Okusunsula obutoffaali mu kkubo ly’ekyama kyetaagisa nnyo okukuuma okugabanya obutoffaali n’okutabuka kw’obutoffaali (homeostasis). Ng’oggyeeko okusunsula okuyita mu bisusunku, omulimu gw’amasavu mu kusunsula kinesin mu nkola y’okutambuza eby’ekyama kibuuzo ekikulu ekimaze ebbanga nga tekinnaddibwamu. Wano, tukola 3D simultaneous multicolor high-resolution real-time imaging okukakasa mu vivo nti obutoffaali obupya obukoleddwa glycosylphosphatidylinositol-immobilized proteins n’ebitundu ebiwanvu ennyo ceramide lipid moieties zikuŋŋaanyizibwa era classified mu specialized endoplasms Net exit site, eky’enjawulo ku ekyo ekikozesebwa transmembrane proteins. Okugatta ku ekyo, tulaga nti obuwanvu bw’olujegere lwa ceramide mu luwuzi lwa endoplasmic reticulum bukulu nnyo mu kulonda kuno okw’okusunsula. Okunoonyereza kwaffe kuwa obujulizi obusooka obutereevu mu vivo okugabanya emigugu gya puloteyina okusinziira ku buwanvu bw’olujegere lw’amasavu mu bifo ebirondeddwa ebweru mu kkubo ly’ekyama.
Mu butoffaali bwa eukaryotic, puloteyina ezikoleddwa mu endoplasmic reticulum (ER) olwo zisunsulwa mu kiseera ky’okutambuza okuyita mu kkubo ery’ekyama okutuusibwa mu kifo kyazo ekituufu eky’obutoffaali (1). Ng’oggyeeko okusunsula okutambulizibwa ekkooti, ​​kyali kiteeberezebwa okumala ebbanga nti lipids ezimu nazo zisobola okukola ng’ebifo ebifuluma okulonda nga zizikuŋŋaanya mu bitundu by’olususu ebitongole nti puloteyina ezenjawulo (2-5). Wabula wakyaliwo obutaba na bujulizi butereevu mu vivo okukakasa enkola eno esoboka eyesigamiziddwa ku lipid. Okusobola okugonjoola ekizibu kino ekikulu, twasoma mu kizimbulukusa engeri glycosylphosphatidylinositol (GPI) anchored proteins (GPI-APs) gye zifulumizibwamu mu ngeri ey’enjawulo okuva mu ER. GPI-APs ze puloteyina ez’enjawulo ezikwatagana n’amasavu ku ngulu w’obutoffaali ( 6, 7). GPI-AP ye puloteyina efulumye eyeegattira ku bipapula eby’ebweru eby’oluwuzi lwa pulasima okuyita mu kitundu kya glycolipid (GPI anchor). Bakkiriza GPI anchors nga conservative post-okuvvuunula enkyukakyuka mu ER lumen (8). Oluvannyuma lw’okugattibwa, GPI-AP eyita mu kyuma kya Golgi (5, 9) okuva mu ER okutuuka ku luwuzi lwa pulasima. Okubeerawo kwa GPI anchors kuleetera GPI-AP okutambuza okwawukana ku transmembrane secreted proteins (nga mw’otwalidde ne plasma membrane proteins endala) okuyita mu kkubo ery’ekyama ( 5 , 9 , 10 ). Mu butoffaali bw’ekizimbulukusa, GPI-APs zaawulwa okuva ku puloteyina endala ezifulumiziddwa mu endoplasmic reticulum, n’oluvannyuma ne zipakibwa mu bikuta eby’enjawulo ebizingiddwako coat protein complex II (COPII) ( 6 , 7 ). Ebisalawo enkola eno ey’okugabanya mu nkola y’okutunda ebweru wa ER tebitegeerekeka bulungi, naye kiteeberezebwa nti enkola eno eyinza okwetaaga amasavu naddala okuddamu okukola enzimba y’ekitundu ky’amasavu eky’ennanga ya GPI ( 5 , 8 ). Mu kizimbulukusa, okuddamu okukola amasavu ga GPI kutandika amangu ddala nga GPI emaze okwekwata, era mu mbeera nnyingi, kireetera ceramide okwesiba ku 26-carbon long-chain saturated fatty acid (C26:0) (11, 12). C26 ceramide ye ceramide enkulu ekoleddwa obutoffaali bw’ekizimbulukusa okutuusa kati. Kisengekebwa mu ER era ekisinga obungi kifulumizibwa mu kyuma kya Golgi nga kiyita mu COPII vesicles (13). Okufulumya ER ebweru wa GPI-AP mu ngeri ey’enjawulo kyetaagisa okusengejja ceramide okugenda mu maaso ( 14 , 15 ), era mu ngeri endala, okukyusa ceramide okudda mu inositol phosphate ceramide (IPC) mu kyuma kya Golgi kisinziira ku GPI anchor synthesis ( 16 ). Okunoonyereza ku biramu n’obuwuka obukolebwa kulaga nti ceramides ez’olujegere lwa acyl oluwanvu ennyo zisobola okwegatta okukola ebitundu ebisengekeddwa n’eby’obutonde eby’enjawulo ( 17 , 18 ). Ebiwandiiko bino bireeta endowooza nti C26 ceramide ne GPI-AP ne C26 ceramide bakozesa eby’obugagga byabwe eby’omubiri okwegatta mu bitundu oba ebitundu ebitegekeddwa mu mbeera y’amasavu g’olususu lwa ER olutabula ennyo. Okusinga kikolebwa glycerolipids ennyimpi n’ezitali zijjudde (C16:1 ne C18:1) (19, 20). Ebitundu bino bijja kulonda okussa essira ku bifo ebitongole ebifuluma ER (ERES), nga ceramide ne ceramide-based GPI-AP bisobola okutambuza awamu okutuuka ku Golgi mu vesicle y’emu eyeetongodde COPII (5).
Mu kunoonyereza kuno, tugezesezza butereevu enkola eno eyesigamiziddwa ku lipid nga tukozesa super-resolution confocal real-time imaging microscopy (SCLIM), nga eno nkola ya microscopy ey’omulembe esobola okwetegereza omulundi gumu puloteyina eziwandiikiddwa mu ngeri ya fluorescent Ebifaananyi ebya langi ssatu n’eby’ebitundu bisatu (3D) birina okusalawo n’obwangu obw’amaanyi ennyo mu butoffaali obulamu (21, 22).
Twasooka kukozesa tekinologiya wa SCLIM okwongera okunnyonnyola engeri GPI-AP eya bulijjo n’ekibinja kya C26 ceramide gye yakeberebwamu okuva mu puloteyina ezifulumizibwa okuyita mu lususu oluvannyuma lw’okuva mu ER mu S. cerevisiae. Okusobola okukebera ensengeka ya ER, twakozesa enkola y’obuzaale esobola okulaba butereevu emigugu emipya egyakolebwa nga giyingira mu ERES mu vivo ( 7 , 23 ). Nga emigugu, twalonda C26 ceramide-based GPI-AP Gas1 ewandiikiddwa ne green fluorescent protein (GFP) ne transmembrane secreted protein Mid2 ewandiikiddwa ne near-infrared fluorescent protein (iRFP), byombi nga bigenderera plasma membrane (24-26). Mu sec31-1 temperature-sensitive mutant, emigugu gino ebiri giragibwa wansi wa galactose-inducible promoter ne constitutive ERES marker. Ku bbugumu erisukkiridde (37°C), kubanga enkyukakyuka ya sec31-1 ekosa omulimu gw’ekitundu ky’ekkanzu ya COPII Sec31 okuziyiza okumera kwa COPII n’okufulumya ER, emigugu emipya egyakolebwa gikuŋŋaanyizibwa ku ER (23). Oluvannyuma lw’okunyogoga okutuuka ku bbugumu erya wansi (24°C), obutoffaali obukyusiddwa sec31-1 bwaddamu okuva mu kitundu eky’ekyama, era emigugu emipya egy’obutonde egyakuŋŋaanyizibwa ne gitandika okufulumizibwa ebweru okuva mu ER. Okulaba kwa CLIM kwalaga nti ebisinga ku Gas1-GFP ne Mid2-iRFP ebipya ebikoleddwa bikyakuŋŋaanyizibwa mu ER y’obutoffaali obukyusiddwa sec31-1 oluvannyuma lw’okufumbirwa ku 37°C n’oluvannyuma ne bifulumizibwa ku 24°C okumala eddakiika 5 (Ekifaananyi 1). Okuva Mid2-iRFP bwe esaasaanyizibwa ku luwuzi lwa ER lwonna, ate Gas1-GFP n’ekuŋŋaanyizibwa era n’ekuŋŋaanyizibwa mu kitundu ky’oluwuzi lwa ER ekitali kigenda mu maaso, okusaasaana kwazo kwa njawulo ddala (Ekifaananyi 1, A okutuuka ku C ne Firimu S1). Okugatta ku ekyo, nga bwe kiragibwa mu kifaananyi 1D, ekibinja kya Gas1-GFP tekirina Mid2-iRFP. Ebivudde mu kunoonyereza kuno biraga nti puloteyina za GPI-AP ne transmembrane zaawulwamu mu bitundu by’olususu lwa ER eby’enjawulo nga bukyali. Ekibinja kya Gas1-GFP kiriraanye ERES eyeetongodde ewandiikiddwa ne mCherry’s COPII coat protein Sec13 (Ekifaananyi 1, E ne F, ne firimu S1) (23).
