Okushoroora puroteyini omu muhanda gw‘okusheeshasheesha ni kikuru munonga omu kuguma n‘obutafaari oburi omu bicweka n‘okuguma omu mwanya. Oyihireho okushoroora okurikurabira omu bigogo, omurimo gw‘amaizi g‘omubiri omu kushoroora kwa kinesin omu nkora y‘okutwara ebishaju n‘ekibuuzo ky‘omutaano ekimazire obwire buraingwa kitakagarukiremu. Aha, nitukora ebishushani bya 3D omu bwire bumwe eby’erangi nyingi eby’omuhendo gw’ahaiguru omu bwire bw’amazima kuhamya omu nkora y’amagara ngu puroteyini ezisya ezikozirwe glycosylphosphatidylinositol-immobilized eziine ebicweka by’amaizi ebiraingwa munonga eby’omubiri nibiteerana kandi bikashoroorwa omu bicweka by’omubiri eby’omutaano Net usembrane exit site, ekiri. Okwongyera ahari ekyo, nitworeka ngu oburaingwa bw’orujegyere rwa ceramide omu nkora ya endoplasmic reticulum nibukuru munonga omu kutoorana oku okushoroora. Okucondooza kwaitu nikuha obuhame bw‘okubanza obuhikire omu nkora y‘okushoroora emigugu ya puroteyini kurugiirira aha bureingwa bw‘enjegyere y‘amashaju omu myanya y‘okutooranamu ey‘okushohoza omu muhanda gw‘okushohoza.
Omu butafaari bwa eukaryotic, puroteyini ezirikukorwa omu endoplasmic reticulum (ER) niziheza zishoroorwa omu bwire bw’okuzitwara kurabira omu muhanda gw’okusheeshasheesha kubaasa kuhikwa ahu zihikire omu butafaari (1). Oyihireho okushoroora okurikukoresibwa ekoti, kikaba nikiteekateekwaho kumara obwire buraingwa ngu amashaju agamwe nigabaasa n‘okukora nk‘enshonga z‘okurugaho ezitoorainwe barikugateerana omu bicweka by‘omubiri ebitari bimwe na bimwe ebirikukora puroteyini ezitari zimwe na zimwe (2-5). Kwonka, hakiriho okubura obuhame obuhikire omu nkora y‘amagara oburikworeka ngu enkora egi neebaasa kubaho erikwegamira aha mafuta. Okubaasa kushoboorora ekizibu eki eky‘omutaano, tukacondooza omu kitumbisa oku puroteyini eziine glycosylphosphatidylinositol (GPI) zirikuteebwa aheeru (GPI-APs) zirikushohozibwa omu miringo etarikushushana kuruga omu ER. GPI-APs ni ebiriisa by‘omubiri ebitari bimwe na bimwe ebirikukwatagana n‘amaizi g‘omubiri ( 6, 7). GPI-AP ni puroteyini erikuruga omu mubiri erikukwatiraine n‘ebipapura by‘aheeru by‘omubiri gw‘omubiri kurabira omu kicweka kya glycolipid (GPI anchor). Nibaikiriza ebikwato bya GPI nk‘okuhindura kw‘okurinda bwanyima y‘okuvunuura omu ER lumen (8). Bwanyima y‘okutaho, GPI-AP neehingura omu kikwato kya Golgi (5, 9) kuruga omu ER kuza omu nkora ya plasma. Okubaho kw‘ebikwato bya GPI nikureetera GPI-AP kutwarwa omu muringo gw‘okwetaanisa aha puroteyini ezirikuruga omu nkora y‘okusheeshasheesha (obariiremu ezindi puroteyini z‘okusheeshasheesha z‘omubiri) aha muhanda gw‘okusheeshasheesha (5, 9, 10). Omu butafaari bw’ekitumbisa, GPI-APs nizitaanisibwa n’ebindi bihingwa ebirikusheesheka omu nkora y’omubiri, reero zipakirwa omu bihimba by’omutaano ebizingirwe puroteyini y’ekijwaro II (COPII) (6, 7). Ebirikumanyisa omuringo gw‘okushoroora ogu omu nkora y‘okushohoza ER tibirikwetegyerezibwa gye, kwonka nikiteekateekwaho ngu omuringo ogu nigubaasa kwetenga amaizi g‘omubiri, namunonga okugaruzaho omu nkora y‘ekicweka ky‘amaizi g‘omubiri eky‘enkora ya GPI (5, 8). Omu kitumbisa, okugaruzaho amashaju ga GPI nikutandika ahonaaho bwanyima ya GPI kukwatira hamwe, kandi omu mbeera nyingi, nikireetera ceramide kukwatira hamwe n‘ekiriisa kya 26-carbon eky‘omurunga muraingwa (C26:0) (11, 12). C26 ceramide niyo ceramide ekuru erikuruga omu butafaari bw’ekitumbisa kuhitsya hati. Nikikorwa omu ER kandi ebingi nibitwarwa omu nkora ya Golgi kurabira omu bihimba bya COPII (13). ER okushohoza GPI-AP buzima nikyetengyesa okugumizamu n‘okukora ebiriisa (14, 15), kandi omu kugarukamu, okuhindura ebiriisa kuba inositol phosphate ceramide (IPC) omu kikwato kya Golgi nikurugiirira aha kuteerana kw‘enkora ya GPI (16). Okucondooza okwakozirwe aha bikwatiraine n’ebihangirwe ebiine amagara n’ebicweka by’omubiri ebikozirwe abantu kworekire ngu acyl chain ceramides eziraingwa munonga nizibaasa kukwatanisa zikore emyanya erikutebeekanisibwa erimu emitwarize y’omubiri ey’omutaano (17, 18). Ebihandiiko ebi nibireetaho okuteekateeka ngu C26 ceramide na GPI-AP na C26 ceramide nibakoresa emitwarize yaabo y‘omubiri kwegaita omu bicweka ebitebeekanisiibwe gye nari ebicweka omu mbeera y‘amaizi g‘omubiri eya ER erimu obutabanguko. Okukira munonga nikiba kirimu ebiriisa bikye kandi ebitarikujwara (C16:1 na C18:1) (19, 20). Ebicweka ebi nibiija kutooranwa kuta omutima aha myanya y‘okuruga omu ER (ERES) emwe, ahu ceramide na ceramide-based GPI-AP birikubaasa kutwarwa hamwe kuza Golgi omu nkora niyo emwe eyehaireyo eya COPII (5).
Omu kucondooza oku, tukagyezaho butoosha omuringo ogu ogurikwegamira aha mafuta g‘omubiri turikukoresa obumanyiso bw‘okukyebera obutafaari bw‘omubiri oburikwetwa super-resolution confocal real-time imaging microscopy (SCLIM), oburikuba buri obukoryo bw‘okukyebera oburikworekwa obw‘omurembe oburikubaasa kureeba omu bwire bumwe puroteyini eziine akamanyiso ka fluorescent Ebirungo by‘erangi ishatu n‘eby‘omubiri biine obubonero bw‘ahaiguru (3D) . (21, 22).
Twabanza twakoresa tekinologiya ya SCLIM kwongyera kushoboorora oku GPI-AP eyaabutoosha n‘ekibiina kya C26 ceramide yaakyeberwa kuruga omu puroteyini ezirikuruga omu nkora y‘okusheeshasheesha bwanyima y‘okuruga omu ER omu S. cerevisiae. Okubaasa kukyebera oku ER erikushoroorwa, tukakoresa enkora y‘obuzaarwa erikubaasa kureeba butunu emigugu ensya ekozirwe erikutaaha omu ERES omu nkora (7, 23). Nk’omugugu, tukatoorana C26 ceramide-based GPI-AP Gas1 ehandiikirweho puroteyini y’ekibira (GFP) na puroteyini erikuruga omu nkora y’okusheeshasheesha Mid2 ehandiikirweho puroteyini y’okusheeshasheesha eri haihi (iRFP), byombi ebirikuta omutima aha nkora ya plasma (24–26). Omu sec31-1 omuhinduzi orikuhurira obutagatsi, emigugu egi ebiri neeyorekwa ahansi y’omuhi w’obuhwezi orikureetaho galactose n’akamanyiso ka ERES akarikureetaho. Omu bwire bw’obwire bw’obwire bw’obwire (37°C), ahabw’okugira ngu okuhinduka kwa sec31-1 nikukwata aha murimo gw’ekicweka kya COPII coat Sec31 okuzibira okukura kwa COPII n’okushohoza ER, emigugu ensya ekozirwe neeteerana aha ER (23). Bwanyima y’okukyendeeza aha bwire bw’obwire bukye (24°C), obutafaari bwa sec31-1 oburikuhinduka bukagaruka omu mwanya gw’okusheeshasheesha, kandi emigugu mirungi ey’obuhangwa eyaabaire eteeraine ekatandika kushohora kuruga omu ER. Okureeba kwa CLIM kukooreka ngu ebingi aha bisya ebikozirwe Gas1-GFP na Mid2-iRFP bikaba bikiri omu ER y’obutafaari bwa sec31-1 oburikuhinduka bwanyima y’okubiteekaho aha 37°C reero byarekurwa aha 24°C kumara edakiika 5 (ekishushani 1). Ahabw‘okugira ngu Mid2-iRFP neejanjaara aha rubaju rwona orwa ER, kandi Gas1-GFP neeteerana kandi neeteerana omu mwanya ogutarikugumizamu ogwa ER membrane, okugabwa kwayo nikutaana kimwe (ekishushani 1, A kuhika C na Movie S1). Okwongyera ahari ekyo, nk‘oku kirikworekwa omu kishushani 1D, ekibiina kya Gas1-GFP tikiine Mid2-iRFP. Ebirugire omu kucondooza oku nibyoreka ngu GPI-AP na puroteyini z‘omubiri ezirikuraba omu nkora y‘omubiri zikaba zitaanisiibwe omu bicweka bya ER membrane bitarikushushana kare. Ekibiina kya Gas1-GFP kiri haihi na ERES emwe ehandiikirweho puroteyini ya mCherry eya COPII ekoti Sec13 (ekishushani 1, E na F, na firimu S1) (23).
