Kit ma ki poko kwede jami ma ki lwongo ni protein i yoo ma ki cwalo iye gin moni pire tek me gwoko cell ma ki poko ki cell ma ki lwongo ni homeostasis. Medo ikom poko kin jami ma ki lwongo ni shell, tic pa lipids i kinesin me poko jami i yub me tero jami ma ki cwalo i kom dano obedo lapeny ma obedo pi kare malac ma pud peya ki gamo. Kany, wa tiimo 3D simultaneous multicolor high-resolution real-resolution imaging me nyutu in vivo ni gin manyen ma ki yubu ma ki lwongo ni glycosylphosphatidylinositol-immobilized proteins ki ceramide lipid moieties mabor ki poko gi i endoplasms Net exit site, ma pat ki proteins ma ki tiyo kwede i transmembrane. Medo ikom meno, wanyutu ni bor pa dul ma ki lwongo ni ceramide i dul kom ma ki lwongo ni endoplasmic reticulum membrane pire tek pi poko jami eni. Kwano wa ni omiiyo caden mukwongo ma rwate ki kwo me poko jami ma ki cwalo ma lubu bor pa dul kom ma ki lwongo ni lipid chain i kabedo ma ki yero me cwalo jami i yoo ma ki cwalo iye jami.
I kom ler ma ki lwongo ni eukaryotic cells, jami ma ki yubu i endoplasmic reticulum (ER) ki poko gi i kare me tero gi ki i yoo ma ki lwongo ni secretory pathway me cwalo gi kama opore (1). Medo ikom poko kin jami ma ki lwongo ni coat, onwongo ki tamo ni jami mogo ma ki lwongo ni lipids twero tic calo ka ma ki yero me aa ki iye kun ki keto gi i dul ma pat pat ma pat pat (2-5). Ento, pud pe tye gin mo ma nyutu ni jami makwo ni twero bedo tye. Me cobo peko man, wa kwano i yeast kit ma glycosylphosphatidylinositol (GPI) anchored proteins (GPI-APs) ki cwalo kwede ki i ER. GPI-APs obedo dul kom ma pat pat ma gikubbe ki moo i kom cell ( 6, 7). GPI-AP obedo gin ma ki cwalo ma mako dul kom ma woko me plasma membrane kun wok ki i dul kom ma ki lwongo ni glycolipid (GPI anchor). Gi ye ni GPI anchors calo gin ma ki loko i nge loko leb i ER lumen (8). Iyonge keto, GPI-AP kato ki i Golgi apparatus (5, 9) ki i ER me cito i plasma membrane. Nongo pa GPI anchors miyo GPI-AP ki tero ma pat ki transmembrane secreted proteins (medo ki plasma membrane proteins mukene) i yoo me secretory (5, 9, 10). I cell me yeast, GPI-APs ki poko ki i kom protein mukene ma ki cwalo i endoplasmic reticulum, ki dong ki keto i vesicles ma pat pat ma ki umo ki coat protein complex II (COPII) (6, 7). Gin ma moko kit me poko kin jami i yub me cwalo jami woko i ER pe tye atir, ento ki tamo ni yoo man twero mito lipids, tutwale yub me yubu dul lipids me GPI anchor (5, 8). I yeast, GPI lipid remodeling cako cut cut ingee GPI omoko, ki i lok mapol, weko ceramide rwate ki 26-carbon long-chain saturated fatty acid (C26:0) (11, 12). C26 ceramide obedo ceramide ma pire tek ma ki yubu ki yeast cells nio wa kombedi. Ki yubu ne i ER ki pol ne ki cwalo i Golgi apparatus kun wok ki i COPII vesicles (13). ER ma ki cwalo woko pa GPI-AP mito ni ki med ceramide synthesis (14, 15), ki dong, loko ceramide me doko inositol phosphate ceramide (IPC) i Golgi apparatus cung i kom GPI anchor synthesis (16). Kwan ma mako kwo pa jami makwo ki jami ma ki yubu ki cing dano onyutu ni acyl chain ceramides mabor twero ribbe me yubu dul ma tye ki kit ma pat pat (17, 18). Lok ma ki nongo ni okelo tam ni C26 ceramide ki GPI-AP ki C26 ceramide tiyo ki jami ma nen me rwate i kabedo ma ki yubu maber onyo kabedo ma tye i ER membrane lipid environment ma pe rwate. En tye ki glycerolipids ma cok ki ma pe opong (C16:1 ki C18:1) (19, 20). Kabedo magi ki bi keto tam gi i kom kabedo ma ki lwongo ni ER exit sites (ERES), kama ceramide ki ceramide-based GPI-AP ki twero tero gi kacel i Golgi i COPII vesicle acel (5).
I kwan man, wa temo jami ma ki lwongo ni lipid-based mechanism kun wa tiyo ki super-resolution confocal real-time imaging microscopy (SCLIM), ma obedo yoo me microscopy ma twero neno jami ma ki lwongo ni fluorescently labeled proteins.
Wa cako tic ki diro me SCLIM me medo gonyo kit ma GPI-AP ma tye ki C26 ceramide group onongo ki kwanyo ki i transmembrane secreted proteins inge weko ER i S. cerevisiae. Me neno kit ma ki poko kwede ER, watiyo ki jami ma ki lwongo ni genetic system ma twero neno jami ma ki yubu manyen ma donyo i ERES in vivo (7, 23). Ma calo gin ma ki cwalo, wa yero C26 ceramide-based GPI-AP Gas1 ma ki keto ki green fluorescent protein (GFP) ki transmembrane secreted protein Mid2 ma ki keto ki near-infrared fluorescent protein (iRFP), gin aryo ni gi keto i kom plasma membrane (24-26). I kom gin ma ki lwongo ni sec31-1 temperature-sensitive mutant, jami aryo ni ki nyutu gi i te galactose-inducible promoter ki constitutive ERES marker. I rwom me lyeto ma malo (37°C), pien sec31-1 ma ki loko ni balo tic pa COPII coat component Sec31 me gengo COPII dongo ki ER ma ki cwalo woko, jami ma ki yubu manyen ni bino i ER (23). Iyonge ki juku ne i lyeto ma lapiny (24°C), sec31-1 mutant cells ni odok cen ki i kabedo ma ki cwalo iye jami, ki jami manyen ma ki yubu ni ocako cwalo woko ki i ER. Neno pa CLIM onyutu ni pol pa Gas1-GFP ki Mid2-iRFP ma ki yubu manyen ni pud tye ka dongo i ER pa sec31-1 mutant cells inge keto i 37°C ki dong ki gonyo i 24°C pi dakika 5 (lanyut 1). Kit ma Mid2-iRFP ki poko i kom ER membrane weny, ki Gas1-GFP ki keto i kom ER membrane ma pe rwate, poko gi pat ataa (lanyut 1, A onyo C ki filim S1). Medo ikom meno, kit ma ki nyutu kwede i cal 1D, dul pa Gas1-GFP pe ki Mid2-iRFP. Adwogi eni nyutu ni GPI-AP ki jami ma ki lwongo ni transmembrane proteins ki poko gi i dul ER membrane ma pat pat con. Dul pa Gas1-GFP tye cok ki ERES ma ki coyo ki mCherry's COPII coat protein Sec13 (lanyut 1, E ki F, ki filim S1) (23).
