Gutondekanya poroteyine mu nzira y’imisemburo ni ingenzi mu kubungabunga ishyirwa mu bice by’uturemangingo no kubungabunga ubuzima bw’uturemangingo. Uretse gutondekanya hakoreshejwe igikoresho cy’ibumba, uruhare rwa lipide mu gutondekanya kinesin mu nzira yo gutwara poroteyine ni ikibazo cy’ibanze kimaze igihe kirekire kitarasubizwa. Hano, dukora isuzuma rya 3D icyarimwe ry’amabara menshi mu buryo bworoshye kugira ngo dugaragaze mu mubiri ko poroteyine nshya za glycosylphosphatidylinositol zidashobora kugenda zifite lipide ndende cyane za ceramide zishyizwe hamwe kandi zigashyirwa mu byiciro byihariye bya endoplasme. Ahantu ho gusohokera, bitandukanye n’aho poroteyine za transmembrane zikoreshwa. Byongeye kandi, tugaragaza ko uburebure bwa ceramide mu membrane ya endoplasmic reticulum ari ingenzi kuri ubu buryo bwo guhitamo. Ubushakashatsi bwacu butanga ibimenyetso bya mbere mu mubiri byo gushyira imizigo ya poroteyine hashingiwe ku burebure bw’urunigi rwa lipide mu bice byihariye byoherezwa mu nzira y’imisemburo.
Mu turemangingo twa eukaryotic, poroteyine zikorwa muri retikulamu ya endoplasmic (ER) zishyirwa mu byiciro mu gihe cyo gutwara mu nzira y’imisemburo kugira ngo zigere aho zigomba kugera (1). Uretse gutondeka hakoreshejwe coat-mediated, byari bimaze igihe kinini byibazwa ko lipide zimwe na zimwe zishobora no gukora nk'ahantu ho gusohoka hifashishijwe kuzishyira mu bice byihariye bya membrane poroteyine runaka (2-5). Ariko, haracyari ibura ry’ibimenyetso bitaziguye mu mubiri bigaragaza ubu buryo bushoboka bushingiye kuri lipide. Kugira ngo dukemure iki kibazo cy’ibanze, twize mu musemburo uburyo poroteyine za glycosylphosphatidylinositol (GPI) zishyirwaho (GPI-APs) zoherezwa mu buryo butandukanye zivuye muri ER. GPI-APs ni ubwoko bwa poroteyine zifitanye isano na lipide (6, 7). GPI-AP ni poroteyine isohoka ifatanye n'udupapuro two hanze twa plasma membrane binyuze mu gice cya glycolipide (GPI anchor). Bemera anchor za GPI nk'impinduka zihoraho nyuma yo guhindura imiterere ya ER (8). Nyuma yo gufatanya, GPI-AP inyura muri Golgi appareil (5, 9) iva kuri ER ijya kuri plasma membrane. Kuba hari GPI anchors bituma GPI-AP itwarwa ukwayo na poroteyine zikomoka kuri transmembrane (harimo n'izindi poroteyine zikomoka kuri plasma membrane) mu nzira y'ibanga (5, 9, 10). Mu turemangingo tw'umusemburo, GPI-APs zitandukanyijwe n'izindi poroteyine zikomoka kuri endoplasmic reticulum, hanyuma zigashyirwa mu bice byihariye bipfunyitse na coat protein complex II (COPII) (6, 7). Ibintu bigena iyi gahunda yo gushyira mu byiciro mu nzira yo kohereza ER ntabwo birasobanutse neza, ariko bikekwa ko iyi nzira ishobora gusaba lipide, cyane cyane kuvugurura imiterere y'igice cya lipide cya GPI anchor (5, 8). Muri yeast, GPI lipide ihinduka itangira ako kanya nyuma yuko GPI ifatanye, kandi mu bihe byinshi, bituma ceramide ifatana na aside fatty saturated ya karuboni 26 (C26: 0) (11, 12). C26 ceramide niyo ceramide nyamukuru ikorwa n’uturemangingo tw’umusemburo kugeza ubu. Ikorwa muri ER kandi inyinshi muri yo yoherezwa mu bikoresho bya Golgi binyuze muri COPII vesicles (13). Kohereza GPI-AP muri ER bisaba cyane cyane ko ikorwa rya ceramide rikomeza (14, 15), kandi, guhindura ceramide muri inositol phosphate ceramide (IPC) muri Golgi biterwa no gukora GPI anchor (16). Ubushakashatsi bwa biophysical ku ruhu rw’ubukorano bwagaragaje ko ceramide ndende cyane za acyl chain zishobora guhuzwa zigakora domaine zateguwe zifite imiterere yihariye (17, 18). Aya makuru atuma habaho igitekerezo cy’uko C26 ceramide na GPI-AP hamwe na C26 ceramide bikoresha imiterere yabyo kugira ngo bihuzwe mu turere cyangwa uturere turi mu gace ka ER membrane lipide karimo akajagari. Igizwe ahanini na glycerolipide ngufi kandi zitarimo (C16: 1 na C18: 1) (19, 20). Utu turere tuzibanda ku buryo bwihariye ahantu hatandukanye ho gusohokera ER (ERES), aho ceramide na ceramide-based GPI-AP bishobora koherezwa hamwe muri Golgi muri vesicle imwe ya COPII (5).
Muri ubu bushakashatsi, twagerageje mu buryo butaziguye ubu buryo bushingiye ku lipide dukoresheje super-resolution confocal real-time imaging microscopy (SCLIM), ikaba ari uburyo bugezweho bwo gusuzuma poroteyine zishobora kubona icyarimwe poroteyine zifite amazina y’urumuri. Amashusho y’amabara atatu n’ay’ibice bitatu (3D) afite ubushobozi bwo kureba no kwihuta cyane mu turemangingo tuzima (21, 22).
Twabanje gukoresha ikoranabuhanga rya SCLIM kugira ngo dusobanure neza uburyo GPI-AP isanzwe hamwe na C26 ceramide group yapimwe muri poroteyine za transmembrane nyuma yo kuva muri ER muri S. cerevisiae. Kugira ngo tugenzure uburyo ER ishyirwa mu byiciro, twakoresheje sisitemu ya genetiki ishobora kwerekana neza imizigo mishya yakozwe yinjira muri ERES mu mubiri (7, 23). Nk'imizigo, twahisemo GPI-AP Gas1 ishingiye kuri C26 ceramide yanditseho poroteyine y'icyatsi kibisi (GFP) na poroteyine ya transmembrane Mid2 yanditseho poroteyine y'urumuri hafi ya infrared (iRFP), byombi bibanda kuri plasma membrane (24–26). Muri mutant ifata ubushyuhe bwa sec31-1, iyi mizigo ibiri igaragara munsi ya galactose-inducible promoter hamwe na constitutive ERES marker. Ku bushyuhe bukabije (37°C), kubera ko sec31-1 mutation igira ingaruka ku mikorere ya COPII coat component Sec31 kugira ngo ibuze COPII kumera no kohereza ER hanze, imizigo mishya yakozwe irakusanya kuri ER (23). Nyuma yo gukonja kugeza ku bushyuhe buke (24°C), uturemangingo twa sec31-1 twimutse twavuye mu gace k’urusobe rw’ibinyabutabire, maze imizigo mishya yakozwe mu buryo bwa sintetike itangira koherezwa mu mahanga iva muri ER. Ishusho ya CLIM yagaragaje ko inyinshi muri Gas1-GFP nshya na Mid2-iRFP bikiri mu ER y’uturemangingo twa sec31-1 twimutse nyuma yo gukubitwa kuri 37°C hanyuma bigasohorwa kuri 24°C mu gihe cy’iminota 5 (Ishusho ya 1). Kubera ko Mid2-iRFP ikwirakwizwa ku ruhu rwose rwa ER, na Gas1-GFP ikaba yibumbiye hamwe kandi ikusanyirizwa mu gace ka ER gatandukanye, uko ikwirakwizwa kwabyo gutandukanye rwose (Ishusho ya 1, A kugeza kuri C na Movie S1). Byongeye kandi, nk’uko bigaragara ku Ishusho ya 1D, itsinda rya Gas1-GFP ntirifite Mid2-iRFP. Ibi bisubizo bigaragaza ko poroteyine za GPI-AP na transmembrane zatandukanyijwe mu turere dutandukanye twa ER hakiri kare. Agace ka Gas1-GFP kari iruhande rw'agace runaka ka ERES kanditseho mCherry's COPII coat protein Sec13 (Ishusho ya 1, E na F, na filime S1) (23).
