He mea nui ka hoʻokaʻawale ʻana o ka protein ma ke ala huna no ka mālama ʻana i ka hoʻokaʻawale ʻana o nā cell a me ka homeostasis. Ma waho aʻe o ka hoʻokaʻawale ʻana i hoʻopili ʻia e ka pūpū, ʻo ke kuleana o nā lipids i ka hoʻokaʻawale ʻana o kinesin i ke kaʻina hana o ka lawe ʻana i nā mea huna he nīnau kumu lōʻihi ia i pane ʻole ʻia. Maanei, hana mākou i ka 3D simultaneous multicolor high-resolution real-time imaging e hōʻoia i loko o vivo ua hui pū ʻia nā protein glycosylphosphatidylinositol-immobilized hou me nā moieties lipid ceramide lōʻihi loa a hoʻokaʻawale ʻia i loko o nā endoplasms kūikawā Net exit site, kahi ʻokoʻa mai ka mea i hoʻohana ʻia e nā protein transmembrane. Eia kekahi, hōʻike mākou he mea koʻikoʻi ka lōʻihi o ke kaulahao o ka ceramide i loko o ka membrane reticulum endoplasmic no kēia koho ʻana o ka hoʻokaʻawale ʻana. Hāʻawi kā mākou haʻawina i ka hōʻike pololei mua in vivo e hoʻokaʻawale i nā ukana protein e pili ana i ka lōʻihi o ke kaulahao lipid i nā wahi hoʻokuʻu koho i ke ala huna.
I loko o nā pūnaewele eukaryotic, ua hoʻokaʻawale ʻia nā protein i hana ʻia i loko o ka endoplasmic reticulum (ER) i ka wā o ka lawe ʻana ma o ke ala huna no ka lawe ʻana i ko lākou wahi kūpono i ke kelepona (1). Ma waho aʻe o ka hoʻokaʻawale ʻana i hoʻopili ʻia e ka ʻaʻahu, ua lōʻihi ka manaʻo e hiki i kekahi mau lipid ke lawelawe ma ke ʻano he mau wahi puka koho ma ka hoʻohui ʻana iā lākou i loko o nā kikowaena membrane kikoʻī e pili ana i nā protein kikoʻī (2-5). Eia nō naʻe, aia nō ka nele o nā hōʻike pololei in vivo e hōʻoia i kēia ʻano hana lipid-based. I mea e hoʻoponopono ai i kēia pilikia kumu, ua aʻo mākou i ka hū pehea e hoʻokuʻu ʻokoʻa ʻia ai nā protein anchored glycosylphosphatidylinositol (GPI) (GPI-APs) mai ka ER. ʻO GPI-APs kahi ʻano o nā protein cell surface i hoʻopili ʻia me ka lipid (6, 7). ʻO GPI-AP kahi protein huna i hoʻopili ʻia i nā leaflet waho o ka membrane plasma ma o ka glycolipid moiety (GPI anchor). ʻAe lākou i nā heleuma GPI ma ke ʻano he mau hoʻololi post-translational conservative i ka lumen ER (8). Ma hope o ka hoʻopili ʻana, hele ʻo GPI-AP ma o ka Golgi apparatus (5, 9) mai ka ER a i ka membrane plasma. ʻO ke alo o nā heleuma GPI ke kumu e lawe ʻia ai ʻo GPI-AP ma kahi kaʻawale mai nā protein i hūnā ʻia e ka transmembrane (me nā protein membrane plasma ʻē aʻe) ma ke ala huna (5, 9, 10). I loko o nā cell yeast, hoʻokaʻawale ʻia nā GPI-AP mai nā protein i hūnā ʻia i loko o ka endoplasmic reticulum, a laila hoʻopili ʻia i loko o nā vesicles kū hoʻokahi i kāʻei ʻia e ka coat protein complex II (COPII) (6, 7). ʻAʻole maopopo nā mea hoʻoholo o kēia kaʻina hana hoʻokaʻawale i ke kaʻina hana hoʻokuʻu aku o ER, akā ua kuhi ʻia e koi paha kēia ʻano hana i nā lipids, ʻoiai ka hoʻoponopono hou ʻana o ka ʻāpana lipid o ka heleuma GPI (5, 8). I loko o ka yeast, hoʻomaka koke ka hoʻoponopono hou ʻana o ka lipid GPI ma hope o ka hoʻopili ʻana o ka GPI, a i nā manawa he nui, hoʻoulu ia i ka ceramide e hoʻopaʻa i ka 26-carbon long-chain saturated fatty acid (C26:0) (11, 12). ʻO C26 ceramide ka ceramide nui i hana ʻia e nā cell yeast a hiki i kēia manawa. Hoʻohui ʻia ia i loko o ka ER a ʻo ka hapa nui o ia mea e hoʻokuʻu ʻia aku i ka Golgi apparatus ma o nā vesicles COPII (13). ʻO ka hoʻokuʻu ʻana aku o ER o GPI-AP e koi pono ana i ka hoʻomau ʻana o ka synthesis ceramide (14, 15), a ma ka huli ʻana, ʻo ka hoʻololi ʻana o ka ceramide i ka inositol phosphate ceramide (IPC) i loko o ka Golgi apparatus e pili ana i ka synthesis anchor GPI (16). Ua hōʻike ʻia nā haʻawina biophysical me nā membrane artificial e hiki i nā ceramides kaulahao acyl lōʻihi loa ke hui pū e hana i nā kikowaena i kauoha ʻia me nā waiwai kino kū hoʻokahi (17, 18). Ke alakaʻi nei kēia mau ʻikepili i ka manaʻo e hoʻohana ana ʻo C26 ceramide a me GPI-AP me C26 ceramide i ko lākou mau waiwai kino e hui pū i nā ʻāpana a i ʻole nā ​​​​ʻāpana i kauoha ʻia i loko o kahi lipid membrane ER haunaele. Hoʻokumu nui ʻia ia me nā glycerolipids pōkole a unsaturated (C16: 1 a me C18: 1) (19, 20). E koho pono ʻia kēia mau ʻāpana ma nā wahi puka ER kikoʻī (ERES), kahi e hiki ai ke lawe pū ʻia ka ceramide a me ka GPI-AP i hoʻokumu ʻia i ka ceramide i ka Golgi ma ka vesicle COPII hoʻolaʻa like (5).
Ma kēia haʻawina, ua hoʻāʻo pololei mākou i kēia ʻano hana lipid-based ma ka hoʻohana ʻana i ka super-resolution confocal real-time imaging microscopy (SCLIM), ʻo ia kahi ʻenehana microscopy ʻoi loa e hiki ai ke nānā like i nā protein fluorescently i kapa ʻia ʻO nā kiʻi ʻekolu kala a me ʻekolu-dimensional (3D) he kiʻekiʻe loa ka hoʻonā a me ka wikiwiki i loko o nā cell ola (21, 22).
Ua hoʻopili mua mākou i ka ʻenehana SCLIM e wehewehe hou aku pehea i nānā ʻia ai ka GPI-AP maʻamau me ka hui ceramide C26 mai nā protein i hūnā ʻia e ka transmembrane ma hope o ka haʻalele ʻana i ka ER ma S. cerevisiae. I mea e nānā ai i ka hoʻokaʻawale ʻana o ER, ua hoʻohana mākou i kahi ʻōnaehana genetic e hiki ke nānā pololei i ka ukana i hoʻohui hou ʻia e komo ana i ka ERES in vivo (7, 23). Ma ke ʻano he ukana, ua koho mākou iā C26 ceramide-based GPI-AP Gas1 i kapa ʻia me ka protein fluorescent ʻōmaʻomaʻo (GFP) a me ka protein i hūnā ʻia e ka transmembrane Mid2 i kapa ʻia me ka protein fluorescent kokoke-infrared (iRFP), ʻo ia mau mea ʻelua e kuhikuhi ana i ka membrane plasma (24-26). I loko o ka mutant wela-sensitive sec31-1, ua hōʻike ʻia kēia mau ukana ʻelua ma lalo o kahi mea hoʻolaha galactose-inducible a me kahi māka ERES constitutive. I ka mahana loa (37°C), no ka mea, ʻo ka mutation sec31-1 e hoʻopilikia i ka hana o ka ʻāpana ʻaʻahu COPII ʻo Sec31 e kāohi i ka germination COPII a me ka ER export, hōʻiliʻili ka ukana i hoʻohui hou ʻia ma ka ER (23). Ma hope o ka hoʻomaʻalili ʻana i kahi mahana haʻahaʻa (24°C), ua hoʻōla ʻia nā pūnaewele mutant sec31-1 mai ka ʻāpana huna, a ua hoʻomaka ʻia ka lawe ʻia aku o ka ukana synthetic hou i hōʻiliʻili ʻia mai ka ER. Ua hōʻike ʻia ka ʻike ʻana o CLIM ua hōʻiliʻili mau ka hapa nui o nā Gas1-GFP a me Mid2-iRFP i hana hou ʻia i loko o ka ER o nā pūnaewele mutant sec31-1 ma hope o ka hoʻoulu ʻana ma 37°C a laila hoʻokuʻu ʻia ma 24°C no 5 mau minuke (Kiʻi 1). ʻOiai ua hoʻolaha ʻia ʻo Mid2-iRFP ma ka membrane ER holoʻokoʻa, a ua hoʻopaʻa ʻia ʻo Gas1-GFP a hōʻiliʻili ʻia ma ka ʻāpana membrane ER discontinuous, ʻokoʻa loa kā lākou hoʻolaha ʻana (Kiʻi 1, A a i C a me Movie S1). Eia kekahi, e like me ka mea i hōʻike ʻia ma ke Kiʻi 1D, ʻaʻohe Mid2-iRFP o ka hui Gas1-GFP. Hōʻike kēia mau hopena ua hoʻokaʻawale ʻia nā protein GPI-AP a me nā protein transmembrane i nā ʻāpana membrane ER like ʻole i ka wā mua. Aia ka hui Gas1-GFP ma kahi kokoke i kahi ERES kikoʻī i lepili ʻia me ka protein uhi COPII o mCherry ʻo Sec13 (Kiʻi 1, E a me F, a me ke kiʻiʻoniʻoni S1) (23).
