Kuronga mapuroteni munzira yekubuda kwemasero kwakakosha pakuchengetedza kupatsanurwa kwemasero uye homeostasis. Pamusoro pekuronga-kugadziriswa kweganda, basa remafuta mukugadzirisa kinesin mukufamba kwemasero inyaya yekare isati yapindurwa. Pano, tinoita 3D panguva imwe chete multicolor high-resolution real-time imaging kuratidza in vivo kuti mapuroteni matsva eglycosylphosphatidylinositol-immobilized ane ceramide lipid moieties refu kwazvo anounganidzwa uye anorongwa muzvikamu zve endoplasms. Net exit site, iyo yakasiyana neiyo inoshandiswa nemapuroteni e transmembrane. Pamusoro pezvo, tinoratidza kuti urefu hwecheni yeceramide mu endoplasmic reticulum membrane hwakakosha pakusarudzwa uku kwekusarudzwa. Chidzidzo chedu chinopa humbowo hwekutanga hwakananga in vivo hwekupatsanura mapuroteni anotakura zvinhu zvichibva pakureba kwecheni yemafuta munzvimbo dzakasarudzwa dzekuburitsa kunze munzira yekubuda kwemasero.
Mumasero eukaryotic, mapuroteni anogadzirwa mu endoplasmic reticulum (ER) anobva arongwa panguva yekufambisa kuburikidza nenzira yekusvinura kuti aendeswe kunzvimbo yawo yakakodzera yemasero (1). Kuwedzera pakurongedza kwakabatana ne coat, zvaifungidzirwa kwenguva refu kuti mamwe mafuta anogonawo kushanda senzvimbo dzekusarudza dzekubuda nekubatanidza munzvimbo dzakatarwa dze membrane kune mapuroteni chaiwo (2-5). Zvisinei, kuchine kushaikwa kwehumbowo hwakananga mu vivo hunoratidza nzira iyi inogona kushandiswa ne lipid-based. Kuti tigadzirise dambudziko iri, takadzidza mune yeast kuti glycosylphosphatidylinositol (GPI) anchored proteins (GPI-APs) dzinotumirwa zvakasiyana kubva kuER. GPI-APs imhando dzakasiyana dzemapuroteni epamusoro emasero akabatana ne lipid (6, 7). GPI-AP ipuroteni inosvinwa yakabatana nemapepa ekunze e plasma membrane kuburikidza ne glycolipid moiety (GPI anchor). Vanogamuchira GPI anchors se conservative post-translational modifications mu ER lumen (8). Mushure mekunamirwa, GPI-AP inopfuura nemuGolgi apparatus (5, 9) kubva kuER kuenda kuplasma membrane. Kuvapo kwezvitsigiro zveGPI kunoita kuti GPI-AP itakurwe zvakasiyana kubva kumapuroteni anoburitswa netransmembrane (kusanganisira mamwe mapuroteni eplasma membrane) munzira yekusvinura (5, 9, 10). Mumasero embiriso, maGPI-AP anoparadzaniswa nemamwe mapuroteni anoburitswa mu endoplasmic reticulum, uye obva aiswa muma vesicles akasiyana akaputirwa ne coat protein complex II (COPII) (6, 7). Zvinokonzera maitiro aya ekupatsanura mu ER export process hazvina kujeka, asi zvinofungidzirwa kuti nzira iyi ingangoda lipids, kunyanya kugadziriswa kwechimiro che lipid chikamu che GPI anchor (5, 8). Mu mbiriso, kugadziriswa kwemafuta eGPI kunotanga pakarepo mushure mekunge GPI yabatana, uye muzviitiko zvakawanda, kunoita kuti ceramide isunge kune 26-carbon long-chain saturated fatty acid (C26:0) (11, 12). C26 ceramide ndiyo ceramide huru inogadzirwa nemasero embiriso kusvika pari zvino. Inogadzirwa muER uye yakawanda yacho inotumirwa kuGolgi apparatus kuburikidza neCOPII vesicles (13). Kutumirwa kweGPI-AP kuER kunoda kuti ceramide irambe ichigadzirwa (14, 15), uye kushandurwa kweceramide kuita inositol phosphate ceramide (IPC) muGolgi apparatus kunoenderana nekugadzirwa kweGPI anchor (16). Zvidzidzo zvebiophysical zvine artificial membranes zvakaratidza kuti acyl chain ceramide dzakareba kwazvo dzinogona kubatana kuti dzigadzire nzvimbo dzakarongwa dzine hunhu hwakasiyana hwepanyama (17, 18). Ruzivo urwu rwunotungamira kufungidziro yekuti C26 ceramide neGPI-AP neC26 ceramide dzinoshandisa hunhu hwadzo hwepanyama kuti dzibatane munzvimbo dzakarongeka kana nzvimbo dziri munzvimbo ine ER membrane lipids dzakaipa. Inonyanya kuumbwa ne glycerolipids pfupi uye dzisina kuguta (C16:1 naC18:1) (19, 20). Nzvimbo idzi dzichanyanya kutariswa panzvimbo dzekubuda dzeER (ERES), uko ceramide ne ceramide-based GPI-AP zvinogona kutakurwa pamwe chete kuenda kuGolgi muchikamu chimwe chete cheCOPII vesicle (5).
Muchidzidzo ichi, takaedza zvakananga nzira iyi ye lipid-based nekushandisa super-resolution confocal real-time imaging microscopy (SCLIM), inova nzira yemazuva ano ye microscopy inogona kuona mapuroteni ane fluorescent panguva imwe chete. Mifananidzo yemavara matatu nemativi matatu (3D) ine resolution yakakwira uye kumhanya kwakanyanya mumasero mapenyu (21, 22).
Takatanga tashandisa tekinoroji yeSCLIM kuti titsanangure kuti GPI-AP yakajairika ine boka reC26 ceramide yaiongororwa sei kubva kumapuroteni anoburitswa netransmembrane mushure mekubuda muER muS. cerevisiae. Kuti tiongorore kupatsanurwa kweER, takashandisa genetic system inogona kuona zvakananga zvinhu zvitsva zvakagadzirwa zvichipinda muERES in vivo (7, 23). Sezvinhu zvinotakura, takasarudza C26 ceramide-based GPI-AP Gas1 yakanyorwa negreen fluorescent protein (GFP) uye transmembrane secreted protein Mid2 yakanyorwa ne near-infrared fluorescent protein (iRFP), zvese zviri zviviri zvinonanga plasma membrane (24–26). Mu sec31-1 temperature-sensitive mutant, zvinhu zviviri izvi zvinoratidzwa pasi pe galactose-inducible promoter uye constitutive ERES marker. Pakupisa kwakanyanya (37°C), nekuti sec31-1 mutation inokanganisa mashandiro eCOPII coat component Sec31 kudzivirira COPII kumera uye ER export, zvinhu zvitsva zvakagadzirwa zvinoungana paER (23). Mushure mekutonhorera kusvika patembiricha yakaderera (24°C), maseru e sec31-1 mutant akadzoka kubva munzvimbo inosvinira, uye zvinhu zvitsva zvakagadzirwa zvakaunganidzwa zvakatanga kutumirwa kunze kubva kuER. Kuona kweCLIM kwakaratidza kuti mazhinji emapuroteni matsva eGas1-GFP neMid2-iRFP achiri kuunganidzwa muER yemasero e sec31-1 mutant mushure mekuiswa mu 37°C uye akazoburitswa pa24°C kwemaminitsi mashanu (Mufananidzo 1). Sezvo Mid2-iRFP ichigoverwa pa membrane yese yeER, uye Gas1-GFP yakabatana uye yakaunganidzwa munzvimbo ye membrane yeER isina kuenderera mberi, kupararira kwavo kwakasiyana zvachose (Mufananidzo 1, A kusvika C uye Movie S1). Pamusoro pezvo, sezvakaratidzwa muMufananidzo 1D, boka reGas1-GFP harina Mid2-iRFP. Mhedzisiro iyi inoratidza kuti mapuroteni eGPI-AP ne transmembrane akaparadzaniswa kuita nzvimbo dzakasiyana dze membrane yeER pakutanga. Chikamu cheGas1-GFP chiri padyo neERES chaiyo yakanyorwa neproteni yeCOPII coat yemCherry Sec13 (Mufananidzo 1, E naF, uye bhaisikopo S1) (23).
