyvinylidene fluoride (PVDF). Iphrotheni yabonakala ngokufaka i-PVDF umbala nge-amide black. Ibhendi ye-Gas1-GFP yasikwa ku-PVDF yagezwa izikhathi ezi-5 nge-methanol kanye kanye ngamanzi e-liquid chromatography-MS (LC-MS). Ngokufaka ulwelwesi lwe-membrane nge-500μl 0.3 M NaOAc (pH 4.0), i-buffer kanye ne-500μl ingxube ye-sodium nitrite engu-1 M esanda kuncibilikiswa ku-37°C amahora ama-3, ingxenye ye-lipid ikhishwa ku-Gas1-GFP futhi ihlikihlwe. Ukukhululwa kwe-inosine phosphate ceramide phakathi kwe-glucosamine ne-inositol (51). Ngemva kwalokho, umugqa we-membrane wagezwa izikhathi ezine ngamanzi e-LC-MS grade, womiswa ekushiseni kwegumbi, futhi wagcinwa emoyeni we-nitrogen ku--80°C kuze kube yilapho kuhlaziywa. Njengokulawula, kwasetshenziswa isampula engenalutho ye-membrane ye-PVDF ekuhlolweni ngakunye. I-lipid ekhishwe ku-Gas1-GFP yabe isihlaziywa yi-MS njengoba kuchaziwe (50). Ngamafuphi, imichilo ye-PVDF equkethe i-GPI-lipid yaphinde yamiswa ku-75μl negative mold solvent [chloroform/methanol (1:2) + 5 mM ammonium acetate] futhi yadlula ku-electrospray ionization (ESI)-MRM/MS Analysis of sphingolipid species (TSQ Vantage). Kulesi simo, idatha ye-MS yatholakala kumodi ye-ion negative.
Njengoba kushiwo ngaphambili, ingxenye ye-lipid ye-anchor ye-GPI yahlukaniswa ne-[3H]-inositol-labeled GPI-AP (16). Ama-lipid ahlukaniswe nge-chromatography enongqimba oluncane kusetshenziswa uhlelo lwe-solvent (55:45:10 chloroform-methanol-0.25% KCl) futhi aboniswa kusetshenziswa i-FLA-7000 (Fujifilm).
Amaseli aveza i-Gas1-GFP (600×107) ahlanzwa kabili nge-TNE buffer ene-TNE buffer, aphulwa ngama-glass beads, abese efakwa i-centrifuge ukuze kususwe udoti wamaseli kanye nama-glass beads. I-supernatant yabe isifakwa i-centrifuge ku-17,000 g ihora eli-1 ku-4°C. I-pellet yahlanzwa ku-TNE futhi yafakwa i-1 U PI-PLC (Invitrogen) ku-TNE equkethe i-0.2% digitalis saponin ihora eli-1 ku-37°C. Ngemva kokwelashwa nge-enzyme, i-membrane yasuswa nge-centrifuge ku-17,000 g ku-4°C ihora eli-1. Ukuze kuvikelwe i-Gas1-GFP, i-supernatant yafakwa i-GFP-Trap_A (ChromoTek) ku-4°C ubusuku bonke. I-Gas1-GFP ehlanziwe ehlukaniswe yi-SDS-PAGE yagcotshwa nge-Coomassie brilliant blue. Ibhendi yokufaka i-Gas1-GFP yanqunywa kumpunga ozungeze umsele wamanzi, kwathi ngemva kokuxubana ne-iodoacetamide kanye nokunciphisa nge-dithiothreitol, kwenziwa ukugaya nge-trypsin ngejeli. Khipha futhi komise ama-peptide e-tryptic nama-peptide nge-GPI-glycans. I-peptide eyomile yancibilikiswa ku-20 μl wamanzi. Faka ingxenye (8μl) ku-LC. Ikholomu ye-octadecylsilane (ODS) (Develosil 300ODS-HG-5; ububanzi bangaphakathi obungu-150 mm×1.0 mm; Nomura Chemical, Aichi Prefecture, Japan) yasetshenziswa ukuhlukanisa ama-peptide ngaphansi kwezimo ezithile ze-gradient. Isigaba esihambayo siyi-solvent A (0.08% formic acid) kanye