sec31-1 cells nyutu galactose-induced secretions, acyl chain mabor (C26) ceramide GPI-AP Gas1-GFP (GPI-AP, green) ki transmembrane protein Mid2-iRFP (TMP, blue) ki man Constructive ERES labeling Sec13-mCherry (E) onongo ki keto i RES i°C dakika, ki dwoko i 24°C, ki ki kwanyo cal ki SCLIM dakika 5 lacen. (A to C) nyutu cal ma ki keto kacel onyo acel me 2D me dege (A), cal me 2D me 10 z-sections (B) onyo cal me 3D cell hemisphere me jami ma ki cwalo ki ERES markers (C). Lapim 1μm (A ki B). Gin ma ki pimo kwede tye 0.551μm (C). Gas1-GFP onongo kinongo i dul ER ma pat pat, kun Mid2-iRFP onongo kinongo ki poko i ER membrane (C). (D) cal ni nyutu rwom me ryeny pa Gas1-GFP ki Mid2-iRFP i dul pa Gas1-GFP ma lubu lanyut ma tar (tung lacuc). AU, gin ma ki lwongo ni arbitrary unit. (E ki F) nyutu cal 3D ma rwate ki jami ki lanyut me ERES. Gas1-GFP clusters ki nongo gi cok ki ERES ma ki ngeyo. Gin ma ki pimo ni tye 0.551μm. (F) Lanyut ma nen calo dul ma ki lwongo ni Gas1-GFP ma rwate ki ERES. Kabedo ma i kine ki tung acuc nyutu cal ma ki medo ki 3D ki neno ma ki loko i kom dul pa Gas1-GFP ma ki yero.
Wat macok ma tye i kin dul pa Gas1-GFP ki ERES ma ki ngeyo nyutu ni Gas1-GFP twero donyo i ERES ma ki yero, ma pat ki yero ma Mid2-iRFP tiyo kwede me aa ki i ER. Me cobo peko man, wa kwano wel ERES pi gin acel onyo aryo keken (lanyut 2, A onyo C). Wanongo ni pol ERES (70%) tye ki jami ma ki cwalo acel keken. Cal ma piny i cal 2C nyutu labol aryo ma rwate pa ERES ki Gas1-GFP keken (lanyut 1) onyo Mid2-iRFP keken (lanyut 2). Ka i poro, ma romo 20% pa ERES tye ki jami aryo ma gi rwate i kabedo acel. Kinongo ni ERES mogo (10%) tye ki jami ma ki cwalo ma pat pat, ento onongo ki poko gii i kabedo ma pat pat. Pi meno, kwan man nyutu ni inge ER ni ki cwalo woko, GPI-AP Gas1-GFP ki transmembrane cargo Mid2-iRFP ki poko gi i ERES ma pat pat (lanyut 2D). Kit me poko kin jami ni rwate ki jami macon ma ki kwano (6) ki kit ma ki ngeyo kwede (7). Wa twero bene neno kit ma jami ma ki gwoko gi tye ka donyo kwede i ERES (lanyut 2E ki filim S2). cal 2E nyutu ni dul matidi pa Gas1-GFP (panel 3) onyo Mid2-iRFP (panel 4) keken aye donyo i ERES ki tung acel ki ki keto i kabedo ma ki poko. Dul me 5 me cal 2E nyutu ni Gas1-GFP ki Mid2-iRFP pol kare ki nongo gi i ERES acel, ento gi donyo ki tung ma pat pat ki gi bedo i kabedo ma pat pat ma twero nyutu COPII vesicles ma pat pat. Wamoko bene ni poko ma ki neno ki poko pa C26 ceramide-based GPI-AP Gas1 ma calo ERES ma ki yero tye ma pat pien gin mukene ma ki cwalo i kom ler, ma ki lwongo ni GFP-tagged plasma membrane protein Axl2 (27 ), ma nyutu kit ma rom ki Mid2-iRFP. (Cal S1 ki Filim S3). Axl2-GFP ma ki yubu manyen ni ki poko i ER membrane calo Mid2-iRFP (lanyut S1, A ki B), ki ki keto kacel ki Mid2-iRFP i ERES mapol (lanyut S1, B onyo D). Dul 1 ki 2 me cal 1. S1C nyutu labol aryo ma rwate pa ERES ma jami aryo ma ki cwalo gi rwate. I lok eni, jami aryo ni weny gi donyo i ERES kacel (lanyut S1E, Panel 3 ki Movie S3).
Gin ma ki lwongo ni sec31-1 ma nyutu gin ma ki lwongo ni galactose inducible secretions, Gas1-GFP (GPI-AP, green) ki Mid2-iRFP (TMP, blue) ki constitutive ERES labeling Sec13-mCherry (ERES, magenta) ki keto gi i 37 Iyonge bedo pi dakika 30 i °C, ki gonyo ion me block ion ki SCLIM ingee dakika 20. (A to C) Cal ma nyutu 2D ma ki nyutu (A; rwom me rwom, 1μm) onyo cal me 3D cell hemisphere (B ki C; rwom me rwom, 0.456μm) pa jami ma ki cwalo ki 10 z-sections ma ki nyutu ki ERES. Kabedo ma piny i (B) ki kabedo ma tye i (C) nyutu cal ma ki yubu me nyutu jami ma tye i ERES (magenta) [Gas1-GFP (gray) ki Mid2-iRFP (light blue)]. (C) Open arrow: ERES tingo gin acel keken (1 onyo 4). Lanyut macol: ERES tye ki jami ma ki poko (5). Lanyut ma nen maber: ERES ma tye ki jami ma ki keto kacel. Piny kany: ERES acel ma ki yero ni tye ki Gas1-GFP (1) onyo Mid2-iRFP (2) keken. Lapim, 100 nm. (D) Kwanyo wel pa cal ma ki nyutu i (C). Pek pa ERES ma tye ki jami acel keken (Gas1-GFP onyo Mid2-iRFP), jami ma ki poko ki jami ma rwate. I kom jami adek ma ki temo, n=432 i cell 54. Bal = SD. Two-tailed unpaired t test. *** P = 0.0002. (E) cal 3D pa ERES ma ki yero me jami ma ki gwoko ma ki keto lanyut ki (C). Gas1-GFP (macol) (3) onyo Mid2-iRFP (bulu) (4) donyo i ERES (magenta) ki tung acel ki ki gengo i kabedo matidi i ERES. I kare mogo, jami ma ki cwalo ni gi donyo i ERES acel (5) ki tung acel ki gi bedo i kabedo ma pat pat i ERES ni. Lapim, 100 nm.