sec31-1 obutafaari nibworeka okushohoza okurikureetwaho galactose, omurunga muraingwa gw’acyl (C26) ceramide GPI-AP Gas1-GFP (GPI-AP, ekinyaatsi) n’ekiriisa ky’omubiri ekirikuhinguraho ebiriisa Mid2-iRFP (TMP, bururu) kandi eki ERES ey’okwombeka ehandiikirwe Sec13-mEScu omu 2012. edakiika 5, zaaza aha 24°C, kandi zaashushanisibwa na SCLIM bwanyima y‘edakiika 5. (A kuhika C) nikyoreka ekishushani ky‘okujwekyera ekigaitsirwe nari ekishushani kimwe kya 2D eky‘enyonyi (A), ekishushani ky‘okureeberaho kya 2D eky‘ebicweka 10 bya z (B) nari ekishushani kya 3D eky‘obutafaari bw‘omubiri eky‘emigugu n‘obumanyiso bwa ERES (C). Omubaro gw‘okupima 1μm (A na B). Ekipimo ky’okupima ni 0.551μm (C). Gas1-GFP ekashangwa omu bicweka bya ER ebitarikushushana nari omu bibumbe, obwo Mid2-iRFP ekashangwa kandi ekajanjaara omu ER membrane yoona (C). (D) Ekishushani nikyoreka obuhango bw‘okujwara kwa Gas1-GFP na Mid2-iRFP omu kigombe kya Gas1-GFP aha rubaju rw‘omurongooti gw‘omushaari ogweru (aha rubaju rwa bumosho). AU, ekicweka ky’obutegyeki. (E na F) nibijwekyera ekishushani kya 3D ekirikugaita ebintu n‘akamanyiso ka ERES. Ebirundo bya Gas1-GFP bikashangwa haihi na ERES emwe. Ekipimo ky’okupima ni 0.551μm. (F) Omusyo ogugumire ogurikwera nigumanyisa ekibiina kya Gas1-GFP ekiine akakwate na ERES. Empapura z‘ahagati n‘eza buryo nizooreka ekishushani kya 3D ekihango ekigaitsirwe hamwe n‘okureeba okuhindukire kw‘ekibiina kya Gas1-GFP ekitoorainwe.
Enkoragana y‘omwanya eri haihi ahagati y‘ekibiina kya Gas1-GFP n‘ERES emwe neeyoreka ngu Gas1-GFP neebaasa kutaaha omu ERES etoorainwe, erikutaana n‘okutoorana okurikukoresibwa Mid2-iRFP kuruga omu ER. Okushoboorora okubaasa oku, tukapima omubaro gwa ERES aha kintu kimwe nari bibiri byonka (ekishushani 2, A kuhika C). Twashanga ngu ERES nyingi (70%) zirimu omuringo gumwe gwonka gw’emigugu. Ekishushani ky‘ahansi omu kishushani 2C nikyoreka eby‘okureeberaho bibiri eby‘omutaano ebya ERES erimu Gas1-GFP yonka (ekishushani 1) nari Mid2-iRFP yonka (ekishushani 2). Okutaana n‘ekyo, enkumi 20% za ERES zirimu emigugu ebiri erikuteerana omu mwanya gumwe. Kikashangwa ngu ERES ezimwe (10%) zikaba zirimu emigugu y’emiringo ebiri, kwonka zikaba zitaanisiibwe omu myanya etari emwe n’emwe erikureebwa gye. Nahabwekyo, okucondooza oku okw‘embariirira nikworeka ngu bwanyima ya ER kushohora, GPI-AP Gas1-GFP n‘omugugu gw‘okuraba omu nkora Mid2-iRFP nibibaganisibwamu ERES zitari zimwe na zimwe (ekishushani 2D). Oburungi bw’okushoroora obu nibukwatagana munonga n’okucondooza okwabandize okwa biochemical (6) n’okumanyisa enkora y’ebihangirwe (7). Nitubaasa n‘okureeba emitwarize y‘emigugu erimu karantiini erikutaaha omu ERES (ekishushani 2E na firimu S2). Ekishushani 2E nikyoreka ngu ekicweka kikye kyonka ekya Gas1-GFP (ekipande kya 3) nari Mid2-iRFP (ekipande kya 4) nikitaaha omu ERES kuruga aha rubaju rumwe kandi kikingirwe omu mwanya ogutarikushushana. Ekipande kya 5 eky‘ekishushani 2E nikyoreka ngu Gas1-GFP na Mid2-iRFP obumwe nizishangwa omu ERES emwe, kwonka nizitaaha kuruga aha mbaju zitari zimwe na zimwe kandi niziteerana omu bicweka bitarikushushana ebirikubaasa kujwekyera obutafaari bwa COPII obutarikushushana. Kandi tukahamya ngu okwahukana n’okushoroora okureebirwe okwa C26 ceramide-based GPI-AP Gas1 nk’okutoorana ERES n’okw’omutaano ahabw’okuba omugugu ogundi ogw’okushohoza ebiri omu nkora y’okusheeshasheesha, puroteyini y’omubiri eya GFP Axl2 (27 ), erikworeka emitwarize erikushushana na Mid2-iRFP. (Ekishushani S1 na Firimu S3). Axl2-GFP ensya ekozirwe neejanjaara kurabira omu nkora ya ER nka Mid2-iRFP (ekishushani S1, A na B), kandi neekora hamwe na Mid2-iRFP omu ERES nyingi (ekishushani S1, B kuhika D). Empapura 1 na 2 z‘ekishushani 1. S1C neeyoreka eby‘okureeberaho bibiri eby‘omutaano ebya ERES ahu emigugu ebiri ey‘okuhinguraho erikuteerana. Omu mbeera ezi, ebintu byombi nibitaaha omu ERES hamwe (ekishushani S1E, ekipande 3 na firimu S3).
Obutafaari bwa sec31-1 oburikworeka ebishaju ebitarikubaasa kureetaho galactose, Gas1-GFP (GPI-AP, kiragara) na Mid2-iRFP (TMP, bururu) n’ebirikuhandiika ERES ebirikukora Sec13-mCherry (ERES, magenta) bikateebwa aha 37 Bwanyima y’okubiteeka aha °C kuhika aha °4 edakiika z’okurekura. block, n‘ekishushani na SCLIM bwanyima y‘edakiika 20. (A kuhika C) Ebishushani by‘okureeberaho eby‘okureeberaho bya 2D (A; omubaro gw‘okupima, 1μm) nari ebishushani by‘ekicweka ky‘obutafaari bwa 3D (B na C; ekicweka ky‘okupima, 0.456μm) eby‘emigugu n‘ebicweka 10 ebya z ebiine akamanyiso ka ERES. Ekipande ky‘ahansi omu (B) n‘ekipande omu (C) nibyoreka ebishushani ebikozirwe kworeka ebintu byonka ebiri omu ERES (erangi erikutukura) [Gas1-GFP (enzirugavu) na Mid2-iRFP (erangi erikutukura)]. (C) Omushaari ogukingire: ERES neetwara omugugu gumwe gwonka (1 kuhika ahari 4). Omushaari gw’enzirugavu: ERES erimu emigugu etaanisiibwe (5). Omushaari ogurikwera ogugumire: ERES erimu emigugu eri omu mwanya gumwe. Ahaifo: ERES emwe etoorainwe erimu Gas1-GFP (1) nari Mid2-iRFP (2) yonka. Omubaro gw’okupima, 100 nm. (D) Okupima omubaro gw’ebishushani ebikye ebishobooroirwe omu (C). Omubaro gwa ERES oguri ahagati y‘emigugu emwe yonka (Gas1-GFP nari Mid2-iRFP), emigugu etaanisiibwe n‘emigugu erikuteerana. Omu kucondooza kwa kashatu okwetongoire, n=432 omu butafaari 54. Enshobi y’omubaro = SD. Ekigyezo kya t ekitaine mikira ebiri. *** P = 0.0002. (E) Ekishushani kya 3D ekya ERES etoorainwe ey‘emigugu eri omu karantiini erimu akamanyiso ka (C). Gas1-GFP (ekibira) (3) nari Mid2-iRFP (bururu) (4) neetaaha omu ERES (erangi erikutukura) kuruga aha rubaju rumwe kandi neegarukira aha mwanya mukye omunda ya ERES. Obumwe, emiringo yombi y‘emigugu neetaaha omu ERES emwe (5) kuruga aha rubaju rumwe kandi ekomirwe omu mwanya ogurikwetaanisa aha bandi omu ERES. Omubaro gw’okupima, 100 nm.