Masero e sec31-1 anoratidza kubuda kwe galactose, ceramide refu ye acyl (C26) GPI-AP Gas1-GFP (GPI-AP, girini) uye transmembrane protein Mid2-iRFP (TMP, bhuruu) uye iyi Constructive ERES labeling Sec13-mCherry (ERES, magenta) yakaiswa pa37°C kwemaminitsi makumi matatu, ikaendeswa ku24°C, uye yakatorwa ne SCLIM maminitsi mashanu gare gare. (A kusvika kuC) inoratidza mufananidzo we 2D wakabatanidzwa kana mumwe chete we plane (A), mufananidzo we 2D projection we 10 z-sections (B) kana mufananidzo we 3D cell hemisphere we cargo ne ERES markers (C). Scale bar 1μm (A na B). Chiyero che 0.551μm (C). Gas1-GFP yakaonekwa munzvimbo dzakasiyana dze ER kana ma clusters, nepo Mid2-iRFP yakaonekwa uye yakaparadzirwa mu ER membrane yese (C). (D) Girafu inoratidza simba reGas1-GFP neMid2-iRFP muboka reGas1-GFP richitevedza mutsetse wemuseve muchena (kuruboshwe). AU, chikamu chisingachinji. (E naF) vanomiririra mufananidzo we3D unosanganisa zvinhu nechiratidzo cheERES. Maboka eGas1-GFP akaonekwa pedyo neERES chaiyo. Chikamu chechikero i0.551μm. (F) Museve wakasimba muchena unoratidza boka reGas1-GFP rakabatana neERES. Mapaneru epakati nerudyi anoratidza mufananidzo wakakurisa we3D wakabatana uye maonero akatenderera eboka reGas1-GFP rakasarudzwa.
Hukama hwepedyo pakati peboka reGas1-GFP neERES chaiyo hunoratidza kuti Gas1-GFP inogona kupinda muERES dzakasarudzwa, izvo zvakasiyana nesarudzo inoshandiswa neMid2-iRFP kuti ibve muER. Kuti tigadzirise mukana uyu, takayera chiyero cheERES chechinhu chimwe chete kana zviviri (Mufananidzo 2, A kusvika C). Takaona kuti maERES mazhinji (70%) ane rudzi rumwe chete rwezvinhu zvinotakura. Mufananidzo wepazasi weMufananidzo 2C unoratidza mienzaniso miviri yakajairika yeERES ine Gas1-GFP chete (Mufananidzo 1) kana Mid2-iRFP chete (Mufananidzo 2). Kusiyana neizvi, inenge 20% yeERES ine zvinhu zviviri zvinotakura zvinopindirana munzvimbo imwe chete. Zvakaonekwa kuti mamwe maERES (10%) aive nemhando mbiri dzezvinhu zvinotakura, asi zvakaparadzaniswa munzvimbo dzakasiyana zvakajeka. Nokudaro, ongororo iyi inoratidza kuti mushure mekunge ER yatumirwa kunze kwenyika, GPI-AP Gas1-GFP ne transmembrane cargo Mid2-iRFP zvakakamurwa kuita maERES akasiyana (Mufananidzo 2D). Kushanda zvakanaka kwekusarudza uku kunoenderana zvikuru neongororo yekare yebiochemical (6) uye kusarudzwa kwechimiro (7). Tinogonawo kuona maitiro emutoro wakavharirwa uchipinda muERES (Mufananidzo 2E neMovie S2). Mufananidzo 2E unoratidza kuti chikamu chidiki cheGas1-GFP (paneli 3) kana Mid2-iRFP (paneli 4) chinopinda muERES kubva kudivi rimwe uye chakavharirwa munzvimbo isina chinhu. Paneli 5 yeMufananidzo 2E inoratidza kuti Gas1-GFP neMid2-iRFP dzimwe nguva zvinowanikwa muERES imwechete, asi zvinopinda kubva kumativi akasiyana uye zvakabatana munzvimbo dzakasiyana dzinogona kumiririra ma COPII vesicles akasiyana. Takasimbisawo kuti kupatsanurwa kwakacherechedzwa uye kurongwa kweC26 ceramide-based GPI-AP Gas1 se ERES yakasarudzwa kwakanangana nekuti imwe transmembrane secretion cargo, iyo GFP-tagged plasma membrane protein Axl2 (27), inoratidza maitiro akafanana neMid2-iRFP. (Mufananidzo S1 neMovie S3). Axl2-GFP ichangobva kugadzirwa inoparadzirwa kuburikidza ne ER membrane seMid2-iRFP (Mufananidzo S1, A naB), uye inowanikwa pamwe chete neMid2-iRFP mu ERES zhinji (Mufananidzo S1, B kusvika D). Mapaneru 1 na2 eMufananidzo 1. S1C inoratidza mienzaniso miviri yakajairika yeERES apo zvinhu zviviri zve transmembrane zvinopindirana. Muzviitiko izvi, zvinhu zvese zvinopinda ERES pamwe chete (Mufananidzo S1E, Panel 3 na Movie S3).