Ma lubo, wa temo lok ma ki waco ni long acyl chain ceramide (C26) ma tye i ER membrane ma weko ki poko ki poko pa Gas1 i ERES ma ki yero. Me agiki, watiyo ki dul tobi ma ki loko GhLag1, ma iye ceramide synthases aryo ma ki lwongo ni Lag1 ki Lac1 ki loko gi ki GhLag1 (lag1 ma rom ki pama), ma okelo dul tobi ma tye ki cell membrane Ceramide strain ma nok loyo wild type (lanyut 3A) (28). Kwano jami ma ki lwongo ni Mass spectrometry (MS) onyutu ni i kom ler ma ki lwongo ni wild-type strains, 95% pa wel ceramide ni tye mabor atika (C26) chain ceramide, kun i GhLag1, 85% pa ceramide ni tye mabor tutwal (C18 ki C16). ), 2% keken pa ceramide tye ma bor (C26) ceramide. Kadi bed ni C18 ki C16 ceramides gubedo ceramides ma ki nongo i GhLag1 membrane nio wa kombedi, MS analysis bene moko ni GPI anchor pa Gas1-GFP ma ki nyutu i GhLag1 tye ki C26 ceramide, ma rom ki wild-type lipids. Kit ma tye kwede rom (Fig. 3A) (26). Pi meno, man tere ni ceramide remodeling enzyme Cwh43 tye ma yero maber pi C26 ceramide, ma ki nyutu i cal 26, en maro keto GPI anchor ki i C26 ceramide manok i GhLag1 strain. S2 (29). Kadi bed kumeno, cell membrane pa GhLag1 tye ki C18-C16 ceramide keken, kun Gas1-GFP pud tye ki C26 ceramide. Lok man miyo dul man bedo gin maber me cobo peko me bor pa acyl chain pa membrane ceramide i ER. Tic ma ki tamo ni tye i dul ki poko jami. I ngeye, wa cako kwano kero pa C26 Gas1-GFP me cokke i dul i GhLag1 ki allele ma loko rwom me lyeto pa sec31-1 kun wok ki i microscopy ma ki lwongo ni conventional fluorescence microscopy, ma dul mabor (C18-C16) keken aye tye i ER membrane Ceramide (Fig. 3). Wa neno ni i sec31-1, pol pa Gas1-GFP onongo tye i dul, kun Gas1-GFP i sec31-1 GhLag1 ki bor (C18-C16) long ceramide ER membrane onongo pe ki dul dok kipoko i ER membrane ducu. Me bedo atir, pien C26 ceramide-based clustering tye macok ki ERES (lanyut 1), wa lubo kwedo ka yub man twero bedo ki tic pa ER export protein mechanism. GPI-AP tiyo ki yub me COPII ma pat pi ER cwalo woko, ma ki doro ki Ted1's ma ki yubu me loko dul kom ma ki lwongo ni glycan me GPI anchor (30, 31). Gin ma ki lwongo ni Recombinant GPI-glycan ni dong ki ngeyo ki transmembrane cargo receptor p24 complex, ma en bene kwanyo Lst1, ma en aye isoform ma pat pat pa COPII cargo binding subunit Sec24, ma yubu GPI-AP-rich COPII Vesicles are necessary (31-33). Pi meno, wa yubu gin ma ki lwongo ni double mutant ma orwate ki kwanyo jami ma ki lwongo ni single proteins (p24 complex component Emp24, GPI-glycan remodeling enzyme Ted1 ki COPII subunit Lst1) ki sec31-1 mutant strain, ki wa kwano gi Tika twere me yubu Gas1-Gluster GFP (Figure 3). Waneno ni i sec31-1emp24Δ ki sec31-1ted1Δ, Gas1-GFP tye ma pe ki poko i kin ER membrane, ma calo ma onongo ki neno i sec31-1 GhLag1, kun i sec31-1lst1Δ, Gas1-GFP Like sec31-1-1. Adwogi eni nyutu ni medo i kom bedo tye pa C26 ceramide i ER membrane, dul pa Gas1-GFP bene mito ni orwate ki p24 complex, ki pi mito Lst1 ma ki ngeyo. I ngeye, wa temo neno kit ma bor pa ceramide i ER membrane twero doro rwate pa Gas1-GFP ki p24. Ento, wanongo ni bedo pa C18-C16 ceramide i membrane pe gudo GPI-glycans ma ki yubu ki p24 complex (lanyut S3 ki S4, A ki B) onyo mako GPI-AP ki cwalo GPI-AP woko. twero. Coy COPII ma tye i dul Lst1 (lanyut S4C). Pi meno, C26 ceramide-dependent clustering pe mito ni jami ma ki lwongo ni protein ni gubed ka tic ki jami ma pat pat ma ki cwalo i ER, ento miiyo kony me poko jami ma pat pat ma lubu bor pa jami ma ki lwongo ni lipid. I ngeye, wa temo ka ce rwom me ceramide acyl chain ma tye i ER membrane pire tek pi poko kin Gas1-GFP ma calo ERES ma ki yero. Kit ma Gas1 ma tye i GhLag1 ma tye ki ceramide ma tye ki dul ma cok ni weko ER ki donyo i plasma membrane (lanyut S5), waye ni ka poko ne ki doro ki bor pa ceramide acyl chain, Gas1 ma tye i GhLag1 twero bedo ma ki loko ki rwate. Jami ma ki lwongo ni ERES ma tye ki dul kom acel.
(A) Gin ma ki lwongo ni cell membrane pa GhLag1 tye ki C18-C16 ceramides ma cok, kun GPI anchor pa Gas1-GFP pud tye ki C26 IPC acel ki cell ma ki lwongo ni wild-type. Ma malo: rwom me acyl chain length pa ceramide i cell membrane pa wild-type (Wt) ki GhLag1p strains ki mass spectrometry (MS). Lok ma ki nongo ni nyutu pacenti pa ceramide weny. I kin jami adek ma ki temo ma pat pat. Bal = SD. Two-tailed unpaired t test. **** P ＜0.0001. Labol ma piny: MS nyamo rwom me acyl chain pa IPC ma tye i Gas1-GFP (GPI-IPC) GPI anchor ma ki nyutu i wild-type ki GhLag1p strains. Lok ma ki nongo ni nyutu wel ma ki nongo ki i IPC. I kin jami abic ma ki temo ma pat pat. Bal = SD. Two-tailed unpaired t test. ns, pe pire tek. P = 0.9134. (B) Fluorescence micrographs pa sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ ki sec31-1lst1Δ ma nyutu galactose-induced Gas1-GFP ki gwoko gi i 37°C pi dakika 30 ki dwoko piny me tic microscopy ingee 24°C. Lanyut ma tar: ER Gas1-GFP cluster. Open arrow: Unclustered Gas1-GFP ki poko i kom ER membrane weny, ma nyutu ER ma nyutu nuclear ring staining. Lapim, 5μm. (C) Kwanyo wel pa cal ma ki nyutu i (B). Wel ma romo pacenti pa cell ma tye ki dul kom ma ki lwongo ni Gas1-GFP. I kom jami adek ma ki temo, n≥300 cells. Bal = SD. Two-tailed unpaired t test. **** P ＜0.0001.