sec31-1 obutoffaali bulaga galactose-induced secretions, olujegere oluwanvu acyl (C26) ceramide GPI-AP Gas1-GFP (GPI-AP, kiragala) ne transmembrane protein Mid2-iRFP (TMP, bbululu) era kino Constructive ERES labeling Sec13-mCherry (ERES, magenta) yafumbirwa ku 37°C okumala eddakiika 30, yasengulwa okutuuka 24°C, era nga kikubiddwa ekifaananyi kya SCLIM oluvannyuma lw’eddakiika 5. (A okutuuka ku C) eraga ekifaananyi ekikiikirira ekigatta oba ekimu ekya 2D eky’ennyonyi (A), ekifaananyi kya 2D eky’okulaga eky’ebitundu 10 ebya z (B) oba ekifaananyi kya 3D cell hemisphere eky’emigugu n’obubonero bwa ERES (C). Ebbaala ya minzaani 1μm (A ne B). Ekitundu kya minzaani kiri 0.551μm (C). Gas1-GFP yazuulibwa mu bitundu bya ER ebitali bimu oba ebibinja, ate Mid2-iRFP yazuulibwa era n’esaasaanyizibwa mu luwuzi lwa ER lwonna (C). (D) Grafu eraga amaanyi g’ekitangaala ekikwatagana (relative fluorescence intensity) ekya Gas1-GFP ne Mid2-iRFP mu kibinja kya Gas1-GFP okuyita ku layini y’obusaale obweru (ku kkono). AU, ekitundu ekitali kya bulijjo. (E ne F) zikiikirira ekifaananyi kya 3D ekigatta ebintu n’akabonero ka ERES. Ebibinja bya Gas1-GFP byazuulibwa okumpi ne ERES entongole. Ekitundu kya minzaani kiri 0.551μm. (F) Akasaale akagumu akazungu kalaga ekibinja kya Gas1-GFP ekikwatagana ne ERES. Ebipande ebiri wakati ne ddyo biraga ekifaananyi kya 3D ekigaziyiziddwa ekigatta n’okulaba okukyusiddwa okw’ekibinja kya Gas1-GFP ekirondeddwa.
Enkolagana ey’okumpi ey’ekifo wakati w’ekibinja kya Gas1-GFP ne ERES eyeetongodde eraga nti Gas1-GFP esobola okuyingira mu ERES erongooseddwa, eyawukana ku kulonda okukozesebwa Mid2-iRFP okuva mu ER. Okusobola okukola ku kino ekisoboka, twapima obungi bw’omugerageranyo gwa ERES ku kintu kimu oba bibiri byokka (Ekifaananyi 2, A okutuuka ku C). Twakizudde nti ERES ezisinga obungi (70%) zirimu ekika ky’emigugu kimu kyokka. Ekifaananyi wansi mu kifaananyi 2C kiraga ebyokulabirako bibiri ebya bulijjo ebya ERES nga erina Gas1-GFP yokka (Ekifaananyi 1) oba Mid2-iRFP yokka (Ekifaananyi 2). Okwawukana ku ekyo, ebitundu nga 20% ebya ERES birimu emigugu ebiri egikwatagana mu kitundu kye kimu. Kyazuulibwa nti ERES ezimu (10%) zaalimu ebika by’emigugu bibiri, naye nga zaawuddwamu mu bitundu eby’enjawulo ebirabika obulungi. N’olwekyo, okwekenneenya kuno okw’ebibalo kulaga nti oluvannyuma lwa ER okufulumizibwa ebweru w’eggwanga, GPI-AP Gas1-GFP n’omugugu oguyita mu lususu Mid2-iRFP byawulwamu ERES ez’enjawulo (Ekifaananyi 2D). Obulung’amu buno obw’okusunsula bukwatagana nnyo n’okwekenneenya kw’ebiramu okwasooka (6) n’okusalawo enkula (7). Tusobola n’okwetegereza enneeyisa y’emigugu egyateekebwa mu kalantiini nga giyingira mu ERES (Ekifaananyi 2E ne Firimu S2). Ekifaananyi 2E kiraga nti ekitundu ekitono kyokka ekya Gas1-GFP (panel 3) oba Mid2-iRFP (panel 4) kye kiyingira mu ERES okuva ku ludda olumu era nga kisibiddwa mu kifo ekitali kimu. Panel 5 ey’ekifaananyi 2E eraga nti Gas1-GFP ne Mid2-iRFP oluusi zisangibwa mu ERES y’emu, naye ziyingira okuva ku njuyi ez’enjawulo era zikuŋŋaanyizibwa mu bitundu eby’enjawulo ebiyinza okukiikirira ebiwujjo bya COPII eby’enjawulo. Twakakasa era nti okwawula okwetegereza n’okugabanya C26 ceramide-based GPI-AP Gas1 nga selective ERES is specific kubanga omugugu omulala transmembrane secretion, GFP-tagged plasma membrane protein Axl2 (27 ), okulaga enneeyisa efaananako ne Mid2-iRFP. (Ekifaananyi S1 ne Firimu S3). Axl2-GFP empya ekoleddwa esaasaanyizibwa okuyita mu luwuzi lwa ER nga Mid2-iRFP (Ekifaananyi S1, A ne B), era esangibwa wamu ne Mid2-iRFP mu ERES ezisinga obungi (Ekifaananyi S1, B okutuuka ku D). Paneli 1 ne 2 ez’ekifaananyi 1. S1C eraga ebyokulabirako bibiri ebimanyiddwa ebya ERES nga emigugu ebiri egy’okuyita mu lususu gikwatagana. Mu mbeera zino, ebyamaguzi byombi biyingira mu ERES wamu (Ekifaananyi S1E, Panel 3 ne Movie S3).
Obutoffaali bwa sec31-1 obulaga galactose inducible secretions, Gas1-GFP (GPI-AP, green) ne Mid2-iRFP (TMP, blue) ne constitutive ERES labeling Sec13-mCherry (ERES, magenta) bwateekebwa ku 37 Oluvannyuma lw’okufumbirwa okumala eddakiika 30 ku °C, bugenda ku 24 °C okufulumya secretion block, n’okukuba ekifaananyi ne SCLIM oluvannyuma Eddakiika 20. (A okutuuka ku C) Ebifaananyi ebikiikirira 2D projection (A; scale bar, 1μm) oba 3D cell hemisphere images (B ne C; scale unit, 0.456μm) eby’omugugu n’ebitundu 10 ebya z ebiteekeddwako akabonero ka ERES. Ekipande ekya wansi mu (B) n’ekipande mu (C) biraga ebifaananyi ebirongooseddwa okulaga ebintu byokka ebiri mu ERES (magenta) [Gas1-GFP (enzirugavu) ne Mid2-iRFP (bbululu omutangaavu)]. (C) Akasaale akaggule: ERES etwala omugugu gumu gwokka (1 okutuuka ku 4). Akasaale enzirugavu: ERES erimu emigugu egyawuddwamu (5). Akasaale akagumu akazungu: ERES erimu emigugu egisangibwa awamu. Wansi: ERES emu erongooseddwa erimu Gas1-GFP (1) oba Mid2-iRFP (2) yokka. Ebbaala ya minzaani, 100 nm. (D) Okugera obungi bw’ekifaananyi ekitono (photomicrograph) ekinyonyoddwa mu (C). Ebitundu bya wakati ebya ERES ebirimu omugugu gumu gwokka (Gas1-GFP oba Mid2-iRFP), emigugu egyawuddwamu n’emigugu egy’okukwatagana. Mu kugezesa okusatu okwetongodde, n=432 mu butoffaali 54. Ebbaala y’ensobi = SD. Okugezesebwa kwa t okutali kwa mikira ebiri. *** P = 0.0002. (E) Ekifaananyi kya 3D ekya ERES ezirondeddwa ez’emigugu egyateekebwa mu kalantiini nga giwandiikiddwako (C). Gas1-GFP (kijanjalo) (3) oba Mid2-iRFP (bbulu) (4) eyingira mu ERES (magenta) okuva ku ludda olumu era ekoma mu kitundu ekitono munda mu ERES. Oluusi, ebika by’emigugu byombi biyingira mu ERES y’emu (5) okuva ku ludda lwe lumu era nga bisibiddwa mu kifo ekyesudde munda mu ERES. Ebbaala ya minzaani, 100 nm.