Ekindi, tukagyezaho enteekateeka ngu acyl chain ceramide (C26) erimu omu ER membrane neevuga okuteerana n‘okushoroora Gas1 omu ERES ezitoorainwe. Okubaasa kuhikiiriza eki, tukakoresa ekitumbisa ekihindwirwemu GhLag1, omu kika kya ceramide synthases ibiri eky’omunda Lag1 na Lac1 kyahindurwa GhLag1 (Lag1 homolog ya pamba), ekyarugiremu ekitumbisa ekiine obutafaari bw’omubiri Ceramide ekika kigufu kukira ekika ky’omukibira (Ekishushani 3A) (2). Okushwijuma kwa Mass spectrometry (MS) kukooreka ngu omu miringo y’omukibira, 95% y’omubaro gwona gw’ebihimba niguba guri mureingwa munonga (C26) omurunga gw’ebihingwa, obwo omu GhLag1, 85% by’ebihimba nibiraingwa munonga (C18 na C16). ), 2% zonka eza ceramide ni ceramide y’omurunga mureingwa munonga (C26). N‘obu C18 na C16 ceramides ziraabe ziri ceramides enkuru ezishangirwe omu nkora ya GhLag1 kuhitsya hati, okucondooza kwa MS nakwo kukahamya ngu GPI anchor ya Gas1-GFP erikworekwa omu nkora ya GhLag1 erimu C26 ceramide, erikugyeragyeranisibwa n‘ebishaju by‘omuringo gw‘omukibira. Omutindo nigwo gumwe (Ekishushani 3A) (26). Nahabwekyo, eki nikimanyisa ngu enzyme erikugaruzaho ceramide Cwh43 neetoorana munonga C26 ceramide, nk‘oku kirikworekwa omu kishushani 26, neekunda kutaho ekikwato kya GPI kuruga omu C26 ceramide nkye omu ruganda rwa GhLag1. S2 (29). Nangwa n’obu kiraabe kiri kityo, obutafaari bw’omubiri obwa GhLag1 omu kubanza burimu C18-C16 yonka, kandi Gas1-GFP ekiine C26 ceramide. Eki nikireetera oruganda oru kuba ekikwato kirungi ky‘okushoboorora buzima ekizibu ky‘oburaingwa bw‘orujegyere rwa acyl rwa membrane ceramide omu ER. Omurimo gw‘okuteekateeka ogwa kiraasi n‘okushoroora. Bwanyima, tukabanza twacondooza aha bushoborozi bwa C26 Gas1-GFP kwerundaana omu bicuncu omu GhLag1 n’ekicweka ky’omubiri ekirikuhinduka ekirikuhurira ebirikuba biri omu bwire bw’obutiti ekya sec31-1 kurabira omu kucondooza okurikukoresibwa omu kucondooza okurikukoresibwa omu kucondooza oku, ahu orujegyere oruraingwa (C18-C16) rwonka ruriho omu ER membrane Ceramide (Fig. 3). Twareeba ngu omu sec31-1, ebingi bya Gas1-GFP bikaba biri omu bicuncu, obwo Gas1-GFP omu sec31-1 GhLag1 erimu ekicweka ky‘omubiri ekiraingwa (C18-C16) ekiraingwa kya ceramide ER okukira munonga kitarikuteerana kandi kigabwa omu Omu kicuncu kyona ekya ER. Kubaasa kushoboorora gye, ahabw‘okugira ngu okuteerana kw‘ebicweka bya C26 ceramide nikukwatagana munonga na ERES emwe (ekishushani 1), tukakuratiraho kucondooza yaaba enkora egi neebaasa n‘okutwariramu omurimo gw‘omuringo gw‘okushohoza puroteyini ya ER. GPI-AP neekozesa enkora ya COPII ey‘omutaano ey‘okushohoza ER, erikutegyekwa munonga Ted1‘s okuhindura omu nkora y‘ekicweka kya glycan eky‘enkomo ya GPI (30, 31). GPI-glycan erikugarurwamu bwanyima neemanyirwa aha nkora y‘okutwara emigugu p24, erikugaruka neetoorana Lst1, eri isoform y‘omutaano ey‘ekicweka kikuru ky‘emigugu ya COPII Sec24, ekirikureetaho GPI-AP-rich COPII Vesicles are necessary (31). Nahabwekyo, tukakora omuhinduzi wa kabiri ogwagaita okusazibwamu kwa puroteyini ezi emwe (ekicweka kya p24 ekigumire Emp24, enzyme erikuhindura GPI-glycan Ted1 n’ekicweka kya COPII ekimanyirwe Lst1) n’omuringo gw’omuhinduzi wa sec31-1, kandi twabyegaho Nikibaasika kuhangaho GFP1-clu3). Twareeba ngu omu kicweka kya sec31-1emp24Δ na sec31-1ted1Δ, Gas1-GFP neekira kuba etarikuteerana kandi neejanjaara omu nkora yoona eya ER, nk’oku kyabaire nikireebeka enyima omu kicweka kya sec31-1 GhLag1, obwo omu kicweka kya sec31-1lst1Δ, Gas1-GFP nk’ekicweka kya sec3. Ebirugire omu kucondooza oku nibyoreka ngu okwiha aha kubaho kwa C26 ceramide omu ER membrane, okuteerana kwa Gas1-GFP nakwo nikwetenga kukwatira hamwe p24 complex, kandi tikurikwetenga okuhandiikwa kwa Lst1 okwa buriijo. Bwanyima, tukacondooza aha kubaasa ngu oburaingwa bw’orujegyere rwa ceramide omu ER membrane nibubaasa kutebeekanisa okuteerana kwa Gas1-GFP na p24. Kwonka tukashanga ngu okubaho kwa C18-C16 ceramide omu nkora y‘okusheeshasheesha tikurikukora aha GPI-glycans ezigarukiremu kwombekwa p24 complex (ebishushani S3 na S4, A na B) nari okukwatira hamwe na GPI-AP n‘okushohoza GPI-AP. okubasya. Okuhandiika COPII omu muringo gw‘ahansi Lst1 (ekishushani S4C). Nahabwekyo, okuteerana kwa C26 ceramide-dependent tikurikwetenga okukoragana kwa puroteyini n‘emiringo etari emwe n‘emwe y‘okushohoza puroteyini za ER, kwonka nikushagika omuringo ogundi gw‘okushoroora ogurikugyenderwaho oburaingwa bw‘amaizi. Bwanyima, tukashwijuma yaaba oburaingwa bw‘orujegyere rwa ceramide acyl omu ER membrane nibukuru omu kushoroora gye Gas1-GFP nk‘ERES erikutooranwa. Ahabw‘okugira ngu Gas1 omu ruganda rwa GhLag1 orurimu ceramide y‘orujegyere rukye neeruga omu ER etaaha omu nkora ya plasma membrane (ekishushani S5), nitwikiriza ngu okushoroora kwaba nikugyendera aha bureingwa bw‘orujegyere rwa ceramide acyl, Gas1 omu ruganda rwa GhLag1 neebaasa kugaruka kuhindurwa kandi egyende. Ebintu bya ERES ebiine omubiri nigwo gumwe.
(A) Obutafaari bw’omubiri bwa GhLag1 okukira munonga burimu obutafaari bwa C18-C16 ceramides obugufu, obwo ekikwato kya GPI ekya Gas1-GFP ekiine C26 IPC nk’obutafaari bw’omuringo gw’omukibira. Aharuguru: okushwijuma oburaingwa bw‘orujegyere rwa acyl rwa ceramide omu nkora y‘obutafaari bw‘omuringo gw‘omukibira (Wt) n‘ebika bya GhLag1p kurabira omu kucondooza kw‘obuhango (MS). Ebihandiiko nibijwekyera ebicweka 10 ahari 100 eby’omubaro gwona ogwa ceramide. Omubaro gw’okugyezesa kwa kashatu okwetongoire. Enshobi y’omubaro = SD. Ekigyezo kya t ekitaine mikira ebiri. **** P <0,0001. Ekipande ky‘ahansi: MS okushwijuma oburaingwa bw‘orujegyere rwa acyl rwa IPC oruriho omu Gas1-GFP (GPI-IPC) GPI anchor erikworekwa omu nkora y‘omukibira n‘eya GhLag1p. Ebihandiiko nibijwekyera ebicweka 10 ahari 100 eby‘akamanyiso ka IPC koona. Omubaro gw’okugyezesa okutaano okwetongoire. Enshobi y’omubaro = SD. Ekigyezo kya t ekitaine mikira ebiri. ns, ti kikuru. P = 0.9134. (B) Ebishushani by’okukyebera eby’omubiri eby’omubiri gw’omuntu, obutafaari bw’omubiri oburikworeka obutafaari oburikworeka Gas1-GFP oburikureetwaho galactose bikateebwa omu nkora y’okukyebera ahansi aha 37°C kumara edakiika 30°C. bwanyima ya 24°C. Omushaari ogurikwera: Ekirundo kya ER Gas1-GFP. Omushaari ogukingire: Gas1-GFP etaine kirungo neegabwa aha rubaju rwona orwa ER, erikworeka okujwara kw‘empeta y‘enyukiriya ey‘obumanyiso bwa ER. Omubaro gw’okupima, 5μm. (C) Okupima omubaro gw’ebishushani ebikye ebishobooroirwe omu (B). Omubaro gw’obutafaari oburimu obubonero bwa Gas1-GFP. Omu kucondooza okushatu okwetongoire, obutafaari n≥300. Enshobi y’omubaro = SD. Ekigyezo kya t ekitaine mikira ebiri. **** P <0,0001.