Me cobo peko eni, wa otimo SCLIM ma nyutu Gas1-GFP ki Mid2-iRFP i GhLag1 ki sec31-1 temperature-sensitive mutant allele (lanyut 4 ki filim S4). Iyonge ma ki gwoko ER i 37°C ki lacen ki gonyo i 24°C, pol pa Gas1-GFP ma ki yubu manyen ni pe ki keto kacel ki poko ne i ER membrane, kit ma ki neno kwede ki microscopes macon (lanyut 4, A ki B ). Medo ikom meno, wel ma dit pa ERES (67%) tye ki jami ma ki cwalo ma tye iye (lanyut 4D). Dul 1 ki 2 me cal 4C nyutu labol aryo ma rwate pa ERES ki Gas1-GFP ki Mid2-GFP ma gi rwate. Medo ikom meno, jami aryo ni ki coyo gi i ERES acel (lanyut 4E, panel 3 ki filim S4). Pi meno, adwogi wa nyutu ni bor pa ceramide acyl chain i ER membrane obedo gin ma pire tek ma moko ER protein aggregation ki poko ne.
Sec31-1 GhLag1 cells ma nyutu galactose-induced secretions, Gas1-GFP (GPI-AP, green) ki Mid2-iRFP (TMP, blue) ki constitutive ERES-labeled Sec13-mCherry (ERES, magenta) Keto i 37°C. Medo pi dakika 30, dwoko piny nio wa i 24°C me gonyo jami ma ki cwalo, ki cal ki SCLIM inge dakika 20. (A to C) Cal ma nyutu 2D (A; rwom me rwom, 1μm) onyo cal me 3D cell hemisphere (B ki C; rwom me rwom, 0.45μm) pa 10 z-sections ma ki nyutu ki cargo ki ERES. Kabedo ma piny i (B) ki kabedo ma tye i (C) nyutu cal ma ki yubu me nyutu jami ma tye i ERES (magenta) [Gas1-GFP (gray) ki Mid2-iRFP (light blue)]. (C) Lanyut ma opong ki ler: ERES, jami ma rwate. Yab lanyut: ERES tye ki gin acel keken. Kabedo ma piny: ERES ma ki yero ni tye ki jami ma rwate (1 ki 2) ma ki keto i (C). Lapim, 100 nm. (D) Kwanyo wel pa cal ma ki nyutu i (C). I sec31-1 ki sec31-1 GhLag1 units, gin acel keken (Gas1-GFP onyo Mid2-iRFP) tye iye, ki wel ma romo ERES pi gin ma ki poko ki gin ma ki cwalo ma rwate. I kom jami adek ma ki temo, n = 432 i 54 cells (sec31-1) ki n = 430 i 47 cells (sec31-1 GhLag1). Bal = SD. Two-tailed unpaired t test. *** P = 0.0002 (sec31-1) ki ** P = 0.0031 (sec31-1 GhLag1). (E) cal 3D pa ERES ma ki yero ki jami ma ki cwalo ma rwate (3) ma ki keto i (C). Gas1-GFP (ma lum) ki Mid2-iRFP (ma bulu) bino bot ERES (magenta) ki tung acel ki gibedo i kabedo acel ma ERES ki gengo. Lapim, 100 nm.
Kwano man miyo caden ma rwate ki jami makwo ma nyutu ni jami ma ki keto i kom jami ma ki lwongo ni lipid-based protein cargoes ki poko gi i kabedo ma ki yero me cwalo jami woko i yoo me cwalo jami, ki nyutu kit ma acyl chain length pire tek kwede pi poko jami. Nono ki diro me microscopy ma tek ki ma ki lwongo ni SCLIM, wanyutu Gas1-GFP ma ki yubu manyen (GPI-AP ma dit ki acyl chain mabor (C26) ceramide lipid portion) i yeast) Kabedo ma ki keto i ERs ma pat pat gikubbe ki EERs ma pat pat, ma nongo proteins ma ki cwalo ki ERRES gi poko i membrane (lanyut 1). Medo ikom meno, jami aryo ni gi donyo i ERES ma pat pat (lanyut 2). Bor pa acyl chain pa cellular ceramide ma tye i membrane ni ki dwoko piny ki C26 me oo C18-C16, Gas1-GFP cluster ni ki balo i ER region ma ki poko, ki Gas1-GFP ki loko me aa ki i ER ki transmembrane protein kun wok ki i ERES acel-lu (lanyut 3 ki cal 3). 4).
Kadi bed ni GPI-AP tiyo ki yoo me protein ma ki ngeyo me aa ki i ER, wanongo ni C26 ceramide-dependent separation pe jenge i kom protein ma pat pat ma twero kelo ERES specialization (lanyut S4 ki S5). Ma ka meno, gin ma wanongo miiyo kony bot yoo mukene me poko kin jami ma ki lwongo ni lipid-based protein clustering ki kwanyo jami mukene. Gin ma waneno nyutu ni Gas1-GFP region onyo dul ma rwate ki ERES mo pe ki transmembrane secreted protein Mid2-iRFP, ma nyutu ni C26 ceramide-dependent GPI-AP cluster obi konyo gi me donyo i ERES ma rwate, ki i kare acel, kwanyo transmembrane woko. Ka i poro, bedo tye pa C18-C16 ceramides i ER membrane pe miyo GPI-AP me yubu kabedo onyo dul, pi meno pe gi kwanyo onyo loko transmembrane secreted proteins i ERES acel-lu (lanyut 3 ki 4). . Pi meno, wa tamo ni C26 ceramide kelo poko kin jami ki poko kin jami ma konyo dongo pa jami ma gikubbe ki ERES ma pat pat.