Ekiddako, twagezesa endowooza nti long acyl chain ceramide (C26) eri mu ER membrane evuga specific clustering n’okusunsula Gas1 mu selective ERES. Ku nkomerero eno, twakozesa ekika ky’ekizimbulukusa ekikyusiddwa GhLag1, nga mu kino endogenous ceramide synthases ebbiri Lag1 ne Lac1 zakyusibwa ne GhLag1 (the Lag1 homolog of cotton), ekyavaamu ekika ky’ekizimbulukusa nga kiriko cell membrane Ceramide ekika ekimpi okusinga ekika ky’omu nsiko (Figure 3A) (28). Okwekenenya kwa Mass spectrometry (MS) kwalaga nti mu bika eby’omu nsiko, ebitundu 95% ku ceramide yonna ye ceramide empanvu ennyo (C26), chain ceramide, ate mu GhLag1, 85% ku ceramide mpanvu nnyo (C18 ne C16). ), ebitundu 2% byokka ebya ceramide ye ceramide ey’olujegere oluwanvu ennyo (C26). Wadde nga C18 ne C16 ceramides ze ceramides enkulu ezizuuliddwa mu GhLag1 membrane okutuusa kati, okwekenneenya MS era kukakasizza nti GPI anchor ya Gas1-GFP eragiddwa mu GhLag1 strain erimu C26 ceramide, egeraageranyizibwa ku lipids ez’ekika ky’omu nsiko. Omutindo gwe gumu (Fig. 3A) (26). N’olwekyo, kino kitegeeza nti enziyiza eddaabiriza ceramide Cwh43 elonda nnyo ku C26 ceramide, nga bwe kiragibwa mu kifaananyi 26, esinga kuyingizaamu ennanga ya GPI okuva mu bungi obutono obwa C26 ceramide mu kika kya GhLag1. S2 (29) (29) nga bwe. Wadde kiri kityo, oluwuzi lw’obutoffaali bwa GhLag1 okusinga lulimu C18-C16 ceramide yokka, ate Gas1-GFP ekyalina C26 ceramide. Ensonga eno efuula ekika kino ekintu ekirungi ennyo okugonjoola mu ngeri ey’enjawulo ekizibu ky’obuwanvu bw’olujegere lwa acyl olwa membrane ceramide mu ER. Omulimu ogw’okuteebereza ogwa kiraasi n’okusunsula. Awo, twasooka okusoma obusobozi bwa C26 Gas1-GFP okukuŋŋaanyizibwa mu bikunta mu GhLag1 n’ebbugumu-sensitive mutant allele ya sec31-1 okuyita mu fluorescence microscopy eya bulijjo, nga olujegere oluwanvu (C18-C16) lwokka lwe luli mu ER membrane Ceramide (Fig. 3). Twalaba nti mu sec31-1, Gas1-GFP ezisinga zaali zikuŋŋaanyiziddwa mu bikunta, ate Gas1-GFP mu sec31-1 GhLag1 n’oluwuzi oluwanvu (C18-C16) oluwanvu ceramide ER okusinga teyali bikuŋŋaanyiziddwa era nga esaasaanyizibwa mu Mu luwuzi lwa ER lwonna. Okubeera omutuufu, olw’okuba C26 ceramide-based clustering ekwatagana nnyo ne ERES entongole (Figure 1), twaddako okunoonyereza oba enkola eno eyinza n’okuzingiramu omulimu gw’enkola ya ER export protein mechanism. GPI-AP ekozesa enkola ya COPII ey’enjawulo ku ER okutunda ebweru, nga eno efugirwa nnyo Ted1′s structural remodeling of the glycan portion of the GPI anchor (30, 31). GPI-glycan ezzeemu okugatta awo emanyibwa ekizibu ky’omugugu ekiyita mu lususu p24, nga kino nakyo kisunsulamu okuwandiika Lst1, nga eno ye isoform eyeetongodde ey’ekitundu ekikulu ekisiba emigugu ekya COPII Sec24, ekikola COPII erimu GPI-AP Vesicles zeetaagibwa (31-33). Nolwekyo, twazimba ekikyukakyuka eky’emirundi ebiri ekyagatta okusazaamu obutoffaali buno obumu (ekitundu ekizibu ekya p24 Emp24, GPI-glycan remodeling enzyme Ted1 ne COPII subunit entongole Lst1) n’ekika ky’enkyukakyuka ya sec31-1, ne tuzisoma Kisoboka okukola Gas1-cluster GFP (Figure 3). Twalaba nti mu sec31-1emp24Δ ne sec31-1ted1Δ, Gas1-GFP okusinga tekwatagana era esaasaanyizibwa mu luwuzi lwa ER lwonna, nga bwe kyalabiddwa emabegako mu sec31-1 GhLag1, ate mu sec31-1lst1Δ, Gas1-GFP Nga sec31-1. Ebivuddemu bino biraga nti ng’oggyeeko okubeerawo kwa C26 ceramide mu luwuzi lwa ER, okukuŋŋaanyizibwa kwa Gas1-GFP nakyo kyetaaga okusiba ku p24 complex, era tekyetaagisa kuwandiikibwa kwa Lst1 okwetongodde. Awo, twanoonyereza ku busobozi nti obuwanvu bw’olujegere lwa ceramide mu luwuzi lwa ER busobola okulungamya okusiba kwa Gas1-GFP ku p24. Wabula twakizuula nti okubeerawo kwa C18-C16 ceramide mu luwuzi tekukosa GPI-glycans ezizzeemu okuzimbibwa p24 complex (Figures S3 ne S4, A ne B) oba okusiba ku GPI-AP n’okufulumya GPI-AP. obusobozi. Okuwandiika ekika kya COPII ekitono Lst1 (Ekifaananyi S4C). N’olwekyo, okukuŋŋaanyizibwa okwesigamiziddwa ku C26 ceramide tekyetaagisa nkolagana ya puloteyina n’enkola za puloteyina ez’enjawulo ez’okufulumya ER, wabula kuwagira enkola endala ey’okusunsula evugirwa obuwanvu bw’amasavu. Awo, twekenneenya oba obuwanvu bw’olujegere lwa ceramide acyl mu luwuzi lwa ER kikulu nnyo mu kugabanya okulungi okwa Gas1-GFP nga ERES elonda. Okuva Gas1 mu kika kya GhLag1 ekirimu ceramide ey’olujegere olumpi bw’ava mu ER n’ayingira mu luwuzi lwa pulasima (Figure S5), tukkiriza nti singa okusunsula kuvugibwa obuwanvu bw’olujegere lwa ceramide acyl, Gas1 mu kika kya GhLag1 esobola okuddamu okulagirwa n’okusalako. Ebyamaguzi bya ERES ebirina oluwuzi lwe lumu.