Okushoboorora ekizibu eki butoosha, tukakora okureeba kwa SCLIM okwa Gas1-GFP na Mid2-iRFP omu GhLag1 na sec31-1 allele erikuhinduka erikuhurira obutagatsi (Ekishushani 4 na Movie S4). Bwanyima ya ER kuguma aha 37°C kandi bwanyima ekarekurwa ahari 24°C, ebingi aha Gas1-GFP eyaabaire eherize kukorwa tibikateeraine kandi byajanjaara omu nkora ya ER yoona, nk’oku kirikureebeka omu bipimo bya microscope ebya butoosha (Ekishushani 4, A na B ). Okwongyera ahari ekyo, ekicweka kihango kya ERES (67%) kirimu emiringo ebiri y‘emigugu erikushangwa hamwe omunda (ekishushani 4D). Empapura 1 na 2 z‘ekishushani 4C nizooreka eby‘okureeberaho bibiri eby‘omutaano ebya ERES ebiine Gas1-GFP na Mid2-GFP ebirikuteerana. Okwongyera ahari ekyo, ebintu byombi bikahandiikwa omu ERES emwe (ekishushani 4E, ekipande 3 na firimu S4). Nahabwekyo, ebyarugamu byaitu nibyoreka ngu oburaingwa bw‘orujegyere rwa ceramide acyl omu ER membrane n‘ekintu kikuru ekirikumanyisa okuteerana n‘okushoroora puroteyini ya ER.
Obutafaari bwa Sec31-1 GhLag1 oburikworeka ebishaju ebirikuruga omu nkora ya galactose, Gas1-GFP (GPI-AP, ekinyaatsi) na Mid2-iRFP (TMP, bururu) hamwe n’ebirikureetaho ERES ebiine akamanyiso ka Sec13-mCherry (ERES, ekitukura) Nibikuubwa aha 37°C. Gumizamu kumara edakiika 30, oshukye aha 24°C kurekura ebishaju, kandi okore ekishushani na SCLIM bwanyima y‘edakiika 20. (A kuhika C) Ebishushani by‘okureeberaho eby‘okureeberaho bya 2D (A; omubaro gw‘okupima, 1μm) nari ebishushani by‘ekicweka ky‘obutafaari bwa 3D (B na C; ekicweka ky‘okupima, 0.45μm) eby‘ebicweka 10 ebya z ebiine akamanyiso k‘emigugu na ERES. Ekipande ky‘ahansi omu (B) n‘ekipande omu (C) nibyoreka ebishushani ebikozirwe kworeka ebintu byonka ebiri omu ERES (erangi erikutukura) [Gas1-GFP (enzirugavu) na Mid2-iRFP (erangi erikutukura)]. (C) Omusyo oguijwire omweru: ERES, ebintu nibiteerana. Omushaari ogukingire: ERES erimu ekintu kimwe kyonka. Ekipande ky‘ahansi: ERES etoorainwe eine ebintu ebirikuteerana (1 na 2) ebihandiikirwe omu (C). Omubaro gw’okupima, 100 nm. (D) Okupima omubaro gw’ebishushani ebikye ebishobooroirwe omu (C). Omu bicweka bya sec31-1 na sec31-1 GhLag1, emigugu emwe yonka (Gas1-GFP nari Mid2-iRFP) neeteebwamu, kandi ebicweka bya ERES eby‘omugati ahabw‘emigugu erikwetaanisa n‘emigugu erikweshushaniriza. Omu kucondooza kwa kashatu okwetongoire, n = 432 omu butafaari 54 (sec31-1) na n = 430 omu butafaari 47 (sec31-1 GhLag1). Enshobi y’omubaro = SD. Ekigyezo kya t ekitaine mikira ebiri. *** P = 0,0002 (ekicweka kya 31-1) na ** P = 0,0031 (ekicweka kya 31-1 GhLag1). (E) Ekishushani kya 3D kya ERES etoorainwe n‘emigugu erikweshushaniriza (3) ehandiikirwe omu (C). Gas1-GFP (ekibira) na Mid2-iRFP (bururu) nizihika ahari ERES (erangi erikutukura) kuruga aha rubaju rumwe kandi ziguma omu mwanya gumwe ogu ERES erikuzibirwa. Omubaro gw’okupima, 100 nm.
Okucondooza oku nikuha obuhame obuhikire omu nkora ya vivo ngu emigugu ya puroteyini erikwegamira aha lipid neeshoroorwa omu myanya y‘okutooranamu ey‘okushohoza omu muhanda gw‘okushohoza, kandi neeyoreka obukuru bw‘oburaingwa bw‘orujegyere rwa acyl ahabw‘okutooranamu okushoroora. Okukozesa omuringo gw’amaani kandi ogw’omurembe gw’okukyebera ebikye ebirikwetwa SCLIM, tukooreka Gas1-GFP ensya eyakozirwe (ekicweka kikuru kya plasma membrane GPI-AP ekiine orujegyere rwa acyl orureingwa munonga (C26) ceramide lipid portion) omu kitumbisa ) Ebicweka ebiteeraine omu puroteyini z’omutaano ezitarikushushana zirikugabwa kurabira omu ER, omubiri gwa ER (ekishushani 1). Okwongyera ahari ekyo, emiringo egi ebiri y‘ebintu neetaaha omu ERES zitari zimwe na zimwe omu kutoorana (ekishushani 2). Oburaingwa bw‘orujegyere rwa acyl orw‘obutafaari bwa ceramide omu nkora y‘omubiri nibukyendeera kuruga ahari C26 kuhika ahari C18-C16, ekirundo kya Gas1-GFP nikiteganisibwa omu kicweka kya ER ekitaine rubaju, kandi Gas1-GFP neehindurwa omuhanda kuruga omu ER n‘ekiriisa ky‘omubiri ekirikuhinguraho kurabira omu ERES niyo emwe (ekishushani 3 n‘ekishushani 3). 4).
N‘obu GPI-AP eraabe neekoresa omuringo gw‘omutaano gwa puroteyini kuruga omu ER, tukashanga ngu okwahukana okurikurugiirira ahari C26 ceramide tikurikwegamira aha nkoragana ya puroteyini etari emwe n‘emwe erikubaasa kureetera ERES kuba ey‘omutaano (ebishushani S4 na S5). Omu mwanya gw‘ekyo, ebi twashanga nibishagika omuringo ogundi gw‘okushoroora ogurikugyenderwaho okuteerana kwa puroteyini ezirikwegamira aha mafuta g‘omubiri n‘okukuratiraho okubinga emigugu endiijo. Ebi twareebire nibyoreka ngu ekicweka kya Gas1-GFP nari ekibiina ekiine akakwate na ERES emwe tikiine puroteyini erikuruga omu nkora ya transmembrane Mid2-iRFP, ekirikworeka ngu ekibiina kya GPI-AP ekirikurugiirira ahari C26 ceramide nikiija kworobya okutaaha kwabo omu ERES erikukwatagana, kandi omu bwire nibwo bumwe, nikiihaho secretions egi erikwetwa transmembrane TheFig1). 2). Okutaana n‘ekyo, okubaho kwa C18-C16 ceramides omu ER membrane tikurikureetera GPI-AP kukora ebicweka nari ebirundo, n‘ahabw‘ekyo tibirikwihaho nari kugaruraho puroteyini ezirikuruga omu ER membrane omu ERES niyo emwe (ebishushani 3 na 4). . Nahabwekyo, nituteekateeka ngu C26 ceramide neereetaho okwahukana n‘okushoroora obwo erikuhwera omu kuteerana puroteyini eziine akakwate na ERES emwe.