Masero e sec31-1 anoratidza galactose inducible secretions, Gas1-GFP (GPI-AP, green) uye Mid2-iRFP (TMP, blue) uye constitutive ERES labeling Sec13-mCherry (ERES, magenta) akaiswa pa37. Mushure mekufukidzwa kwemaminitsi makumi matatu pa °C, enda ku24 °C kuti ubvise secretion block, uye mufananidzo neSCLIM mushure memaminitsi makumi maviri. (A kusvika C) Mifananidzo ye2D projection (A; scale bar, 1μm) kana 3D cell hemisphere mifananidzo (B na C; scale unit, 0.456μm) yecargo uye 10 z-sections dzakanyorwa neERES. Panel yepasi mu (B) uye panel mu (C) zvinoratidza mifananidzo yakagadziriswa kuratidza chete zvinhu zviripo muERES (magenta) [Gas1-GFP (grey) uye Mid2-iRFP (light blue)]. (C) Museve wakavhurika: ERES inotakura chidimbu chimwe chete chemutoro (1 kusvika 4). Museve wegrey: ERES ine mutoro wakaparadzaniswa (5). Museve muchena wakasimba: ERES ine mutoro uri panzvimbo imwe chete. Pazasi: ERES imwe chete yakasarudzwa ine Gas1-GFP (1) kana Mid2-iRFP (2). Scale bar, 100 nm. (D) Kuyera kwephotomicrograph yakatsanangurwa mu (C). Avhareji yezana yeERES ine mutoro mumwe chete (Gas1-GFP kana Mid2-iRFP), mutoro wakaparadzaniswa uye mutoro unopindirana. Mukuyedza katatu kwakazvimirira, n=432 mumasero makumi mashanu nemana. Error bar = SD. T test isina kupatsanurwa miviri. *** P = 0.0002. (E) Mufananidzo we3D weERES yakasarudzwa yemutoro wakaparadzaniswa wakanyorwa ne (C). Gas1-GFP (girini) (3) kana Mid2-iRFP (bhuruu) (4) inopinda muERES (magenta) kubva kudivi rimwe uye inongogumira kunzvimbo diki mukati meERES. Dzimwe nguva, mhando dzese dziri mbiri dzezvinhu zvinotakura zvinhu dzinopinda muERES imwechete (5) kubva kudivi rimwe chete uye dzinovharirwa munzvimbo yakaparadzana mukati meERES. Scale bar, 100 nm.
Tevere, takaedza fungidziro yekuti long acyl chain ceramide (C26) iri mu ER membrane inofambisa clustering chaiyo uye kurongeka kweGas1 mu ERES dzakasarudzwa. Kuti tiite izvi, takashandisa modified yeast strain GhLag1, umo maviri endogenous ceramide synthases Lag1 neLac1 akatsiviwa neGhLag1 (Lag1 homologue yedonje), zvichikonzera yeast strain ine cell membrane Ceramide strain pfupi pane wild type (Mufananidzo 3A) (28). Mass spectrometry (MS) analysis yakaratidza kuti mu wild-type strains, 95% ye total ceramide yakareba kwazvo (C26) chain ceramide, nepo muGhLag1, 85% yeceramide yakareba kwazvo (C18 neC16). ), 2% chete yeceramide yakareba kwazvo (C26) chain ceramide. Kunyangwe C18 neC16 ceramide dziri idzo ceramide huru dzakaonekwa muGhLag1 membrane kusvika pari zvino, ongororo yeMS yakasimbisawo kuti GPI anchor yeGas1-GFP inoratidzwa muGhLag1 strain ine C26 ceramide, iyo inofananidzwa nemafuta emhando yemusango. Hunhu hwayo hwakafanana (Mufananidzo 3A) (26). Saka, izvi zvinoreva kuti ceramide remodeling enzyme Cwh43 inonyanya kusarudza C26 ceramide, sezvakaratidzwa muMufananidzo 26, inonyanya kusanganisira GPI anchor kubva muchikamu chidiki cheC26 ceramide muGhLag1 strain. S2 (29). Kunyange zvakadaro, cell membrane yeGhLag1 ine C18-C16 ceramide chete, nepo Gas1-GFP ichiri neC26 ceramide. Izvi zvinoita kuti strain iyi ive chishandiso chakakodzera chekugadzirisa dambudziko re acyl chain length ye membrane ceramide muER. Basa rekufungidzira rekirasi uye kurongeka. Zvadaro, takatanga tadzidza kugona kweC26 Gas1-GFP kuungana mumasumbu muGhLag1 neallele inochinja tembiricha ye sec31-1 kuburikidza ne microscopy ye fluorescence, uko chete chain refu (C18-C16) iripo mu ER membrane Ceramide (Mufananidzo 3). Takaona kuti mu sec31-1, Gas1-GFP yakawanda yaive yakabatana mumasumbu, nepo Gas1-GFP mu sec31-1 GhLag1 ine membrane refu (C18-C16) ceramide ER haina kuunganidzwa uye yakagoverwa mu ER membrane yese. Kutaura chokwadi, nekuti C26 ceramide-based clustering yakabatana zvakanyanya ne ERES chaiyo (Mufananidzo 1), takazoongorora kana maitiro aya angasanganisirawo basa re ER export protein mechanism. GPI-AP inoshandisa system yakakosha yeCOPII ye ER export, iyo inodzorwa zvakanyanya neTed1's structural remodeling ye glycan chikamu che GPI anchor (30, 31). GPI-glycan inozoonekwa ne transmembrane cargo receptor p24 complex, iyo inozosarudza kutora Lst1, inova isoform chaiyo ye COPII cargo binding subunit Sec24, ichigadzira GPI-AP-rich COPII Vesicles inodiwa (31-33). Saka, takagadzira double mutant yakabatanidza kubviswa kwemapuroteni aya ega (p24 complex component Emp24, GPI-glycan remodeling enzyme Ted1 uye specific COPII subunit Lst1) ne sec31-1 mutant strain, uye takaidzidza. Zvinokwanisika here kugadzira Gas1-cluster GFP (Mufananidzo 3). Takaona kuti mu sec31-1emp24Δ uye sec31-1ted1Δ, Gas1-GFP haina kuunganidzwa uye yakapararira mu ER membrane yese, sezvakaonekwa kare mu sec31-1 GhLag1, nepo mu sec31-1lst1Δ, Gas1-GFP Sezvakaita sec31-1. Mhedzisiro iyi inoratidza kuti pamusoro pekuvapo kweC26 ceramide mu ER membrane, kuunganidzwa kweGas1-GFP kunofanirawo kubatana ne p24 complex, uye hakudi kuunganidzwa kweLst1 chaiyo. Zvadaro, takaongorora mukana wekuti urefu hwecheni yeceramide mu ER membrane hunogona kudzora kusungwa kweGas1-GFP ku p24. Zvisinei, takaona kuti kuvapo kweC18-C16 ceramide mu membrane hakukanganisi GPI-glycans dzakagadziriswazve ne p24 complex (Mifananidzo S3 na S4, A na B) kana kubatana ne GPI-AP uye kutumira GPI-AP kunze kwenyika. Recruit COPII subtype Lst1 (Mufananidzo S4C). Naizvozvo, C26 ceramide-dependent clustering haidi kudyidzana kwemapuroteni ne ER export protein mechanisms dzakasiyana, asi inotsigira imwe nzira yekuronga inotungamirwa nehurefu hwemafuta. Zvadaro, takaongorora kana urefu hwe ceramide acyl chain mu ER membrane hwakakosha pakupatsanurwa kwakanaka kweGas1-GFP se ERES yakasarudzwa. Sezvo Gas1 iri muGhLag1 strain ine short-chain ceramide ichibuda muER yopinda muplasma membrane (Mufananidzo S5), tinotenda kuti kana kupatsanura kwacho kuchitungamirirwa nehurefu hweceramide acyl chain, Gas1 iri muGhLag1 strain inogona kutungamirirwa uye kuyambuka. ERES zvinhu zvine membrane imwechete.