Selile za sec31-1 zigaragaza imisemburo iterwa na galactose, umuyoboro muremure wa acyl (C26) ceramide GPI-AP Gas1-GFP (GPI-AP, icyatsi kibisi) na poroteyine ya transmembrane Mid2-iRFP (TMP, ubururu) kandi iyi nyuguti ya Constructive ERES Sec13-mCherry (ERES, magenta) yashyizwe kuri 37°C mu gihe cy'iminota 30, yimurirwa kuri 24°C, hanyuma ifatwa na SCLIM nyuma y'iminota 5. (A kugeza kuri C) yerekana ishusho ya 2D ihujwe cyangwa imwe y'indege (A), ishusho ya 2D y'ibice 10 by'iz (B) cyangwa ishusho ya 3D selile hemisphere y'imizigo n'ibimenyetso bya ERES (C). Umwanya wa scale 1μm (A na B). Igice cya scale ni 0.551μm (C). Gas1-GFP yabonetse mu turere twa ER cyangwa mu matsinda atandukanye, mu gihe Mid2-iRFP yabonetse kandi ikwirakwizwa mu membrane ya ER (C). (D) Imbonerahamwe igaragaza ubukana bw'urumuri rwa Gas1-GFP na Mid2-iRFP mu itsinda rya Gas1-GFP ku murongo w'umwambi w'umweru (ibumoso). AU, igice cy'ubudasa. (E na F) bihagarariye ishusho ya 3D ihuza ibicuruzwa n'ikimenyetso cya ERES. Amatsinda ya Gas1-GFP yabonetse hafi ya ERES yihariye. Igice cy'ubunini ni 0.551μm. (F) Akambi k'umweru gakomeye kagaragaza itsinda rya Gas1-GFP rifitanye isano na ERES. Uduce two hagati n'iburyo tugaragaza ishusho ya 3D yahujwe hamwe n'ishusho izunguruka y'itsinda rya Gas1-GFP ryatoranijwe.
Isano iri hagati y’itsinda rya Gas1-GFP n’itsinda ryihariye rya ERES igaragaza ko Gas1-GFP ishobora kwinjira muri ERES zitoranijwe, bitandukanye n’uburyo Mid2-iRFP ikoresha kugira ngo ive muri ER. Kugira ngo dukemure ibi bishoboka, twapimye igipimo cya ERES ku bicuruzwa kimwe cyangwa bibiri gusa (Ishusho ya 2, A kugeza kuri C). Twasanze ERES nyinshi (70%) zirimo ubwoko bumwe bw’imizigo. Ishusho yo hasi y’Ishusho ya 2C igaragaza ingero ebyiri zisanzwe za ERES zifite Gas1-GFP gusa (Ishusho ya 1) cyangwa Mid2-iRFP gusa (Ishusho ya 2). Mu buryo bunyuranye, hafi 20% bya ERES zirimo imizigo ibiri ihurirana mu gace kamwe. Byagaragaye ko zimwe muri ERES (10%) zari zirimo ubwoko bubiri bw’imizigo, ariko zari zaratandukanijwe mu duce dutandukanye cyane. Kubwibyo, iri sesengura ry’imibare rigaragaza ko nyuma yo kohereza ER mu mahanga, GPI-AP Gas1-GFP n’imizigo ya transmembrane Mid2-iRFP bigabanyijemo ERES zitandukanye (Ishusho ya 2D). Ubu buryo bwo gutondekanya buhuye cyane n’isesengura rya biochemical ryabanje (6) n’igenwa ry’imiterere (7). Dushobora kandi kureba imyitwarire y’imizigo yashyizwe mu kato yinjira muri ERES (Ishusho ya 2E na Movie S2). Ishusho ya 2E igaragaza ko igice gito cya Gas1-GFP (igice cya 3) cyangwa Mid2-iRFP (igice cya 4) gusa cyinjira muri ERES giturutse ku ruhande rumwe kandi gifungiranye mu gace katandukanye. Igice cya 5 cy’Ishusho ya 2E kigaragaza ko Gas1-GFP na Mid2-iRFP rimwe na rimwe biboneka muri ERES imwe, ariko binjira mu mpande zitandukanye kandi byiganje mu turere dutandukanye dushobora kugaragaza vesicles zitandukanye za COPII. Twemeje kandi ko gutandukanya no gushyira mu byiciro C26 ceramide-based GPI-AP Gas1 nka ERES itoranyije ari ERES yihariye kuko indi mizigo ya transmembrane secretion, poroteyine ya plasma membrane ya GFP yitwa Axl2 (27), igaragaza imyitwarire isa na Mid2-iRFP. (Ishusho ya S1 na Movie S3). Axl2-GFP nshya yakozwe ikwirakwizwa binyuze mu ruhu rw'umuyoboro wa ER nka Mid2-iRFP (Ishusho S1, A na B), kandi ihuzwa na Mid2-iRFP muri ERES nyinshi (Ishusho S1, B kugeza D). Uduce twa 1 na 2 tw'Ishusho ya 1. S1C igaragaza ingero ebyiri zisanzwe za ERES aho imizigo ibiri ya transmembrane ihurira. Muri ibi bihe, ibicuruzwa byombi byinjira hamwe na ERES (Ishusho S1E, Uduce twa 3 na Movie S3).
Uturemangingo twa sec31-1 tugaragaza galactose inducible secretions, Gas1-GFP (GPI-AP, icyatsi kibisi) na Mid2-iRFP (TMP, ubururu) hamwe na constitutive ERES label Sec13-mCherry (ERES, magenta) byashyizwe kuri 37 Nyuma yo guterura iminota 30 kuri °C, jya kuri 24 °C kugira ngo urekure secretion block, hanyuma ushyireho SCLIM nyuma y'iminota 20. (A kugeza kuri C) Amashusho ya 2D projection (A; scale bar, 1μm) cyangwa amashusho ya 3D cell hemisphere (B na C; scale unit, 0.456μm) y'imizigo n'ibice 10 bya z byashyizweho ikimenyetso cya ERES. Igice cyo hasi muri (B) n'igice muri (C) byerekana amashusho yatunganyijwe kugira ngo yerekane gusa ibicuruzwa biri muri ERES (magenta) [Gas1-GFP (imvi) na Mid2-iRFP (ubururu bworoheje)]. (C) Akambi gafunguye: ERES itwara igice kimwe cy'umuzigo (1 kugeza kuri 4). Akambi k'ikijuju: ERES irimo imizigo itandukanye (5). Akambi k'umweru gakomeye: ERES irimo imizigo iherereye hamwe. Hasi: ERES imwe yatoranijwe irimo Gas1-GFP (1) cyangwa Mid2-iRFP (2). Akambi k'ubunini, 100 nm. (D) Ingano ya mikorografi ya foto ivugwa muri (C). Ijanisha mpuzandengo rya ERES ririmo imizigo imwe gusa (Gas1-GFP cyangwa Mid2-iRFP), imizigo itandukanye n'imizigo ihurirana. Mu bushakashatsi butatu bwigenga, n=432 mu turemangingo 54. Akambi k'ikosa = SD. Ikizamini cya t kidahujwe n'imirizo ibiri. *** P = 0.0002. (E) Ishusho ya 3D ya ERES yatoranijwe y'imizigo yashyizwe mu kato yashyizweho ikimenyetso cya (C). Gas1-GFP (icyatsi kibisi) (3) cyangwa Mid2-iRFP (ubururu) (4) yinjira muri ERES (magenta) uhereye ku ruhande rumwe kandi igenzurwa n'agace gato muri ERES. Hari igihe ubwoko bwombi bw'imizigo bwinjira muri ERES imwe (5) buturutse ku ruhande rumwe kandi bugashyirwa ahantu hiherereye muri ERES. Umugozi w'ingufu, 100 nm.