(A) Olususu lw’obutoffaali bwa GhLag1 okusinga lulimu ceramides ennyimpi eza C18-C16, ate nga GPI anchor ya Gas1-GFP ekyalina C26 IPC y’emu n’obutoffaali obw’ekika ky’omu nsiko. Waggulu: okwekenneenya obuwanvu bw’olujegere lwa acyl olwa ceramide mu luwuzi lw’obutoffaali bw’ebika by’ekika ky’omu nsiko (Wt) ne GhLag1p nga bakozesa mass spectrometry (MS). Data ekiikirira ebitundu ku kikumi ebya ceramide yonna. Average y’okugezesa okusatu okwetongodde. Ebbaala y’ensobi = SD. Okugezesebwa kwa t okutali kwa mikira ebiri. **** P <0.0001. Ekipande ekya wansi: Okwekenenya kwa MS okw’obuwanvu bw’olujegere lwa acyl olwa IPC eri mu Gas1-GFP (GPI-IPC) GPI anchor eragiddwa mu bika by’omu nsiko ne GhLag1p. Data ekiikirira ebitundu ku kikumi ebya siginiini ya IPC yonna. Average ya kugezesa kutaano okwetongodde. Ebbaala y’ensobi = SD. Okugezesebwa kwa t okutali kwa mikira ebiri. ns, si kikulu. P = 0.9134. (B) Ebifaananyi ebitonotono eby’okumasamasa ebya sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ ne sec31-1lst1Δ obutoffaali obulaga galactose-induced Gas1-GFP byafumbirwa ku 37°C okumala eddakiika 30 ne biyisibwa wansi okukola routine fluorescence microscopy oluvannyuma 24°C. Akasaale akazungu: Ekibinja kya ER Gas1-GFP. Akasaale akaggule: Unclustered Gas1-GFP esaasaanyizibwa ku ER membrane yonna, eraga ER characteristic nuclear ring staining. Ebbaala ya minzaani, 5μm. (C) Okugera obungi bw’ekifaananyi ekitono ennyo (photomicrograph) ekinyonyoddwa mu (B). Ebitundu bya wakati eby’obutoffaali obulina ensengekera ya Gas1-GFP eya punctate. Mu kugezesa okusatu okwetongodde, obutoffaali n≥300. Ebbaala y’ensobi = SD. Okugezesebwa kwa t okutali kwa mikira ebiri. **** P <0.0001.
Okugonjoola butereevu ekizibu kino, twakola SCLIM visualization ya Gas1-GFP ne Mid2-iRFP mu GhLag1 ne sec31-1 temperature-sensitive mutant allele (Figure 4 ne Movie S4). Oluvannyuma lwa ER okukuumibwa ku 37°C n’oluvannyuma n’efulumizibwa ku 24°C, ebisinga ku Gas1-GFP eyaakakolebwa tebyakuŋŋaanyizibwa era ne bisaasaanyizibwa mu luwuzi lwa ER lwonna, nga bwe kyalabiddwa mu microscopes eza bulijjo (Figure 4, A and B ). Okugatta ku ekyo, ebitundu ebinene ku buli kikumi ebya ERES (67%) birimu ebika by’emigugu bibiri ebisangibwa awamu mu yo (Ekifaananyi 4D). Paneli 1 ne 2 ez’ekifaananyi 4C ziraga ebyokulabirako bibiri ebya bulijjo ebya ERES nga bikwatagana Gas1-GFP ne Mid2-GFP. Okugatta ku ekyo, ebyamaguzi byombi byawandiikibwa mu ERES y’emu (Figure 4E, panel 3 and movie S4). N’olwekyo, ebivudde mu kunoonyereza kwaffe biraga nti obuwanvu bw’olujegere lwa ceramide acyl mu luwuzi lwa ER kye kikulu ekisalawo okukuŋŋaanyizibwa n’okugabanya kwa puloteyina za ER.
Obutoffaali bwa Sec31-1 GhLag1 obulaga ebifulumizibwa ebiva ku galactose, Gas1-GFP (GPI-AP, kiragala) ne Mid2-iRFP (TMP, bbulu) n’ensengekera ya ERES-labeled Sec13-mCherry (ERES, magenta) Fumbira ku 37°C. Weeyongere okumala eddakiika 30, wansi ku 24°C okufulumya ebifuluma, era ekifaananyi ne SCLIM oluvannyuma lw’eddakiika 20. (A okutuuka ku C) Ebifaananyi ebikiikirira 2D projection (A; scale bar, 1μm) oba 3D cell hemisphere images (B ne C; scale unit, 0.45μm) eby’ebitundu 10 ebya z ebiteekeddwako akabonero k’emigugu ne ERES. Ekipande ekya wansi mu (B) n’ekipande mu (C) biraga ebifaananyi ebirongooseddwa okulaga ebintu byokka ebiri mu ERES (magenta) [Gas1-GFP (enzirugavu) ne Mid2-iRFP (bbululu omutangaavu)]. (C) Akasaale akajjudde enjeru: ERES, ebyamaguzi bikwatagana. Open arrow: ERES erimu ekintu kimu kyokka. Ekipande ekya wansi: ERES erongooseddwa erina ebintu ebikwatagana (1 ne 2) ebiwandiikiddwa mu (C). Ebbaala ya minzaani, 100 nm. (D) Okugera obungi bw’ekifaananyi ekitono (photomicrograph) ekinyonyoddwa mu (C). Mu yuniti za sec31-1 ne sec31-1 GhLag1, omugugu gumu gwokka (Gas1-GFP oba Mid2-iRFP) gwe guteekebwamu, n’ebitundu bya ERES ebya wakati ku migugu egyawuddwamu n’emigugu egy’okukwatagana. Mu kugezesa okusatu okwetongodde, n = 432 mu butoffaali 54 (sec31-1) ne n = 430 mu butoffaali 47 (sec31-1 GhLag1). Ebbaala y’ensobi = SD. Okugezesebwa kwa t okutali kwa mikira ebiri. *** P = 0.0002 (ekitundu 31-1) ne ** P = 0.0031 (ekitundu 31-1 GhLag1). (E) Ekifaananyi kya 3D ekya ERES erongooseddwa nga kiriko emigugu egy’okukwatagana (3) egyateekebwako akabonero mu (C). Gas1-GFP (kijanjalo) ne Mid2-iRFP (bbulu) zisemberera ERES (magenta) okuva ku ludda lwe lumu ne zisigala mu kitundu kye kimu ekikugirwa ERES. Ebbaala ya minzaani, 100 nm.
Okunoonyereza kuno kuwa obujulizi obutereevu mu vivo nti emigugu gya puloteyina egyesigama ku lipid gisengekebwa mu bifo ebisunsuddwa ebweru mu kkubo ly’ekyama, era kulaga obukulu bw’obuwanvu bw’olujegere lwa acyl okusobola okulonda okugabanya. Nga tukozesa enkola ey’amaanyi era ey’omulembe ey’okukebera obuwuka obutonotono eyitibwa SCLIM, twalaga Gas1-GFP (a major plasma membrane GPI-AP with a very long acyl chain (C26) ceramide lipid portion) mu kizimbulukusa ) Ebitundu ebikuŋŋaanyiziddwa mu ERs ezitali zimu bikwatagana ne ERES ezenjawulo, ate nga puloteyina ezifulumizibwa okuyita mu lususu zisaasaanyizibwa mu luwuzi lwa ER lwonna (Ekifaananyi 1). Okugatta ku ekyo, ebika by’ebyamaguzi bino ebibiri biyingira mu ERES ez’enjawulo mu ngeri elondamu (Ekifaananyi 2). Obuwanvu bw’olujegere lwa acyl olwa ceramide y’obutoffaali mu luwuzi bukendeezebwa okuva ku C26 okutuuka ku C18-C16, ekibinja kya Gas1-GFP kitaataaganyizibwa mu kitundu kya ER ekitali kimu, era Gas1-GFP ekyusibwa okuva mu ER ne puloteyina eyita mu luwuzi okuyita mu ERES y’emu (Ekifaananyi 3 n’Ekifaananyi 3). 4).
Newankubadde nga GPI-AP ekozesa enkola ya puloteyina ey’enjawulo okuva mu ER, twakizuula nti okwawukana okwesigamye ku C26 ceramide tekwesigama ku nkolagana za puloteyina ez’enjawulo eziyinza okuvaako okukuguka kwa ERES (Ebifaananyi S4 ne S5). Wabula, bye twazudde biwagira enkola endala ey’okugabanyaamu evugirwa okukuŋŋaanyizibwa kwa puloteyina okwesigamiziddwa ku lipid n’oluvannyuma okuggyibwako emigugu emirala. Okwetegereza kwaffe kulaga nti ekitundu kya Gas1-GFP oba ekibinja ekikwatagana ne ERES entongole tekirina puloteyina efuluma mu lususu Mid2-iRFP, ekiraga nti ekibinja kya C26 ceramide-dependent GPI-AP kijja kwanguyiza okuyingira kwabwe mu ERES ekwatagana, era mu kiseera kye kimu, okuggyako transmembrane Ebifulumizibwa biyingira mu ERES eno entongole (Ebifaananyi 1 ne 2). Okwawukana ku ekyo, okubeerawo kwa C18-C16 ceramides mu ER membrane tekireetera GPI-AP kukola bitundu oba ebibinja, kale teziggyako oba tezikyusa puloteyina ezifulumizibwa okuyita mu lususu mu ERES y’emu (Figures 3 and 4). . Nolwekyo, tuteesa nti C26 ceramide evuga okwawula n’okugabanya nga eyamba okukuŋŋaanyizibwa kwa puloteyina ezikwatagana ne ERES entongole.