Hōʻike nā pūnaewele sec31-1 i nā mea huna i hoʻokomo ʻia e ka galactose, kahi ceramide GPI-AP Gas1-GFP (GPI-AP, ʻōmaʻomaʻo) a me ka protein transmembrane Mid2-iRFP (TMP, polū) a ua hoʻomoʻa ʻia kēia lepili ERES Constructive Sec13-mCherry (ERES, magenta) ma 37°C no 30 mau minuke, neʻe ʻia i 24°C, a kiʻi ʻia e SCLIM 5 mau minuke ma hope. Hōʻike ʻo (A a C) i kahi kiʻi 2D i hui pū ʻia a hoʻokahi paha o kahi mokulele (A), kahi kiʻi projection 2D o 10 mau ʻāpana z (B) a i ʻole kahi kiʻi hemisphere kelepona 3D o ka ukana a me nā māka ERES (C). Pā unahi 1μm (A a me B). ʻO ke ana unahi he 0.551μm (C). Ua ʻike ʻia ʻo Gas1-GFP ma nā ʻāpana ER a i ʻole nā ​​​​hui, ʻoiai ua ʻike ʻia a hoʻolaha ʻia ʻo Mid2-iRFP ma o ka membrane ER (C). (D) Hōʻike ka pakuhi i ka ikaika fluorescence pili o Gas1-GFP a me Mid2-iRFP i loko o ka hui Gas1-GFP ma ka laina pua keʻokeʻo (hema). AU, ʻāpana kūʻokoʻa. Hōʻike ʻo (E a ​​me F) i ke kiʻi 3D e hoʻohui i nā waiwai a me ka māka ERES. Ua ʻike ʻia nā hui Gas1-GFP kokoke i ka ERES kikoʻī. ʻO ke anakahi unahi he 0.551μm. (F) Hōʻailona ka pua keʻokeʻo paʻa i ka hui Gas1-GFP e pili ana me ERES. Hōʻike nā panela waena a me ka ʻākau i ke kiʻi 3D i hoʻonui ʻia a me kahi hiʻohiʻona wili ʻia o ka hui Gas1-GFP i koho ʻia.
ʻO ka pilina pili loa ma waena o ka hui Gas1-GFP a me kahi ERES kikoʻī e hōʻike ana hiki iā Gas1-GFP ke komo i ka ERES koho, kahi ʻokoʻa mai ke koho ʻana i hoʻohana ʻia e Mid2-iRFP e haʻalele i ka ER. No ka hoʻoponopono ʻana i kēia hiki, ua helu mākou i ka lakio ERES no hoʻokahi a ʻelua paha waiwai (Kiʻi 2, A a C). Ua ʻike mākou ʻo ka hapa nui o ERES (70%) he hoʻokahi wale nō ʻano ukana. Hōʻike ke kiʻi ma lalo o ke Kiʻi 2C i ʻelua mau hiʻohiʻona maʻamau o ERES me Gas1-GFP wale nō (Kiʻi 1) a i ʻole Mid2-iRFP wale nō (Kiʻi 2). I ka hoʻohālikelike ʻana, ma kahi o 20% o ERES he ʻelua mau ukana e pili ana ma kahi like. Ua ʻike ʻia aia kekahi mau ERES (10%) he ʻelua ʻano ukana, akā ua hoʻokaʻawale ʻia lākou ma nā wahi like ʻole. No laila, hōʻike kēia loiloi helu ma hope o ka hoʻokuʻu ʻia ʻana o ka ER, ua māhele ʻia ʻo GPI-AP Gas1-GFP a me ka ukana transmembrane Mid2-iRFP i nā ERES like ʻole (Kiʻi 2D). He kūlike loa kēia pono hoʻokaʻawale ʻana me ka loiloi biochemical ma mua (6) a me ka hoʻoholo morphological (7). Hiki iā mākou ke nānā i ke ʻano o ka ukana i hoʻokaʻawale ʻia e komo ana i ka ERES (Kiʻi 2E a me Movie S2). Hōʻike ka Kiʻi 2E he hapa liʻiliʻi wale nō o Gas1-GFP (panel 3) a i ʻole Mid2-iRFP (panel 4) e komo ana i ka ERES mai kekahi ʻaoʻao a ua hoʻopaʻa ʻia i kahi ʻāpana discrete. Hōʻike ka Panel 5 o ka Kiʻi 2E i kekahi manawa loaʻa ʻo Gas1-GFP a me Mid2-iRFP i ka ERES like, akā komo lākou mai nā ʻaoʻao like ʻole a ua hoʻopaʻa ʻia i nā wahi kaʻawale e hōʻike ana i nā vesicles COPII like ʻole. Ua hōʻoia pū mākou ʻo ka hoʻokaʻawale ʻana a me ka hoʻokaʻawale ʻana o C26 ceramide-based GPI-AP Gas1 ma ke ʻano he ERES koho he kikoʻī no ka mea ʻo kekahi ukana huna transmembrane ʻē aʻe, ʻo ka protein membrane plasma i hōʻailona ʻia e GFP ʻo Axl2 (27), e hōʻike ana i ke ʻano like me Mid2-iRFP. (Kiʻi S1 a me Movie S3). Hoʻolaha ʻia ka Axl2-GFP i hana hou ʻia ma o ka membrane ER e like me Mid2-iRFP (Kiʻi S1, A a me B), a ua hui pū ʻia me Mid2-iRFP ma ka hapa nui o ERES (Kiʻi S1, B a i D). Nā Panel 1 a me 2 o ke Kiʻi 1. Hōʻike ʻo S1C i ʻelua mau hiʻohiʻona maʻamau o ERES kahi e hoʻopili ai ʻelua mau ukana transmembrane. I kēia mau hihia, komo pū nā waiwai ʻelua i ka ERES (Kiʻi S1E, Panel 3 a me Movie S3).
Ua kau ʻia nā pūnaewele sec31-1 e hōʻike ana i nā mea huna galactose inducible, ʻo Gas1-GFP (GPI-AP, ʻōmaʻomaʻo) a me Mid2-iRFP (TMP, polū) a me ka lepili ERES constitutive ʻo Sec13-mCherry (ERES, magenta) ma 37 Ma hope o ka hoʻomoʻa ʻana no 30 mau minuke ma °C, e neʻe i 24 °C e hoʻokuʻu i ka poloka huna, a kiʻi me SCLIM ma hope o 20 mau minuke. (A a i C) Nā kiʻi hoʻolālā 2D hōʻike (A; pā unahi, 1μm) a i ʻole nā ​​kiʻi hemisphere kelepona 3D (B a me C; ʻāpana unahi, 0.456μm) o ka ukana a me 10 mau ʻāpana z i hōʻailona ʻia e ERES. Hōʻike ka panela haʻahaʻa ma (B) a me ka panela ma (C) i nā kiʻi i hana ʻia e hōʻike wale i nā waiwai i loaʻa ma ERES (magenta) [Gas1-GFP (hina) a me Mid2-iRFP (polū māmā)]. (C) Pua hāmama: Hoʻokahi wale nō ʻāpana ukana a ERES e lawe ai (1 a 4). Pua hina: Loaʻa iā ERES ka ukana i hoʻokaʻawale ʻia (5). Pua paʻa keʻokeʻo: Loaʻa iā ERES ka ukana i hoʻokaʻawale ʻia. Ma lalo: ʻO ka ERES hoʻokahi i koho ʻia aia wale nō ʻo Gas1-GFP (1) a i ʻole Mid2-iRFP (2). Pā unahi, 100 nm. (D) Ka helu ʻana o ka photomicrograph i wehewehe ʻia ma (C). ʻO ka pakeneka awelika o ERES nona hoʻokahi ukana wale nō (Gas1-GFP a i ʻole Mid2-iRFP), ka ukana i hoʻokaʻawale ʻia a me ka ukana e pili ana. I loko o ʻekolu mau hoʻokolohua kūʻokoʻa, n=432 ma 54 mau cell. Pā hewa = SD. Hoʻāʻo t ʻelua huelo ʻole. *** P = 0.0002. (E) Kiʻi 3D o ERES i koho ʻia o ka ukana i hoʻokaʻawale ʻia i hōʻailona ʻia me (C). ʻO Gas1-GFP (ʻōmaʻomaʻo) (3) a i ʻole Mid2-iRFP (uliuli) (4) e komo i loko o ERES (magenta) mai kekahi ʻaoʻao a ua kaupalena ʻia i kahi wahi liʻiliʻi i loko o ERES. I kekahi manawa, komo nā ʻano ukana ʻelua i ka ERES like (5) mai ka ʻaoʻao like a ua hoʻopaʻa ʻia i kahi kaʻawale i loko o ka ERES. Pā unahi, 100 nm.