Hanyuma, twagerageje igitekerezo cy'uko umuyoboro muremure wa acyl (C26) uboneka mu ruhu rw'umukara (ER membrane) utuma Gas1 ishyirwa mu byiciro byihariye n'uburyo ishyirwa mu byiciro bya ERES. Muri urwo rwego, twakoresheje ubwoko bw'umusemburo bwahinduwe bwa GhLag1, aho Lag1 na Lac1 byasimbuwe na GhLag1 (uburyo bwa Lag1 bw'ipamba), bigatuma habaho ubwoko bw'umusemburo bufite uruhu rw'uturemangingo rwa Ceramide rugufi kurusha ubwoko bw'umusemburo (Ishusho ya 3A) (28). Isesengura rya Mass spectrometry (MS) ryagaragaje ko mu bwoko bw'umusemburo, 95% bya ceramide yose ni ndende cyane (C26) chain ceramide, mu gihe muri GhLag1, 85% bya ceramide ni ndende cyane (C18 na C16). ), 2% bya ceramide ni ndende cyane (C26) chain ceramide. Nubwo C18 na C16 ceramide ari zo ceramide z’ingenzi zabonetse mu membrane ya GhLag1 kugeza ubu, isesengura rya MS ryemeje kandi ko icyuma cya GPI cya Gas1-GFP kigaragara mu bwoko bwa GhLag1 kirimo C26 ceramide, igereranywa na lipide zo mu bwoko bwa wild-type. Ubwiza ni bumwe (Ishusho ya 3A) (26). Kubwibyo, ibi bivuze ko enzyme ya ceramide Cwh43 ihitamo cyane C26 ceramide, nk'uko bigaragara ku Ishusho ya 26, ikoresha cyane icyuma cya GPI giturutse ku gipimo gito cya C26 ceramide mu bwoko bwa GhLag1. S2 (29). Nyamara, membrane y'uturemangingo ya GhLag1 muri rusange irimo C18-C16 ceramide gusa, mu gihe Gas1-GFP igifite C26 ceramide. Ibi bituma iyi membrane iba igikoresho cyiza cyo gukemura by'umwihariko ikibazo cy'uburebure bw'uruhererekane rwa acyl rwa membrane ceramide muri ER. Uruhare rw'icyitegererezo rw'ubwoko n'uburyo bwo gutondeka. Hanyuma, twabanje kwiga ubushobozi bwa C26 Gas1-GFP bwo kwikusanya mu matsinda muri GhLag1 hamwe na allele ya mutant ihindagurika ya sec31-1 binyuze muri mikroskopi isanzwe ya fluorescence, aho urunigi rurerure (C18-C16) ruboneka gusa muri ER membrane Ceramide (Ishusho ya 3). Twabonye ko muri sec31-1, Gas1-GFP nyinshi zari zigizwe n'amatsinda, mu gihe Gas1-GFP muri sec31-1 GhLag1 hamwe na ER membrane ndende (C18-C16) ntabwo ahanini zari zigizwe n'amatsinda kandi zigakwirakwizwa muri ER membrane yose. Mu by'ukuri, kubera ko C26 ceramide-based clustering ifitanye isano rya hafi na ERES yihariye (Ishusho ya 1), twakurikiranyeho gusuzuma niba iyi nzira ishobora no kuba ikubiyemo imikorere ya ER export protein mechanism. GPI-AP ikoresha sisitemu yihariye ya COPII yo kohereza ER, igenzurwa cyane na Ted1's structure renovation y'igice cya glycan cy'umuyoboro wa GPI (30, 31). Hanyuma, GPI-glycan ivuguruye imenyekanishwa na transmembrane cargo receptor p24 complex, nayo igahitamo Lst1, ikaba ari isoform yihariye y’igice kinini cya COPII gihuza imizigo Sec24, igakora GPI-AP-rich COPII Vesicles ni ngombwa (31-33). Kubwibyo, twakoze mutant ebyiri zahuje gusiba izi poroteyine imwe (igice cya p24 complex Emp24, enzyme Ted1 yo guhindura GPI-glycan n’igice cyihariye cya COPII Lst1) hamwe n’ubwoko bwa sec31-1 mutant, maze tubyiga. Ese birashoboka gukora Gas1-cluster GFP (Ishusho ya 3). Twabonye ko muri sec31-1emp24Δ na sec31-1ted1Δ, Gas1-GFP ahanini idacucitse kandi ikwirakwizwa mu membrane ya ER, nk'uko byagaragaye mbere muri sec31-1 GhLag1, mu gihe muri sec31-1lst1Δ, Gas1-GFP Kimwe na sec31-1. Ibi bisubizo bigaragaza ko uretse kuba hari C26 ceramide mu ruhu rw'umukara, guhuza Gas1-GFP nabyo bigomba gufatana na p24 complex, kandi ntibisaba Lst1 yihariye. Hanyuma, twasuzumye uburyo uburebure bwa ceramide mu ruhu rw'umukara bushobora kugenzura uburyo Gas1-GFP ifatana na p24. Ariko, twasanze kuba hari C18-C16 ceramide mu ruhu rw'umukara bitagira ingaruka kuri GPI-glycans zongeye kubakwa na p24 complex (Ishusho S3 na S4, A na B) cyangwa gufatana na GPI-AP no kohereza GPI-AP. Recruit COPII subtype Lst1 (Ishusho S4C). Kubwibyo, guhuza C26 ceramide-dependent clustering ntibisaba imikoranire ya poroteyine n'uburyo butandukanye bwa ER poroteyine zoherezwa mu mahanga, ahubwo bishyigikira ubundi buryo bwo gutondeka buyobowe n'uburebure bwa lipide. Hanyuma, twasesenguye niba uburebure bwa ceramide acyl mu ruhu rw'umukara rw'umukara ari ingenzi kugira ngo Gas1-GFP ishyirwe mu byiciro byiza nka ERES zitoranijwe. Kubera ko Gas1 mu bwoko bwa GhLag1 hamwe na ceramide ngufi iva muri ER ikinjira muri plasma membrane (Ishusho S5), twizera ko niba uburyo bwo gutondekanya buyobowe n'uburebure bw'umuyoboro wa ceramide acyl, Gas1 mu bwoko bwa GhLag1 ishobora koherezwa no kwambutswa. Ibicuruzwa bya ERES bifite membrane imwe.
(A) Uduce tw’uturemangingo twa GhLag1 ahanini tuba dufite ceramide ngufi za C18-C16, mu gihe icyuma gikingira GPI cya Gas1-GFP kigifite C26 IPC imwe n’uturemangingo two mu bwoko bwa wild-type. Hejuru: isesengura ry’uburebure bwa acyl chain ya ceramide mu membrane y’uturemangingo two mu bwoko bwa wild-type (Wt) na GhLag1p hifashishijwe spectrometry ya mass (MS). Amakuru agaragaza ijanisha rya ceramide yose. Impuzandengo y’igerageza ritatu ryigenga. Umurongo w’ikosa = SD. Ikizamini cya t gifite imirizo ibiri idahujwe. **** P ＜0.0001. Igice cyo hasi: Isesengura rya MS ry’uburebure bwa acyl chain ya IPC buri muri Gas1-GFP (GPI-IPC) GPI agaragara mu bwoko bwa wild-type na GhLag1p. Amakuru agaragaza ijanisha ry’ikimenyetso cyose cya IPC. Impuzandengo y’igerageza ritanu ryigenga. Umurongo w’ikosa = SD. Ikizamini cya t gifite imirizo ibiri idahujwe. ns, ntabwo ari ngombwa. P = 0.9134. (B) Mikorogarafi z'urumuri rw'izuba za sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ na sec31-1lst1Δ zigaragaza Gas1-GFP iterwa na galactose zashyizwe mu bushyuhe bwa 37°C mu gihe cy'iminota 30 hanyuma zishyirwa mu bikorwa mikorogarafi y'urumuri rw'izuba nyuma ya 24°C. Umwambi w'umweru: ER Gas1-GFP cluster. Umwambi ufunguye: Gas1-GFP idakora ikwirakwizwa ku ruhu rw'izuba rwose rwa ER, igaragaza ibara ry'impeta ya kirimbuzi ya ER. Umwambi w'ingero, 5μm. (C) Ingano ya mikorogarafi ya foto ivugwa muri (B). Ijanisha ry'impuzandengo y'uturemangingo dufite imiterere ya Gas1-GFP. Mu bushakashatsi butatu bwigenga, uturemangingo twa n≥300. Umwambi w'ikosa = SD. Ikizamini cya t gifite imirizo ibiri idakora. **** P ＜0.0001.