Oyinza otya okutuuka ku C26 ceramide-dependent clustering eno mu kitundu kya ER ekigere? Omuze gwa membrane ceramide okwawukana ku bbali guyinza okuleetera GPI-AP ne C26 ceramide okukola lipids entono era eziragirwa mu kaseera ako mu mbeera y’amasavu esinga obutaba ya bulijjo ey’oluwuzi lwa ER olulimu glycerolipids ennyimpi era ezitajjula. Ebibinja by’omutindo (17, 18). Ebibinja bino ebitono eby’ekiseera bisobola okwongera okuyungibwa mu bibinja ebinene, ebinywevu oluvannyuma lw’okusiba ku p24 complex ( 34 ). Okukwatagana ne kino, twalaga nti C26 Gas1-GFP yeetaaga okukwatagana ne p24 complex okukola ebibinja ebinene ebirabika (Figure 3). Ekizibu kya p24 ye oligomer ya heterozygous ekoleddwamu puloteyina nnya ez’enjawulo eza p24 transmembrane mu kizimbulukusa ( 35 ), ekiwa okusiba kwa multivalent, ekiyinza okuvaako okusalasala kw’ebibinja ebitono ebya GPI-AP, bwe kityo ne kivaamu ekibinja ekinene Stable ( 34 ). Enkolagana wakati wa protein ectodomains za GPI-APs nayo eyinza okuyamba mu kugatta kwazo, nga bwe kiragibwa mu kiseera ky’entambula yaabwe eya Golgi mu mammalian polarized epithelial cells ( 36 ). Naye, C18-C16 ceramide bw’eba mu luwuzi lwa ER, ekizibu kya p24 bwe kikwatagana ne Gas1-GFP, ebibinja ebinene eby’enjawulo tebijja kutondebwa. Enkola eyali wansi eyinza okusinziira ku butonde obw’enjawulo obw’obutonde n’eddagala lya ceramide ey’olujegere oluwanvu olwa acyl. Okunoonyereza ku biophysical membranes kulaga nti wadde nga zombi empanvu (C24) n’ennyimpi (C18-C16) acyl chain ceramides zisobola okuleeta phase separation, long acyl chain ceramides (C24) zokka ze zisobola okutumbula high Curvature ne film bending okuddamu okubumba firimu. Okuyita mu kujulizagana (17, 37, 38). Kizuuliddwa nti transmembrane helix ya TMED2, homologue y’omuntu eya Emp24, elonda okukwatagana ne C18 ceramide-based sphingomyelin mu cytoplasmic lobules ( 39 ). Nga tukozesa molecular dynamics (MD) simulations, twakizuula nti C18 ne C26 ceramides zombi zikuŋŋaanyizibwa okwetoloola cytoplasmic lobules za Emp24 transmembrane helix, era zirina bye zifaanagana (Figure S6). Kinajjukirwa nti kino kiraga nti transmembrane helix ya Emp24 esobola okuvaako okusaasaana kw’amasavu mu membrane mu ngeri etali ya kigero. Kino kyavaamu gye buvuddeko nga kyesigamiziddwa ku butoffaali bw’ebisolo ebiyonka. Okukoppa kwa MD okufaananako era kulaga okubeerawo kw’amasavu ga ether (40) . N’olwekyo, tuteebereza nti C26 ceramide mu lobules ebbiri eza ER26 egaggawalidde mu kitundu. GPI-AP mu luminal lobules bwesiba butereevu ku multivalent p24 n’okukuŋŋaanyizibwa kwa C26 ceramide okwetoloola p24 mu cytoplasmic lobules, esobola okutumbula okukuŋŋaanyizibwa kwa Protein okuwerekera n’okukoona kw’olususu bikolebwa okuyita mu ngalo ( 41 ), ekivaako GPI-AP okwawukana mu bitundu ebitali bimu ebiriraanye ERES, era nga kino kisinga kwagala ebikoona ebinene ebitundu by’oluwuzi lwa ER ( 42 ). Lipoota ezaaliwo emabega zawagira enkola eyateesebwako ( 43 , 44 ). Okusiba kwa oligolectins, obuwuka oba obuwuka obuziyiza endwadde ku ceramide-based glycosphingolipids (GSL) ku luwuzi lwa plasma kuleeta okukuŋŋaanyizibwa kwa GSL okunene, kwongera okwawukana kwa phase era kuleeta okukyukakyuka kw’olususu n’okuyingira munda ( 44 ). Iwabuchi etc. (43) Kyazuulibwa nti mu kubeerawo kw’enjegere za acyl empanvu (C24) naye nga si nnyimpi (C16), ligandi ey’obungi (multivalent ligand) esibiddwa ku GSL lactosylceramide yaleetera okutondebwawo kw’ebibinja ebinene n’okuyingira mu luwuzi, era okukyusakyusa obubonero obutambulira ku cytoplasm Lyn ku bipapula kuyungibwamu enjegere za acyl ezigatta ebiwuka ebiyitibwa neutrophils.
Mu mammalian polarized epithelial cells, okukuŋŋaanyizibwa kw’omukutu oguziyiza Golgi (TGN) okutuuka ku ddaala ly’oluwuzi lwa pulasima olw’oku ntikko kufuga okwawukana n’okusunsula GPI-AP ( 10 , 45 ). Okugatta kuno kuvugibwa GPI-AP oligomerization ( 36 ), naye era kuyinza okusinziira ku buwanvu bw’olujegere lwa ceramide lwe tusanga mu kizimbulukusa. Newankubadde nga mammalian GPI-AP erina ether lipid-based anchor, era ensengekera yaayo ey’eddagala ya njawulo nnyo ku acyl chain ceramide empanvu ennyo, okunoonyereza okwakakolebwa kwazuula nti lipids zombi zirina evolutionarily similar physical and chemical properties And function ( 40 ). N’olwekyo, ekitundu kya ether lipid mu butoffaali bw’ebisolo ebiyonka kiyinza okufaananako ne C26 ceramide mu kizimbulukusa, era omulimu gwayo kwe kukwatagana ne ceramide ey’olujegere oluwanvu mu luwuzi okutumbula okukuŋŋaanyizibwa n’okusunsula GPI-AP. Newankubadde nga kino kikyalina okugezesebwa butereevu, ebizuuliddwa emabegako biwagira nti okutambuza ceramide ow’olujegere oluwanvu olwa acyl okutuuka mu mubiri gwa Golgi tekukolebwa puloteyini ezitambuza obutoffaali, wabula kisinziira ku kusengejja ennanga za GPI nga ekizimbulukusa. Nolwekyo, enkola y’okukuuma eby’enkulaakulana erabika ng’esobola okulonda okutambuza awamu ceramide ey’olujegere lwa acyl oluwanvu ennyo ne GPI-AP (13, 16, 20, 46, 47) mu kisenge ky’entambula kye kimu.
Mu nkola z’obutoffaali bw’ekizimbulukusa n’ebisolo ebiyonka (mammalian polarized epithelial cell systems), okukuŋŋaanyizibwa kwa GPI-AP n’okwawukana ku puloteyina endala ez’oluwuzi lwa pulasima byonna bibaawo nga tebinnaba kutuuka ku ngulu w’obutoffaali. Paladino ne banne. . Obutoffaali kungulu. Mu kizimbulukusa, okunoonyereza kuno kwalaga nti ekibinja kya GPI-AP ekisinziira ku C26 ceramide ku ER kisobola okulungamya enteekateeka y’ekibinja n’emirimu gy’emirimu gya GPI-AP ku luwuzi lwa plasma ( 24 , 49 ). Okukwatagana n’ekyokulabirako kino, obutoffaali bwa GhLag1 bulina allergy eri GPI inhibitors oba eddagala erikosa cell wall integrity ( 28 ), era obwetaavu bw’okukola Gas1-GFP clusters ( 49 ) tip ceramide projected mu mating of yeast cells kiraga G Possible physiological consequences of hLag1 cells. Ensobi ya GPI-AP. Naye, okwongera okugezesa oba ensengeka y’emirimu gy’obutaffaali ebadde etegekeddwa okuva mu ER mu nkola ey’okusunsula okusinziira ku buwanvu bw’amasavu kijja kuba nsonga y’okunoonyereza kwaffe mu biseera eby’omu maaso.