(A) Kamudzira kesero reGhLag1 kanonyanya kunge kaine maceramides mapfupi eC18-C16, nepo GPI anchor yeGas1-GFP ichiri neC26 IPC yakafanana nemasero emhando yemusango. Pamusoro: acyl chain length analysis yeceramide mu cell membrane yemhando yemusango (Wt) neGhLag1p ne mass spectrometry (MS). Data rinomiririra chikamu checeramide yese. Avhareji yemiedzo mitatu yakazvimirira. Error bar = SD. T-tailed unpaired t test. **** P ＜0.0001. Pazasi panel: MS analysis ye acyl chain length yeIPC iripo muGas1-GFP (GPI-IPC) GPI anchor inoratidzwa mumhando yemusango neGhLag1p. Data rinomiririra chikamu chechiratidzo chese cheIPC. Avhareji yemiedzo mishanu yakazvimirira. Error bar = SD. T-tailed unpaired t test. ns, hazvina kukosha. P = 0.9134. (B) Ma micrograph eFluorescence e sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ uye sec31-1lst1Δ masero anoratidza galactose-induced Gas1-GFP akaiswa mu incubation pa37°C kwemaminitsi makumi matatu ndokuendeswa kuIt routine fluorescence microscopy mushure me24°C. Museve muchena: ER Gas1-GFP cluster. Museve wakavhurika: Unclustered Gas1-GFP inoparadzirwa pa ER membrane yese, ichiratidza ER hunhu nuclear ring staining. Scale bar, 5μm. (C) Kuyera kwe photomicrograph yakatsanangurwa mu (B). Avhareji yezana remasero ane punctate Gas1-GFP structure. Muzviedzo zvitatu zvakazvimiririra, n≥300 masero. Error bar = SD. T-tailed unpaired t test. **** P ＜0.0001.
Kuti tigadzirise dambudziko iri zvakananga, takaita SCLIM visualization yeGas1-GFP neMid2-iRFP muGhLag1 ne sec31-1 temperature-sensitive mutant allele (Mufananidzo 4 neMovie S4). Mushure mekunge ER yachengetwa pa37°C uye yakazoburitswa pa24°C, Gas1-GFP yakawanda itsva yakagadzirwa haina kuunganidzwa uye kuparadzirwa muER membrane yese, sezvakaonekwa nema microscopes akajairwa (Mufananidzo 4, A naB). Pamusoro pezvo, chikamu chikuru cheERES (67%) chinosanganisira mhando mbiri dzecargo dziri mairi (Mufananidzo 4D). Mapaneru 1 na2 eMufananidzo 4C anoratidza mienzaniso miviri yakajairika yeERES ine Gas1-GFP inopindirana neMid2-GFP. Pamusoro pezvo, zvinhu zvese zvakatorwa muERES imwechete (Mufananidzo 4E, panel 3 namovie S4). Nokudaro, zvatakawana zvinoratidza kuti kureba kweceramide acyl chain muER membrane chinhu chakakosha chinoratidza kuunganidzwa kweprotein yeER uye kupatsanurwa.
Masero eSec31-1 GhLag1 anoratidza kubuda kwegalactose, Gas1-GFP (GPI-AP, girini) uye Mid2-iRFP (TMP, bhuruu) uye constitutive ERES-labeled Sec13-mCherry (ERES, magenta) Inwai pa37°C. Rambai kwemaminitsi makumi matatu, donhedzai kusvika ku24°C kuti mubudise kubuda, uye mufananidzo neSCLIM mushure memaminitsi makumi maviri. (A kusvika kuC) Mifananidzo ye2D projection (A; scale bar, 1μm) kana 3D cell hemisphere mifananidzo (B naC; scale unit, 0.45μm) yezvikamu gumi zve z zvakaratidzwa necargo neERES. Panel yepasi mu (B) nepanel mu (C) zvinoratidza mifananidzo yakagadziriswa kuratidza chete zvinhu zviripo muERES (magenta) [Gas1-GFP (grey) uye Mid2-iRFP (light blue)]. (C) Museve wakachena wakazadzwa: ERES, zvinhu zvinopindirana. Museve wakashama: ERES ine chinhu chimwe chete. Panera repasi: ERES yakasarudzwa ine zvinhu zvinopindirana (1 na2) zvakanyorwa mu (C). Scale bar, 100 nm. (D) Kuyera kwephotomicrograph yakatsanangurwa mu (C). Mu sec31-1 uye sec31-1 GhLag1 units, cargo imwe chete (Gas1-GFP kana Mid2-iRFP) inosanganisirwa, uye avhareji yeERES ye cargo yakaparadzana uye cargo inopindirana. Muzviedzo zvitatu zvakazvimiririra, n = 432 mumasero makumi mashanu nemana (sec31-1) uye n = 430 mumasero makumi mana nemanomwe (sec31-1 GhLag1). Error bar = SD. T test isina kuvharwa miviri. *** P = 0.0002 (sec31-1) uye ** P = 0.0031 (sec31-1 GhLag1). (E) Mufananidzo we3D weERES yakasarudzwa ine cargo inopindirana (3) zvakanyorwa mu (C). Gas1-GFP (yegirini) uye Mid2-iRFP (yebhuruu) dzinosvika kuERES (magenta) kubva kudivi rimwe chete uye dzinogara munzvimbo imwecheteyo yeERES yakaganhurirwa. Scale bar, 100 nm.
Chidzidzo ichi chinopa humbowo hwakananga mu vivo hwekuti zvinhu zvinotakura mapuroteni zvinobva mumafuta zvinoiswa munzvimbo dzakasarudzwa dzekutumira kunze munzira yekuburitsa, uye zvinoratidza kukosha kwehurefu hweacyl chain pakusarudza kupatsanura. Tichishandisa nzira ine simba uye yepamusoro inonzi SCLIM, takaratidza Gas1-GFP itsva yakagadzirwa (membrane huru yeplasma GPI-AP ine chikamu chakareba cheacyl chain (C26) ceramide lipid) mumbiriso ) Nzvimbo dzakaunganidzwa mu ER dzakasiyana dzakabatana neERES chaiyo, nepo mapuroteni anoburitswa mumembrane anoparadzirwa mu ER yese (Mufananidzo 1). Pamusoro pezvo, mhando mbiri idzi dzezvinhu dzinopinda muERES dzakasiyana nenzira yakasarudzwa (Mufananidzo 2). Hurefu hweacyl chain hwe cellular ceramide mu membrane hunoderedzwa kubva kuC26 kuenda kuC18-C16, Gas1-GFP cluster inokanganiswa kuenda munzvimbo ye ER yakasiyana, uye Gas1-GFP inodzoserwa kuti ibve mu ER ne transmembrane protein kuburikidza neERES imwechete (Mufananidzo 3 neMufananidzo 3). 4).
Kunyangwe GPI-AP ichishandisa nzira yakasarudzika yeprotein kuti ibude muER, takaona kuti C26 ceramide-dependent separation haitsamiri pakudyidzana kwemapuroteni akasiyana anogona kutungamira kuERES specialization (Mifananidzo S4 naS5). Pane kudaro, zvatakawana zvinotsigira imwe nzira yekupatsanura inotungamirirwa ne lipid-based protein clustering uye mushure mekubviswa kwezvimwe zvinhu. Zvatakaona zvinoratidza kuti dunhu reGas1-GFP kana cluster yakabatana neERES chaiyo haina transmembrane secreted protein Mid2-iRFP, izvo zvinoratidza kuti C26 ceramide-dependent GPI-AP cluster ichaita kuti ipinde muERES yakakodzera, uye panguva imwe chete, kubvisa transmembrane. Kubuda kunopinda muERES iyi chaiyo (Mifananidzo 1 na2). Kusiyana neizvi, kuvapo kweC18-C16 ceramide muER membrane hakuiti kuti GPI-AP iumbe nzvimbo kana mapoka, saka haabvisi kana kutsiva mapuroteni akabudiswa mutransmembrane muERES imwechete (Mifananidzo 3 na4). Saka, tinokurudzira kuti C26 ceramide itungamirire kupatsanurwa nekurongwa kwezvikamu nekubatsira kuunganidzwa kwemapuroteni akabatana neERES chaiyo.