ne-solvent B (0.15% formic acid ku-80% acetonitrile). Uhlelo lwe-Accela HPLC (i-Thermo Fisher Scientific, eBoston, eMassachusetts) lusetshenziswe ukukhipha ikholomu nge-solvent A zingakapheli imizuzu engu-55 ngesilinganiso sokugeleza esingu-50 μl min-1 imizuzu emi-5, bese kuthi ukuhlushwa kwe-solvent B kwandiswe kwaba ngu-40%., e-United States). I-eluate yafakwa njalo emthonjeni we-ESI ion, kanti ama-peptide e-tryptic nama-peptide ane-GPI-glycans ahlaziywa yi-LTQ Orbitrap XL (i-hybrid linear ion trap-orbitrap mass spectrometer; i-Thermo Fisher Scientific). Ekusethweni kwe-MS, i-voltage yomthombo we-capillary isethwe ku-4.5 kV, kanti izinga lokushisa le-capillary yokudlulisa ligcinwe ku-300°C. I-voltage ye-capillary kanye ne-voltage yelensi yetyhubhu isethwe ku-15 V kanye no-50 V, ngokulandelana. Idatha ye-MS itholakale kumodi ye-ion elungile (isisombululo esingu-60,000; ukunemba kwesisindo sezingxenye eziyi-10 ngesigidi) kububanzi besisindo esingu-300/m/z mass/charge ratio (m/z) 3000. Idatha ye-MS/MS itholakala nge-ion trap ku-LTQ Orbitrap XL [izinombolo zokuqala ezintathu lapho idatha incike khona, i-collision induced dissociation (CID)].
Ukulingisa kwe-MD kwenziwa kusetshenziswa isofthiwe ye-GROMACS (52) kanye nensimu yamandla ye-MARTINI 2 (53-55). I-CHARMM GUI Membrane Builder (56, 57) yabe isisetshenziswa ukwakha ungqimba olune-bilayer oluqukethe i-dioleoylphosphatidylcholine (DOPC) kanye ne-Cer C18 noma i-DOPC kanye ne-Cer C26. I-topology kanye nezixhumanisi ze-Cer C26 zithathwe ku-DXCE ngokususa ubuhlalu obengeziwe emsileni we-sphingosine. Sebenzisa inqubo echazwe ngezansi ukuze ulinganise ungqimba oluphindwe kabili bese ulusebenzisa, bese usebenzisa izixhumanisi zokugcina zesistimu ukwakha uhlelo oluqukethe i-Emp24. Isizinda se-transmembrane se-yeast Emp24 (izinsalela 173 kuya ku-193) sakhiwa njenge-α-helix kusetshenziswa isakhiwo sama-molecule sethuluzi le-visual MD (VMD) (58). Ngemuva kwalokho, ngemuva kokususa ama-lipid agqagqene, iphrotheni yaqoqwa kancane kancane yafakwa kungqimba olune-bilayer kusetshenziswa i-CHARMM GUI. Uhlelo lokugcina luqukethe i-1202 DOPC kanye ne-302 Cer C26 noma i-1197 DOPC kanye ne-295 Cer C18 kanye ne-Emp24. Faka i-ion ohlelweni ekugxilweni okungu-0.150M. Kwenziwe amakhophi amane azimele ezingxenyeni ezimbili ze-bilayer.
I-lipid bilayer ilinganiswe kusetshenziswa inqubo ye-CHARMM GUI, ehilela ukunciphisa bese kulinganiswa izinyathelo ezingu-405,000, lapho imikhawulo yesikhundla incishiswa kancane kancane futhi isuswa, futhi isinyathelo sesikhathi sandiswa kusuka ku-0.005 ps kuya ku-0.02 ps. Ngemva kokulinganisa, ikhiqiza ama-µs angu-6 ngesinyathelo sesikhathi esingu-0.02 ps. Ngemva kokufaka i-Emp24, sebenzisa inqubo efanayo ye-CHARMM GUI ukunciphisa nokulinganisela uhlelo, bese isebenza imizuzwana engu-8 ekukhiqizeni.