Ebika bya Saccharomyces cerevisiae ebikozesebwa mu mulimu guno biwandiikiddwa mu kipande S1. Ebika bya MMY1583 ne MMY1635 ebya SCLIM okukuba ebifaananyi by’obutoffaali obulamu byazimbibwa mu mugongo gwa W303. Ebika bino ebiraga Sec13-mCherry n’akabonero ka puloteyina eyaka (fluorescent protein tag) byazimbibwa nga bakozesa enkola eyesigamiziddwa ku polymerase chain reaction (PCR) nga pFA6a plasmid ye template ( 23 ). Ekika ekiraga Mid2-iRFP ekiwandiikiddwako puloteyina eyaka wansi w’okufugibwa GAL1 promoter kyazimbibwa bwe kiti. PCR okugaziya omutendera gwa iRFP-KanMx okuva mu pKTiRFP-KAN vector (ekirabo kya E. O’Shea, Addgene plasmid nnamba 64687; http://n2t.net/addgene: 64687; ekizuula eby’obugagga by’okunoonyereza (RRID): Addgene_64687) Era ne kiyingizibwa mu C-terminus ya endogenous Mid2. Oluvannyuma lw’omutendera gw’ensengekera y’ensengekera y’obutonde (genome sequence) ogwa Mid2-iRFP okugaziwa n’okukolebwa mu pulomoota wa GAL1, gwagattibwa mu kifo kya Not I-Sac I ekya plasmid y’okugatta pRS306. Plasmid eyavaamu pRGS7 ya linearized ne Pst I okukwatagana mu URA3 locus.
Ensengekera ya Gas1-GFP fusion gene eraga wansi w’okufugibwa GAL1 promoter mu centromere (CEN) plasmid, ezimbiddwa bwe ziti. Omutendera gwa Gas1-GFP gwayongezebwa mu PCR okuva mu pRS416-GAS1-GFP plasmid (24) (ekirabo kya L. Popolo) ne cloned mu kifo kya Xma I–Xho I ekya CEN plasmid pBEVY-GL LEU2 (ekirabo kya C) . Miller; Addgene plasmid nnamba 51225; Enkola y’okukuuma obutonde bw’ensi: 51225; RRID: Okwongerako_51225). Plasmid eyavaamu yatuumibwa erinnya pRGS6. Ensengekera y’obutonde (Axl2-GFP fusion gene) nayo eraga wansi w’okufugibwa GAL1 promoter ya pBEVY-GL LEU2 vector, era enzimba yaayo eri bweti. Omutendera gwa Axl2-GFP gwayongezebwa okuva mu pRS304-p2HSE-Axl2-GFP plasmid (23) nga gukozesebwa PCR, era ne gukolebwa mu kifo kya Bam HI-Pst I ekya pBEVY-GL LEU2 vector. Plasmid eyavaamu yatuumibwa erinnya pRGS12. Ensengekera ya oligonucleotides ezikozesebwa mu kunoonyereza kuno ewandiikiddwa mu kipande S2.
Ekika kino kyayongerwako 0.2% adenine ne 2% glucose [YP-dextrose (YPD)], 2% raffinose [YP-raffinose] rich yeast extract protein p (YP) medium (1 % Ekizimbulukusa ekiggiddwamu ne 2% protein ept). (YPR)] oba 2% galactose [YP-galactose (YPG)] nga ensibuko ya kaboni, oba mu kifo ekitono eky’obutonde (0.15% yeast nitrogen base ne 0.5% ammonium sulfate) okugatta ku amino acids ne bases ezisaanidde ebyetaagisa mu mmere , Era nga mulimu 2% glucose (synthetic glucose minimal medium) oba 2% galactose (synthetic galactose minimal medium) nga ensibuko ya kaboni.
Ku lw’okukuba ebifaananyi mu kiseera ekituufu, obutoffaali obukyusiddwa sec31-1 obukwata ku bbugumu obulaga ekizimbisibwa wansi wa GAL1 promoter bwakuzibwa mu YPR medium ku 24°C ekiro okutuuka mu mid-log phase. Oluvannyuma lw’okuyingizibwa mu YPG ku 24°C okumala essaawa 1, obutoffaali bwafumbirwa mu SG ku 37°C okumala eddakiika 30, oluvannyuma ne bukyusibwa okudda ku 24°C okufuluma okuva mu bulooka y’okufulumya. Concanavalin A yakozesebwa okutereeza obutoffaali ku slayidi y’endabirwamu era n’ekubwa ebifaananyi ne SCLIM. SCLIM ye nkola ya Olympus IX-71 inverted fluorescence microscope ne UPlanSApo 100×1.4 numerical aperture oil lens (Olympus), sikaani ya sipiidi ey’amaanyi n’ey’omugerageranyo gwa siginiini n’amaloboozi amangi ekyukakyuka disiki confocal scanner (Yokogawa Electric), custom spectrometer, n’okunyogoza okw’enjawulo Ekyuma ekinyweza ebifaananyi eky’enkola eno (Hamamatsu Photonics) esobola okuwa enkola ya lenzi ekuza nga erina okukuza okusembayo okwa ×266.7 ne kkamera y’ekyuma ekiyungiddwa ku chajingi ekubisaamu obusannyalazo (Hamamatsu Photonics) (21). Okufuna ebifaananyi kukolebwa pulogulaamu ya custom software (Yokogawa Electric). Ku bifaananyi bya 3D, twakozesa ekintu ekikoleddwa ku mutindo gwa piezoelectric actuator okukankanya lenzi y’ekigendererwa mu vertikal, era ne tukuŋŋaanya ebitundu by’amaaso nga byawukana nm 100 mu mugogo. Ekifaananyi kya Z-stack kikyusibwa ne kifuulibwa data ya 3D voxel, era omulimu gw’okusaasaanya ensonga ogw’enzikiriziganya ogukozesebwa ku microscope ya disiki ekyukakyuka (rotating disc confocal microscope) gukozesebwa mu kukola deconvolution nga gukozesebwa pulogulaamu ya Volocity (PerkinElmer). Nga bakozesa pulogulaamu ya Volocity okutuuka ku threshold mu ngeri ey’otoma okwekenneenya ebifo ebiri awamu, ERES omuli n’emigugu yapimibwa. Okwekenenya layini ya sikaani kwakolebwa nga tukozesa pulogulaamu ya MetaMorph (Molecular Devices).
Kozesa pulogulaamu ya GraphPad Prism okuzuula obukulu mu bibalo. Ku kigezo kya Student’s t-test eky’emikira ebiri n’ekigezo kya bulijjo eky’okukebera enjawulo mu ngeri emu (ANOVA), enjawulo wakati w’ebibinja zitwalibwa okuba n’akakwate akakulu ku P <0.05 (*).
Ku fluorescence microscopy ya Gas1-GFP, obutoffaali log phase bwakuzibwa ekiro mu YPD ne bukuŋŋaanyizibwa nga centrifugation, okunaaba emirundi ebiri ne phosphate buffered saline, ne incubated ku ice okumala waakiri eddakiika 15, n’oluvannyuma okugenda mu maaso wansi wa microscope nga bwe kyayogerwako emabegako Check (24). Ekyuma ekirabika obulungi ekya Leica DMi8 microscope (HCX PL APO 1003/1.40 oil PH3 CS) nga kirimu lenzi egenderera, ekyuma ekisengejja L5 (GFP), kkamera ya Hamamatsu ne pulogulaamu ya Application Suite X (LAS X) kye kyakozesebwa okugula. .