Nzira yekuwana sei kuungana uku kweC26 ceramide munzvimbo chaiyo yeER? Katsika ke membrane ceramide kekuparadzana kanogona kukonzera kuti GPI-AP neC26 ceramide zvigadzire ma lipid madiki uye akarongwa pakarepo munzvimbo isina kurongeka ye lipid ye ER membrane ine glycerolipids pfupi uye isina kuguta. Mapoka emhando yepamusoro (17, 18). Mapoka madiki aya enguva pfupi anogona kusanganiswa kuita mapoka makuru, akasimba mushure mekusunga kune p24 complex (34). Zvichienderana neizvi, takaratidza kuti C26 Gas1-GFP inofanira kushanda ne p24 complex kuti igadzire mapoka makuru anooneka (Mufananidzo 3). P24 complex i heterozygous oligomer inoumbwa nemapuroteni mana akasiyana e p24 transmembrane mu yeast (35), iyo inopa multivalent binding, izvo zvinogona kutungamira mukusangana kwemapoka madiki eGPI-AP, nokudaro zvichigadzira Stable cluster yakakura (34). Kudyidzana pakati pemapuroteni ectodomains eGPI-APs kunogonawo kubatsira mukuungana kwawo, sezvakaratidzwa panguva yekufambisa kwawo Golgi mumasero eepithelial ane polarized mammalian (36). Zvisinei, kana C18-C16 ceramide iripo muER membrane, kana p24 complex yakabatana neGas1-GFP, mapoka makuru akasiyana haazoumbwe. Maitiro ari pasi anogona kutsamira pane chaiwo physical uye chemical properties ye long acyl chain ceramide. Zvidzidzo zvebiophysical zve artificial membranes zvinoratidza kuti kunyangwe ese ari maviri marefu (C24) uye mapfupi (C18-C16) acyl chain ceramide anogona kukonzera kupatsanurwa kwechikamu, marefu acyl chain ceramide (C24) chete ndiwo anogona kukurudzira Curvature nefirimu kukotama kuti pavezve chimiro chefirimu. Kuburikidza nekutarisana (17, 37, 38). Zvakaratidzwa kuti transmembrane helix yeTMED2, iyo homologue yevanhu yeEmp24, inosangana neC18 ceramide-based sphingomyelin mu cytoplasmic lobules (39). Tichishandisa molecular dynamics (MD) simulations, takaona kuti ese ari maviri C18 neC26 ceramide anoungana akatenderedza cytoplasmic lobules yeEmp24 transmembrane helix, uye ane zvaanoda zvakafanana (Mufananidzo S6). Zvakakosha kuziva kuti izvi zvinoratidza kuti transmembrane helix yeEmp24 inogona kutungamira mukupararira kwemafuta mu membrane asymmetric. Uyu mhedzisiro yazvino yakavakirwa pamasero emhuka dzinoyamwisa. MD simulations dzakafanana dzinoratidzawo kuvapo kwe ether lipids (40). Saka, tinofungidzira kuti C26 ceramide mu ma lobule maviri eER26 yakapfuma munharaunda. Kana GPI-AP iri mumaluminal lobules ikabatana zvakananga ne multivalent p24 uye kuunganidzwa kweC26 ceramide yakatenderedza p24 mu cytoplasmic lobules, inogona kukurudzira kuunganidzwa kweProtein uye membrane curvature inogadzirwa kuburikidza neminwe (41), zvichiita kuti GPI-AP iparadzane kuita nzvimbo dzakasiyana dziri pedyo neERES, iyo inofarirawo nzvimbo dzakakombama dze ER membrane (42). Mishumo yapfuura yakatsigira nzira yakarongwa (43, 44). Kusungana kwema oligolectins akawanda, hutachiona kana ma antibodies kune ceramide-based glycosphingolipids (GSL) pa plasma membrane kunokonzera kuunganidzwa kukuru kweGSL, kunowedzera kupatsanurwa kwechikamu uye kunokonzera membrane deformation uye internalization (44). Iwabuchi nezvimwewo (43) Zvakaonekwa kuti kana paine macheni marefu (C24) asi asiri mapfupi (C16), ligand ine simba rakawanda yakasungirirwa kuGSL lactosylceramide yakakonzera kuumbwa kwemasumbu makuru uye membrane invagination, uye cytoplasm Lyn-mediated signal transduction pamapepa inosanganiswa nemacheni eacyl muma neutrophils akabatana.
Mumasero e epithelial ane polarized mammalia, kuwanda kwe anti-Golgi network (TGN) kusvika padanho re apical plasma membrane kunodzora kupatsanurwa nekusarudzwa kweGPI-AP (10, 45). Kuunganidzwa uku kunotungamirirwa ne GPI-AP oligomerization (36), asi zvinogonawo kutsamira pakureba kwe ceramide chain yatinowana mu mbiriso. Kunyangwe GPI-AP yemamalia ine ether lipid-based anchor, uye chimiro chayo chemakemikari chakasiyana zvikuru ne acyl chain ceramide yakareba kwazvo, ongororo yakaitwa munguva pfupi yapfuura yakawana kuti ma lipid ese ane hunhu hwakafanana hwepanyama uye hwemakemikari uye basa (40). Nokudaro, chikamu che ether lipid mumasero emammalia chingave chakafanana ne C26 ceramide mu mbiriso, uye basa rayo nderekubatana ne long-chain ceramide mu membrane kukurudzira GPI-AP aggregation nekusarudzwa. Kunyangwe mukana uyu uchiri kuda kuongororwa zvakananga, zvakawanikwa kare zvinotsigira kuti kutakurwa kwe long acyl chain ceramide kuenda kumuviri weGolgi hakuitwi nema cytoplasmic transfer proteins, asi zvinoenderana nekugadzirwa kwe GPI anchors dzakadai se yeast. Nokudaro, evolutionary conservative mechanism inoita seinokwanisa kutakura acyl chain ceramide yakareba kwazvo uye GPI-AP (13, 16, 20, 46, 47) mu transport vesicle imwe chete.
Mumasystem emasero eepithelial ane mbiriso uye emhuka dzinoyamwisa, kuunganidzwa kweGPI-AP nekuparadzaniswa kubva kune mamwe mapuroteni eplasma membrane zvese zvinoitika zvisati zvasvika pamusoro pesero. Paladino et al. (48) vakaona kuti paTGN yemasero eepithelial ane mbiriso, kuunganidzwa kweGPI-AP hakungodi chete pakusarudzwa kweGPI-APs kune apical plasma membrane, asiwo kunodzora kurongeka kweGPI-APs uye basa rayo rezvehupenyu. Pamusoro pesero. Muyeast, chidzidzo ichi chakaratidza kuti C26 ceramide-dependent GPI-AP cluster paER inogona kudzora kurongeka kweboka uye basa reGPI-AP paplasma membrane (24, 49). Zvichienderana nemuenzaniso uyu, masero eGhLag1 ane allergy kune GPI inhibitors kana mishonga inokanganisa madziro emasero (28), uye kudiwa kwemasumbu anoshanda eGas1-GFP (49) etip ceramide anofungidzirwa mukubatana kwemasero embiriso kunoratidza G​​ mhedzisiro inogona kuitika yemuviri yemasero ehLag1. GPI-AP error. Zvisinei, kuongorora zvakare kana kurongeka kwebasa repamusoro pesero kwakarongwa kubva kuER nenzira yekuronga yakavakirwa pahurefu hwemafuta ndiko kuchave musoro weongororo yedu yeramangwana.