Nitubaasa kuhikiiriza tuta okuteerana oku okurikurugiirira aha C26 ceramide omu mwanya gwa ER ogumanyirwe? Omuze gwa membrane ceramide kutaanisa aha rubaju nigubaasa kureetera GPI-AP na C26 ceramide kukora obutafaari bukye kandi obutebeekanisiibwe ahonaaho omu mwanya gw‘amaizi ogutarikuhikaana n‘obutoosha ogwa ER membrane ogurimu glycerolipids ngufu kandi ezitarikujwara. Ebirundo by’omutindo (17, 18). Obucweka obu obukye obw‘akaire kakye nibubaasa kwongyera kugaita ebicweka bihango kandi ebigumire bwanyima y‘okukwatiraine n‘ekicweka kya p24 (34). Okurugiirira ahari eki, tukooreka ngu C26 Gas1-GFP neeyetenga kukwatagana na p24 complex kubaasa kuhanga ebirundo bihango ebirikureebeka (ekishushani 3). Ekicweka kya p24 ni heterozygous oligomer erimu puroteyini ina zitari zimwe na zimwe eza p24 transmembrane omu kitumbisa (35), ekirikuha okuteerana kw’ebintu bingi, ekirikubaasa kureetera okuteerana kw’ebicweka bikye bya GPI-AP, obwo bikareetaho ekicweka kihango kya Stable (34). Enkoragana ahagati y‘ebicweka bya puroteyini ectodomains bya GPI-APs nabyo nibibaasa kureetaho okuteerana kwabyo, nk‘oku kirikworekwa omu bwire bw‘okutwarwa kwabyo okwa Golgi omu butafaari bw‘ebihaha oburimu ebiriisa (36). Kwonka C18-C16 ceramide ku erikuba eriho omu ER membrane, p24 complex ku erikuba neekwatanisa na Gas1-GFP, ebirundo bihango eby’omutaano tibirikwija kubaho. Enkora eri ahansi neebaasa kurugiirira aha mitwarize y‘omubiri n‘eby‘obuhangwa eby‘omutaano eby‘omurunga gwa acyl ceramide. Okucondooza okwakozirwe aha bihangirwe ebiine amagara (Biophysical) aha bihimba ebikozirwe abantu nikworeka ngu n‘obu ebicweka by‘omubiri ebiraingwa (C24) n‘ebigufu (C18-C16) biraabe nibibaasa kureetera okutaana kw‘ebicweka, ebicweka by‘omubiri ebiraingwa (C24) byonka nibyo birikubaasa kutunguura Curvature y‘ahaiguru n‘okugoma kwa firimu kugaruka kuhindura firimu. Kurabira omu kugaaniira (17, 37, 38). Kikashobororwa ngu ekicweka ky‘omubiri ekirikuhinguraho TMED2, ekirikushushana n‘omuntu ekya Emp24, nikikwatagana omu muringo gw‘okutooranamu na C18 ceramide-based sphingomyelin omu bihimba by‘omubiri (39). Twakozesa okugyeragyeranisa kw‘eby‘obuhangwa (MD), tukashanga ngu C18 na C26 ceramides zombi niziteerana okwehinguririza ebicweka by‘omubiri eby‘omubiri eby‘omubiri gw‘omubiri (cytoplasmic lobules) eby‘omubiri gw‘omubiri (cytoplasmic lobules) eby‘omubiri gw‘omubiri (Emp24 transmembrane helix), kandi ziine ebi barikukunda ebirikushushana (ekishushani S6). Nikirungi kumanya ngu eki nikyoreka ngu transmembrane helix ya Emp24 neebaasa kureetera okugabwa kw‘amaizi g‘omubiri omu nkora y‘okujanjaara okutarikushushana. Eki n’ekirugiremu ekihererukireyo kurugiirira aha butafaari bw’ebinyonyi ebirikunywa amaizi. Okugyeragyeranisa kwa MD okurikushushana nakwo nikworeka okubaho kw’ebiriisa bya ether (40) . Nahabwekyo, nituteekateeka ngu C26 ceramide omu lobules ibiri eza ER26 neetunga eby’obugaiga omu mwanya. GPI-AP omu luminal lobules ku erikuba neekwatanisa butoosha na multivalent p24 n‘okuteerana kwa C26 ceramide okwehinguririza p24 omu cytoplasmic lobules, nikibaasa kutunguura okuteerana kwa puroteyini okurikuhererukayo n‘okugoma kw‘ebihaha nikuruga omu minwa (41), ekirikureetera GPI-AP kutaanisa omu kicweka ky‘ahaiguru adja . ebicweka by‘omubiri gw‘omubiri (42). Ripoota ezabandize zikashagika enkora etebekanisiibwe (43, 44). Okukwatira hamwe kwa oligolectins, obukooko oburikureeta endwara nari obutafaari oburikurwanisa glycosphingolipids (GSL) oburikukoresibwa ceramide aha rubaju rwa plasma nikureetaho okuteerana kwa GSL kwingi, kwongyera aha kwahukana kw‘ebicweka kandi kureetera orubaju kuhinduka n‘okutaaha omunda (44). Iwabuchi etc. (43) Kikashangwa ngu omu kubaho kw’enjegyere z’acyl eziraingwa (C24) kwonka zitari ngufu (C16), ligand y’ebintu bingi erikukwatanisa na GSL lactosylceramide ekareetaho okutandika kw’ebirundo bihango n’okutaaha omu nkora kw’ebicweka by’omubiri, kandi okuhindura kw’obumanyiso bw’ebicweka by’omubiri omu nkora ya Lyn-mediated cells ahabw’ebipapura bya chagittropyl ni ac.
Omu butafaari bw‘omubiri oburikwetwa polarized epithelial network, okuteerana kw‘omutimbagano gw‘okurwanisa Golgi (TGN) kuhika aha rurengo rw‘omubiri gw‘ahaiguru nigutegyeka okwahukana n‘okushoroora GPI-AP (10, 45). Okuteerana oku nikutegyekwa GPI-AP oligomerization (36), kwonka nikibaasa n‘okurugiirira aha bureingwa bw‘orujegyere rwa ceramide oru turikushanga omu kitumbisa. N‘obu GPI-AP y‘ebinyonyi eraabe eine ekikwato ekirikuhamya ether lipid, kandi n‘omutindo gwayo gw‘eby‘obuhangwa nigutaana munonga n‘omutindo gw‘eby‘obuhangwa oguraingwa munonga ogwa acyl chain ceramide, okucondooza okukozirwe juba kukashanga ngu eby‘okurya byombi biine emitwarize y‘omubiri n‘eby‘obuhangwa erikushushana omu kutunguuka N‘emirimo (40). Nahabwekyo, ekicweka kya ether lipid omu butafaari bw’ebinyonyi nikibaasa kushushana na C26 ceramide omu kitumbisa, kandi omurimo gwakyo n’okukwatanisa na ceramide ey’orujegyere ruraingwa omu nkora y’okutunguura okuteerana kwa GPI-AP n’okushoroora. N‘obu okubaasa oku kuraabe nikwetenga kugyezibwa butunu, ebyarugamu enyima nibishagika ngu okutwarwa kw‘omurunga gw‘obwire buraingwa ogwa acyl chain ceramide kuza omu mubiri gwa Golgi tikurikukorwa puroteyini z‘okuhingisa eza cytoplasmic, kureka nikurugiirira aha kuteerana kw‘ebikwato bya GPI nk‘ekitumbisa. Nahabwekyo, omuringo gw‘okurinda eby‘obuhangwa nigushusha ngu nigubaasa kutoorana hamwe kutwara hamwe acyl chain ceramide n‘orujegyere ruraingwa munonga na GPI-AP (13, 16, 20, 46, 47) omu nkora y‘okutwara emwe.
Omu nkora y‘obutafaari bw‘omubiri oburikwetwa yeast n‘obw‘ebinyonyi, okuteerana kwa GPI-AP n‘okwahukana n‘ebindi bihingwa bya plasma membrane byona nibibaho bitakahikire aha mutwe gw‘obutafaari. Paladino n’abandi. (48) bakashanga ngu aha TGN y‘obutafaari bw‘omubiri oburikwetwa mammalian polarized epithelial cells, okuteerana kwa GPI-AP tikurikwetengyesa ahabw‘okutoorana GPI-APs kuhika aha apical plasma membrane kwonka, kureka n‘okutebeekanisa okuteerana kwa GPI-APs n‘emirimo yaayo ey‘ebihangwa. Aha mutwe gw’obutafaari. Omu kitumbisa, okucondooza oku kukooreka ngu ekibiina kya GPI-AP ekirikurugiirira ahari C26 ceramide aha ER nikibaasa kutebeekanisa entebeekanisa y‘ekibiina n‘emirimo y‘emirimo ya GPI-AP aha nkora ya plasma (24, 49). Okurugiirira aha muringo ogu, obutafaari bwa GhLag1 nibugira allergy aha bizibu bya GPI nari emibazi erikuteganisa obuhame bw‘ekisiika ky‘obutafaari (28), kandi okwetenga kw‘ebirundo bya Gas1-GFP ebirikukora (49) eby‘omutwe ceramide ebirikuteekateekwaho omu kuteerana kw‘obutafaari bw‘ekitumbisa nibyoreka G Ebirikubaasa kuruga omu butafaari bw‘omubiri bwa hLag1. Enshobi ya GPI-AP. Kwonka, okwongyera kugyezaho kureeba yaaba entebeekanisa y‘emirimo y‘aha mutwe gw‘obutafaari ebaire etebeekanisiibwe kuruga omu ER omu muringo gw‘okushoroora kurugiirira aha bureingwa bw‘amaizi g‘omubiri nikiija kuba ekicweka ky‘okucondooza kwaitu okw‘omumaisho.