ʻO ka mea aʻe, ua hoʻāʻo mākou i kahi kuhiakau e hoʻokele ka ceramide acyl chain lōʻihi (C26) i loko o ka membrane ER i ka clustering kikoʻī a me ka hoʻokaʻawale ʻana o Gas1 i loko o ERES koho. No kēia hopena, ua hoʻohana mākou i kahi ʻano hū i hoʻololi ʻia ʻo GhLag1, kahi i pani ʻia ai nā synthases ceramide endogenous ʻelua ʻo Lag1 lāua ʻo Lac1 e GhLag1 (ka homolog Lag1 o ka pulupulu), e hopena ana i kahi ʻano hū me kahi membrane cell Ceramide strain pōkole ma mua o ke ʻano hihiu (Kiʻi 3A) (28). Ua hōʻike ka loiloi spectrometry mass (MS) i loko o nā ʻano hihiu, 95% o ka ceramide holoʻokoʻa he ceramide kaulahao lōʻihi loa (C26), ʻoiai ma GhLag1, 85% o ka ceramide he lōʻihi loa (C18 a me C16). ), 2% wale nō o ka ceramide he ceramide kaulahao lōʻihi loa (C26). ʻOiai ʻo C18 a me C16 ceramides nā ceramides nui i ʻike ʻia ma ka membrane GhLag1 a hiki i kēia manawa, ua hōʻoia pū ka loiloi MS aia ka heleuma GPI o Gas1-GFP i hōʻike ʻia ma ke ʻano GhLag1 i loko o ka C26 ceramide, kahi e like me nā lipids wild-type. Ua like ke ʻano (Fig. 3A) (26). No laila, ʻo ia hoʻi, ʻo ka enzyme remodeling ceramide Cwh43 he koho nui loa no C26 ceramide, e like me ka mea i hōʻike ʻia ma ke Kiʻi 26, makemake nui ia e hoʻokomo i ka heleuma GPI mai kahi liʻiliʻi o C26 ceramide i loko o ke ʻano GhLag1. S2 (29). Eia naʻe, ʻo ka membrane cell o GhLag1 wale nō ka C18-C16 ceramide, ʻoiai ʻo Gas1-GFP he C26 ceramide nō. ʻO kēia ʻoiaʻiʻo e hoʻolilo i kēia ʻano i mea hana kūpono e hoʻoponopono pono i ka pilikia o ka lōʻihi o ke kaulahao acyl o ka membrane ceramide i loko o ka ER. ʻO ke kuleana hypothetical o ka papa a me ka hoʻokaʻawale ʻana. A laila, ua aʻo mua mākou i ka hiki o C26 Gas1-GFP ke hōʻiliʻili i loko o nā hui ma GhLag1 me kahi allele mutant wela-sensitive o sec31-1 ma o ka microscopy fluorescence maʻamau, kahi wale nō ke kaulahao lōʻihi (C18-C16) i loaʻa i loko o ka membrane ER Ceramide (Kiʻi 3). Ua ʻike mākou ma sec31-1, ʻo ka hapa nui o Gas1-GFP i hoʻopaʻa ʻia i loko o nā hui, ʻoiai ʻo Gas1-GFP ma sec31-1 GhLag1 me ka membrane ceramide ER lōʻihi (C18-C16) ʻaʻole i hoʻopaʻa ʻia a hoʻolaha ʻia i loko o ka membrane ER holoʻokoʻa. I mea e pololei ai, no ka mea, pili loa ka clustering C26 ceramide i nā ERES kikoʻī (Kiʻi 1), ua noiʻi hou mākou inā paha e pili pū kēia kaʻina hana i ka hana o ka mīkini protein export ER. Hoʻohana ʻo GPI-AP i kahi ʻōnaehana COPII kūikawā no ka export ER, kahi i hoʻoponopono ikaika ʻia e ka hoʻoponopono hou ʻana o Ted1 i ka ʻāpana glycan o ka heleuma GPI (30, 31). A laila ʻike ʻia ka GPI-glycan recombinant e ka transmembrane cargo receptor p24 complex, kahi e koho ai iā Lst1, ʻo ia kahi isoform kikoʻī o ka subunit hoʻopaʻa ukana COPII nui ʻo Sec24, e hana ana i kahi GPI-AP-rich COPII Vesicles pono (31-33). No laila, ua kūkulu mākou i kahi mutant pālua i hoʻohui i ka holoi ʻana o kēia mau protein hoʻokahi (ka ʻāpana paʻakikī p24 ʻo Emp24, GPI-glycan remodeling enzyme Ted1 a me ka subunit COPII kikoʻī ʻo Lst1) me ke ʻano mutant sec31-1, a ua aʻo iā lākou Hiki ke hana i ka Gas1-cluster GFP (Kiʻi 3). Ua ʻike mākou ma sec31-1emp24Δ a me sec31-1ted1Δ, ʻo Gas1-GFP ka mea nui i hoʻopili ʻole ʻia a hoʻolaha ʻia ma loko o ka membrane ER, e like me ka mea i ʻike mua ʻia ma sec31-1 GhLag1, ʻoiai ma sec31-1lst1Δ, Gas1-GFP E like me sec31-1. Hōʻike kēia mau hopena ma waho aʻe o ke alo o C26 ceramide i loko o ka membrane ER, pono pū ka clustering o Gas1-GFP e hoʻopaʻa i ka complex p24, a ʻaʻole pono ka hoʻolimalima Lst1 kikoʻī. A laila, ua ʻimi mākou i ka hiki ke hiki i ka lōʻihi o ke kaulahao o ceramide i loko o ka membrane ER ke hoʻoponopono i ka hoʻopaʻa ʻana o Gas1-GFP i p24. Eia naʻe, ua ʻike mākou ʻaʻole pili ka alo o C18-C16 ceramide i loko o ka membrane i ka GPI-glycans i kūkulu hou ʻia e ka complex p24 (Nā Kiʻi S3 a me S4, A a me B) a i ʻole ka hoʻopaʻa ʻana iā GPI-AP a me ka hoʻokuʻu ʻana aku iā GPI-AP. hiki. Hoʻolimalima i ka subtype COPII Lst1 (Kiʻi S4C). No laila, ʻaʻole koi ka clustering C26 ceramide-dependent i nā pilina protein me nā ʻano hana protein hoʻokuʻu ER like ʻole, akā kākoʻo i kahi ʻano hana hoʻokaʻawale ʻē aʻe i hoʻokele ʻia e ka lōʻihi lipid. A laila, ua kālailai mākou inā he mea nui ka lōʻihi o ke kaulahao ceramide acyl i loko o ka membrane ER no ka hoʻokaʻawale pono ʻana o Gas1-GFP ma ke ʻano he ERES koho. ʻOiai ʻo Gas1 i loko o ke ʻano GhLag1 me ka ceramide kaulahao pōkole e haʻalele ana i ka ER a komo i loko o ka membrane plasma (Kiʻi S5), ke manaʻoʻiʻo nei mākou inā e hoʻokele ʻia ka hoʻokaʻawale ʻana e ka lōʻihi o ke kaulahao ceramide acyl, hiki ke hoʻohuli hou ʻia a keʻa ʻia ka Gas1 i loko o ke ʻano GhLag1. Nā waiwai ERES me ka membrane like.