Kit me nongo C26 ceramide-dependent clustering i ER area mo? Kit ma membrane ceramide twero poko kwede twero weko GPI-AP ki C26 ceramide me yubu lipids ma tiino ki ma ki keto i rwom ma lapiny i ER membrane ma tye ki glycerolipids ma tidi ki ma pe opong. Dul maber (17, 18). Dul matino tino magi ki twero medo gi me doko dul madongo, ma ocung matek inge ribbe ki p24 complex (34). Ma rwate ki lok man, wanyutu ni C26 Gas1-GFP mito tic ki p24 me yubu dul ma nen ma dit (lanyut 3). Gin ma ki lwongo ni p24 ni tye gin ma ki lwongo ni heterozygous oligomer ma tye ki p24 ma pat pat ma tye i yeast (35), ma miyo multivalent binding, ma twero kelo cross-linking pa GPI-AP clusters ma nok, ma weko kelo Stable cluster madit (34). Kit ma dul kom ma ki lwongo ni protein ectodomains pa GPI-APs gi rwate kwede bene twero konyo me ribbe kacel, ma ki nyutu i kare ma gi tero Golgi i kom ler ma ki lwongo ni mammalian polarized epithelial cells (36). Ento, ka C18-C16 ceramide tye i ER membrane, ka p24 ma rwate ki Gas1-GFP, dul madongo ma pat pat pe obi bedo. Kit ma tic kwede twero cung i kom kit ma kom ki jami ma tye i kom gin ma ki lwongo ni long acyl chain ceramide ni tye kwede. Kwano jami makwo i kom jami ma ki yubu ki cing dano nyutu ni kadi bed ni acyl chain ceramides mabor (C24) ki ma cok (C18-C16) twero kelo apoka poka, acyl chain ceramides mabor (C24) keken aye twero kelo Curvature ma malo ki film bending me loko kit ma film ni nen kwede. Ki ikom rwate (17, 37, 38). Ki nyutu ni transmembrane helix pa TMED2, ma obedo dano ma rom ki Emp24, tiyo kacel ki C18 ceramide-based sphingomyelin i cytoplasmic lobules (39). Nono jami ma ki lwongo ni molecular dynamics (MD) simulations, wanongo ni C18 ki C26 ceramides gi rwate i kom cytoplasmic lobules me Emp24 transmembrane helix, ki gi tye ki miti marom (lanyut S6). Ber me poyo ni man nyutu ni transmembrane helix pa Emp24 twero kelo poko ma pe rwate pa lipids i membrane. Man obedo adwogi manyen ma jenge i kom cell pa ler ma nywalo lutino. Labol me MD ma rom bene nyutu bedo tye pa ether lipids (40) . Pi meno, wa tamo ni C26 ceramide ma tye i lobule aryo pa ER26 tye ma ki medo iye. Ka GPI-AP i luminal lobules rwate ki multivalent p24 ki rwom me C26 ceramide ma orumo p24 i cytoplasmic lobules, twero kelo rwate pa Protein ki membrane curvature ma ki yubu ki cing (41), ma weko GPI-AP poko i kabedo ma pat pat macok ki ERES, ma bene konyo kabedo ma tye ki curvature ma malo ER membrane (42). Ripot mukato ni ocung i kom yoo ma ki waco ni (43, 44). Kit ma oligolectins, pathogens onyo antibodies gi rwate kwede ki ceramide-based glycosphingolipids (GSL) i kom plasma membrane kelo GSL madongo, medo poko kin dul kom ki kelo alokaloka i membrane ki donyo i iye (44). Iwabuchi ki mukene. (43) Kinongo ni i kare ma tye ki acyl chains mabor (C24) ento pe ma cok (C16), gin ma ki lwongo ni multivalent ligand ma ki keto i GSL lactosylceramide okelo dongo pa dul madongo ki membrane invagination, ki cytoplasm Lyn-mediated signal transduction i kom leaflets ni ki doro ki acyl chains ma rwate ki neutrophils.
I kom ler ma ki lwongo ni mammalian polarized epithelial cells, dwong pa anti-Golgi network (TGN) me oo i rwom me apical plasma membrane loyo poko ki poko pa GPI-AP (10, 45). Dul man ki kelo ki GPI-AP oligomerization (36), ento bene twero cung i kom bor pa ceramide ma wanongo i yeast. Kadi bed ni GPI-AP pa ler ma ki lwongo ni mammalian tye ki gin ma ki lwongo ni ether lipid-based anchor, ki kit ma kome nen kwede pat ki ceramide ma tye ki acyl chain mabor, kwan ma ki timo cok cok ni onongo ni lipids aryo ni tye ki kit ma rwate ki kit ma gi tiyo kwede (40). Pi meno, dul ma ki lwongo ni ether lipid ma tye i kom ler ma ki lwongo ni mammalian twero bedo rom ki C26 ceramide ma tye i yeast, ki tic ne tye me ribe ki ceramide ma tye ki dul mabor ma tye i membrane me kelo dongo pa GPI-AP ki poko ne. Kadi bed ni twero man pud mitte ni ki temo, gin ma kinongo con nyutu ni tero acyl chain ceramide mabor me cito i kom Golgi pe ki tiimo ki cytoplasmic transfer proteins, ento cung i kom yubu pa GPI anchors calo yeast. Pi meno, yub me gwoko kwo ma nen calo twero yero me tero jami ma ki lwongo ni acyl chain ceramide ki GPI-AP (13, 16, 20, 46, 47) i yoo acel.
I kom jami ma ki lwongo ni yeast ki mammalian polarized epithelial cell systems, GPI-AP ma gi ribbe ki poko gi ki i kom jami ma ki lwongo ni plasma membrane proteins weng time ma peya oo i kom cell. Paladino ki luwote (48) onongo ni i kom TGN pa ler ma ki lwongo ni mammalian polarized epithelial cells, GPI-AP clustering pe mitte keken pi poko GPI-APs i apical plasma membrane, ento bene doro yub me clustering pa GPI-APs ki tic ne. Kom cell. I yeast, kwan man onyutu ni C26 ceramide-dependent GPI-AP cluster i kom ER twero doro yub pa cluster ki tic pa GPI-AP i kom plasma membrane (24, 49). Ma rwate ki labol man, GhLag1 cells gi tye ki two ma gengo GPI onyo yadi ma balo cell wall integrity (28), ki miti pi tic pa Gas1-GFP clusters (49) me tip ceramide ma ki yubu i mating pa yeast cells nyutu G     Adwogi ma twero bedo tye i kom hLag1 cells. GPI-AP bal. Ento, medo temo ka ce yub me tic pa cell ma ki yubu ki i ER ki yoo me poko ma lubu bor pa lipid obi bedo gin ma wa bi kwano i anyim.
Gin ma ki lwongo ni Saccharomyces cerevisiae ma ki tiyo kwede i tic man ki keto gi i Table S1. MMY1583 ki MMY1635 ma ki lwongo ni SCLIM pi neno cell ma kwo ki yubu gi i nge W303. Gin ma nyutu Sec13-mCherry ki lanyut ma ryeny ki yubu kun kitiyo ki yoo me polymerase chain reaction (PCR) ki pFA6a plasmid ma calo labol (23). Gin ma nyutu Mid2-iRFP ma ki keto i kom gin ma ki lwongo ni fluorescent protein ma tye i te loc pa GAL1 promoter ni ki yubu kit man. PCR amplification me iRFP-KanMx sequence ki pKTiRFP-KAN vector (mic pa E. O'Shea, Addgene plasmid number 64687; http://n2t.net/addgene: 64687; research resource identifier (RRID): Addgene_64687) Ki keto i C-termind-endous pa Midgene. Iyonge ma Mid2-iRFP genome sequence ni ki medo ki ki keto i GAL1 promoter, ki keto i Not I-Sac I site me integration plasmid pRS306. Gin ma ki nongo ni pRGS7 ki keto i rwom acel ki Pst I me donyo i URA3 locus.