Kugira ngo dukemure iki kibazo mu buryo butaziguye, twakoze isuzuma rya SCLIM rya Gas1-GFP na Mid2-iRFP muri GhLag1 hamwe na allele ya sec31-1 itagaragaza ubushyuhe (Ishusho ya 4 na Movie S4). Nyuma y'uko ER iguma kuri 37°C hanyuma ikarekurwa kuri 24°C, Gas1-GFP nyinshi nshya zakozwe ntizashyizwe hamwe ngo zikwirakwizwe mu membrane ya ER, nk'uko byagaragaye kuri mikorosikopi zisanzwe (Ishusho ya 4, A na B). Byongeye kandi, igipimo kinini cya ERES (67%) kirimo ubwoko bubiri bw'imizigo ihuriweho (Ishusho ya 4D). Uduce twa 1 na 2 tw'Ishusho ya 4C tugaragaza ingero ebyiri zisanzwe za ERES hamwe na Gas1-GFP na Mid2-GFP bihurirana. Byongeye kandi, ibicuruzwa byombi byakusanyijwe muri ERES imwe (Ishusho ya 4E, agace ka 3 na movie S4). Kubwibyo, ibisubizo byacu bigaragaza ko uburebure bw'urunigi rwa ceramide acyl mu membrane ya ER ari ikintu cy'ingenzi kigena iteranywa rya poroteyine ya ER n'ishyirwa mu byiciro.
Uturemangingo twa Sec31-1 GhLag1 tugaragaza imisemburo iterwa na galactose, Gas1-GFP (GPI-AP, icyatsi kibisi) na Mid2-iRFP (TMP, ubururu) hamwe na Sec13-mCherry (ERES, magenta) ifite ikirango cya ERES (ERES, magenta) irabika kuri 37°C. Komeza iminota 30, umanuke kuri 24°C kugira ngo urekure imisemburo, hanyuma ushushanye na SCLIM nyuma y'iminota 20. (A kugeza kuri C) Amashusho yerekana 2D projection (A; scale bar, 1μm) cyangwa amashusho ya 3D cell hemisphere (B na C; scale unit, 0.45μm) y'ibice 10 bya z byagaragajwe na cargo na ERES. Igice cyo hasi muri (B) n'igice muri (C) bigaragaza amashusho yatunganyijwe kugira ngo yerekane gusa ibicuruzwa biri muri ERES (magenta) [Gas1-GFP (ikijuju) na Mid2-iRFP (ubururu bworoheje)]. (C) Akambi k'umweru kuzuye: ERES, ibicuruzwa birahurirana. Akambi gafunguye: ERES irimo ikintu kimwe gusa. Igice cyo hasi: ERES yatoranijwe ifite ibicuruzwa bihurirana (1 na 2) byanditseho (C). Umwanya w'ibipimo, 100 nm. (D) Ingano ya mikorografi ya foto ivugwa muri (C). Mu bice bya sec31-1 na sec31-1 GhLag1, harimo imizigo imwe gusa (Gas1-GFP cyangwa Mid2-iRFP), hamwe n'impuzandengo ya ERES ku mizigo yihariye n'imizigo ihurirana. Mu bushakashatsi butatu bwigenga, n = 432 mu turemangingo 54 (sec31-1) na n = 430 mu turemangingo 47 (sec31-1 GhLag1). Umwanya w'ikosa = SD. Ikizamini cya t kidahujwe n'imirizo ibiri. *** P = 0.0002 (sec31-1) na ** P = 0.0031 (sec31-1 GhLag1). (E) Ishusho ya 3D ya ERES yatoranijwe ifite imizigo ihurirana (3) yanditseho (C). Gas1-GFP (icyatsi kibisi) na Mid2-iRFP (ubururu) byegera ERES (magenta) ku ruhande rumwe kandi bikaguma mu gace kamwe ka ERES. Umurongo wa scale, 100 nm.
Ubu bushakashatsi butanga ibimenyetso bitaziguye byerekana ko poroteyine zishingiye kuri lipide zishyirwa mu bice by’ingenzi byoherezwa mu mahanga mu nzira y’ubuso, kandi bugaragaza akamaro k’uburebure bw’umunyururu wa acyl mu guhitamo ibice. Dukoresheje uburyo bukomeye kandi bugezweho bwa microscopy bwitwa SCLIM, twerekanye Gas1-GFP nshya yakozwe (urusobe rw’amaraso rwa GPI-AP rufite igice kirekire cyane cya acyl chain (C26) ceramide lipide) mu musemburo) Uduce twishyize hamwe muri ER zitandukanye dufitanye isano na ERES yihariye, mu gihe poroteyine zikomoka kuri transmembrane zikwirakwizwa mu rusobe rw’amaraso rwa ER (Ishusho ya 1). Byongeye kandi, ubwo bwoko bubiri bw’ibintu bwinjira muri ERES zitandukanye mu buryo butandukanye (Ishusho ya 2). Uburebure bw’umunyururu wa acyl wa ceramide y’ingirabuzimafatizo mu rusobe rw’amaraso bugabanuka kuva kuri C26 kugera kuri C18-C16, itsinda rya Gas1-GFP rigahungabana mu gace ka ER gatandukanye, maze Gas1-GFP igahindurirwa inzira kugira ngo isige ER hamwe na poroteyine ya transmembrane binyuze muri ERES imwe (Ishusho ya 3 n’Ishusho ya 3). 4).
Nubwo GPI-AP ikoresha uburyo bwihariye bwa poroteyine kugira ngo isohoke muri ER, twasanze gutandukana kw’ibikomoka kuri ceramide kuri C26 bidashingiye ku mikoranire itandukanye ya poroteyine bishobora gutuma habaho kwihitiramo ERES (Ishusho S4 na S5). Ahubwo, ibyo twabonye bishyigikira ubundi buryo bwo gushyira mu byiciro buterwa no gukusanya poroteyine zishingiye kuri lipide no gukuraho indi mizigo. Ibyo twabonye bigaragaza ko agace cyangwa itsinda rya Gas1-GFP rifitanye isano na ERES runaka ridafite poroteyine ikomoka kuri membrane Mid2-iRFP, bigaragaza ko itsinda rya GPI-AP rishingiye kuri ceramide kuri C26 rizaborohereza kwinjira muri ERES ijyanye nayo, kandi icyarimwe, rikuraho transmembrane. Ibice bivamo byinjira muri iyi ERES yihariye (Ishusho ya 1 na 2). Mu buryo bunyuranye, kuba hari ceramide za C18-C16 mu membrane ya ER ntibituma GPI-AP ikora uturere cyangwa amatsinda, bityo ntibikuraho cyangwa ngo bisimbuze poroteyine zikomoka kuri transmembrane muri ERES imwe (Ishusho ya 3 na 4). Kubwibyo, turasaba ko C26 ceramide ituma habaho gutandukanya no gushyira mu byiciro binyuze mu koroshya ikusanyirizo rya poroteyine zifitanye isano na ERES runaka.