Mhando dzeSaccharomyces cerevisiae dzakashandiswa mubasa iri dzakanyorwa muTable S1. Mhando dzeMMY1583 neMMY1635 dzeSCLIM dzemifananidzo yema cell live cell dzakagadzirwa kumashure kweW303. Mhando idzi dzinoburitsa Sec13-mCherry ne fluorescent protein tag dzakagadzirwa uchishandisa nzira yepolymerase chain reaction (PCR) ine pFA6a plasmid se template (23). Mhando iyi inoratidza Mid2-iRFP yakanyorwa ne fluorescent protein pasi pekutonga kweGAL1 promoter yakagadzirwa seinotevera. Kuwedzerwa kwePCR kwe iRFP-KanMx sequence kubva ku pKTiRFP-KAN vector (chipo che E. O'Shea, Addgene plasmid nhamba 64687; http://n2t.net/addgene: 64687; research resource identifier (RRID): Addgene_64687) Uye yakaiswa muC-terminus ye endogenous Mid2. Mushure mekunge Mid2-iRFP genome sequence yawedzerwa uye yabatanidzwa muGAL1 promoter, yakabatanidzwa munzvimbo yeNot I-Sac I ye integration plasmid pRS306. Plasmid pRGS7 yakabuda yakagadziriswa nePst I kuti ibatanidzwe munzvimbo yeURA3.
Jini reGas1-GFP fusion rinoratidzwa pasi pekutonga kweGAL1 promoter mu centromere (CEN) plasmid, iyo yakagadzirwa seinotevera. Gas1-GFP sequence yakawedzerwa nePCR kubva pa pRS416-GAS1-GFP plasmid (24) (chipo cha L. Popolo) uye yakaunganidzwa munzvimbo yeXma I–Xho I yeCEN plasmid pBEVY-GL LEU2 (chipo cha C). Miller; Addgene plasmid nhamba 51225; http://n2t.net/addgene: 51225; RRID: Addgene_51225). Plasmid yakabuda yakatumidzwa zita rekuti pRGS6. Jini reAxl2-GFP fusion rinoratidzwawo pasi pekutonga kweGAL1 promoter ye pBEVY-GL LEU2 vector, uye kuvakwa kwayo ndekwekuti. Kutevedzana kweAxl2-GFP kwakawedzerwa kubva pa pRS304-p2HSE-Axl2-GFP plasmid (23) ne PCR, uye kwakaunganidzwa munzvimbo yeBam HI-Pst I ye pBEVY-GL LEU2 vector. Plasmid yakabuda yakatumidzwa zita rekuti pRGS12. Kutevedzana kwe oligonucleotides yakashandiswa muchidzidzo ichi kwakanyorwa muTable S2.
Mhando iyi yakawedzerwa ne 0.2% adenine uye 2% glucose [YP-dextrose (YPD)], 2% raffinose [YP-raffinose] rich yeast extract protein p (YP) medium (1% Yeast extract uye 2% protein ept). (YPR)] kana 2% galactose [YP-galactose (YPG)] senzvimbo yekabhoni, kana mune imwe nzvimbo yekabhoni (0.15% yeast nitrogen base uye 0.5% ammonium sulfate) kuwedzera maamino acids nemabhesi akakodzera anodiwa pakudya, Uye ine 2% glucose (synthetic glucose minimal medium) kana 2% galactose (synthetic galactose minimal medium) senzvimbo yekabhoni.
Kuti zvionekwe panguva chaiyo, masero e sec31-1 mutant anonzwa tembiricha airatidza chimiro pasi peGAL1 promoter akakurira munzvimbo yeYPR iri pakati pe24°C husiku hwese kusvika pakati pelog phase. Mushure mekuiswa muYPG pa24°C kweawa imwe chete, masero acho akaiswa muSG pa37°C kwemaminitsi makumi matatu, ndokuzoendeswa ku24°C kuti abude kubva musecretion block. Concanavalin A yakashandiswa kugadzirisa masero pagirazi slide uye yakatorwa neSCLIM. SCLIM musanganiswa weOlympus IX-71 inverted fluorescence microscope uye UPlanSApo 100×1.4 numeral aperture oil lens (Olympus), high-speed uye high-signal-to-noise ratio rotating disc confocal scanner (Yokogawa Electric), custom spectrometer, uye custom cooling. Iyo system's image intensifier (Hamamatsu Photonics) inogona kupa magnifying lens system ine final magnification ye ×266.7 uye charge-coupled device camera inowedzera maelectron (Hamamatsu Photonics) (21). Kuwana mifananidzo kunoitwa necustom software (Yokogawa Electric). Pamifananidzo ye3D, takashandisa piezoelectric actuator yakagadzirwa kuti tideb objective lens tichibva tadzika, uye takaunganidza optical parts 100 nm akaparadzana mu stack. Mufananidzo weZ-stack unoshandurwa kuita 3D voxel data, uye theoretical point spread function inoshandiswa kurotation disc confocal microscope inoshandiswa pa deconvolution processing neVolocity software (PerkinElmer). Nekushandisa software yeVolocity kuti iongorore otomatiki nzvimbo, ERES kusanganisira zvinhu zvakatakura yakayerwa. Kuongororwa kwemutsara kwakaitwa uchishandisa software yeMetaMorph (Molecular Devices).
Shandisa software yeGraphPad Prism kuti uone kukosha kwenhamba. Pabvunzo yeT-yeMudzidzi ine miswe miviri uye bvunzo yekuongorora kwevariance yenzira imwe chete (ANOVA), mutsauko uripo pakati pemapoka unoonekwa seune simba guru paP <0.05 (*).
Pakuongorora fluorescence microscopy yeGas1-GFP, masero e log phase akakura husiku hwese muYPD ndokuunganidzwa ne centrifugation, akagezwa kaviri ne phosphate buffered saline, ndokuiswa mu incubation kwemaminitsi gumi nemashanu, uye ozoenderera mberi nekushandisa microscope sezvakatsanangurwa kare Tarisa (24). Maikorosikopu yeLeica DMi8 (HCX PL APO 1003/1.40 oil PH3 CS) yakashongedzerwa ne objective lens, L5 (GFP) filter, Hamamatsu camera uye Application Suite X (LAS X) software zvakashandiswa pakutenga.