Sampuli zalongoosebwa ne SDS sample buffer ku 65°C okumala eddakiika 10, oluvannyuma ne zaawulwamu nga zikozesa SDS-polyacrylamide gel electrophoresis (PAGE). Ku kwekenneenya immunoblotting, 10 μl za sampuli zatikkibwa buli lane. Antibody enkulu: Kozesa rabbit polyclonal anti-Gas1 ku dilution ya 1:3000, rabbit polyclonal anti-Emp24 ku dilution ya 1:500, ne rabbit polyclonal anti-GFP (ekirabo okuva ewa H. Riezman) ku dilution ya 1:3000. Antibody ya mouse monoclonal anti-Pgk1 yakozesebwa ku dilution ya 1:5000 (ekirabo okuva ewa J. de la Cruz). Obuziyiza obw’okubiri: Horseradish peroxidase (HRP) conjugated goat anti-rabbit immunoglobulin G (IgG) ekozesebwa ku dilution ya 1:3000 (Pierce). HRP-conjugated goat anti-mouse IgG yakozesebwa ku dilution ya 1:3000 (Pierce). Ekitundu ky’abaserikale b’omubiri kyatunuulirwa mu nkola ya chemiluminescence eya SuperSignal West Pico reagent (Thermo Fisher Scientific).
Nga bwe kyayogerwako mu (31), okugezesa okw’obutonde okw’obusimu obuziyiza endwadde kwakolebwa ku kitundu kya ER ekigaggawalidde. Mu bufunze, okunaaba obutoffaali bw’ekizimbulukusa ne TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride ne protease inhibitor mixture) ku 600 nm ( OD600) ku 100 optical density emirundi ebiri. Yamenyeka n’obululu bw’endabirwamu, oluvannyuma ebisasiro by’obutoffaali n’obululu bw’endabirwamu ne biggyibwawo nga bakozesa okufuumuula. Oluvannyuma amazzi agasukkulumye ku mazzi gafuumuulwa ku 17,000 g okumala eddakiika 15 ku 4°C. Ekikuta kyaddamu okusibwa mu TNE era digitalis saponin n’eteekebwamu okutuuka ku kigero ekisembayo ekya 1%. Ekiyimiriziddwa kyafumbirwa okumala essaawa 1 nga kikyusibwakyusibwa ku 4°C, olwo ebitundu ebitasaanuuka ne biggyibwamu nga bifuumuulwa ku 13,000 g ku 4°C okumala eddakiika 60. Ku Gas1-GFP immunoprecipitation, sooka osooka kufumbira sampuli n’obululu bwa agarose empty (ChromoTek) ku 4°C okumala essaawa 1, n’oluvannyuma ofumbe ne GFP-Trap_A (ChromoTek) ku 4°C okumala essaawa 3. Ebimuli ebya immunoprecipitated byanaazibwa emirundi etaano ne TNE erimu 0.2% digoxigenin, ne bifuumuulwa ne SDS sample buffer, ne byawulwa ku SDS-PAGE, ne byekenneenyezebwa mu immunoblotting.
Nga bwe kyayogerwako mu (31), okusalawo okusalasala kwakolebwa ku kitundu kya ER ekigaggawalidde. Mu bufunze, ekitundu kya ER ekyagaggawalidde kyafumbirwa ne 0.5 mM dithiobis(succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, USA; 20°C, eddakiika 20). Enkola y’okusalasala yazikizibwa nga bongerako glycine (50 mM final concentration, eddakiika 5, 20°C).
Nga bwe kyayogerwako emabegako (50), okwekenneenya MS kwa ceramide mu bika by’omu nsiko ne GhLag1 kwakolebwa. Mu bufunze, obutoffaali bwakuzibwa okutuuka ku exponential phase (3 ku 4 OD600 units/ml) mu YPD ku 30°C, era obutoffaali 25×107 bwakungula. Enkyukakyuka yazo ezikizibwa ne asidi wa trichloroacetic. Kozesa ekizimbulukusa ekiggyamu [ethanol, amazzi, ether, pyridine ne 4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] ne 1.2 nmol ez’omutindo ogw’omunda ogwa C17 ceramide (860517, Avanti polar lipid) omutindo). Kozesa ekirungo kya monomethylamine [methanol, amazzi, n-butanol ne methylamine solution (4:3:1:5 v/v)] okukola mild alkaline hydrolysis y’ekirungo ekiggiddwamu, n’oluvannyuma kozesa n-butanol erimu amazzi okuggyamu omunnyo. Mu nkomerero, ekiggiddwamu kyaddamu okusibwa mu kizimbulukusa eky’engeri ennungi [chloroform/methanol/amazzi (2:7:1) + 5 mM ammonium acetate] ne kifukibwa mu kipima ekipima ekinene. Okulondoola ebikolwa ebingi (MRM) kwakolebwa okuzuula n’okugera obungi bwa molekyu za sphingolipid. Ekipima obuzito bwa TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) kirimu ensibuko ya robotic nanoflow ion source Nanomate HD (Advion Biosciences, Ithaca, NY) okwekenneenya amasavu. Amasoboza g’okutomeragana galongooseddwa ku buli mutendera gwa ceramide. MS data yafunibwa mu mbeera ya positive. Ku buli biological replicate, akabonero k’amasavu ye median y’ebipimo bisatu ebyetongodde.
Nga bwe kyayogerwako mu (31), obutoffaali (800×107) obulaga Gas1-GFP bwakolebwako immunoprecipitation ey’obutonde. Gas1-GFP erongooseddwa yayawulwamu SDS-PAGE n’ekyusibwa n’eyingizibwa mu luwuzi lwa polyvinylidene fluoride (PVDF). Puloteeni eno yalabibwa mu birowoozo nga basiiga PVDF ne amide black. Bandi ya Gas1-GFP yasalibwa okuva mu PVDF ne kinaazibwa emirundi 5 ne methanol ate omulundi gumu n’amazzi aga liquid chromatography-MS (LC-MS) grade. Nga tufumbira oluwuzi olusiba ne 500μl 0.3 M NaOAc (pH 4.0), buffer ne 500μl omutabula gwa 1 M sodium nitrite ogwakasaanuuka ku 37°C okumala essaawa 3, ekitundu ky’amasavu kifulumizibwa okuva mu Gas1-GFP ne kisengejebwa Okufulumya inosine phosphate ceramide wakati wa glucosamine ne inositol (51). Oluvannyuma lw’ekyo, ekitundu ky’olususu kyanaazibwa emirundi ena n’amazzi aga ddaala lya LC-MS, ne kikalira ku bbugumu erya bulijjo, ne kiterekebwa mu bbanga lya nayitrojeni ku -80°C okutuusa lwe kyekenneenyezebwa. Nga ekifuga, sampuli etaliiko kintu kyonna eya PVDF membrane yakozesebwa ku buli kugezesa. Amasavu agaggibwa mu Gas1-GFP olwo ne geekenneenyezebwa MS nga bwe kyayogerwako ( 50 ). Mu bufunze, PVDF strips ezirimu GPI-lipid zaddamu okusibwa mu 75μl negative mold solvent [chloroform/methanol (1:2) + 5 mM ammonium acetate] ne ziyita mu electrospray ionization (ESI)-MRM/MS Analysis of sphingolipid species (TSQ Vantage). Mu mbeera eno, data ya MS yafunibwa mu mbeera ya ion negativu.
Nga bwe kyayogeddwako emabegako, ekitundu ky’amasavu eky’ennanga ya GPI kyawulwa okuva ku [3H]-inositol-labeled GPI-AP ( 16 ). Amasavu gaawulwamu nga tukozesa enkola ya thin-layer chromatography nga tukozesa enkola y’ekizimbulukusa (55:45:10 chloroform-methanol-0.25% KCl) ne galabibwa nga tukozesa FLA-7000 (Fujifilm).