(A) ʻO ka membrane cell o GhLag1 ka mea nui loa i nā ceramides C18-C16 pōkole, ʻoiai ʻo ka heleuma GPI o Gas1-GFP e loaʻa mau ana ka C26 IPC like me nā cell wild-type. Ma luna: ka loiloi lōʻihi o ke kaulahao acyl o ka ceramide i loko o ka membrane cell o nā ʻano wild-type (Wt) a me GhLag1p e ka spectrometry mass (MS). Hōʻike ka ʻikepili i ka pakeneka o ka ceramide holoʻokoʻa. ʻO ka awelika o ʻekolu mau hoʻokolohua kūʻokoʻa. Kahe hewa = SD. Hoʻāʻo t ʻelua-tailed unpaired. **** P <0.0001. Panel lalo: Ka loiloi MS o ka lōʻihi o ke kaulahao acyl o ka IPC i loaʻa i loko o ka heleuma Gas1-GFP (GPI-IPC) GPI i hōʻike ʻia i loko o nā ʻano wild-type a me GhLag1p. Hōʻike ka ʻikepili i ka pakeneka o ka hōʻailona IPC holoʻokoʻa. ʻAwelike o ʻelima mau hoʻokolohua kūʻokoʻa. Kahe hewa = SD. Hoʻāʻo t ʻelua-tailed unpaired. ns, ʻaʻole koʻikoʻi. P = 0.9134. (B) Ua hoʻoulu ʻia nā micrographs fluorescence o sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ a me sec31-1lst1Δ e hōʻike ana i ka Gas1-GFP i hoʻokomo ʻia i ka galactose ma 37°C no 30 mau minuke a ua hoʻoili ʻia i lalo e Hana i ka microscopy fluorescence maʻamau ma hope o 24°C. Pua keʻokeʻo: ER Gas1-GFP cluster. Pua hāmama: Ua hoʻolaha ʻia ʻo Unclustered Gas1-GFP ma ka membrane ER holoʻokoʻa, e hōʻike ana i ke ʻano o ka ER nuclear ring staining. Pā unahi, 5μm. (C) Ka helu ʻana o ka photomicrograph i wehewehe ʻia ma (B). ʻO ka pakeneka awelika o nā cell me ka punctate Gas1-GFP structure. I loko o ʻekolu mau hoʻokolohua kūʻokoʻa, n≥300 mau cell. Pā hewa = SD. Hoʻāʻo t ʻelua-tailed unpaired. **** P ＜0.0001.
No ka hoʻoponopono pololei ʻana i kēia pilikia, ua hana mākou i ka nānā ʻana o SCLIM o Gas1-GFP a me Mid2-iRFP ma GhLag1 me ka allele mutant sec31-1 temperature-sensitive (Kiʻi 4 a me Movie S4). Ma hope o ka mālama ʻia ʻana o ka ER ma 37°C a ma hope iho ua hoʻokuʻu ʻia ma 24°C, ʻaʻole i hui pū ʻia a hoʻolaha ʻia ka hapa nui o ka Gas1-GFP i hana hou ʻia ma loko o ka membrane ER, e like me ka mea i ʻike ʻia e nā microscopes maʻamau (Kiʻi 4, A a me B). Eia kekahi, ʻo ka pakeneka nui o ERES (67%) e komo pū ana me ʻelua ʻano ukana i hoʻonoho pū ʻia ma loko (Kiʻi 4D). Hōʻike nā Panels 1 a me 2 o ke Kiʻi 4C i ʻelua mau hiʻohiʻona maʻamau o ERES me ka Gas1-GFP a me Mid2-GFP e uhi ana. Eia kekahi, ua hoʻopaʻa inoa ʻia nā waiwai ʻelua i loko o ka ERES like (Kiʻi 4E, panel 3 a me ke kiʻiʻoniʻoni S4). No laila, hōʻike kā mākou mau hopena ʻo ka lōʻihi o ke kaulahao ceramide acyl i loko o ka membrane ER he mea nui ia e hoʻoholo ai i ka hōʻuluʻulu ʻana a me ka hoʻokaʻawale ʻana o ka protein ER.
ʻO nā pūnaewele Sec31-1 GhLag1 e hōʻike ana i nā mea huna i hoʻokomo ʻia e ka galactose, Gas1-GFP (GPI-AP, ʻōmaʻomaʻo) a me Mid2-iRFP (TMP, polū) a me ka constitutive ERES-labeled Sec13-mCherry (ERES, magenta) E hoʻomoʻa ma 37°C. E hoʻomau no 30 mau minuke, e hoʻohaʻahaʻa i 24°C e hoʻokuʻu i nā mea huna, a kiʻi me SCLIM ma hope o 20 mau minuke. (A a i C) Nā kiʻi hoʻolālā 2D hōʻike (A; pā unahi, 1μm) a i ʻole nā ​​kiʻi hemisphere kelepona 3D (B a me C; ʻāpana unahi, 0.45μm) o nā ʻāpana z 10 i hōʻailona ʻia e ka ukana a me ERES. ʻO ka panela haʻahaʻa ma (B) a me ka panela ma (C) e hōʻike i nā kiʻi i hana ʻia e hōʻike wale i nā waiwai i loaʻa ma ERES (magenta) [Gas1-GFP (hina) a me Mid2-iRFP (polū māmā)]. (C) Pua piha keʻokeʻo: ERES, pili nā waiwai. Pua hāmama: Hoʻokahi wale nō mea i loko o ERES. Panel haʻahaʻa: Loaʻa i ka ERES i koho ʻia nā waiwai e pili ana (1 a me 2) i hōʻailona ʻia ma (C). Pā unahi, 100 nm. (D) Ka helu ʻana o ka photomicrograph i wehewehe ʻia ma (C). Ma nā ʻāpana sec31-1 a me sec31-1 GhLag1, hoʻokahi wale nō ukana (Gas1-GFP a i ʻole Mid2-iRFP) i hoʻokomo ʻia, a me ka pakeneka awelika o ERES no ka ukana i hoʻokaʻawale ʻia a me ka ukana e pili ana. Ma nā hoʻokolohua kūʻokoʻa ʻekolu, n = 432 ma 54 mau pūnaewele (sec31-1) a me n = 430 ma 47 mau pūnaewele (sec31-1 GhLag1). Pā hewa = SD. Hoʻāʻo t ʻelua huelo ʻole. *** P = 0.0002 (sec31-1) a me ** P = 0.0031 (sec31-1 GhLag1). (E) Kiʻi 3D o ERES i koho ʻia me ka ukana e pili ana (3) i hōʻailona ʻia ma (C). Hoʻokokoke ʻo Gas1-GFP (ʻōmaʻomaʻo) a me Mid2-iRFP (uliuli) iā ERES (magenta) mai ka ʻaoʻao like a noho ma ka wahi i kaupalena ʻia ʻo ERES like. Pā unahi, 100 nm.
Hāʻawi kēia haʻawina i nā hōʻike pololei in vivo e hoʻokaʻawale ʻia nā ukana protein lipid-based i nā wahi hoʻokuʻu koho ma ke ala huna, a hōʻike i ke koʻikoʻi o ka lōʻihi o ke kaulahao acyl no ke koho ʻana i ka hoʻokaʻawale ʻana. Ma ka hoʻohana ʻana i kahi ʻenehana microscopy ikaika a ʻoi loa i kapa ʻia ʻo SCLIM, ua hōʻike mākou i ka Gas1-GFP i hana hou ʻia (kahi membrane plasma nui GPI-AP me kahi ʻāpana lipid ceramide acyl chain (C26) lōʻihi loa) i loko o ka hū) ʻO nā wahi i hui pū ʻia i loko o nā ER discrete e pili ana me nā ERES kikoʻī, ʻoiai ua hoʻolaha ʻia nā protein i hūnā ʻia e ka transmembrane ma o ka membrane ER (Kiʻi 1). Eia kekahi, komo kēia mau ʻano waiwai ʻelua i nā ERES like ʻole me ke koho (Kiʻi 2). Hoʻemi ʻia ka lōʻihi o ke kaulahao acyl o ka ceramide cellular i loko o ka membrane mai C26 a i C18-C16, ua hoʻopau ʻia ka hui Gas1-GFP i loko o ka ʻāpana ER discrete, a ua hoʻololi ʻia ʻo Gas1-GFP e haʻalele i ka ER me ka protein transmembrane ma o ka ERES like (Kiʻi 3 a me ke Kiʻi 3). 4).
ʻOiai ʻo GPI-AP e hoʻohana ana i kahi ʻano protein kūikawā e puka i waho o ER, ua ʻike mākou ʻaʻole hilinaʻi ka hoʻokaʻawale ʻana o C26 ceramide-dependent i nā pilina protein like ʻole e alakaʻi ai i ka hoʻokaʻawale ʻana o ERES (Nā Kiʻi S4 a me S5). Akā, kākoʻo kā mākou mau ʻike i kahi ʻano hoʻokaʻawale ʻē aʻe i hoʻokele ʻia e ka hui ʻana o ka protein lipid-based a me ka hoʻokaʻawale ʻana o nā ukana ʻē aʻe. Hōʻike kā mākou mau nānā ʻana ʻaʻole i loaʻa i ka ʻāpana Gas1-GFP a i ʻole ka hui e pili ana me kahi ERES kikoʻī ka protein transmembrane secreted Mid2-iRFP, e hōʻike ana e kōkua ka hui GPI-AP C26 ceramide-dependent i ko lākou komo ʻana i loko o ka ERES pili, a i ka manawa like, e kāpae i ka transmembrane. Hoʻokomo nā mea huna i kēia ERES kikoʻī (Nā Kiʻi 1 a me 2). I ka hoʻohālikelike ʻana, ʻo ke alo o nā ceramides C18-C16 i loko o ka membrane ER ʻaʻole ia e hoʻokumu i ka GPI-AP i nā ʻāpana a i ʻole nā ​​hui, no laila ʻaʻole lākou e kāpae a pani paha i nā protein transmembrane secreted i loko o ka ERES like (Nā Kiʻi 3 a me 4). . No laila, ke hāpai nei mākou e hoʻokele ʻo C26 ceramide i ka hoʻokaʻawale ʻana a me ka hoʻokaʻawale ʻana ma o ka hoʻomaʻamaʻa ʻana i ka hui ʻana o nā protein i hoʻopili ʻia i nā ERES kikoʻī.