Ebika bya Saccharomyces cerevisiae ebyakozesiibwe omu murimo ogu biri omu runyiriri rwa S1. Ebika bya MMY1583 na MMY1635 ebya SCLIM eby’okuteera ebishushani by’obutafaari obuhuriire bikakorwa omu rubaju rwa W303. Ebika ebi ebirikworeka Sec13-mCherry ebiine akamanyiso ka puroteyini erikumurinkana bikakorwa barikukoresa omuringo gw’okugarukamu kw’omurunga (PCR) n’ekirungo kya pFA6a nk’eky’okureeberaho (23). Omuringo ogurikworeka Mid2-iRFP oguhandiikirweho puroteyini erikumurinkana ahansi y’obushoboorozi bw’omuhi w’obuhwezi wa GAL1 gukozirwe nk’oku gurikukurataho. PCR amplification y’omubaro gwa iRFP-KanMx kuruga omu pKTiRFP-KAN vector (ekiconco kya E. O’Shea, Addgene plasmid namba 64687; Bwanyima y’okukuratana kw’ebicweka by’omubiri ebya Mid2-iRFP kutunguuka kandi bikahindurwa omu nkora ya GAL1, bikateebwa omu mwanya gwa Not I-Sac I ogw’okuteerana plasmid pRS306. Plasmid eyarugiremu pRGS7 ekateebwa omu nkora ya Pst I kubaasa kuza omu mwanya gwa URA3.
Omubazi gw’okuteerana ogwa Gas1-GFP nigumanyisibwa ahansi y’obushoboorozi bw’omuhi w’obuhwezi wa GAL1 omu plasmid ya centromere (CEN), eyombekirwe nk’oku eri. Omubaro gwa Gas1-GFP gukagarurwamu amaani na PCR kuruga omu pRS416-GAS1-GFP (24) (ekiconco kya L. Popolo) kandi gwahindurwa omu mwanya gwa Xma I–Xho I ogwa CEN pBEVY-GL LEU2 (ekiconco kya C) . Omugabe; Namba y’ekirungo ky’omubiri 51225; 51225; RRID: Enyuguta_51225). Plasmid eyarugiremu ekaheebwa eiziina pRGS6. Omubazi gw’okuteerana ogwa Axl2-GFP nagwo nigworeka ahansi y’obushoboorozi bw’omuhi w’obuhwezi wa GAL1 ow’omubazi gwa pBEVY-GL LEU2, kandi okwombeka kwagwo kuri nk’oku. Omubaro gwa Axl2-GFP gukagarurwamu amaani kuruga omu pRS304-p2HSE-Axl2-GFP (23) kurabira omu PCR, kandi gwakorwa omu mwanya gwa Bam HI-Pst I ogw’omubazi gwa pBEVY-GL LEU2. Plasmid eyarugiremu ekaheebwa eiziina rya pRGS12. Omukuratana gw‘ebicweka by‘omubiri ebirikwetwa oligonucleotides ebyakozesiibwe omu kucondooza oku biri omu runyiriri rwa S2.
Omuringo ogu gukaba gwongyerwamu 0.2% adenine na 2% shukaari [YP-dextrose (YPD)], 2% raffinose [YP-raffinose] ebiriisa by’ekitumbisa ebirimu ebiriisa p (YP) ebiri ahagati (1 % ebiriisa by’ekitumbisa na 2% ebiriisa ept). (YPR)] nari 2% galactose [YP-galactose (YPG)] nk’entandikwa ya kaboni, nari omu nkora y’okukora ebintu ebikye munonga (0.15% ekitumbisa ky’omubazi gw’okukyebera na 0.5% eky’omubazi gw’okukyebera) kwongyera aha amino asidi n’ebirungo by’omubiri ebihikire ebirikwetengyesa omu by’okurya , Kandi ebirimu 2% y’ekitumbisa ky’omubazi gw’okukyebera2 nari shukaari y’omubiri ( (ekirungo kya galactose ekikozirwe omu nkora y’obuhangwa) nk’entandikwa ya kaboni.
Aha kushushanisa omu bwire obwabuzima, obutafaari oburikuhinduka oburikuhurira obutagatsi oburikworeka ebyombeko ebiri ahansi y’omuhi w’obuhwezi wa GAL1 bukakurira omu mwanya gwa YPR ahari 24°C ekiro kyona kuhika aha nkora ya mid-log. Bwanyima y’okuteebwa omu YPG ahari 24°C kumara eshaaha 1, obutafaari bukateebwa omu SG ahari 37°C kumara edakiika 30, reero bwatwarwa omu 24°C kubaasa kuruga omu secretion block. Concanavalin A ekakozesibwa kuboneza obutafaari aha rupapura rw’endabirwamu kandi ekashushanisibwa na SCLIM. SCLIM n’omugaati gw’ekikwato ky’okureeba ebiri omu mushana ekya Olympus IX-71 n’ekikwato ky’okureeba ebiri omu mushana ekya UPlanSApo 100×1.4 (Olympus), ekikwato ky’okupima ebiri omu sipiidi mpango kandi eky’omubaro gw’obumanyiso n’eiraka ry’ahaiguru ekirikuhindura ebiri omu itaka (Yokogawa Electric n’ekikwato ky’okupima ebiri omu itaka ekirikukoresibwa omu muringo gw’omutaano). (Hamamatsu Photonics) neebaasa kuha enkora y’okureeba ebiri omu maisho n’okutunguura okurikuhererukayo okwa ×266.7 hamwe na kamera y’ekikwato ekirimu eshagama erikukanyisa obutafaari bw’omubiri (Hamamatsu Photonics) (21). Okutunga ebishushani nikukorwa puroguraamu y’omutaano (Yokogawa Electric). Aha bishushani bya 3D, tukakoresa ekikwato ky’amashanyarazi ekikozirwe omu muringo gw’omutaano kutengeeta ekishushani ky’ekigyendererwa omu nkora, kandi twateerana ebicweka by’amaisho 100 nm kuruga ahari buri kimwe omu nshaho. Ekishushani kya Z-stack nikihindurwa omu 3D voxel data, kandi omurimo gw‘okujanjaaza enshonga z‘ebiteekateeko ogurikukoresibwa omu kucondooza kwa disiki erikwehindura nigukoresibwa omu kucondooza okurikukoresibwa puroguraamu ya Volocity (PerkinElmer). Omu kukozesa puroguraamu ya Volocity kukyebera omutindo gw‘okweshwijuma kw‘omwanya gumwe, ERES obariiremu emigugu ekapimwa. Okushwijuma kwa line scan kukakorwa barikukoresa puroguraamu ya MetaMorph (Ebyoma bya Molecular).
Koresa puroguraamu ya GraphPad Prism kubaasa kumanya obukuru bw‘embariirira. Aha kigyezo kya Student‘s t-test eky‘emikira ebiri n‘ekigyezo kya buriijo eky‘okushwijuma okuhinduka kw‘omuringo gumwe (ANOVA), entaaniso ahagati y‘ebicweka nibitwarwa nk‘ebiine akakwate kakuru na P <0.05 (*).
Aha nkora ya fluorescence microscopy ya Gas1-GFP, obutafaari bwa log phase bukakuzibwa ekiro kyona omu YPD kandi bwatorotoorwa omu nkora ya centrifugation, bwayozibwa emirundi ebiri na phosphate buffered saline, kandi bwateebwa aha ice kumara haakiri edakiika 15, reero bwagumizamu ahansi y’okukyebera nk’oku kyashoboororwa enyima (Check24). Ekikwato kya Leica DMi8 (HCX PL APO 1003/1.40 amajuta PH3 CS) ekirimu ekishushani ky’okureeba, ekicweka ky’okushwijuma L5 (GFP), kamera ya Hamamatsu hamwe na puroguraamu ya Application Suite X (LAS X) bikakoresibwa omu kutunga. .
Sampuli zikahindurwa omu nkora ya SDS sampuli buffer aha 65°C kumara edakiika 10, reero bwanyima zataanisibwa n’okukoresa SDS-polyacrylamide gel electrophoresis (PAGE). Aha kucondooza kwa immunoblotting, 10 μl y‘eky‘okureeberaho ekaba erimu buri layini. Omubazi gw’okubanza: Koresa omubazi gw’okurwanisa Gas1 ogw’akame aha nkora ya 1:3000, omubazi gw’okurwanisa Emp24 ogw’akame aha nkora ya 1:500, n’omubazi gw’okurwanisa GFP (ekiconco kuruga ahari H. Riezman) aha nkora ya 1:3000. Omubazi gw’embeba ogurikurwanisa Pgk1 gukakoresibwa omu nkora ya 1:5000 (ekiconco kuruga ahari J. de la Cruz). Ekirungo kya kabiri: Ekirungo ky’embuzi ekirikurwanisa obukooko G (IgG) ekirikukoresibwa omu nkora ya 1:3000 (Pierce). IgG y’embuzi erikurwanisa embeba erimu HRP ekakoresibwa aha nkora ya 1:3000 (Pierce). Ekicweka ky‘okugarukamu kw‘obutafaari bw‘omubiri kikakyeberwa omu muringo gw‘okukyebera ebiri omu mubiri ogwa SuperSignal West Pico (Thermo Fisher Scientific).