Ni gute wagera kuri iyi cluster ishingiye kuri C26 ceramide mu gace runaka ka ER? Uburyo membrane ceramide yitandukanya ku ruhande bushobora gutuma GPI-AP na C26 ceramide bikora lipide nto kandi zigahita zishyirwaho mu buryo butunguranye mu gace ka lipide ka ER karimo glycerolipide ngufi kandi zitarimo. Cluster nziza (17, 18). Izi cluster nto z'igihe gito zishobora kongera guhuzwa mu macluster manini kandi ahamye nyuma yo gufatana na p24 complex (34). Dukurikije ibi, twagaragaje ko C26 Gas1-GFP igomba gukorana na p24 complex kugira ngo ikore cluster nini zigaragara (Ishusho ya 3). P24 complex ni heterozygous oligomer igizwe na poroteyine enye zitandukanye za p24 transmembrane mu musemburo (35), itanga uburyo bwinshi bwo gufatana, bishobora gutuma cluster nto za GPI-AP zihurirana, bityo bigatuma cluster nini ikomera (34). Imikoranire hagati ya ectodomains za poroteyine za GPI-APs ishobora no kugira uruhare mu kwishyira hamwe kwazo, nk'uko byagaragajwe mu gihe cyo gutwara Golgi mu turemangingo tw’inyamabere twitwa polarized epithelial cells (36). Ariko, iyo C18-C16 ceramide iri mu membrane ya ER, iyo p24 complex ifatanye na Gas1-GFP, amatsinda manini atandukanye ntabwo azaba avutse. Uburyo bw'ibanze bushobora guterwa n'imiterere yihariye y'umubiri n'iya shimi ya ceramide ndende ya acyl chain. Inyigo za biophysical ku membrane z'ubukorano zigaragaza ko nubwo ceramide ndende (C24) n'igufi (C18-C16) acyl chain zishobora gutera gutandukana kw'ibyiciro, ceramide ndende ya acyl chain (C24) gusa ni zo zishobora gutuma curvature nini na filime bihindagurika kugira ngo bihindure filime. Binyuze mu kugereranya (17, 37, 38). Byagaragaye ko helix ya transmembrane ya TMED2, ihuza abantu na Emp24, ikorana neza na sphingomyelin ishingiye kuri C18 ceramide muri lobules za cytoplasmic (39). Dukoresheje simulation za molekile (MD), twasanze ceramide za C18 na C26 zirundanye hafi ya lobules za cytoplasmic za Emp24 transmembrane helix, kandi zifite ibyo zikunda bisa (Ishusho S6). Ni ngombwa kumenya ko ibi bigaragaza ko helix ya transmembrane ya Emp24 ishobora gutuma lipide zikwirakwira mu membrane zitangana. Iki ni igisubizo giherutse gutangwa gishingiye ku tunyangingo tw’inyamabere. Simulation za MD nk’izo zigaragaza kandi ko hari lipide za ether (40). Kubwibyo, dutekereza ko ceramide ya C26 muri lobules ebyiri za ER26 ikungahaye mu gace runaka. Iyo GPI-AP muri lobules za luminal ifatanye neza na p24 nyinshi kandi C26 ceramide ikagira ubwinshi hafi ya p24 muri lobules za cytoplasmic, ishobora gutuma poroteyine iterana kandi membrane curvature ikorwa binyuze mu ntoki (41), bigatuma GPI-AP yitandukanya mu duce dutandukanye twegeranye na ERES, ibyo bikaba binafasha uturere twinshi twa ER membrane (42). Raporo zabanjirije iyi zashyigikiye uburyo bwatanzwe (43, 44). Guhuza oligolectine, pathogens cyangwa antibody kuri ceramide-based glycosphingolipids (GSL) kuri plasma membrane bitera GSL nini, byongera itandukana ry’ibyiciro kandi bigatera guhinduka kw’membrane no kwinjizwa mu mubiri (44). Iwabuchi n'ibindi (43) Byagaragaye ko iyo hari imiyoboro miremire ya acyl (C24) ariko itari migufi (C16), ligand nyinshi ihujwe na GSL lactosylceramide yatumye habaho amatsinda manini n'ingufu, kandi uburyo bwa cytoplasm Lyn-mediated signal transduction ku dupapuro tw'amakuru buvangwa n'imiyoboro ya acyl muri neutrophils zifatanye.
Mu tunyangingo tw’inyamabere twitwa polarized epithelial cells, ubwinshi bw’urusobe rw’amaraso rurwanya Golgi (TGN) kugeza ku rwego rwa apical plasma membrane bugenzura itandukana n’uburyo GPI-AP ikorwa (10, 45). Uku kwishyira hamwe guterwa na GPI-AP oligomerization (36), ariko bishobora kandi guterwa n’uburebure bw’urunigi rwa ceramide dusanga mu musemburo. Nubwo GPI-AP y’inyamabere ifite ether lipid-based anchor, kandi imiterere yayo ya shimi itandukanye cyane n’iya acyl chain ceramide ndende cyane, ubushakashatsi buherutse gukorwa bwagaragaje ko lipide zombi zifite imiterere n’imikorere isa mu buryo bw’umubiri n’ubwa shimi (40). Kubwibyo, igice cya ether lipide mu tunyangingo tw’inyamabere gishobora kuba gisa na C26 ceramide mu musemburo, kandi uruhare rwacyo ni uguhuza na ceramide y’urunigi rurerure mu musemburo kugira ngo habeho kwishyira hamwe no gutondeka kwa GPI-AP. Nubwo ubu buryo bugikeneye kugeragezwa mu buryo butaziguye, ibyavumbuwe mbere byemeza ko gutwara ceramide ndende ya acyl chain ceramide mu mubiri wa Golgi bidakorwa na poroteyine zo kohereza cytoplasmic, ahubwo biterwa no gukorana kwa GPI anchors nk'umusemburo. Kubwibyo, uburyo bwo gukosora ibintu bugaragara nk'aho bushobora gutwara ceramide ndende cyane ya acyl chain na GPI-AP (13, 16, 20, 46, 47) mu gice kimwe cyo gutwara.
Mu mikorere y’uturemangingo tw’imisemburo n’uturemangingo tw’inyamabere, kwishyira hamwe no gutandukana kwa GPI-AP n’izindi poroteyine z’uturemangingo tw’inyamabere byose bibaho mbere yuko bigera ku buso bw’uturemangingo. Paladino n’abandi (48) basanze kuri TGN y’uturemangingo tw’inyamabere tw’inyamabere, kwishyira hamwe kwa GPI-AP ntabwo ari ngombwa gusa kugira ngo hashyirwe mu byiciro bya GPI-APs ku buso bw’uturemangingo tw’inyamabere, ahubwo binagenzura imiterere y’uturemangingo twa GPI-AP n’imikorere yatwo. Ubuso bw’uturemangingo. Mu imisemburo, ubu bushakashatsi bwagaragaje ko itsinda rya GPI-AP rishingiye kuri C26 ceramide kuri ER rishobora kugenzura imiterere y’uturemangingo n’imikorere ya GPI-AP ku buso bw’uturemangingo (24, 49). Dukurikije ubu buryo, uturemangingo twa GhLag1 dufite allergie ku miti ibuza GPI cyangwa imiti igira ingaruka ku busugire bw’uturemangingo (28), kandi gukenera uduce tw’inyamabere twa Gas1-GFP (49) twa ceramide igaragara mu guhuza uturemangingo tw’inyamabere bigaragaza G​​Ingaruka zishobora kubaho ku mubiri w’uturemangingo twa hLag1. Ikosa rya GPI-AP. Ariko, gupima niba imikorere y’ubuso bw’uturemangingo yarateguwe uhereye kuri ER hakoreshejwe uburyo bwo gutondeka bushingiye ku burebure bwa lipide bizaba ari byo bizakorwa mu bushakashatsi bwacu bw’ejo hazaza.