Pehea e hoʻokō ai i kēia hui ʻana o C26 ceramide-dependent i loko o kahi ER kikoʻī? ʻO ke ʻano o ka membrane ceramide e hoʻokaʻawale i ka ʻaoʻao e hiki ai iā GPI-AP a me C26 ceramide ke hana i nā lipids liʻiliʻi a hoʻonohonoho koke ʻia i loko o kahi lipid ʻoi aku ka maʻamau ʻole o ka membrane ER e loaʻa ana nā glycerolipids pōkole a unsaturated. Nā hui maikaʻi (17, 18). Hiki ke hoʻohui hou ʻia kēia mau hui liʻiliʻi liʻiliʻi i loko o nā hui nui a paʻa ma hope o ka hoʻopaʻa ʻana i ka hui p24 (34). E kūlike me kēia, ua hōʻike mākou e pono ʻo C26 Gas1-GFP e launa pū me ka hui p24 e hana i nā hui ʻike nui aʻe (Kiʻi 3). ʻO ka hui p24 he oligomer heterozygous i haku ʻia me ʻehā mau protein transmembrane p24 like ʻole i loko o ka hū (35), kahi e hāʻawi ai i ka hoʻopaʻa multivalent, hiki ke alakaʻi i ka cross-linking o nā hui GPI-AP liʻiliʻi, no laila e hana ana i ka hui Stable nui aʻe (34). Hiki i ka pilina ma waena o nā ectodomains protein o GPI-APs ke kōkua i kā lākou hōʻuluʻulu ʻana, e like me ka mea i hōʻike ʻia i ka wā o kā lākou lawe ʻana iā Golgi i loko o nā pūnaewele epithelial polarized mammalian (36). Eia naʻe, i ka wā e loaʻa ai ka ceramide C18-C16 i loko o ka membrane ER, i ka wā e hoʻopaʻa ai ka hui p24 iā Gas1-GFP, ʻaʻole e hoʻokumu ʻia nā hui nui kaʻawale. Hiki ke hilinaʻi ʻia ke ʻano hana ma lalo o nā waiwai kino a me nā kemika kikoʻī o ka ceramide kaulahao acyl lōʻihi. Hōʻike nā haʻawina biophysical o nā membrane artificial ʻoiai ʻo nā ceramide kaulahao acyl lōʻihi (C24) a me ka pōkole (C18-C16) hiki ke hoʻokaʻawale i ka pae, ʻo nā ceramide kaulahao acyl lōʻihi wale nō (C24) hiki ke hoʻolaha i ka Curvature kiʻekiʻe a me ke kūlou kiʻiʻoniʻoni e hoʻoponopono hou i ke kiʻiʻoniʻoni. Ma o ka ʻōlelo like ʻana (17, 37, 38). Ua hōʻike ʻia ʻo ka helix transmembrane o TMED2, ka homologue kanaka o Emp24, e launa pū me ka sphingomyelin C18 ceramide-based i loko o nā lobules cytoplasmic (39). Ma ka hoʻohana ʻana i nā simulations molecular dynamics (MD), ua ʻike mākou ua hōʻiliʻili nā ceramides C18 a me C26 a puni nā lobules cytoplasmic o ka Emp24 transmembrane helix, a he like ko lākou makemake (Kiʻi S6). He mea kūpono ke hoʻomaopopo ʻana e hōʻike ana kēia e hiki i ka transmembrane helix o Emp24 ke alakaʻi i ka hoʻolaha asymmetric o nā lipids i loko o ka membrane. He hopena hou kēia e pili ana i nā cell mammalian. Hōʻike pū nā simulations MD like i ke alo o nā lipids ether (40). No laila, ke kuhi nei mākou ua hoʻonui ʻia ka C26 ceramide i loko o nā lobules ʻelua o ER26 ma kahi kūloko. Ke hoʻopaʻa pololei ʻo GPI-AP i loko o nā lobules luminal i ka multivalent p24 a me ka hōʻiliʻili ʻana o C26 ceramide a puni p24 i loko o nā lobules cytoplasmic, hiki iā ia ke hoʻolaha i ka hui pū ʻana o ka Protein a me ka curvature membrane i hana ʻia ma o nā manamana lima (41), e hoʻokaʻawale ana iā GPI-AP i nā ʻāpana discrete e pili ana iā ERES, kahi e makemake nui ai i nā ʻāpana curvature kiʻekiʻe o ka membrane ER (42). Ua kākoʻo nā hōʻike mua i ke ʻano hana i manaʻo ʻia (43, 44). ʻO ka hoʻopaʻa ʻana o nā oligolectins, nā pathogens a i ʻole nā ​​​​antibodies i nā glycosphingolipids (GSL) i hoʻokumu ʻia i ka ceramide ma ka membrane plasma e hoʻoulu ai i ka hoʻohuihui GSL nui, hoʻonui i ka hoʻokaʻawale ʻana o ka pae a hoʻoulu i ka deformation membrane a me ka internalization (44). ʻO Iwabuchi etc. (43) Ua ʻike ʻia i ke alo o nā kaulahao acyl lōʻihi (C24) akā ʻaʻole pōkole (C16), ʻo ka ligand multivalent i hoʻopaʻa ʻia i ka GSL lactosylceramide i hoʻoulu ai i ka hoʻokumu ʻana o nā hui nui a me ka membrane invagination, a ʻo ka cytoplasm Lyn-mediated signal transduction ma nā leaflet ua interdigitated e nā kaulahao acyl i loko o nā neutrophils i hui pū ʻia.
I loko o nā pūnaewele epithelial polarized mammalian, ʻo ka nui o ka pūnaewele anti-Golgi (TGN) i ka pae o ka membrane plasma apical e kāohi i ka hoʻokaʻawale ʻana a me ka hoʻokaʻawale ʻana o GPI-AP (10, 45). Hoʻokele ʻia kēia hōʻuluʻulu ʻana e ka oligomerization GPI-AP (36), akā hiki ke hilinaʻi ʻia i ka lōʻihi o ke kaulahao ceramide a mākou e ʻike ai i ka hū. ʻOiai ʻo ka GPI-AP mammalian he heleuma ether lipid-based, a ʻokoʻa loa kona ʻano kemika mai ka ceramide kaulahao acyl lōʻihi loa, ua ʻike ʻia kahi noiʻi hou nei he mau waiwai kino a me ke kemika like nā lipids ʻelua a me ka hana (40). No laila, ʻo ka ʻāpana lipid ether i loko o nā pūnaewele mammalian e like paha me ka ceramide C26 i ka hū, a ʻo kāna kuleana ke hui pū me ka ceramide kaulahao lōʻihi i loko o ka membrane e hoʻolaha i ka hōʻuluʻulu ʻana a me ka hoʻokaʻawale ʻana o GPI-AP. ʻOiai pono e hoʻāʻo pololei ʻia kēia hiki, kākoʻo nā ʻike mua ʻaʻole i lawe ʻia ka lawe ʻana o ka ceramide kaulahao acyl lōʻihi i ke kino Golgi e nā protein hoʻoili cytoplasmic, akā hilinaʻi ʻia i ka synthesis o nā heleuma GPI e like me ka hū. No laila, ʻike ʻia ka ʻano hana conservative evolutionary e hiki ke koho like i ka lawe ʻana i ka ceramide acyl chain lōʻihi loa a me GPI-AP (13, 16, 20, 46, 47) i loko o ka vesicle lawe like.
I loko o nā ʻōnaehana cell epithelial polarized yeast a me mammalian, ʻo ka hōʻuluʻulu ʻana o GPI-AP a me ka hoʻokaʻawale ʻana mai nā protein membrane plasma ʻē aʻe e hana ʻia ma mua o ka hiki ʻana i ka ʻili o ke kelepona. Ua ʻike ʻo Paladino et al. (48) ma ka TGN o nā cell epithelial polarized mammalian, ʻaʻole pono wale ka hui ʻana o GPI-AP no ka hoʻokaʻawale koho ʻana o GPI-APs i ka membrane plasma apical, akā hoʻoponopono pū i ka hoʻonohonoho hui ʻana o GPI-APs a me kāna hana olaola. ʻIli o ke kelepona. I loko o ka yeast, ua hōʻike kēia haʻawina e hiki i ka hui GPI-AP e hilinaʻi ana i ka ceramide C26 ma ka ER ke hoʻoponopono i ka hoʻonohonoho hui a me ka hana hana o GPI-AP ma ka membrane plasma (24, 49). E kūlike me kēia kumu hoʻohālike, he maʻi āpau nā cell GhLag1 i nā mea hoʻopaʻa GPI a i ʻole nā ​​​​lāʻau lapaʻau e hoʻopilikia i ka pono o ka paia o ke kelepona (28), a ʻo ka pono no nā hui Gas1-GFP hana (49) o ka tip ceramide i hoʻolālā ʻia i ka hui ʻana o nā cell yeast e hōʻike ana i nā hopena physiological hiki o nā cell hLag1. hewa GPI-AP. Eia nō naʻe, ʻo ka hoʻāʻo hou ʻana inā ua hoʻolālā ʻia ka hoʻonohonoho hana o ka ʻili o ke kelepona mai ka ER e kahi ʻano hoʻokaʻawale e pili ana i ka lōʻihi o ka lipid ke kumuhana o kā mākou noiʻi e hiki mai ana.