Gin ma ki lwongo ni Gas1-GFP fusion gene ni ki nyutu i te loc pa GAL1 promoter i centromere (CEN) plasmid, ma ki yubu kit man. Gin ma ki lwongo ni Gas1-GFP ni ki medo ki PCR ki i pRS416-GAS1-GFP plasmid (24) (mic pa L. Popolo) ki ki keto i Xma I-Xho I kabedo pa CEN plasmid pBEVY-GL LEU2 (mic pa C). Miller; Addgene plasmid namba 51225; http://n2t.net/addgene: 51225; RRID: Addgene_51225). Gin ma ki nongo ni ki lwongo ni pRGS6. Gin ma ki lwongo ni Axl2-GFP fusion gene bene ki nyutu i te loc pa GAL1 promoter me pBEVY-GL LEU2 vector, ki kit ma ki yubu kwede tye kit man. Gin ma ki lwongo ni Axl2-GFP ni ki medo ki pRS304-p2HSE-Axl2-GFP plasmid (23) ki PCR, ki ki keto i Bam HI-Pst I kabedo pa pBEVY-GL LEU2 vector. Gin ma ki nongo ni ki lwongo ni pRGS12. Kit ma oligonucleotides ma ki tiyo kwede i kwan man tye kwede i Table S2.
Kit ma ki medo kwede ki 0.2% adenine ki 2% glucose [YP-dextrose (YPD)], 2% raffinose [YP-raffinose] ma tye ki yeast extract protein p (YP) medium (1 % Yeast extract ki 2% protein ept). (YPR)] onyo 2% galactose [YP-galactose (YPG)] ma calo gin ma kelo carbon, onyo i gin ma ki yubu ma nok (0.15% yeast nitrogen base ki 0.5% ammonium sulfate) me medo amino acids ki bases ma mite pi cam, ki tye ki 2% glucose (glucose glucose) onyo minimal medium galactose (gin ma ki yubu ki galactose ma nok) ma calo gin ma kelo yamo.
Pi neno cal i kare kikome, jami ma ki lwongo ni temperature-sensitive sec31-1 mutant cells ma nyutu jami ma ki yubu i te GAL1 promoter ki dongo gi i YPR medium i 24°C pi dyewor nio wa i mid-log phase. Iyonge keto i YPG i 24°C pi cawa 1, ki gwoko cell ni i SG i 37°C pi dakika 30, ki dong ki dwoko gi i 24°C me gonyo ki i gin ma ki keto iye. Concanavalin A onwongo ki tiyo kwede me yubu cell ni i kom gin ma ki lwongo ni glass slide ki ki kwanyo cal ne ki SCLIM. SCLIM obedo dul pa Olympus IX-71 ma ki lwongo ni inverted fluorescence microscope ki UPlanSApo 100×1.4 numerical aperture oil lens (Olympus), ma rwom ne lamal ki ma tye ki rwom ma lamal me loko gin ma ki lwongo ni rotating disc confocal scanner (Yokogawa Electric), custom spectrometer, ki jami ma ki lwongo ni intensive systems (Hamamatsu Photonics) twero miiyo gin ma ki lwongo ni magnifying lens ki magnifying me agiki me ×266.7 ki jami ma ki lwongo ni charge-coupled device camera ma medo electrons (Hamamatsu Photonics) (21). Kwanyo cal ki tiimo ki jami ma ki yubu pi ngat acel acel (Yokogawa Electric). Pi cal 3D, wa tiyo ki gin ma ki yubu me loko gin ma ki neno ni, ki wa kwanyo jami ma ki neno ni 100 nm ki i kin gii. Cal ma ki lwongo ni Z-stack ni ki loko ne i 3D voxel data, ki gin ma ki lwongo ni theoretical point spread function ma ki tiyo kwede pi rotating disc confocal microscope ki tiyo kwede me deconvolution processing ki Volocity software (PerkinElmer). Nono ki tic ki jami ma ki lwongo ni Volocity me keto rwom me tic pa co-location, ERES ma tye ki jami ma ki cwalo ki pimo. Ki tiyo ki jami ma ki lwongo ni MetaMorph software (Molecular Devices).
Tii ki jami ma ki lwongo ni GraphPad Prism me nongo gin ma pire tek. Pi two-tailed Student's t-test ki ordinary one-way analysis of variance (ANOVA) test, apokapoka ma tye i kin gurup ki ngeyo ni tye ki adwogi madit i kom P <0.05 (*).
Pi fluorescence microscope pa Gas1-GFP, log phase cells ni ki dongo gii dyewor i YPD ki ki kwanyo gi ki centrifugation, ki lwoko tyen aryo ki phosphate buffered saline, ki ki keto i kom ice pi dakika ma romo 15, ki dong ki medo ki microscope kit ma onongo kiloko kwede con ni Check (24). Gin ma ki lwongo ni Leica DMi8 microscope (HCX PL APO 1003/1.40 oil PH3 CS) ma ki keto iye gin ma ki lwongo ni objective lens, L5 (GFP) filter, Hamamatsu camera ki Application Suite X (LAS X) software aye ki tiyo kwede me nongo jami. .
Kamu kene ni ki loko ki SDS sample buffer i 65°C pi dakika 10, ki dong ki poko ki SDS-polyacrylamide gel electrophoresis (PAGE). Pi kwedo jami ma ki lwongo ni immunoblotting, 10 μl me gin ma ki nyutu ni ki keto i lane acel acel. Gin ma ki lwongo ni Primary antibody: Tii ki rabbit polyclonal anti-Gas1 i rwom me 1:3000, rabbit polyclonal anti-Emp24 i rwom me 1:500, ki rabbit polyclonal anti-GFP (mic pa H. Riezman) i rwom me 1:3000. Gin ma ki lwongo ni mouse monoclonal anti-Pgk1 antibody ni ki tiyo kwede i rwom me 1:5000 (mic ma aa ki bot J. de la Cruz). Gin ma ki lwongo ni Secondary antibody: Horseradish peroxidase (HRP) ma ki keto i kom ler ma ki lwongo ni anti-rabbit immunoglobulin G (IgG) ma ki tiyo kwede i rwom me 1:3000 (Pierce). HRP-conjugated goat anti-mouse IgG onongo kitiyo kwede i rwom me 1:3000 (Pierce). Kabedo ma kom dano gamo iye two ni ki neno ki yoo me chemiluminescence ma ki lwongo ni SuperSignal West Pico reagent (Thermo Fisher Scientific).