Nk‘oku kyashoboororwa omu (31), okucondooza kw‘obuhangwa okwa immunoprecipitation kukakorwa aha kicweka kya ER ekigaigahire. Omu bugufu, oyoze obutafaari bw’ekitumbisa n’ekirungo kya TNE [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM n’omubazi gw’okuzibira puroteyini) aha 600 nm ( OD600) aha 100 mM emirundi ebiri. Kikaba kihendagurika n’ebirungo by’endabirwamu, reero ebisigarira by’obutafaari n’ebirungo by’endabirwamu bikaihwaho barikukoresa okubiteera omu nkora ya centrifugation. Bwanyima, ekirungo eki kikaba kikyeberwa aha 17,000 g kumara edakiika 15 ahari 4°C. Empeho ekagaruka yateebwa omu TNE kandi digitalis saponin ekayongyerwamu kuhika aha nkora y’aha muheru eya 1%. Ekirungo eki kikateebwa omu nkora kumara eshaaha 1 n’okukihindura aha 4°C, reero ebicweka ebitarikubaasa kusheesheka bikaihwaho omu kukyebera aha 13,000 g ahari 4°C kumara edakiika 60. Aha kukyebera obutafaari bw’omubiri oburikurwanisa endwara ya Gas1-GFP, banza oteere sampo n’ebihimba bya agarose ebitarimu kintu kyona (ChromoTek) aha 4°C kumara eshaaha 1, reero oteere GFP-Trap_A (ChromoTek) aha 4°C kumara eshaaha 3. Ebirungo ebyabaire birimu obutafaari bw’omubiri bikayozibwa emirundi etaano na TNE erimu 0.2% digoxigenin, byakyeberwa na SDS sample buffer, byataanisibwa aha SDS-PAGE, kandi byakyeberwa omu muringo gw’okukyebera obutafaari bw’omubiri.
Nk‘oku kyashoboororwa omu (31), okucondooza okurikukwatanisa kukakorwa aha kicweka kya ER ekigaigahire. Omu bugufu, ekicweka kya ER ekigaigahire kikateebwa omu nkora ya 0.5 mM dithiobis (succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, USA; 20°C, edakiika 20). Enkora y‘okuteerana ekazimwa ahabw‘okutaho glycine (50 mM ey‘aha muheru, edakiika 5, 20°C).
Nkoku kyashobooroirwe enyima (50), okucondooza kwa MS okwa ceramide omu miringo y‘omukibira na GhLag1 kukakorwa. Omubugufu, obutafaari bukakura kuhika aha nkora y‘okukyebera (3 kuhika 4 OD600 units/ml) omu YPD ahari 30°C, kandi obutafaari 25×107 bukasharurwa. Okuhindura kwabyo omu mubiri nikuzimwa n’ekiriisa kya trichloroacetic acid. Koresa ekirungo ky‘okushohoza [etanoli, amaizi, eteri, piridiini na 4.2 N y‘omubazi gw‘okushohoza (15:15:5:1:0.018 v/v)] na 1.2 nmol y‘omutindo gw‘omunda C17 ceramide (860517, Avanti polar lipid) omutindo). Koresa ekirungo kya monomethylamine [methanol, amaizi, n-butanol n’omubazi gwa methylamine (4:3:1:5 v/v)] kukora okucondooza okukye okw’amaizi g’omubiri, reero okozese n-butanol erimu amaizi kusheesha omunyugunyu. Eky’aha muheru, ekicweka eki kikagaruka kyateebwa omu nkora nungi y’okushohoza [klorofoomu/methanol/amaizi (2:7:1) + 5 mM ammonium asetate] kandi kyateebwa omu nkora y’okupima obuhango bw’ebintu. Okucondooza aha nkora nyingi (MRM) kukakorwa ahabw‘okumanya n‘okupima obuhango bw‘ebicweka bya sphingolipid. Ekikwato kya TSQ Vantage ekya kashatu eky’okupima obuhango bw’omubiri (Thermo Fisher Scientific) kiine roboti y’okukyebera amaizi g’omubiri. Amaani g’okuteerana nigabonezibwa ahari buri kicweka kya ceramide. MS data ekatunga omu muringo murungi. Aha buri kimwe ekirikugarukamu omu bihangirwe, akamanyiso k‘amaizi g‘omubiri niko kari ahagati y‘ebipimo bishatu eby‘okwepimira.
Nkoku kyashoboororwa omu (31), obutafaari (800×107) oburikworeka Gas1-GFP bukateebwa omu nkora y‘obuhangwa ey‘okurwanisa endwara. Gas1-GFP eyashemeziibwe ekataanisibwa SDS-PAGE kandi yatwarwa omu nkora y’okusheeshasheesha (PVDF). Puroteni ekareebeka omu maisho barikugijwanzya PVDF n’erangi erikwiragura eya amide. Ekipande kya Gas1-GFP kikasharaho kuruga omu PVDF kandi kyayozibwa emirundi 5 na methanol kandi omurundi gumwe n’amaizi g’omutindo gw’okukyebera-MS (LC-MS). Omu kuteeka omubiri gw’omubiri n’ebicweka 500 μl 0.3 M NaOAc (pH 4.0), ekirungo ky’okukyebera n’ekirungo ky’okukyebera 500 μl ky’omubazi gw’okukyebera 1 M ogwa sodium nitrite 1 M oguherize kusheesheka aha 37°C kumara eshaaha 3, ekicweka ky’amaizi g’omubiri nikiruga omu Gas1-GFP n’ekirungo ky’okusheeshasheesha ahagati y’ekirungo ky’okusheeshasheesha. (51) Bwanyima y’ekyo, orunyiriri rwa membrane rukanaabwa emirundi ena n’amaizi g’omutindo gwa LC-MS, rwayomezibwa aha bwire bw’obutagatsi bw’omu kishengye, kandi rwabiikwa omu mwanya ogurimu nitrogen ahari -80°C kuhitsya obu rwakyeberwa. Nk‘okukyebera, sampo y‘ekibunu kya PVDF etaine kintu kyona ekakoresibwa ahari buri kugyezesa. Amashaju agarugire omu Gas1-GFP bwanyima gakakyeberwa MS nk‘oku kyashoboororwa (50). Omu bugufu, ebipande bya PVDF ebirimu GPI-lipid bikagaruka byateebwa omu 75μl y‘ekirungo ky‘ebizimba ebitari birungi [klorofoomu/methanol (1:2) + 5 mM ammonium asetate] kandi byahinguraho omu kucondooza kw‘amashanyarazi (ESI)-MRM/MS Okushwijuma ebika by‘amaizi g‘omubiri (TSQ Vantage). Omu muringo ogu, MS data ekatunga omu muringo gwa ion ezitari nungi.
Nk‘oku kyagambirweho enyima, ekicweka ky‘amaizi g‘omubiri eky‘enkomo ya GPI kikataanisibwa na [3H]-inositol-ekihandiikirweho GPI-AP (16). Amashaju gakataanisibwa omu nkora y’okukyebera ebiri omu nkora y’okukyebera (55:45:10 klorofoomu-methanol-0.25% KCl) kandi gakareebeka omu kureeba barikukoresa FLA-7000 (Fujifilm).