Ua helu ʻia nā ʻano Saccharomyces cerevisiae i hoʻohana ʻia ma kēia hana ma ka Papa S1. Ua kūkulu ʻia nā ʻano MMY1583 a me MMY1635 o SCLIM no ke kiʻi ʻana i nā cell ola ma ke kua o W303. Ua kūkulu ʻia kēia mau ʻano e hōʻike ana iā Sec13-mCherry me kahi hōʻailona protein fluorescent me ka hoʻohana ʻana i kahi ʻano hana polymerase chain reaction (PCR) me ka pFA6a plasmid ma ke ʻano he template (23). Ua kūkulu ʻia ke ʻano e hōʻike ana iā Mid2-iRFP i lepili ʻia me ka protein fluorescent ma lalo o ka mana o ka mea hoʻolaha GAL1 penei. Hoʻonui ʻia ka PCR o ka moʻo iRFP-KanMx mai ka vector pKTiRFP-KAN (makana a E. O'Shea, helu plasmid Addgene 64687; http://n2t.net/addgene: 64687; mea hōʻike kumuwaiwai noiʻi (RRID): Addgene_64687) A hoʻokomo ʻia i loko o ka C-terminus o ka endogenous Mid2. Ma hope o ka hoʻonui ʻia ʻana o ka moʻo genome Mid2-iRFP a me ke cloning ʻia i loko o ka mea hoʻolaha GAL1, ua hoʻohui ʻia i loko o ka pūnaewele Not I-Sac I o ka plasmid hoʻohui pRS306. Ua hoʻohālikelike ʻia ka plasmid hopena pRGS7 me Pst I e hoʻohui i loko o ka locus URA3.
Ua hōʻike ʻia ka gene fusion Gas1-GFP ma lalo o ka mana o ka mea hoʻolaha GAL1 i loko o ka plasmid centromere (CEN), i kūkulu ʻia penei. Ua hoʻonui ʻia ka moʻo Gas1-GFP e PCR mai ka plasmid pRS416-GAS1-GFP (24) (makana a L. Popolo) a ua cloned ʻia i loko o ka pūnaewele Xma I–Xho I o ka plasmid CEN pBEVY-GL LEU2 (makana a C). Miller; helu plasmid Addgene 51225; http://n2t.net/addgene: 51225; RRID: Addgene_51225). Ua kapa ʻia ka plasmid hopena ʻo pRGS6. Ua hōʻike pū ʻia ka gene fusion Axl2-GFP ma lalo o ka mana o ka mea hoʻolaha GAL1 o ka vector pBEVY-GL LEU2, a penei kona kūkulu ʻana. Ua hoʻonui ʻia ke kaʻina Axl2-GFP mai ka plasmid pRS304-p2HSE-Axl2-GFP (23) e PCR, a ua cloned ʻia i loko o ka Bam HI-Pst I site o ka vector pBEVY-GL LEU2. Ua kapa ʻia ka plasmid hopena ʻo pRGS12. Ua helu ʻia ke kaʻina o nā oligonucleotides i hoʻohana ʻia ma kēia haʻawina ma ka Papa S2.
Ua hoʻohui ʻia ke ʻano me 0.2% adenine a me 2% glucose [YP-dextrose (YPD)], 2% raffinose [YP-raffinose] rich yeast extract protein p (YP) medium (1% Yeast extract a me 2% protein ept). (YPR)] a i ʻole 2% galactose [YP-galactose (YPG)] ma ke ʻano he kumu kalapona, a i ʻole ma kahi medium minimal synthetic (0.15% yeast nitrogen base a me 0.5% ammonium sulfate) e hoʻohui i nā waikawa amino kūpono a me nā kumu e pono ai no ka meaʻai, a loaʻa iā ia ka 2% glucose (synthetic glucose minimal medium) a i ʻole 2% galactose (synthetic galactose minimal medium) ma ke ʻano he kumu kalapona.
No ke kiʻi ʻana i ka manawa maoli, ua ulu ʻia nā pūnaewele mutant sec31-1 i ʻike ʻia i ka mahana e hōʻike ana i ke kūkulu ʻana ma lalo o ka mea hoʻolaha GAL1 i loko o ka medium YPR ma 24°C i ka pō a hiki i ka wā waena o ka log. Ma hope o ka hoʻokomo ʻia ʻana i loko o YPG ma 24°C no 1 hola, ua hoʻomoʻa ʻia nā pūnaewele i loko o SG ma 37°C no 30 mau minuke, a laila ua hoʻoili ʻia i 24°C e hoʻokuʻu mai ka poloka huna. Ua hoʻohana ʻia ʻo Concanavalin A e hoʻopaʻa i nā pūnaewele ma kahi slide aniani a kiʻi ʻia e SCLIM. ʻO SCLIM kahi hui pū ʻana o ka microscope fluorescence inverted Olympus IX-71 a me ka UPlanSApo 100 × 1.4 numerical aperture oil lens (Olympus), ka mīkini hoʻopili disc rotating wikiwiki a me ka ratio kiʻekiʻe-signal-to-noise (Yokogawa Electric), spectrometer maʻamau, a me ka hoʻoluʻu maʻamau. Hiki i ka mea hoʻoikaika kiʻi o ka ʻōnaehana (Hamamatsu Photonics) ke hāʻawi i kahi ʻōnaehana lens hoʻonui me ka hoʻonui hope loa o × 266.7 a me kahi kāmela hāmeʻa i hoʻopili ʻia e hoʻonui i nā electrons (Hamamatsu Photonics) (21). Hana ʻia ka loaʻa ʻana o ke kiʻi e ka polokalamu maʻamau (Yokogawa Electric). No nā kiʻi 3D, ua hoʻohana mākou i kahi actuator piezoelectric i hana ʻia e hoʻoluliluli i ka lens objective vertical, a hōʻiliʻili i nā ʻāpana optical 100 nm ke kaʻawale i loko o kahi stack. Hoʻololi ʻia ke kiʻi Z-stack i ʻikepili voxel 3D, a ʻo ka hana theoretical point spread i hoʻohana ʻia no ka microscope confocal disc rotating e hoʻohana ʻia no ka hana deconvolution e ka polokalamu Volocity (PerkinElmer). Ma ka hoʻohana ʻana i ka polokalamu Volocity e hoʻopaʻa ponoʻī i ka palena no ka nānā ʻana i ka wahi like, ua ana ʻia ʻo ERES me ka ukana. Ua hana ʻia ka nānā ʻana i ka laina scan me ka hoʻohana ʻana i ka polokalamu MetaMorph (Molecular Devices).
E hoʻohana i ka polokalamu GraphPad Prism e hoʻoholo ai i ke koʻikoʻi helu. No ka hoʻāʻo t-ʻelua-tailed Student a me ka hoʻāʻo loiloi hoʻokahi ala maʻamau o ka variance (ANOVA), ua manaʻo ʻia nā ʻokoʻa ma waena o nā hui he hopena koʻikoʻi ma P <0.05 (*).
No ka microscopy fluorescence o Gas1-GFP, ua ulu ʻia nā pūnaewele log phase i ka pō ma YPD a hōʻiliʻili ʻia ma o ka centrifugation, holoi ʻia i ʻelua manawa me ka phosphate buffered saline, a hoʻomoʻa ʻia ma luna o ka hau no ka liʻiliʻi he 15 mau minuke, a laila hoʻomau ʻia ma lalo o ka microscope e like me ka mea i wehewehe mua ʻia Check (24). Ua hoʻohana ʻia ka microscope Leica DMi8 (HCX PL APO 1003/1.40 aila PH3 CS) i lako me kahi lens objective, kānana L5 (GFP), kāmela Hamamatsu a me ka polokalamu Application Suite X (LAS X) no ka loaʻa ʻana. .
Ua hoʻokaʻawale ʻia nā laʻana me ka SDS sample buffer ma 65°C no 10 mau minuke, a laila hoʻokaʻawale ʻia e ka SDS-polyacrylamide gel electrophoresis (PAGE). No ka nānā ʻana i ka immunoblotting, ua hoʻouka ʻia he 10 μl o ka laʻana no kēlā me kēia ala. ʻO ka antibody mua: E hoʻohana i ka rabbit polyclonal anti-Gas1 ma kahi dilution o 1:3000, rabbit polyclonal anti-Emp24 ma kahi dilution o 1:500, a me ka rabbit polyclonal anti-GFP (he makana mai H. Riezman) ma kahi dilution o 1:3000. Ua hoʻohana ʻia ka antibody monoclonal anti-Pgk1 mouse ma kahi dilution o 1:5000 (he makana mai J. de la Cruz). ʻO ka antibody lua: Horseradish peroxidase (HRP) conjugated goat anti-rabbit immunoglobulin G (IgG) i hoʻohana ʻia ma kahi dilution o 1:3000 (Pierce). Ua hoʻohana ʻia ka HRP-conjugated goat anti-mouse IgG ma kahi dilution o 1:3000 (Pierce). Ua ʻike ʻia ka ʻāpana pane pale ma o ke ʻano chemiluminescence o ka reagent SuperSignal West Pico (Thermo Fisher Scientific).