Kit ma ki waco kwede i (31), gin ma ki lwongo ni natural immunoprecipitation experiment ni kitimo i kom dul ER ma ki medo iye. Ki lok macek, lwoko cell me yeast ki TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride ki protease inhibitor mixture) i 600 nm (OD600) i 100 optical density tyen aryo. Ki turo ki jami ma ki lwongo ni glass beads, ki dong ki kwanyo jami ma odong ki jami ma ki lwongo ni glass beads ki centrifugation. Gin ma odong ni ki keto i 17,000 g pi dakika 15 i 4°C. Gin ma ki lwongo ni pellet ni ki dwoko cen i TNE ki digitalis saponin ki medo i rwom me agiki me 1%. Gin ma ki keto ni ki gwoko pi cawa 1 ki loko i 4°C, ki dong jami ma pe romo gonyo ni ki kwanyo ki centrifugation i 13,000 g i 4°C pi dakika 60. Pi Gas1-GFP immunoprecipitation, mukwongo omyero i ket gin ma ki nyutu ni i 4°C pi cawa 1, ki dong i ket GFP-Trap_A (ChromoTek) i 4°C pi cawa 3. Gin ma ki lwongo ni immunoprecipitated beads ni ki lwoko tyen abic ki TNE ma tye ki 0.2% digoxigenin, ki kwanyo ki SDS sample buffer, ki poko i SDS-PAGE, ki ki kwano ki immunoblotting.
Kit ma ki waco kwede i (31), moko rwom me rwate ki timo i kom dul ER ma ki medo iye. Ki lok macek, dul ER ma ki medo ni ki keto i 0.5 mM dithiobis(succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, USA; 20°C, 20 min). Gin ma ki lwongo ni crosslinking reaction ni ki juku ki medo glycine (50 mM me agiki, dakika 5, 20°C).
Kit ma onongo kiloko kwede con (50), MS analysis pa ceramide i wild-type ki GhLag1 strains ki otimo. Ki lok macek, ki dongo cell nio wa i rwom ma lamal (3 onyo 4 OD600 units/ml) i YPD i 30°C, ki 25×107 cell ki kwanyo. Kit ma kom gi tiyo kwede ki neko ki trichloroacetic acid. Tii ki gin ma kwanyo pii [ethanol, pii, ether, pyridine ki 4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] ki 1.2 nmol me internal standard C17 ceramide (860517, Avanti polar lipid) quality). Tii ki monomethylamine reagent [methanol, pii, n-butanol ki methylamine solution (4:3:1:5 v/v)] me timo gin ma ki lwongo ni mild alkaline hydrolysis me gin ma ki kwanyo, ki ingeye tic ki n-butanol ma opong ki pii me kwanyo kadu. Me agiki ne, gin ma ki kwanyo ni ki dwoko cen i gin ma ki lwongo ni positive mode solvent [chloroform/methanol/water (2:7:1) + 5 mM ammonium acetate] ki ki keto i mass spectrometer. Kwedo jami mapol (MRM) ki otimo me nongo ki kwano wel jami ma ki lwongo ni sphingolipid molecules. Gin ma ki lwongo ni TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) tye ki gin ma ki lwongo ni robotic nanoflow ion source Nanomate HD (Advion Biosciences, Ithaca, NY) pi nyamo pek pa moo. Teko me rwate ni ki yubu pi dul ceramide acel acel. Ngec pa MS onongo ki nongo i kit maber. Pi jami makwo acel acel, lanyut me moo tye i kin rwom adek ma ki pimo ma pat pat.
Kit ma ki waco kwede i (31), cell (800×107) ma nyutu Gas1-GFP ki keto i tic me immunoprecipitation. Gas1-GFP ma ki yubu ni ki poko ki SDS-PAGE ki ki keto i polyvinylidene fluoride (PVDF) membrane. Gin ma ki lwongo ni protein ni ki neno ki keto rangi i kom PVDF ki amide macol. Gin ma ki lwongo ni Gas1-GFP ki kwanyo ki i PVDF ki ki lwoko tyen 5 ki methanol ki tyen acel ki pii ma ki lwongo ni liquid chromatography-MS (LC-MS). Ki gwoko gin ma ki lwongo ni membrane strip ki 500μl 0.3 M NaOAc (pH 4.0), buffer ki 500μl ma ki juku manyen 1 M sodium nitrite ma ki rubu i 37°C pi cawa 3, dul ma ki lwongo ni lipid ni ki gonyo ki i Gas1-GFP ki ki gonyo in ininphoosphamine i kin gluemi ki glue (51). Iyonge meno, gin ma ki lwongo ni membrane strip ni ki lwoko tyen angwen ki pii ma rwom ne tye LC-MS, ki juku i rwom me lyeto me ot, ki ki gwoko i yamo ma tye ki nitrogen i -80°C nio ka ki temo. Ma calo gin ma ki temo, ki tiyo ki gin ma ki lwongo ni PVDF membrane pi temo acel acel. Gin ma ki kwanyo ki i Gas1-GFP ki dong ki kwano ki MS kit ma ki waco kwede (50). Ki lok macek, PVDF strips ma tye ki GPI-lipid ki dwoko gi i 75μl me gin ma ki lwongo ni negative mold solvent [chloroform/methanol (1:2) + 5 mM ammonium acetate] ki ki kato ki i electrospray ionization (ESI)-MRM/MS Analysis of sphingolipid species (TSQ Vantage). I kom lok man, ngec pa MS onongo ki nongo i kit me ion ma pe ber.
Kit ma kiloko kwede con, dul ma ki lwongo ni lipid ma tye i GPI anchor ni ki poko ki [3H]-inositol-labeled GPI-AP (16). Ki poko jami ma ki lwongo ni lipids ni ki tic ki jami ma ki lwongo ni solvent system (55:45:10 chloroform-methanol-0.25% KCl) ki ki neno ki tic ki FLA-7000 (Fujifilm).