Obutoffaali obulaga Gas1-GFP (600×107) bwanaazibwa emirundi ebiri ne TNE buffer ne TNE buffer, ne bumenyebwa n’obululu bw’endabirwamu, oluvannyuma ne bufuumuulwa okuggyawo ebisasiro by’obutoffaali n’obululu bw’endabirwamu. Oluvannyuma amazzi agasukkulumye ku mazzi gafuumuulwa ku 17,000 g okumala essaawa emu ku 4°C. Ekikuta kyanaazibwa mu TNE ne kifumbirwa ne 1 U PI-PLC (Invitrogen) mu TNE erimu 0.2% digitalis saponin okumala essaawa emu ku 37°C. Oluvannyuma lw’okulongoosa enziyiza, oluwuzi lwaggyibwawo nga luyita mu kusengejja ku g 17,000 ku 4°C okumala essaawa emu. Okusobola okuziyiza Gas1-GFP, amazzi agasukkulumye ku mazzi gafumbirwa ne GFP-Trap_A (ChromoTek) ku 4°C okumala ekiro. Gas1-GFP erongooseddwa eyawulwamu SDS-PAGE yasiigibwa langi ya Coomassie brilliant blue. Gas1-GFP staining band yasalibwako okuva ku nzirugavu eyeetoolodde omukutu gw’amazzi, n’oluvannyuma oluvannyuma lw’okugifuula alkylation ne iodoacetamide n’okukendeeza ne dithiothreitol, okugaaya mu gel ne trypsin kwakolebwa. Ggyayo n’okukaza tryptic peptides ne peptides ne GPI-glycans. Peptide enkalu yasaanuusibwa mu 20 μl z’amazzi. Fuyira ekitundu (8μl) mu LC. Empagi ya octadecylsilane (ODS) (Develosil 300ODS-HG-5; obuwanvu obw’omunda mm 150×1.0 mm; Nomura Chemical, Aichi Prefecture, Japan) yakozesebwa okwawula peptides mu mbeera ezenjawulo ez’okukyukakyuka. Ekitundu ekitambula kye kizimbulukusa A (0.08% formic acid) ne solvent B (0.15% formic acid mu 80% acetonitrile). Enkola ya Accela HPLC (Thermo Fisher Scientific, Boston, Massachusetts) yakozesebwa okulongoosa empagi n’ekizimbulukusa A mu ddakiika 55 ku kigero ky’okukulukuta ekya 50 μl min-1 okumala eddakiika 5, n’oluvannyuma ekirungo ky’ekizimbulukusa B ne kyongerwa okutuuka ku bitundu 40%. , Amerika). Eluate yayingizibwa obutasalako mu nsibuko ya ion ya ESI, era tryptic peptides ne peptides ezirina GPI-glycans zaakeberebwa mu LTQ Orbitrap XL (hybrid linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific). Mu nteekateeka ya MS, vvulovumenti y’ensibuko y’emisuwa yateekebwa ku 4.5 kV, era ebbugumu ly’emisuwa egy’okutambuza lyakuumibwa ku 300°C. Voltage ya capillary ne voltage ya tube lens zateekebwa ku 15 V ne 50 V, okusinziira ku. MS data yafunibwa mu positive ion mode (resolution ya 60,000; mass accuracy ya 10 parts per million) mu mass range ya 300/m/z mass/charge ratio (m/z) 3000. Data ya MS/MS efunibwa okuyita mu ion trap mu LTQ Orbitrap XL [digito 3 ezisooka data kwe yeesigamye, okutomeragana okuleetebwa dissociation (CID)].
Okukoppa kwa MD kwakolebwa nga tukozesa pulogulaamu ya GROMACS (52) ne MARTINI 2 force field (53-55). Olwo CHARMM GUI Membrane Builder (56, 57) yakozesebwa okuzimba bilayer erimu dioleoylphosphatidylcholine (DOPC) ne Cer C18 oba DOPC ne Cer C26. Topology ne coordinates za Cer C26 ziggibwa mu DXCE nga tuggyawo obululu obw’enjawulo okuva mu mukira gwa sphingosine. Kozesa enkola eyogeddwako wansi okutebenkeza layeri ey’emirundi ebiri n’okugiddukanya, olwo okozese ensengekera ezisembayo ez’enkola okuzimba enkola erimu Emp24. Ekitundu ekiyita mu lususu (transmembrane domain) eky’ekizimbulukusa Emp24 (ebisigadde 173 okutuuka ku 193) kyazimbibwa nga α-helix nga tukozesa ensengekera ya molekyu y’ekintu ekirabika ekya MD (VMD) ( 58 ). Olwo, oluvannyuma lw’okuggyawo amasavu agakwatagana, puloteyina yalongoosebwa mu ngeri ya coarsely granulated era n’eyingizibwa mu bilayer nga tukozesa CHARMM GUI. Enkola esembayo erimu DOPC 1202 ne Cer C26 302 oba DOPC 1197 ne Cer C18 ne Emp24 295. Ioniize enkola eno okutuuka ku kigero kya 0.150M. Ebikoppi bina ebyetongodde byakolebwa ku bitonde bibiri ebya bilayer.
Lipid bilayer esengekebwa nga tukozesa enkola ya CHARMM GUI, erimu okukendeeza n’oluvannyuma okutebenkeza emitendera 405,000, nga ebiziyiza ekifo bikendeezebwa mpolampola ne biggyibwawo, ate omutendera gw’obudde ne gwongerwa okuva ku 0.005 ps okutuuka ku 0.02 ps. Oluvannyuma lw’okutebenkera, efulumya 6 μs nga erina omutendera gw’ekiseera ogwa 0.02 ps. Oluvannyuma lw’okuyingiza Emp24, kozesa enkola y’emu eya CHARMM GUI okukendeeza n’okutebenkeza enkola, n’oluvannyuma odduke okumala s 8 mu kukola.
Ku nkola zonna, mu nkola y’okutebenkeza, puleesa efugibwa Berendsen barostat (59), ate mu nkola y’okufulumya, puleesa efugibwa Parrinello-Rahman barostat (60). Mu mbeera zonna, puleesa eya wakati eri bbaala 1 era ensengekera y’okuyunga puleesa ya semi-isotropic ekozesebwa. Mu nkola ya bbalansi n’okufulumya, thermostat (61) erimu speed recalibration ekozesebwa okugatta ebbugumu ly’obutundutundu bwa protein, lipid ne solvent. Mu kiseera kyonna eky’okukola, ebbugumu erigendererwa liri 310K. Enkolagana etali ya kukwatagana ebalwa nga tukola olukalala lw’okugatta nga tukozesa enkola ya Verlet nga erina 0.005 buffer tolerance. Ekiseera kya Coulomb kibalirirwa nga tukozesa ekifo ky’ensengekera n’ebanga ery’okusalako erya 1.1 nm. Ekigambo kya Vander Waals kikozesa ensengekera y’okusalako nga erina ebanga ly’okusalako erya 1.1 nm, ate enkola ya Verlet ey’okusalako ekozesebwa ku potential drift (62).
Nga tukozesa VMD, obuwanvu bw’amayengo agasala wakati wa DOPC phosphate beads oba ceramide AM1 beads ne protein buba 0.7 nm, era omuwendo gw’amasavu agakwatagana ne protein gubalirirwa. Okusinziira ku nsengekera eno wammanga, bala ensonga y’okukendeeza-okugaggawaza (DE) nga mu (63): Ensonga ya DE = (omuwendo gw’amasavu gonna mu puloteyina 0.7) mu puloteyina 0.7 (omuwendo gwa Cer mu masavu gonna) .
Omuwendo ogwaloopebwa gufunibwa nga wakati, era ebbaala z’ensobi kkopi nnya ezeetongodde eza SE. Obukulu bw’emitindo gya DE factor bubalirirwa n’okugezesebwa kwa t [(averageDE-factor-1)/SE]. Bala omuwendo gwa P okuva mu kugabanya kw’omukira gumu.
Ekintu kya GROMACS kyakozesebwa okubala maapu ya 2D lateral density ey’enkola erimu Emp24 mu ns 250 ezisembayo ez’okulondoola. Okusobola okufuna maapu y’okugaggawaza/okukendeera kwa ceramide, maapu ya density ya Cer egabanyizibwamu omugatte gwa maapu ya Cer ne DOPC, n’oluvannyuma n’egabanyizibwamu n’obungi bwa Cer mu mubiri. Minzaani ya maapu ya langi y’emu ekozesebwa.
Okumanya ebikozesebwa eby’okugatta ku kiwandiiko kino, laba 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-Romero, Ana Maria Perez -Linero), Sergio Lopez (Sergio Lopez), Miho Waga (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
3D high-resolution real-time imaging eraga obukulu bw’obuwanvu bw’olujegere lwa ceramide okusunsula puloteyina mu bifo ebisunsuddwamu ebifulumizibwa.
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 -Linero), Sergio Lopez (Sergio Lopez), Miho Waga (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
3D high-resolution real-time imaging eraga obukulu bw’obuwanvu bw’olujegere lwa ceramide okusunsula puloteyina mu bifo ebisunsuddwamu ebifulumizibwa.
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