E like me ka mea i wehewehe ʻia ma (31), ua hana ʻia kahi hoʻokolohua immunoprecipitation kūlohelohe ma ka ʻāpana ER i hoʻonui ʻia. I ka pōkole, holoi i nā cell yeast me ka TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride a me ka hui protease inhibitor) ma 600 nm (OD600) ma 100 optical density ʻelua. Ua wāwahi ʻia me nā momi aniani, a laila ua wehe ʻia nā ʻōpala cell a me nā momi aniani ma o ka centrifugation. A laila ua centrifuged ʻia ka supernatant ma 17,000 g no 15 mau minuke ma 4°C. Ua hoʻokuʻu hou ʻia ka pellet ma TNE a ua hoʻohui ʻia ka digitalis saponin i kahi ʻano hope loa o 1%. Ua hoʻomoʻa ʻia ka suspension no 1 hola me ka rotation ma 4°C, a laila ua wehe ʻia nā ʻāpana insoluble ma o ka centrifugation ma 13,000 g ma 4°C no 60 mau minuke. No ka immunoprecipitation Gas1-GFP, e hoʻomoʻa mua i ka laʻana me nā momi agarose hakahaka (ChromoTek) ma 4°C no 1 hola, a laila e hoʻomoʻa me GFP-Trap_A (ChromoTek) ma 4°C no 3 hola. Ua holoi ʻia nā momi immunoprecipitated i ʻelima mau manawa me TNE e loaʻa ana he 0.2% digoxigenin, eluted me ka SDS sample buffer, hoʻokaʻawale ʻia ma SDS-PAGE, a kālailai ʻia e ka immunoblotting.
E like me ka mea i wehewehe ʻia ma (31), ua hana ʻia ka hoʻoholo ʻana o ke cross-linking ma ka ʻāpana ER i hoʻonui ʻia. I ka pōkole, ua hoʻoulu ʻia ka ʻāpana ER i hoʻonui ʻia me 0.5 mM dithiobis (succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, USA; 20°C, 20 min). Ua hoʻopau ʻia ka hopena crosslinking ma ka hoʻohui ʻana i ka glycine (50 mM hope loa, 5 mau minuke, 20°C).
E like me ka mea i wehewehe mua ʻia (50), ua hana ʻia ka loiloi MS o ka ceramide i nā ʻano wild-type a me GhLag1. I ka pōkole, ua ulu ʻia nā cell i ka pae exponential (3 a 4 OD600 units/ml) ma YPD ma 30°C, a ua ʻohi ʻia he 25 × 107 mau cell. Hoʻopau ʻia kā lākou metabolism me ka trichloroacetic acid. E hoʻohana i ka mea hoʻoheheʻe unuhi [ethanol, wai, ether, pyridine a me 4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] a me 1.2 nmol o ka maʻamau kūloko C17 ceramide (860517, Avanti polar lipid) maikaʻi). E hoʻohana i ka monomethylamine reagent [methanol, wai, n-butanol a me ka methylamine solution (4:3:1:5 v/v)] e hana i ka hydrolysis alkaline akahai o ka extract, a laila e hoʻohana i ka n-butanol i hoʻomaʻemaʻe ʻia i ka wai e hoʻopau i ka paʻakai. ʻO ka mea hope loa, ua hoʻoulu hou ʻia ka ʻāpana i loko o kahi mea hoʻoheheʻe ʻano maikaʻi [chloroform/methanol/wai (2:7:1) + 5 mM ammonium acetate] a ua hoʻokomo ʻia i loko o ka spectrometer mass. Ua hana ʻia ka nānā ʻana i ka Multi-reaction (MRM) no ka ʻike ʻana a me ka helu ʻana o nā molekala sphingolipid. Ua lako ka TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) me kahi kumu ion nanoflow robotic Nanomate HD (Advion Biosciences, Ithaca, NY) no ka nānā ʻana i ka lipid. Ua hoʻonohonoho ʻia ka ikehu collision no kēlā me kēia māhele ceramide. Ua loaʻa ka ʻikepili MS ma ke ʻano maikaʻi. No kēlā me kēia replicate biological, ʻo ka hōʻailona lipid ka waena o ʻekolu mau ana kūʻokoʻa.
E like me ka mea i wehewehe ʻia ma (31), ua hoʻokomo ʻia nā cell (800 × 107) e hōʻike ana iā Gas1-GFP i ka immunoprecipitation kūlohelohe. Ua hoʻokaʻawale ʻia ka Gas1-GFP i hoʻomaʻemaʻe ʻia e SDS-PAGE a ua hoʻoili ʻia i kahi membrane polyvinylidene fluoride (PVDF). Ua ʻike ʻia ka protein ma ka hoʻoluʻu ʻana i ka PVDF me ka ʻeleʻele amide. Ua ʻoki ʻia ke kāʻei Gas1-GFP mai ka PVDF a holoi ʻia i 5 mau manawa me ka methanol a hoʻokahi manawa me ka wai chromatography-MS (LC-MS). Ma ka hoʻomoʻa ʻana i ka ʻāpana membrane me 500μl 0.3 M NaOAc (pH 4.0), buffer a me 500μl hui hou ʻia 1 M sodium nitrite ma 37 ° C no 3 mau hola, ua hoʻokuʻu ʻia ka ʻāpana lipid mai Gas1-GFP a ua lysed Hoʻokuʻu ʻia o ka inosine phosphate ceramide ma waena o glucosamine a me inositol (51). Ma hope o kēlā, ua holoi ʻia ka ʻāpana membrane ʻehā manawa me ka wai LC-MS, hoʻomaloʻo ʻia i ka mahana o ka lumi, a mālama ʻia i loko o kahi lewa nitrogen ma -80°C a hiki i ka nānā ʻana. Ma ke ʻano he kaohi, ua hoʻohana ʻia kahi hāpana hakahaka o ka membrane PVDF no kēlā me kēia hoʻokolohua. A laila ua kālailai ʻia ka lipid i unuhi ʻia mai Gas1-GFP e MS e like me ka mea i wehewehe ʻia (50). I ka pōkole, ua hoʻoulu hou ʻia nā ʻāpana PVDF i loaʻa iā GPI-lipid i loko o 75μl solvent mold maikaʻi ʻole [chloroform/methanol (1:2) + 5 mM ammonium acetate] a ua hala i ka electrospray ionization (ESI)-MRM/MS Analysis o nā ʻano sphingolipid (TSQ Vantage). I kēia hihia, ua loaʻa ka ʻikepili MS ma ke ʻano ion maikaʻi ʻole.
E like me ka mea i ʻōlelo ʻia ma mua, ua hoʻokaʻawale ʻia ka ʻāpana lipid o ka heleuma GPI mai ka GPI-AP i hōʻailona ʻia me ka [3H]-inositol (16). Ua hoʻokaʻawale ʻia nā lipid e ka chromatography papa lahilahi me ka hoʻohana ʻana i kahi ʻōnaehana solvent (55:45:10 chloroform-methanol-0.25% KCl) a ua nānā ʻia me ka hoʻohana ʻana iā FLA-7000 (Fujifilm).