Ubwoko bwa Saccharomyces cerevisiae bwakoreshejwe muri iki gikorwa bugaragara muri Table S1. Ubwoko bwa MMY1583 na MMY1635 bwa SCLIM bwo gufata amashusho y’uturemangingo tuzima bwakozwe inyuma ya W303. Ubu bwoko bugaragaza Sec13-mCherry bufite tag ya poroteyine irabagirana bwubatswe hakoreshejwe uburyo bwa polymerase chain reaction (PCR) hamwe na pFA6a plasmid nk'icyitegererezo (23). Ubwoko bugaragaza Mid2-iRFP bwanditseho poroteyine irabagirana munsi y’ubuyobozi bwa GAL1 promoter bwakozwe ku buryo bukurikira. Kuzamura PCR ya iRFP-KanMx sequence kuva kuri pKTiRFP-KAN vector (impano ya E. O'Shea, Addgene plasmid nomero 64687; http://n2t.net/addgene: 64687; identifier y’ubushakashatsi (RRID): Addgene_64687) Kandi yinjijwe muri C-terminus ya Mid2 y’umwimerere. Nyuma y’uko uruhererekane rwa genome rwa Mid2-iRFP rwongerewe kandi rugashyirwa muri GAL1 promoter, rwashyizwe mu kibanza cya Not I-Sac I cya plasmid pRS306 integration. Plasmid pRGS7 yavuyemo yashyizwe mu murongo umwe na Pst I kugira ngo yinjizwe mu gace ka URA3.
Ingirabuzimafatizo ya Gas1-GFP igaragazwa munsi y’ubuyobozi bwa promoteri ya GAL1 muri plasmide ya centromere (CEN), ikaba yakozwe ku buryo bukurikira. Uruhererekane rwa Gas1-GFP rwongerewemo na PCR ivuye muri plasmide ya pRS416-GAS1-GFP (24) (impano ya L. Popolo) maze ishyirwa mu kibanza cya Xma I–Xho I cya CEN plasmid pBEVY-GL LEU2 (impano ya C). Miller; Addgene plasmid nimero 51225; http://n2t.net/addgene: 51225; RRID: Addgene_51225). Plasmide yavuyemo yiswe pRGS6. Ingirabuzimafatizo ya Axl2-GFP nayo igaragazwa munsi y’ubuyobozi bwa promoteri ya GAL1 ya vector ya pBEVY-GL LEU2, kandi imiterere yayo ni iyi ikurikira. Urukurikirane rwa Axl2-GFP rwongereweho kuva kuri plasmid ya pRS304-p2HSE-Axl2-GFP (23) hakoreshejwe PCR, maze rushyirwa mu gace ka Bam HI-Pst I ka vector ya pBEVY-GL LEU2. Plasmid yavuyemo yiswe pRGS12. Urukurikirane rwa oligonucleotides rwakoreshejwe muri ubu bushakashatsi ruri mu mbonerahamwe ya S2.
Ubwoko bw’ibinyabutabire bwongewemo 0.2% adenine na 2% glucose [YP-dextrose (YPD)], 2% raffinose [YP-raffinose] protein ikungahaye kuri yeast extract p (YP) medium (1% Yeast extract na 2% protein ept). (YPR)] cyangwa 2% galactose [YP-galactose (YPG)] nk'isoko ya karuboni, cyangwa mu buryo buciriritse bwa sintetike (0.15% yeast azote base na 0.5% ammonium sulfate) kugira ngo yongere aside amine n'ibice bikenewe mu mirire, kandi irimo 2% glucose (synthetic glucose minimum medium) cyangwa 2% galactose (synthetic galactose minimum medium) nk'isoko ya karuboni.
Kugira ngo hakorwe amashusho mu buryo bwihuse, uturemangingo twa sec31-1 twita ku bushyuhe tugaragaza imiterere yatwo munsi ya promoter ya GAL1 twakuriye mu buryo bw’ubushyuhe bwa YPR kuri 24°C nijoro kugeza hagati mu gihe cy’isaha imwe. Nyuma yo kwinjizwa muri YPG kuri 24°C mu gihe cy’isaha imwe, uturemangingo twashyizwe muri SG kuri 37°C mu gihe cy’iminota 30, hanyuma twimurirwa kuri 24°C kugira ngo tuve mu gice cy’isohoka ry’uturemangingo. Concanavalin A yakoreshejwe mu gufunga uturemangingo ku kirahuri kandi ifatwa ishusho na SCLIM. SCLIM ni uruvange rwa mikorosikopi ya Olympus IX-71 inverted fluorescence na lensi y'amavuta ya UPlanSApo 100 × 1.4 (Olympus), scanner ya disiki ya confocal izunguruka yihuta cyane kandi ifite ikigereranyo cyo hejuru cy'urusaku (Yokogawa Electric), spectrometer yihariye, no gukonjesha yihariye. Ingufu zo kongera ishusho muri sisitemu (Hamamatsu Photonics) zishobora gutanga sisitemu yo kongera ishusho ifite ubwiyongere bwa nyuma bwa ×266.7 na kamera ihuza amashanyarazi ikuba electron (Hamamatsu Photonics) (21). Gufata amashusho bikorwa na porogaramu yihariye (Yokogawa Electric). Ku mashusho ya 3D, twakoresheje icyuma gikoresha piezoelectric cyakozwe mu buryo bwihariye kugira ngo tuzunguze lensi ihagaze, kandi twakusanyije ibice by'urumuri 100 nm mu gice kimwe. Ishusho ya Z-stack ihindurwamo amakuru ya voxel ya 3D, kandi imikorere yo gukwirakwiza ingingo ikoreshwa kuri mikorosikopi ya disiki ya confocal izunguruka ikoreshwa mu gutunganya deconvolution na porogaramu ya Volocity (PerkinElmer). Hakoreshejwe porogaramu ya Volocity kugira ngo ikoreshwe mu buryo bwikora kugira ngo habeho isesengura ry’aho ibintu biri, ERES harimo n’imizigo yarapimwe. Isesengura ry’umurongo ryakozwe hakoreshejwe porogaramu ya MetaMorph (Ibikoresho bya Molecular).
Koresha porogaramu ya GraphPad Prism kugira ngo umenye akamaro k'ibarurishamibare. Ku kizamini cya T cy'Umunyeshuri gifite imirizo ibiri n'ikizamini gisanzwe cy'isesengura ry'uburyo butandukanye (ANOVA), itandukaniro hagati y'amatsinda rifatwa nk'irigira ingaruka zikomeye kuri P <0.05 (*).
Ku bijyanye na mikorosikopi ya fluorescence ya Gas1-GFP, uturemangingo tw’urutonde rw’amabara twakuriwe mu ijoro ryose muri YPD hanyuma tugakusanywa hakoreshejwe centrifugation, twozwa kabiri na phosphate buffered saline, hanyuma tugashyirwa ku rubura byibuze iminota 15, hanyuma tugakomeza munsi ya mikorosikopi nk'uko byasobanuwe mbere Reba (24). Mikorosikopi ya Leica DMi8 (HCX PL APO 1003/1.40 oil PH3 CS) ifite lens ikora, filter ya L5 (GFP), kamera ya Hamamatsu na porogaramu ya Application Suite X (LAS X) byakoreshejwe mu kugura.
Ingero zakuwemo SDS sample buffer kuri 65°C mu gihe cy'iminota 10, hanyuma zitandukanywa na SDS-polyacrylamide gel electrophoresis (PAGE). Mu gusesengura immunoblotting, hashyizwemo 10 μl by'icyitegererezo kuri buri murongo. Antibody y'ibanze: Koresha anti-Gas1 y'urukwavu ku kigero cya 1:3000, anti-Emp24 y'urukwavu ku kigero cya 1:500, na anti-GFP y'urukwavu ku kigero cya 1:3000. Antibody ya monoclonal anti-Pgk1 y'imbeba yakoreshejwe ku kigero cya 1:5000 (impano ya J. de la Cruz). Antibody ya kabiri: Horseradish peroxidase (HRP) conjugated goat anti-rabbit immunoglobulin G (IgG) yakoreshejwe ku kigero cya 1:3000 (Pierce). IgG yo mu bwoko bwa HRP-conjugated goat anti-mouse yakoreshejwe ku gipimo cya 1:3000 (Pierce). Agace k'ubudahangarwa bw'umubiri kagaragaye hakoreshejwe uburyo bwa chemiluminescence bwa SuperSignal West Pico reagent (Thermo Fisher Scientific).