Cell ma nyutu Gas1-GFP (600×107) ki lwoko tyen aryo ki TNE buffer ki TNE buffer, ki ki turo ki gilaci, ki dong ki keto i centrifuge me kwanyo jami ma obale ki gilaci. Gin ma odong ni ki keto i 17,000 g pi cawa 1 i 4°C. Gin ma ki lwongo ni pellet ni ki lwoko i TNE ki ki keto i 1 U PI-PLC (Invitrogen) i TNE ma tye ki 0.2% digitalis saponin pi cawa 1 i 37°C. Iyonge tic ki enzyme, ki kwanyo membrane ni ki centrifugation i 17,000 g i 4°C pi cawa 1. Me gengo Gas1-GFP, gin ma odong ni ki gwoko ki GFP-Trap_A (ChromoTek) i 4°C pi dyewor. Gin ma ki lwongo ni Gas1-GFP ma ki poko ki SDS-PAGE ki keto rangi macol ma ki lwongo ni Coomassie brilliant blue. Gin ma ki lwongo ni Gas1-GFP ni ki kwanyo ki i kom rangi ma orumo aqueduct, ki ingeye inge alkylation ki iodoacetamide ki dwoko piny ki dithiothreitol, in-gel digestion ki trypsin ki otimo. Kwanyo ki juku tryptic peptides ki peptides ki GPI-glycans. Gin ma ki lwongo ni peptide ni ki juku ne i 20 μl me pii. Ket dul (8μl) i LC. Gin ma ki lwongo ni octadecylsilane (ODS) column (Develosil 300ODS-HG-5; inner diameter 150 mm×1.0 mm; Nomura Chemical, Aichi Prefecture, Japan) onwongo ki tiyo kwede me poko peptides i te rwom ma pat pat. Gin ma ki lwongo ni mobile phase tye solvent A (0.08% formic acid) ki solvent B (0.15% formic acid in 80% acetonitrile). Gin ma ki lwongo ni Accela HPLC system (Thermo Fisher Scientific, Boston, Massachusetts) ki tiyo kwede me kwanyo gin ma ki lwongo ni column ki solvent A i kin dakika 55 ki rwom me mol pa 50 μl min-1 pi dakika 5, ki dong ki medo rwom me solvent B nio wa i 40%. , lobo Amerika). Gin ma ki lwongo ni eluate ni ki keto i ESI ion source, ki tryptic peptides ki peptides ki GPI-glycans ki kwano ki LTQ Orbitrap XL (hybrid linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific). I yub me MS, teko pa gin ma ki lwongo ni capillary ni ki keto i 4.5 kV, ki rwom me lyeto pa gin ma ki cwalo ni ki gwoko i 300°C. Kit ma ki keto kwede teko pa capillary ki teko pa lens ma ki keto i 15 V ki 50 V, kit meno. Ngec me MS onongo ki nongo i kit me ion maber (rwom me 60,000; rwom me pek pa 10 dul acel acel) i rwom me pek pa 300/m/z pek/charge ratio (m/z) 3000. Ngec me MS/MS ki nongo ki i ion trap ma tye i LTQ Orbit [XL, data mukwongo, ma jenge i kom namba mukwongo collision induced dissociation (CID)].
Kitimo simulation me MD kun kitiyo ki GROMACS (52) software ki MARTINI 2 force field (53-55). CHARMM GUI Membrane Builder (56, 57) ki tiyo kwede me yubu gin ma tye ki dioleoylphosphatidylcholine (DOPC) ki Cer C18 onyo DOPC ki Cer C26. Kit ma ki lwongo ni Topology ki coordinates pa Cer C26 ki nongo ki bot DXCE kun ki kwanyo jami mukene ma tye i kom sphingosine tail. Tii ki yoo ma ki waco piny kany me rwate ki dul aryo ni ki tic kwede, ki dong tii ki rwom me agiki me yub ni me yubu yub ma tye ki Emp24. Gin ma ki lwongo ni transmembrane domain pa yeast Emp24 (residues 173 to 193) ki yubu calo α-helix kun kitiyo ki visual MD (VMD) tool molecular structure (58). I ngeye, ingee kwanyo jami ma ki lwongo ni lipids, gin ma ki lwongo ni protein ni ki poko ne ki keto i bilayer kun kitiyo ki CHARMM GUI. Gin me agiki tye ki 1202 DOPC ki 302 Cer C26 onyo 1197 DOPC ki 295 Cer C18 ki Emp24. Med ion i kom gin ni me bedo 0.150M. Gin ma ki lwongo ni replicates angwen ma pat pat ki otimo pi jami aryo ma ki lwongo ni bilayer.
Gin ma ki lwongo ni lipid bilayer ni ki rwate kun kitiyo ki yoo me CHARMM GUI, ma kwako dwoko piny ki dwoko rwom me 405,000 steps, ma kama ki dwoko piny mot mot ki kwanyo, ki rwom me kare ni ki medo ki 0.005 ps me oo 0.02 ps. Iyonge rwom, en kelo 6 μs ki kare me 0.02 ps. Iyonge keto Emp24, tii ki CHARMM GUI acel-lu me dwoko piny ki rwate ki jami ni, ki ingeye tii pi 8 s i yubu ne.
Pi jami weny, i kare me rwate, rwom me tic pa Berendsen barostat (59), ki i kare me yubu, rwom me tic pa Parrinello-Rahman barostat (60). I lok weng, rwom me yamo tye 1 bar ki yoo me keto yamo ma rwate ki rwom ma lapiny ki tiyo kwede. I yub me rwom ki yubu jami, gin ma ki lwongo ni thermostat (61) ki speed recalibration ki tiyo kwede me keto rwom me lyeto pa protein, lipid ki solvent particles. I kare me tic ni weny, rwom me lyeto ma ki miito tye 310K. Kit ma pe ki rwate kwede ki kwano kun ki yubu dul ma ki keto kacel kun kitiyo ki yub me Verlet ki 0.005 buffer tolerance. Coulomb term ki kwano kun kitiyo ki reaction field ki cut-off distance me 1.1 nm. Lok pa Vander Waals tiyo ki yub me poko kin piny ma tye ki bor ma romo 1.1 nm, ki yub me poko kin Verlet kitiyo kwede pi rwom me ngwec (62).
Nono VMD, rwom me rwom ma tye i kin DOPC phosphate beads onyo ceramide AM1 beads ki protein ni tye 0.7 nm, ki wel lipids ma gi rwate ki protein ni ki kwano. Malube ki formula ma lubo ni, kwano gin ma ki lwongo ni depletion-enrichment (DE) ma calo i (63): DE = (wel pa moo ma tye i protein 0.7) i protein 0.7 (wel pa Cer ma tye i moo)
wel ma ki coyo ni ki nongo calo wel ma tye i kin piny, ki bal ma ki nongo ni tye kopi angwen ma pat pat pa SE. Kit ma DE factor ma pire tek kwede ki kwano ki t test [(averageDE-factor-1)/SE]. Kwan wel P ki ikom poko ma tye ki dul acel.
Gin ma ki lwongo ni GROMACS ni ki tiyo kwede me kwano 2D lateral density map pa jami ma tye ki Emp24 i kin 250 ns me agiki me trace. Me nongo map me dongo/rweny pa ceramide, map me pek pa Cer ki poko ki wel map pa Cer ki DOPC, ki dong ki poko ki dwong pa Cer i kom. Kit ma ki tiyo kwede ki kala acel-lu.
Pi jami ma ki medo i kom pwony man, nen http://advances.sciencemag.org/cgi/content/full/6/50/eaba8237/DC1
Man obedo gin ma dano twero donyo iye ma ki poko i te cik me Creative Commons Attribution-Non-Commercial License, ma ye ni ki ti kwede, poko ne ki dwoko ne i yoo mo keken, ka ce tic kwede me agiki pe pi nongo cente ki lok ni tic ma ki coyo ni tye kakare. Ka neno.
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Lapeny man ki tiyo kwede me temo ka in itye lalim ki gengo cwalo jami ma pi rwate.
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 -Lingio Serero, Lopez (Serhope), (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
3D ma ki neno maber i kare kikome nyutu teko pa ceramide chain length pi poko protein i kabedo ma ki yero.
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 -Lingio Serero, Lopez (Serhope), (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
3D ma ki neno maber i kare kikome nyutu teko pa ceramide chain length pi poko protein i kabedo ma ki yero.
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