Ua holoi ʻia nā pūnaewele e hōʻike ana i ka Gas1-GFP (600 × 107) i ʻelua manawa me ka TNE buffer me ka TNE buffer, a ua wāwahi ʻia me nā momi aniani, a laila centrifuged e wehe i nā ʻōpala cell a me nā momi aniani. A laila centrifuged ka supernatant ma 17,000 g no 1 hola ma 4 ° C. Ua holoi ʻia ka pellet ma TNE a hoʻomoʻa ʻia me 1 U PI-PLC (Invitrogen) ma TNE e loaʻa ana ka 0.2% digitalis saponin no 1 hola ma 37 ° C. Ma hope o ka mālama ʻana i ka enzyme, ua wehe ʻia ka membrane ma ka centrifugation ma 17,000 g ma 4 ° C no 1 hola. No ka immunoprecipitate Gas1-GFP, ua hoʻomoʻa ʻia ka supernatant me GFP-Trap_A (ChromoTek) ma 4 ° C i ka pō. Ua hoʻoluʻu ʻia ka Gas1-GFP i hoʻomaʻemaʻe ʻia i hoʻokaʻawale ʻia e SDS-PAGE me ka uliuli ʻālohilohi Coomassie. Ua ʻoki ʻia ke kāʻei hoʻoluʻu Gas1-GFP mai ka hina e hoʻopuni ana i ka aqueduct, a laila ma hope o ka alkylation me ka iodoacetamide a me ka hoʻemi ʻana me ka dithiothreitol, ua hana ʻia ka ʻeli ʻana i loko o ka gel me ka trypsin. E unuhi a hoʻomaloʻo i nā peptides tryptic a me nā peptides me GPI-glycans. Ua hoʻoheheʻe ʻia ka peptide maloʻo i loko o 20 μl o ka wai. E hoʻokomo i kahi ʻāpana (8μl) i loko o ka LC. Ua hoʻohana ʻia kahi kolamu octadecylsilane (ODS) (Develosil 300ODS-HG-5; ke anawaena o loko 150 mm × 1.0 mm; Nomura Chemical, Aichi Prefecture, Iapana) e hoʻokaʻawale i nā peptides ma lalo o nā kūlana gradient kikoʻī. ʻO ka pae mobile he solvent A (0.08% formic acid) a me ka solvent B (0.15% formic acid i loko o 80% acetonitrile). Ua hoʻohana ʻia kahi ʻōnaehana Accela HPLC (Thermo Fisher Scientific, Boston, Massachusetts) e hoʻokahe i ke kolamu me ka mea hoʻoheheʻe A i loko o 55 mau minuke ma ka nui o ke kahe ʻana o 50 μl min-1 no 5 mau minuke, a laila ua hoʻonui ʻia ka nui o ka mea hoʻoheheʻe B i 40%. , ʻAmelika Hui Pū ʻIa). Ua hoʻokomo mau ʻia ka eluate i loko o ke kumu ion ESI, a ua kālailai ʻia nā peptides tryptic a me nā peptides me GPI-glycans e LTQ Orbitrap XL (hybrid linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific). I ka hoʻonohonoho MS, ua hoʻonohonoho ʻia ke anakahi uila o ke kumu capillary i 4.5 kV, a ua mālama ʻia ka mahana o ke capillary hoʻoili ma 300°C. Ua hoʻonohonoho ʻia ke anakahi uila capillary a me ke anakahi uila lens tube i 15 V a me 50 V, kēlā me kēia. Ua loaʻa ka ʻikepili MS ma ke ʻano ion maikaʻi (hoʻonā o 60,000; pololei o ka nuipa he 10 ʻāpana no ka miliona) ma kahi pae nuipa o 300/m/z lakio nuipa/hoʻopiʻi (m/z) 3000. Loaʻa ka ʻikepili MS/MS ma o ka pahele ion ma ka LTQ Orbitrap XL [nā huahelu 3 mua e hilinaʻi ai ka ʻikepili, collision induced dissociation (CID)].
Ua hana ʻia nā hoʻohālikelike MD me ka hoʻohana ʻana i ka polokalamu GROMACS (52) a me ke kahua ikaika MARTINI 2 (53-55). A laila ua hoʻohana ʻia ka CHARMM GUI Membrane Builder (56, 57) e kūkulu i kahi bilayer e loaʻa ana ka dioleoylphosphatidylcholine (DOPC) a me Cer C18 a i ʻole DOPC a me Cer C26. Loaʻa ka topology a me nā hoʻonohonoho o Cer C26 mai DXCE ma ka wehe ʻana i nā beads keu mai ka huelo sphingosine. E hoʻohana i ke kaʻina hana i wehewehe ʻia ma lalo nei e kaulike i ka papa pālua a holo iā ia, a laila e hoʻohana i nā hoʻonohonoho hope loa o ka ʻōnaehana e kūkulu i kahi ʻōnaehana e loaʻa ana iā Emp24. Ua kūkulu ʻia ka ʻāpana transmembrane o ka yeast Emp24 (nā koena 173 a 193) ma ke ʻano he α-helix me ka hoʻohana ʻana i ka visual MD (VMD) tool molecular structure (58). A laila, ma hope o ka wehe ʻana i nā lipids overlapping, ua granulated coarsely ka protein a hoʻokomo ʻia i loko o ka bilayer me ka hoʻohana ʻana iā CHARMM GUI. Aia i loko o ka ʻōnaehana hope loa he 1202 DOPC a me 302 Cer C26 a i ʻole 1197 DOPC a me 295 Cer C18 a me Emp24. E hoʻohui i ka ʻōnaehana i kahi nui o 0.150M. Ua hana ʻia ʻehā mau hana hoʻohālikelike kūʻokoʻa no nā haku mele bilayer ʻelua.
Hoʻohālikelike ʻia ka lipid bilayer me ka hoʻohana ʻana i ke kaʻina hana CHARMM GUI, kahi e pili ana i ka hoʻēmi ʻana a laila ke kaulike ʻana i 405,000 mau ʻanuʻu, kahi e hoʻemi mālie ʻia ai nā palena kūlana a hoʻopau ʻia, a hoʻonui ʻia ka ʻanuʻu manawa mai 0.005 ps a i 0.02 ps. Ma hope o ke kaulike ʻana, hana ia i 6 µs me kahi ʻanuʻu manawa o 0.02 ps. Ma hope o ka hoʻokomo ʻana iā Emp24, e hoʻohana i ke kaʻina hana CHARMM GUI like e hoʻēmi a kaulike i ka ʻōnaehana, a laila holo no 8 s i ka hana ʻana.
No nā ʻōnaehana āpau, i ka wā o ke kaʻina hana kaulike, ua kāohi ʻia ke kaomi e ka barostat Berendsen (59), a i ka wā o ke kaʻina hana, ua kāohi ʻia ke kaomi e ka barostat Parrinello-Rahman (60). I nā hihia āpau, ʻo ke kaomi awelika he 1 bar a hoʻohana ʻia kahi papahana hoʻopili kaomi semi-isotropic. I ke kaʻina hana kaulike a me ka hana ʻana, hoʻohana ʻia kahi thermostat (61) me ka recalibration wikiwiki e hoʻopili i ka mahana o ka protein, lipid a me nā ʻāpana solvent. I ka wā o ka hana holoʻokoʻa, ʻo ka mahana i manaʻo ʻia he 310K. Ua helu ʻia ka pilina non-bonding ma ka hana ʻana i kahi papa inoa pairing me ka hoʻohana ʻana i ka papahana Verlet me ka 0.005 buffer tolerance. Ua helu ʻia ka huaʻōlelo Coulomb me ka hoʻohana ʻana i ke kahua hopena a me kahi mamao ʻoki o 1.1 nm. Hoʻohana ka huaʻōlelo Vander Waals i kahi papahana ʻoki me kahi mamao ʻoki o 1.1 nm, a hoʻohana ʻia ka papahana ʻoki Verlet no ka drift hiki (62).
Me ka hoʻohana ʻana iā VMD, ʻo ka nalu ʻoki ma waena o nā beads phosphate DOPC a i ʻole nā ​​beads ceramide AM1 a me ka protein he 0.7 nm, a ua helu ʻia ka helu o nā lipids e launa pū me ka protein. Wahi a ke ʻano hana ma lalo nei, e helu i ka depletion-enrichment (DE) factor e like me (63): DE factor = (ka nui o nā lipids āpau i loko o ka protein 0.7) i loko o ka protein 0.7 (ka nui o Cer i loko o nā lipids āpau)
Loaʻa ka waiwai i hōʻike ʻia ma ke ʻano he awelika, a ʻo nā kaha hewa he ʻehā kope kūʻokoʻa o SE. Ua helu ʻia ke koʻikoʻi helu o ka kumu DE e ka hoʻāʻo t [(averageDE-factor-1)/SE]. E helu i ka waiwai P mai ka hoʻolaha hoʻokahi-tailed.
Ua hoʻohana ʻia ka mea hana GROMACS e helu i ka palapala ʻāina density 2D o ka ʻōnaehana e loaʻa ana iā Emp24 i loko o nā 250 ns hope loa o ke kaha. I mea e loaʻa ai ka palapala ʻāina enrichment/depletion o ceramide, ua puʻunaue ʻia ka palapala ʻāina density o Cer e ka huina o ka palapala ʻāina o Cer a me DOPC, a laila ua puʻunaue ʻia e ka nui o Cer i loko o ke kino. Hoʻohana ʻia ka unahi palapala ʻāina kala like.
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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 Wagez -Linero (Sergio Lopez), Misaho Lopez (Sergio Lopez) Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Hōʻike ka kiʻi manawa maoli kiʻekiʻe kiʻekiʻe 3D i ke koʻikoʻi o ka lōʻihi o ke kaulahao ceramide no ka hoʻokaʻawale ʻana i nā protein ma nā wahi hoʻopuka koho.
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 Wagez -Linero (Sergio Lopez), Misaho Lopez (Sergio Lopez) Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Hōʻike ka kiʻi manawa maoli kiʻekiʻe kiʻekiʻe 3D i ke koʻikoʻi o ka lōʻihi o ke kaulahao ceramide no ka hoʻokaʻawale ʻana i nā protein ma nā wahi hoʻopuka koho.
©2020 ʻAmelika Hui Pū ʻIa no ka holomua o ka ʻepekema. Ua mālama ʻia nā pono āpau. ʻO AAAS he hoa pili o HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef a me COUNTER. ScienceAdvances ISSN 2375-2548.