Masampuleti akabviswa muchimiro neSDS sample buffer pa65°C kwemaminitsi gumi, ndokuzopatsanurwa neSDS-polyacrylamide gel electrophoresis (PAGE). Kuti pave nekuongorora kweimmunoblotting, 10 μl yesampule yakaiswa panzira imwe neimwe. Primary antibody: Shandisa rabbit polyclonal anti-Gas1 pakusanganiswa kwe1:3000, rabbit polyclonal anti-Emp24 pakusanganiswa kwe1:500, uye rabbit polyclonal anti-GFP (chipo chakabva kuna H. Riezman) pakusanganiswa kwe1:3000. Mouse monoclonal anti-Pgk1 antibody yakashandiswa pakusanganiswa kwe1:5000 (chipo chakabva kuna J. de la Cruz). Secondary antibody: Horseradish peroxidase (HRP) conjugated goat anti-rabbit immunoglobulin G (IgG) yakashandiswa pakusanganiswa kwe1:3000 (Pierce). HRP-conjugated goat anti-mouse IgG yakashandiswa pakudiridza kwe1:3000 (Pierce). Nzvimbo yekudzivirira muviri yakaonekwa nekushandisa chemiluminescence method yeSuperSignal West Pico reagent (Thermo Fisher Scientific).
Sezvakatsanangurwa muna (31), kuyedza kwekudzivirira muviri kwakaitwa pachikafu cheER chakapfuma. Muchidimbu, gezai masero embiriso neTNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride uye protease inhibitor mixture) pa600 nm (OD600) pa100 optical density kaviri. Yakapwanywa negirazi beads, uye ipapo marara emasero nemagirazi beads zvakabviswa ne centrifugation. Supernatant yakazoiswa centrifuge pa17,000 g kwemaminitsi gumi nemashanu pa4°C. Pellet yakadzoserwa muTNE uye digitalis saponin yakawedzerwa kuhuwandu hwekupedzisira hwe1%. Kumiswa kwacho kwakaiswa muincubation kweawa imwe nekutenderera pa4°C, uye zvikamu zvisinganyunguduke zvakabviswa ne centrifugation pa13,000 g pa4°C kwemaminitsi makumi matanhatu. Kana muchida immunoprecipitation yeGas1-GFP, tangai maisa sample yacho nema empty agarose beads (ChromoTek) pa4°C kweawa imwe chete, mozoisa neGFP-Trap_A (ChromoTek) pa4°C kwemaawa matatu. Ma immunoprecipitated beads akagezwa kashanu neTNE ine 0.2% digoxigenin, akabviswa neSDS sample buffer, akaparadzaniswa paSDS-PAGE, uye akaongororwa ne immunoblotting.
Sezvakatsanangurwa muna (31), kuongorora kwekubatanidza kwakaitwa pachikamu cheER chakapfuma. Muchidimbu, chikamu cheER chakapfuma chakaiswa muchikamu che 0.5 mM dithiobis(succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, USA; 20°C, 20 min). Kugadzirisa kwekubatanidza kwakaderedzwa nekuwedzera glycine (50 mM final concentration, 5 minutes, 20°C).
Sezvakatsanangurwa kare (50), ongororo yeMS yeceramide mumhando dzemusango nedzeGhLag1 yakaitwa. Muchidimbu, masero akakura kusvika padanho re exponential (3 kusvika 4 OD600 units/ml) muYPD pa30°C, uye masero 25×107 akatorwa. Kugayiwa kwawo kunodzimwa ne trichloroacetic acid. Shandisa extraction solvent [ethanol, water, ether, pyridine uye 4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] uye 1.2 nmol ye internal standard C17 ceramide (860517, Avanti polar lipid) quality). Shandisa monomethylamine reagent [methanol, water, n-butanol uye methylamine solution (4:3:1:5 v/v)] kuti uite mild alkaline hydrolysis ye extract, uye wobva washandisa water-saturated n-butanol kubvisa salt. Pakupedzisira, chidimbu chacho chakaiswazve mu positive mode solvent [chloroform/methanol/water (2:7:1) + 5 mM ammonium acetate] ndokupinzwa mu mass spectrometer. Kuongororwa kweMulti-reaction (MRM) kwakaitwa kuti paonekwe uye pave ne sphingolipid molecules. TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) ine robotic nanoflow ion source Nanomate HD (Advion Biosciences, Ithaca, NY) yekuongorora lipids. Simba rekukanganisika rakagadzirirwa yega yega ceramide category. Data reMS rakawanikwa mu positive mode. Kune yega yega biological replica, lipid signal ipakati yezviyero zvitatu zvakazvimiririra.
Sezvakatsanangurwa mu (31), masero (800×107) airatidza Gas1-GFP akaiswa muchimiro chekudzivirira muviri. Gas1-GFP yakacheneswa yakaparadzaniswa neSDS-PAGE ndokuendeswa kune polyvinylidene fluoride (PVDF) membrane. Puroteni iyi yakaonekwa nekuisa PVDF neamide black. Gas1-GFP band yakachekwa kubva kuPVDF ndokugezwa kashanu ne methanol uye kamwe chete nemvura yemhando ye liquid chromatography-MS (LC-MS). Nekuisa membrane strip ne 500μl 0.3 M NaOAc (pH 4.0), buffer uye 500μl 1 M sodium nitrite musanganiswa mutsva pa37°C kwemaawa matatu, lipid fraction inoburitswa kubva kuGas1-GFP uye lysed Kuburitswa kwe inosine phosphate ceramide pakati peglucosamine ne inositol (51). Mushure meizvozvo, jira re membrane rakagezwa kane nemvura yeLC-MS grade, rikaomeswa patembiricha yemumba, uye rakachengetwa mumhepo ine nitrogen pa -80°C kusvika raongororwa. Senzira yekudzora, sampuro isina chinhu ye PVDF membrane yakashandiswa pakuyedza kwega kwega. Mafuta akatorwa kubva kuGas1-GFP akazoongororwa neMS sezvakatsanangurwa (50). Muchidimbu, zvidimbu zvePVDF zvine GPI-lipid zvakadzoserwa mu 75μl negative mold solvent [chloroform/methanol (1:2) + 5 mM ammonium acetate] uye zvakapfuudzwa ne electrospray ionization (ESI)-MRM/MS Analysis ye sphingolipid species (TSQ Vantage). Muchiitiko ichi, data reMS rakawanikwa mu negative ion mode.
Sezvambotaurwa, chikamu chemalipidi cheGPI anchor chakaparadzaniswa kubva ku [3H]-inositol-labeled GPI-AP (16). Malipidi akaparadzaniswa ne thin-layer chromatography uchishandisa solvent system (55:45:10 chloroform-methanol-0.25% KCl) uye akatariswa achishandisa FLA-7000 (Fujifilm).