Kuzo zonke izinhlelo, ngesikhathi senqubo yokulinganisa, ingcindezi ilawulwa yi-barostat ye-Berendsen (59), kanti ngesikhathi senqubo yokukhiqiza, ingcindezi ilawulwa yi-barostat ye-Parrinello-Rahman (60). Kuzo zonke izimo, ingcindezi emaphakathi iyibha eli-1 futhi kusetshenziswa uhlelo lokuhlanganisa ingcindezi oluyi-semi-isotropic. Enqubweni yokulinganisela nokukhiqiza, i-thermostat (61) enokulungiswa kabusha kwesivinini isetshenziswa ukuhlanganisa izinga lokushisa lama-protein, ama-lipid kanye nezinhlayiya ze-solvent ngokulandelana. Phakathi nokusebenza konke, izinga lokushisa eliqondiwe lingu-310K. Ukusebenzisana okungenazibopho kubalwa ngokukhiqiza uhlu lokubhangqa kusetshenziswa uhlelo lwe-Verlet olunokubekezelelana kwe-buffer okungu-0.005. Igama le-Coulomb libalwa kusetshenziswa insimu yokusabela kanye nebanga elinqunyiwe elingu-1.1 nm. Igama le-Vander Waals lisebenzisa uhlelo olunqunyiwe olunebanga elinqunyiwe elingu-1.1 nm, kanti uhlelo olunqunyiwe lwe-Verlet lusetshenziselwa ukukhukhuleka okungenzeka (62).
Ukusebenzisa i-VMD, ubude be-cutoff phakathi kwama-DOPC phosphate beads noma ama-ceramide AM1 beads kanye neprotheni bungu-0.7 nm, futhi inani lama-lipids asebenzisana neprotheni liyabalwa. Ngokwefomula elandelayo, bala i-depletion-enrichment (DE) factor njengaku-(63): i-DE factor = (inani lama-lipids aphelele kuprotheni 0.7) kuprotheni 0.7 (inani le-Cer kuma-lipids aphelele)
Inani elibikiwe litholakala njengesilinganiso, kanti imigoqo yamaphutha iyikhophi ezine ezimele ze-SE. Ukubaluleka kwezibalo kwe-DE factor kubalwa ngokuhlolwa kwe-t [(isilinganiso se-DE-factor-1)/SE]. Bala inani le-P kusukela ekusabalalisweni komsila owodwa.
Ithuluzi le-GROMACS lisetshenziswe ukubala imephu yobuningi be-2D eseceleni yesistimu equkethe i-Emp24 ngaphakathi kwama-250 ns okugcina womkhondo. Ukuze kutholakale imephu yokucebisa/yokunciphisa i-ceramide, imephu yobuningi be-Cer ihlukaniswa ngesamba semephu ye-Cer ne-DOPC, bese ihlukaniswa ngokuhlushwa kwe-Cer emzimbeni. Kusetshenziswa isikali semephu yombala ofanayo.
Ukuze uthole izinto ezengeziwe zalesi sihloko, sicela ubheke ku-http://advances.sciencemag.org/cgi/content/full/6/50/eaba8237/DC1
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Lo mbuzo usetshenziselwa ukuhlola ukuthi uyisivakashi yini futhi uvimbele ukuthunyelwa kogaxekile okuzenzakalelayo.
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 Perezgier Lopero, Ana Maria Perezgier Lopero Waga (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Ukuthwebula izithombe ngesikhathi sangempela okunesisombululo esiphezulu se-3D kwembula ukubaluleka kobude beketanga le-ceramide ekuhlungeni amaprotheni ezindaweni zokukhipha ezikhethiwe.
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 Perezgier Lopero, Ana Maria Perezgier Lopero Waga (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Ukuthwebula izithombe ngesikhathi sangempela okunesisombululo esiphezulu se-3D kwembula ukubaluleka kobude beketanga le-ceramide ekuhlungeni amaprotheni ezindaweni zokukhipha ezikhethiwe.
©2020 Inhlangano YaseMelika Yokuthuthukiswa Kwesayensi. wonke Amalungelo Agodliwe. I-AAAS inguzakwethu we-HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef kanye ne-COUNTER. I-ScienceAdvances ISSN 2375-2548.