Obutafaari oburikworeka Gas1-GFP (600×107) bukanaabibwa emirundi ebiri na TNE buffer na TNE buffer, kandi bwahendagurika n’ebirungo by’endabirwamu, reero bwateebwa omu centrifuge kwihaho ebisigarira by’obutafaari n’ebirungo by’endabirwamu. Ekicweka ekirikuruga ahaiguru kikaba kikyeberwa aha 17,000 g kumara eshaaha 1 ahari 4°C. Ekirungo eki kikanaabwa omu TNE kandi kyateebwa omu nkora ya 1 U PI-PLC (Invitrogen) omu TNE erimu 0.2% ya digitali saponin kumara eshaaha 1 ahari 37°C. Bwanyima y’okujanjaba enzyme, omubiri gukaihwaho omu kukyebera aha 17,000 g ahari 4°C kumara eshaaha 1. Okubaasa kukyebera Gas1-GFP, ebiri aha mutwe bikateebwa omu nkora ya GFP-Trap_A (ChromoTek) aha 4°C ekiro kyona. Gas1-GFP eshemeziibwe eyataanisiibweho SDS-PAGE ekaba erimu erangi ya bururu erikumurinkana eya Coomassie. Ekipande ky’okutaho erangi ekya Gas1-GFP kikasharaho kuruga omu rangi erikutukura eyehinguririize omugyera gw’amaizi, reero bwanyima y’okukora alkylation na iodoacetamide n’okukyendeeza na dithiothreitol, okugabura omu gel na trypsin kukakorwa. Okurugamu kandi oyome ebihimba by’omubiri n’ebihimba ebirimu GPI-glycans. Peptide eyomire ekashohozibwa omu maizi 20 μl. Tamu ekicweka (8μl) omu LC. Enyomyo ya octadecylsilane (ODS) (Develosil 300ODS-HG-5; oburaingwa bw’omunda 150 mm×1.0 mm; Nomura Chemical, Aichi Prefecture, Japan) ekakoresibwa kutaanisa ebicweka by’omubiri ebiri omu itaka ahansi y’embeera z’okuhinduka ezitari zimwe na zimwe. Omuringo ogurikugyendera aha simu n‘ekirungo A (0.08% asidi ya formiki) n‘ekirungo B (0.15% asidi ya formiki omu 80% asetonitiri). Enkora ya Accela HPLC (Thermo Fisher Scientific, Boston, Massachusetts) ekakoresibwa kusheesha enyomyo n’ekirungo A omu dakiika 55 aha nkora y’okusheeshasheesha 50 μl min-1 kumara edakiika 5, reero bwanyima omubaro gw’ekirungo B gukeeyongyera kuhika ahari 40%. , omuri Amerika). Eluate ekaguma neetaaha omu nshonga y’eby’okurya bya ESI, kandi ebiriisa by’omubiri n’ebiriisa by’omubiri ebiine GPI-glycans bikakyeberwa LTQ Orbitrap XL (ekikwato ky’okupima obuhango bw’eby’okurya ebirimu ebiriisa by’omubiri; Thermo Fisher Scientific). Omu ntebeekanisa ya MS, amashanyarazi g‘enshonga y‘okusheeshasheesha gakateebwa ahari 4.5 kV, kandi obutagatsi bw‘okusheeshasheesha bw‘okuhingisa bukaguma ahari 300°C. Amashanyarazi g’omubiri (capillary) n’amaani g’omubiri (tube lens) gakateebwa ahari 15 V na 50 V, nk’oku biri. Ebihandiiko bya MS bikatunga omu muringo gwa ion nungi (okushoboorora kwa 60,000; okuhikiirira kw‘oburemeezi bw‘ebicweka 10 ahari buri miriyoni) omu nkora y‘oburemeezi bwa 300/m/z oburemeezi/omubaro gw‘okushashura (m/z) 3000. Ebihandiiko bya MS/MS nibitunga kurabira omu mutego gwa ion omu XLthe datas3 ey‘okubanza aha [1]. okutaana okurikureetwaho okuteerana (CID)].
Okugyeragyeranisa kwa MD kukakorwa barikukoresa puroguraamu ya GROMACS (52) n‘omusiri gw‘amaani gwa MARTINI 2 (53-55). Okwombeka kw’omubiri okwa CHARMM GUI (56, 57) bwanyima kukakoresibwa kwombeka omubiri gw’omubiri ogurimu ebiriisa bibiri (DOPC) na Cer C18 nari DOPC na Cer C26. Topology na coordinates za Cer C26 niziruga omu DXCE barikwihaho obubare oburikwongyerwaho kuruga omu mukira gwa sphingosine. Kozesa omuringo ogushobooroirwe ahaifo okugyeragyeranisa omutindo gw’emirundi ebiri kandi ogugyendese, reero okozese enkoragana z’aha muheru z’enkora kwombeka enkora erimu Emp24. Ekicweka ky’omubiri eky’ekitumbisa Emp24 (ebisigarira 173 kuhika 193) kikakorwa nk’ekicweka kya α-helix barikukoresa ekikwato ky’okureeba MD (VMD) eky’omutindo gw’ebicweka by’omubiri (58). Bwanyima, bwanyima y’okuihaho amashaju agarikuteerana, puroteyini ekakyeberwa omu muringo gw’obujuma kandi yaateebwa omu bilayer barikukoresa CHARMM GUI. Enkora y'aha muheru erimu DOPC 1202 na Cer C26 302 nari DOPC 1197 na Cer C18 na Emp24 295. Ionize enkora kuhika aha nkora ya 0.150M. Ebishushani bina eby’okwetongora bikakorwa ahabw’ebihandiiko bibiri ebya bilayer.
Ekicweka kya lipid bilayer nikigyeragyeranisibwa barikukoresa enkora ya CHARMM GUI, erimu okukyendeeza kandi bwanyima bakagyeragyeranisa amatembezo 405,000, ahu oburemeezi bw‘omwanya burikukyendeera mporampora kandi bukahwaho, kandi eitembezo ry‘obwire niriyongyerwa kuruga ahari 0.005 ps kuhika ahari 0.02 ps. Bwanyima y‘okugyeragyeranisa, nikireetaho 6 μs n‘eitembezo ry‘obwire rya 0.02 ps. Bwanyima y’okuta Emp24, kozesa omuringo nigwo gumwe ogwa CHARMM GUI kukyendeeza n’okugyeragyeranisa enkora, reero ogyende kumara 8 s omu kuhanga.
Aha nkora zoona, omu bwire bw‘okugyeragyeranisa, omuhimbo nigutegyekwa barostati ya Berendsen (59), kandi omu bwire bw‘okukora, omuhimbo nigutegyekwa barostati ya Parrinello-Rahman (60). Omu mbeera zoona, omubaro gw‘okuteerana ogwagati ni 1 bar kandi enkora y‘okuteerana omubaro gw‘okuteerana ogwa semi-isotropic neekoresibwa. Omu nkora y‘okugyeragyeranisa n‘okukora, ekikwato ky‘okugyeragyeranisa obutagatsi (61) ekiine okugaruka kugyeragyeranisa omuvuyo nikikoresibwa kugaita obutagatsi bwa puroteyini, amashaju n‘ebicweka by‘okushohoza nk‘oku biri. Omu bwire bwona obw’okukora, obutagatsi obugyendereirwe ni 310K. Enkoragana etari y‘okuteerana neebarwa omu kureetaho orunyiriri rw‘okuteerana barikukoresa enteekateeka ya Verlet erimu okugumisiriza kwa 0.005 buffer. Ekigambo kya Coulomb nikibarwa barikukoresa omusiri gw’okugarukamu n’oburaingwa bw’okusharaho 1.1 nm. Ekigambo Vander Waals nikikozesa enkora y‘okusharaho erimu orubaju rw‘okusharaho rwa 1.1 nm, kandi enkora y‘okusharaho eya Verlet neekoresibwa ahabw‘okusheesheka okurikubaasa kubaho (62).
Okukozesa VMD, oburaingwa bw‘omuyaga oburikusharaho ahagati y‘ebirungo bya DOPC phosphate nari ebirungo bya ceramide AM1 na puroteyini ni 0.7 nm, kandi omubaro gw‘amaizi agarikukwatagana na puroteyini nigubarwa. Okurugirira aha nkora egi, barira enshonga y‘okukyendeeza-okugaita (DE) nk‘oku eri omu (63): DE factor = (omubaro gw‘amaizi goona omu puroteyini 0.7) omu puroteyini 0.7 (omuhendo gwa Cer omu puroteyini yoona) .
Omuhendo ogurikugambwaho nigutunga nk‘omubaro gw‘ahagati, kandi emibaro y‘enshobi ni kopi ina ezirikwetongora eza SE. Obukuru bw‘embariirira y‘enshonga ya DE nibubarwa omu kukyebera t [(omubaro gwa DE-enshonga-1)/SE]. Barira omuhendo gwa P kuruga omu kugabanisa kw‘omukira gumwe.
Ekikwato kya GROMACS kikakozesibwa kubara maapu ya 2D lateral density y‘enkora erimu Emp24 omu bwire bwa 250 ns ezirikuhererukayo z‘okukuratira. Okubaasa kutunga maapu y‘okugaita/okukyendeeza aha ceramide, maapu y‘obuhango bwa Cer neebaganisibwamu omubaro gwa maapu ya Cer na DOPC, reero egabwamu n‘okuteerana kwa Cer omu mubiri. Ekipimo kya maapu y’erangi niyo emwe nikikoresibwa.
Okumanya ebihandiiko by’okwongeraho aha kihandiiko eki, reeba aha http://advances.sciencemag.org/cgi/ebirimu/omu bwijwire/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 Lorgihope -Linero), Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Ebishushani bya 3D eby‘obwire bw‘amazima eby‘omutindo gw‘ahaiguru nibyoreka obukuru bw‘oburaingwa bw‘orujegyere rwa ceramide ahabw‘okushoroora puroteyini omu myanya y‘okutooranamu ey‘okushohoza.
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 Lorgihope -Linero), Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Ebishushani bya 3D eby‘obwire bw‘amazima eby‘omutindo gw‘ahaiguru nibyoreka obukuru bw‘oburaingwa bw‘orujegyere rwa ceramide ahabw‘okushoroora puroteyini omu myanya y‘okutooranamu ey‘okushohoza.
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