Masero ane Gas1-GFP (600×107) akagezwa kaviri neTNE buffer neTNE buffer, ndokupwanywa negirazi beads, ndokuzoiswa centrifuge kubvisa marara emasero nemagirazi beads. Supernatant yakazoiswa centrifuge pa17,000 g kweawa imwe pa4°C. Pellet yakagezwa muTNE ndokuiswa 1 U PI-PLC (Invitrogen) muTNE ine 0.2% digitalis saponin kweawa imwe pa37°C. Mushure mekurapwa neenzyme, membrane yakabviswa ne centrifuge pa17,000 g pa4°C kweawa imwe. Kuti immunoprecipitate Gas1-GFP iparadzike, supernatant yakaiswa GFP-Trap_A (ChromoTek) pa4°C husiku hwese. Gas1-GFP yakacheneswa yakaparadzaniswa neSDS-PAGE yakapendwa neCoomassie brilliant blue. Bhandi reGas1-GFP rakachekwa kubva pagrey rakatenderedza aqueduct, uye mushure mekusanganiswa ne iodoacetamide uye kuderedzwa ne dithiothreitol, kugaya mu-gel ne trypsin kwakaitwa. Kubvisa nekuomesa tryptic peptides nema peptides ne GPI-glycans. Peptide yakaomeswa yakanyungudutswa mu 20 μl yemvura. Isa chikamu (8μl) mu LC. Chinyorwa che octadecylsilane (ODS) (Develosil 300ODS-HG-5; dhayamita yemukati 150 mm×1.0 mm; Nomura Chemical, Aichi Prefecture, Japan) chakashandiswa kuparadzanisa ma peptides pasi pemamiriro chaiwo e gradient. Chikamu che mobile is solvent A (0.08% formic acid) uye solvent B (0.15% formic acid mu 80% acetonitrile). Sisitimu yeAccela HPLC (Thermo Fisher Scientific, Boston, Massachusetts) yakashandiswa kubvisa column ne solvent A mukati memaminitsi makumi mashanu nemashanu pa flow rate ye 50 μl min-1 kwemaminitsi mashanu, uye ipapo concentration ye solvent B yakawedzerwa kusvika 40%., United States). Iyo eluate yakaramba ichiiswa muESI ion source, uye tryptic peptides uye peptides ane GPI-glycans zvakaongororwa neLTQ Orbitrap XL (hybrid linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific). Mu MS setup, voltage ye capillary source yakaiswa pa 4.5 kV, uye tembiricha ye transfer capillary yakachengetwa pa 300°C. Capillary voltage uye tube lens voltage yakaiswa pa 15 V uye 50 V, zvichiteerana. Data reMS rakawanikwa mu positive ion mode (resolution ye60,000; mass accuracy yezvikamu gumi pamiriyoni) muhukuru hwe300/m/z mass/charge ratio (m/z) 3000. Data reMS/MS rinowanikwa kuburikidza ne ion trap muLTQ Orbitrap XL [manhamba matatu ekutanga ayo data rinoenderana nawo, collision induced dissociation (CID)].
Kutevedzera MD kwakaitwa uchishandisa software yeGROMACS (52) uye MARTINI 2 force field (53-55). CHARMM GUI Membrane Builder (56, 57) yakazoshandiswa kugadzira bilayer ine dioleoylphosphatidylcholine (DOPC) uye Cer C18 kana DOPC uye Cer C26. Topology uye coordinates dzeCer C26 dzinobva kuDXCE nekubvisa mabeads ekuwedzera kubva kumuswe wesphingosine. Shandisa maitiro anotsanangurwa pazasi kuti uenzanise layer mbiri uye uishandise, wobva washandisa coordinates dzekupedzisira dzesystem kuvaka system ine Emp24. Nzvimbo ye transmembrane ye yeast Emp24 (masara 173 kusvika 193) yakagadzirwa se α-helix uchishandisa chimiro che molecular tool che visual MD (VMD) (58). Zvadaro, mushure mekubvisa lipids dzinopindirana, protein yakaputirwa zvakanyanya ndokuiswa mu bilayer uchishandisa CHARMM GUI. Sisitimu yekupedzisira ine 1202 DOPC uye 302 Cer C26 kana 1197 DOPC uye 295 Cer C18 uye Emp24. Isa iyo sisitimu kusvika pakuwanda kwe0.150M. Makopi mana akazvimiririra akagadzirwa kune maviri maviri ezvikamu zviviri.
Mafuta asina kuenzana anoenzaniswa uchishandisa nzira yeCHARMM GUI, iyo inosanganisira kuderedza uye kuyera matanho 405,000, uko zvipingamupinyi zvenzvimbo zvinoderedzwa zvishoma nezvishoma uye zvinobviswa, uye nguva inowedzerwa kubva pa0.005 ps kusvika pa0.02 ps. Mushure mekuenzanisa, inoburitsa 6 µs nedanho renguva re0.02 ps. Mushure mekuisa Emp24, shandisa maitiro akafanana eCHARMM GUI kuderedza uye kuenzanisa sisitimu, uye wobva wamhanya kwemasekondi masere mukugadzirwa.
Kune ese masisitimu, panguva yekuenzanisa, kudzvanywa kunodzorwa neBerendsen barostat (59), uye panguva yekugadzira, kudzvanywa kunodzorwa neParrinello-Rahman barostat (60). Muzviitiko zvese, avhareji yekudzvanywa i1 bar uye semi-isotropic pressure coupling scheme inoshandiswa. Mukugadzirisa nekugadzirisa, thermostat (61) ine speed recalibration inoshandiswa kubatanidza tembiricha yeprotein, lipid uye solvent particles zvakateerana. Munguva yese yekushanda, tembiricha yakatarwa i310K. Kubatana kusina kubatana kunoverengerwa nekugadzira runyorwa rwekubatanidza uchishandisa Verlet scheme ine 0.005 buffer tolerance. Izwi reCoulomb rinoverengerwa uchishandisa reaction field uye cut-off distance ye1.1 nm. Izwi reVander Waals rinoshandisa cut-off scheme ine cut-off distance ye1.1 nm, uye Verlet cut-off scheme inoshandiswa kune potential drift (62).
Uchishandisa VMD, kureba kwemafungu pakati peDOPC phosphate beads kana ceramide AM1 beads neprotein i0.7 nm, uye huwandu hwemafuta anosangana neprotein hunoverengerwa. Zvichienderana nefomura inotevera, verenga depletion-enrichment (DE) factor sezviri mu (63): DE factor = (huwandu hwemafuta ese muprotein 0.7) muprotein 0.7 (huwandu hweCer mumafuta ese)
Kukosha kwakataurwa kunowanikwa seavhareji, uye mitsetse yekukanganisa ikopi ina dzakazvimiririra dzeSE. Kukosha kwehuwandu hweDE factor kunoverengerwa ne t test [(averageDE-factor-1)/SE]. Verenga kukosha kweP kubva pakugoverwa kwemuswe mumwe chete.
Chishandiso cheGROMACS chakashandiswa kuverenga mepu ye2D lateral density yesystem ine Emp24 mukati me250 ns yekupedzisira yetrace. Kuti uwane mepu yekuwedzera/kuderera kweceramide, mepu yedensity yeCer inokamurwa nehuwandu hwemepu yeCer neDOPC, uye yozokamurwa nehuwandu hweCer mumuviri. Chiyero chemepu yeruvara rumwe chete chinoshandiswa.
Kuti uwane zvimwe zvinyorwa zvechinyorwa chino, ndapota tarisai http://advances.sciencemag.org/cgi/content/full/6/50/eaba8237/DC1
Ichi chinyorwa chakavhurika chinogoverwa pasi pemitemo yeCreative Commons Attribution-Non-Commercial License, iyo inobvumira kushandiswa, kugoverwa uye kuburitswazve mune chero nzira, chero bedzi kushandiswa kwekupedzisira kusiri kwekutsvaga purofiti uye pfungwa ndeyekuti basa rekutanga rakarurama. Reference.
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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-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
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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-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
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