Nkuko byasobanuwe muri (31), igerageza ry’ubudahangarwa bw’umubiri ryakozwe ku gice cya ER cyuzuyemo ubushyuhe. Muri make, oza utunyangingo tw’umusemburo ukoresheje TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride na protease inhibitor uruvange) kuri 600 nm (OD600) kuri 100 optical density kabiri. Byamenetse hakoreshejwe amasaro y’ikirahure, hanyuma imyanda y’utunyangingo n’amasaro y’ikirahure bikurwaho hakoreshejwe centrifugation. Supernatant yashyizwe kuri centrifugation kuri 17.000 g mu gihe cy’iminota 15 kuri 4°C. Agace gato kongerewemo muri TNE maze digitalis saponin yongerwaho 1%. Agace kometseho kashyizwe mu cyuma mu gihe cy’isaha imwe gazunguruka kuri 4°C, hanyuma ibice bidashongeshwa bikurwaho hakoreshejwe centrifugation kuri 13.000 g kuri 4°C mu gihe cy’iminota 60. Ku bijyanye no kuvura indwara za Gas1-GFP, banza ushyiremo agace k'ibice bya agarose (ChromoTek) biri ku bushyuhe bwa 4°C mu gihe cy'isaha 1, hanyuma ushyiremo GFP-Trap_A (ChromoTek) kuri 4°C mu gihe cy'amasaha 3. Amasaro yakuwemo ubudahangarwa yogejwe inshuro eshanu na TNE irimo 0.2% digoxigenin, akurwamo agace k'ibice bya SDS, atandukanywa kuri SDS-PAGE, hanyuma asesengurwa hakoreshejwe uburyo bwo kuvura indwara.
Nkuko byasobanuwe muri (31), gusuzuma ihuriro ry’ibice bya ER byashyizwemo ibintu byinshi. Muri make, ihuriro ry’ibice bya ER byashyizwemo 0.5 mM dithiobis (succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, USA; 20°C, iminota 20). Uburyo bwo guhuza ibintu bwarangiye hakongerwamo glycine (50 mM final concentration, iminota 5, 20°C).
Nkuko byasobanuwe mbere (50), hakozwe isesengura rya MS rya ceramide mu bwoko bwa wild-type na GhLag1. Muri make, uturemangingo twakuze kugeza ku rugero rwo hejuru (3 kugeza kuri 4 OD600 units/ml) muri YPD kuri 30°C, hanyuma uturemangingo 25×107 turasarurwa. Ihinduka ryabyo rihagarikwa na aside trichloroacetic. Koresha solvent yo gukuramo [ethanol, amazi, ether, pyridine na 4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] na 1.2 nmol y’ubwiza bwa C17 ceramide (860517, Avanti polar lipid)). Koresha monomethylamine reagent [methanol, amazi, n-butanol na methylamine solution (4:3:1:5 v/v)] kugira ngo ukore hydrolysis yoroheje ya alkaline y’ibikomoka kuri ayo mavuta, hanyuma ukoreshe n-butanol yuzuye mu mazi kugira ngo uvanemo umunyu. Amaherezo, icyo gikoresho cyongeye gushyirwa mu cyuma gipima neza [chloroform/methanol/water (2:7:1) + 5 mM ammonium acetate] hanyuma gishyirwa muri spectrometer y’uburemere. Hakozwe igenzura ry’uburyo bwinshi (MRM) kugira ngo hamenyekane kandi hamenyekane neza molecule za sphingolipide. TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) ifite robot nanoflow ion source Nanomate HD (Advion Biosciences, Ithaca, NY) kugira ngo hasesengurwe lipide. Ingufu zo kugongana zinozwa kuri buri cyiciro cya ceramide. Amakuru ya MS yabonetse mu buryo bwiza. Kuri buri cyuma gipima neza, ikimenyetso cya lipide ni ikigereranyo cy’ibipimo bitatu byigenga.
Nkuko byasobanuwe muri (31), uturemangingo (800×107) twagaragaje Gas1-GFP twashyizwe mu buryo bw’imyororokere karemano. Gas1-GFP yasukuwe yatandukanyijwe na SDS-PAGE maze yimurirwa mu ruhu rw’umukara rwa polyvinylidene (PVDF). Poroteyine yagaragajwe hakoreshejwe irangi rya PVDF n’umukara wa amide. Agace ka Gas1-GFP kaciwe muri PVDF maze kozwa inshuro 5 na methanol, hanyuma rimwe na rimwe hagakoreshwa amazi y’icyitegererezo cya chromatography-MS (LC-MS). Mu gushyira mu ruhu rw’umukara uruvange rwa 500μl 0.3 M NaOAc (pH 4.0), buffer na 500μl uruvange rwa sodium nitrite ya 1 M ku bushyuhe bwa 37°C mu gihe cy’amasaha 3, igice cya lipide gisohoka muri Gas1-GFP hanyuma kigashyirwa mu ruhu. Irekurwa rya inosine phosphate ceramide hagati ya glucosamine na inositol (51). Nyuma y’ibyo, agace k’urukiramende kakarabaga inshuro enye n’amazi ya LC-MS, kakamishwa ku bushyuhe bw’icyumba, hanyuma kabikwa mu kirere cya azote kuri -80°C kugeza igihe gasesenguwe. Nk’igenzura, icyitegererezo cy’urukiramende rwa PVDF cyakoreshejwe kuri buri gerageza. Ibinure byakuwe muri Gas1-GFP byasesenguwe na MS nk’uko byasobanuwe (50). Muri make, uduce twa PVDF turimo GPI-lipide twongeye gushyirwa mu cyuma cya 75μl negative mold solvent [chloroform/methanol (1:2) + 5 mM ammonium acetate] hanyuma hagatangwa electrospray ionization (ESI)-MRM/MS Analysis of sphingolipide species (TSQ Vantage). Muri iki kibazo, amakuru ya MS yabonetse mu buryo bwa negative ion.
Nkuko byavuzwe mbere, igice cya lipide cya GPI anchor cyatandukanijwe na [3H]-inositol-labeled GPI-AP (16). Filime zatandukanijwe hakoreshejwe chromatography y'urwego ruto hakoreshejwe sisitemu ya solvent (55:45:10 chloroform-methanol-0.25% KCl) kandi zigaragazwa hakoreshejwe FLA-7000 (Fujifilm).
Uturemangingo twa Gas1-GFP (600×107) twogejwe kabiri hakoreshejwe TNE buffer hamwe na TNE buffer, hanyuma tuvunjwa n'udusaro tw'ikirahure, hanyuma dushyirwa muri centrifuge kugira ngo dukureho imyanda y'uturemangingo n'udusaro tw'ikirahure. Supernatant yashyizwe muri centrifuge kuri 17.000 g mu gihe cy'isaha imwe kuri 4°C. Agace gato kogejwe muri TNE hanyuma gashyirwamo 1 U PI-PLC (Invitrogen) muri TNE irimo 0.2% digitalis saponin mu gihe cy'isaha imwe kuri 37°C. Nyuma yo kuvurwa na enzyme, membrane yakuweho hakoreshejwe centrifuge kuri 17.000 g kuri 4°C mu gihe cy'isaha imwe. Kugira ngo Gas1-GFP ikomeze kuvura, supernatant yogejwe muri GFP-Trap_A (ChromoTek) ku bushyuhe bwa 4°C mu ijoro ryose. Gas1-GFP isukuye yatandukanijwe na SDS-PAGE yasizwe irangi ry'ubururu bwiza bwa Coomassie. Umugozi wo gusiga irangi rya Gas1-GFP waciwe ku ibara ry'umukara rikikije umuyoboro w'amazi, hanyuma nyuma yo gukoresha iodoacetamide no kugabanya dithiothreitol, hakozwe igogora rya trypsin. Hakozwe isuku ya peptides na peptides za tryptic na GPI-glycans. Peptide yumye yashongeshejwe muri 20 μl y'amazi. Shyiramo igice (8μl) muri LC. Inkingi ya octadecylsilane (ODS) (Develosil 300ODS-HG-5; umurambararo w'imbere 150 mm × 1.0 mm; Nomura Chemical, Aichi Prefecture, mu Buyapani) yakoreshejwe mu gutandukanya peptides mu bihe byihariye. Icyiciro cyo kugenda ni solvent A (0.08% formic acid) na solvent B (0.15% formic acid muri 80% acetonitrile). Sisitemu ya Accela HPLC (Thermo Fisher Scientific, Boston, Massachusetts) yakoreshejwe mu gukuramo inkingi hamwe na solvent A mu minota 55 ku muvuduko wa 50 μl min-1 mu minota 5, hanyuma igipimo cya solvent B cyongerwa kigera kuri 40%., Leta Zunze Ubumwe za Amerika). Eluate yinjijwe mu isoko ya ESI ion buri gihe, maze peptides za tryptic na peptides zifite GPI-glycans zasesengurwa na LTQ Orbitrap XL (hybrid linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific). Muri MS setup, voltage y'isoko ya capillary yashyizwe kuri 4.5 kV, kandi ubushyuhe bwa capillary yo kohereza bwagumishijwe kuri 300°C. Voltage ya capillary na voltage ya tube lens byashyizwe kuri 15 V na 50 V, uko bikurikirana. Amakuru ya MS yabonetse mu buryo bwa positive ion (resolution ya 60.000; faithfulness of small parts 10 per milion) mu rugero rwa 300/m/z mass/charge ratio (m/z) 3000. Amakuru ya MS/MS aboneka binyuze mu mutego wa ion muri LTQ Orbitrap XL [imibare 3 ya mbere amakuru ashingiraho, collision induced dissociation (CID)].
Gupima MD byakozwe hakoreshejwe porogaramu ya GROMACS (52) na MARTINI 2 force field (53-55). Hanyuma, CHARMM GUI Membrane Builder (56, 57) yakoreshejwe mu kubaka bilayer irimo dioleoylphosphatidylcholine (DOPC) na Cer C18 cyangwa DOPC na Cer C26. Topology na coordinates bya Cer C26 bikomoka kuri DXCE binyuze mu gukuraho amasaro y'inyongera ku murizo wa sphingosine. Koresha inzira ivugwa hano hepfo kugira ngo uhuze urwego rw'ibice bibiri hanyuma ubyikoreshe, hanyuma ukoreshe coordinates za nyuma za sisitemu kugira ngo wubake sisitemu irimo Emp24. Transmembrane domain ya yeast Emp24 (ibisigazwa 173 kugeza 193) yubatswe nk'ikimenyetso cya α-helix hakoreshejwe imiterere ya molecular ya MD (VMD) (58). Hanyuma, nyuma yo gukuraho lipide zifatanye, poroteyine yashyizwemo ibice byinshi hanyuma ishyirwa muri bilayer hakoreshejwe CHARMM GUI. Sisitemu ya nyuma irimo 1202 DOPC na 302 Cer C26 cyangwa 1197 DOPC na 295 Cer C18 na Emp24. Shyira sisitemu kuri 0.150M. Hakozwe kopi enye zigenga ku buryo bubiri bw'ibice bibiri.
Urusobe rw'ibinure rushyirwa kuringaniza hakoreshejwe uburyo bwa CHARMM GUI, bukubiyemo kugabanya no kuringaniza intambwe 405.000, aho inzitizi z'aho ibintu biherereye zigabanuka buhoro buhoro kandi zikavanwaho, kandi intambwe y'igihe ikongerwa kuva kuri 0.005 ps kugera kuri 0.02 ps. Nyuma yo kuringaniza, itanga µs 6 hamwe n'intambwe y'igihe ya 0.02 ps. Nyuma yo gushyiramo Emp24, koresha uburyo bumwe bwa CHARMM GUI kugira ngo ugabanye kandi utunganye sisitemu, hanyuma ukore iminota 8 mu gukora.
Ku buryo bwose, mu gihe cyo kuringaniza, umuvuduko ugenzurwa na barostat ya Berendsen (59), naho mu gihe cyo gukora, umuvuduko ugenzurwa na barostat ya Parrinello-Rahman (60). Muri rusange, umuvuduko mpuzandengo ni bar 1 kandi hakoreshwa gahunda yo guhuza umuvuduko wa semi-isotropic. Mu buryo bwo kuringaniza no gukora, thermostat (61) ifite umuvuduko wo gusubiramo ikoreshwa kugira ngo ihuze ubushyuhe bwa poroteyine, lipide n'uduce twa solvent. Mu gihe cyose cy'imikorere, ubushyuhe bw'intego ni 310K. Ihuriro ritahuza ribarwa hakoreshejwe urutonde rw'imibanire hakoreshejwe gahunda ya Verlet ifite ubushobozi bwo kwihanganira 0.005. Ijambo Coulomb ribarwa hakoreshejwe urubuga rw'ibizamini n'intera yo gukata ya 1.1 nm. Ijambo Vander Waals rikoresha gahunda yo gukata ifite intera yo gukata ya 1.1 nm, naho gahunda yo gukata ya Verlet ikoreshwa mu guhindagurika kw'ibipimo (62).
Ukoresheje VMD, uburebure bw'uburebure hagati y'udupira twa DOPC phosphate cyangwa utupira twa ceramide AM1 na poroteyine ni 0.7 nm, kandi umubare wa lipide zikorana na poroteyine urabarirwa. Dukurikije formula ikurikira, bara factor depletion-enrichment (DE) nkuko biri muri (63): factor ya DE = (ingano ya lipide zose muri poroteyine 0.7) muri poroteyine 0.7 (ingano ya Cer muri lipide zose)
Agaciro kavuzwe kaboneka nk'impuzandengo, kandi utubari tw'amakosa ni kopi enye zigenga za SE. Akamaro k'imibare ka factor ya DE kabarwa hakoreshejwe ikizamini cya t [(impuzandengo ya DE-factor-1) / SE]. Kubara agaciro ka P uhereye ku igabanywa ry'umurizo umwe.
Igikoresho cya GROMACS cyakoreshejwe mu kubara ikarita y’ubucucike bwa 2D ku ruhande rw’ubuso bw’isi irimo Emp24 mu ntera ya 250 ns ya nyuma y’aho iherereye. Kugira ngo haboneke ikarita yo kongera/kugabanya ubucucike bwa ceramide, ikarita y’ubucucike bwa Cer igabanywa n’igiteranyo cy’ikarita ya Cer na DOPC, hanyuma igagabanywa n’ubucucike bwa Cer mu mubiri. Ikarita imwe y’amabara irakoreshwa.
Ku bindi bikoresho by'inyongera kuri iyi nkuru, reba http://advances.sciencemag.org/cgi/content/full/6/50/eaba8237/DC1
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Sofia Rodriguez-Gallardo, Kazuo Kurokawa, Susana Sabido-Bozo, Alejandro Cortez · Gomez (Alejandro Cortes-Gomez), Atsuko Ikeda (Atsuko Ikeda), Valeria Zoni (Valeria Zoni), Auxiliadora Aguilera-Romerez, Anailera-Romero, Ana Lopez), Miho Waga (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Ishusho ya 3D ifite ubushobozi bwo gupima mu gihe nyacyo igaragaza akamaro k'uburebure bw'urunigi rwa ceramide mu gutondekanya poroteyine ahantu hatandukanye hasohoka umusaruro.
Sofia Rodriguez-Gallardo, Kazuo Kurokawa, Susana Sabido-Bozo, Alejandro Cortez · Gomez (Alejandro Cortes-Gomez), Atsuko Ikeda (Atsuko Ikeda), Valeria Zoni (Valeria Zoni), Auxiliadora Aguilera-Romerez, Anailera-Romero, Ana Lopez), Miho Waga (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Ishusho ya 3D ifite ubushobozi bwo gupima mu gihe nyacyo igaragaza akamaro k'uburebure bw'urunigi rwa ceramide mu gutondekanya poroteyine ahantu hatandukanye hasohoka umusaruro.
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