Kusalazya ma protein munzila yakugusya ma protein nchintu chipati loko mukubamba maseelo kuti atabi kabotu. Mukuyungizya kukusalazya kwiinda mukubelesya chikumba, mulimo wamafuta mukusalazya kinesin munzila yakunyamuna zintu zili mumubili mubuzyo mupati uutanaingulwa kwachiindi chilamfu. Aawa, twachita zifwanikiso zya 3D zyamibala myiingi ziboneka kabotu muchiindi cheenzu kutondezya mumubili kuti ma protein mapya aapangidwe a glycosylphosphatidylinositol-immobilized ajisi zibeela zilamfwu zya ceramide lipid zilabungikwa akubikkwa muzibeela zya endoplasms zilibedelede Net exit site, eeyo yiindene aya ma protein aabelesyegwa a transmembrane. Mukuyungizya, tutondezya kuti bulamfwu bwaketani ya ceramide mumubili wa endoplasmic reticulum bulayandika kapati mukusala ooku. Buvwuntauzi bwesu bupa bumboni bwakusaanguna bwakusandula ma protein kweendelanya abwaanda bwamafuta muzibeela zyakutumizya kunze munzila yakuzwa.
Mumaseelo aa eukaryotic, ma protein akapangwa mu endoplasmic reticulum (ER) mpawo asalazyigwa muchiindi chakweenzya kwiinda munzila ya secretory kuti asike kubusena bwawo (1). Mukuyungizya kukusalazya kwiinda mukubelesya chikumba, kwakali kuyeeyelwa kwachiindi chilamfu kuti mafuta amwi alakonzya kubeleka mbuli nzila zyakuzwa kwiinda mukuzibikka antoomwe muzibeela zya membrane zyama puloteni amwi (2-5). Nokuba boobo, kuchili kubula bumboni bwakusinizya kuti nzila eeyi ilakonzya kuchitwa amafuta. Kuti tumanizye penzi eeli, twakaiya mu yisiti mbuli glycosylphosphatidylinositol (GPI) anchored proteins (GPI-APs) mbozigwisigwa kuzwa ku ER. GPI-APs ninzila zyaandeene zya ma protein aali atala aamaseelo ( 6, 7). GPI-AP nipuloteni iizwa anze ya plasma kwiinda mu glycolipid moiety (GPI anchor). Balatambula GPI anchors mbuli kusanduka kwakumbele kwakusandululwa mu ER lumen (8). Nikwakamana kujatanisigwa, GPI-AP yainda muGolgi apparatus (5, 9) kuzwa ku ER kuya ku plasma membrane. Kubawo kwa GPI anchors kupa kuti GPI-AP itolwe kaandaanizyidwe kuzwa kuma protein azwa mumubili (kubikkilizya ama puloteni aambi aa plasma membrane) munzila yakuzwa (5, 9, 10). Mumaseelo aa yisiti, GPI-APs zilaandaanizyigwa kuzwa kuli zimwi zipuloteni zili mu endoplasmic reticulum, mpawo zilabikkwa muzibeela zilibedelede zizingulukidwe a coat protein complex II (COPII) (6, 7). Zintu zipa kuti kube nzila eeyi yakusanganya munzila yakutumina kunze aa ER tazili aantangalala pe, asi kuli kuyeeyelwa kuti nzila eeyi ilakonzya kuyandika mafuta, ikapati kubamba bupange bwacibeela camafuta cha GPI anchor (5, 8). Mubumena, kubambulula mafuta aa GPI kutalika mpawo-mpawo GPI niyakamana, alimwi muziindi zyiingi, kupa kuti ceramide ijatane a 26-carbon long-chain saturated fatty acid (C26:0) (11, 12). C26 ceramide ngo ceramide mupati uupangwa amaseelo aa yisiti kusikila lino. Ilapangwa mu ER mpawo bunji bwayo ilatumwa ku Golgi apparatus kwiinda mu COPII vesicles (13). Kutuminwa kwa ER kwa GPI-AP kuyandika kuzumanana kupanga ceramide (14, 15), mpawo, kusanduka kwa ceramide kuya ku inositol phosphate ceramide (IPC) muGolgi apparatus kuyeeme akupanga GPI anchor (16). ziiyo zya biophysical aama membrane aapangidwe zyakatondezya kuti ma acyl chain ceramides malamfu loko alakonzya kuswaangana kuchita zibeela zijisi bube bulibedelede (17, 18). Eeyi data yakapa kuti kube muzeezo wakuti C26 ceramide antoomwe a GPI-AP a C26 ceramide babelesya bube bwazyo kuti baswaangane mumasena aali kabotu naakuti mumasena aali mubusena butakwe ntenda bwa ER membrane lipid. Ikapati ilijisi ma glycerolipids mafwaafwi alimwi aatakwe busani (C16:1 a C18:1) (19, 20). Masena aaya ayoobikkilwa maanu kumasena amwi aakuzwa mu ER (ERES), ooko ceramide a ceramide-based GPI-AP nkozikonzya kunyangwa antoomwe ku Golgi mu COPII vesicle yomwe (5).
Mukuvwuntauzya ooku, twakasunka nzila eeyi iibelesya mafuta kwiinda mukubelesya super-resolution confocal real-time imaging microscopy (SCLIM), eeyo ninzila ya microscopy iikonzya kubona ma protein aalembedwe a fluorescent aciindi comwe.
Twakasaanguna kubelesya nzila ya SCLIM kuti tuyungizye kupandulula mbuli GPI-AP iili kabotu antoomwe a C26 ceramide group mboyakali kuvwuntauzyigwa kuzwa kuma protein aakazwa mumubili nikwakazwa mu ER mu S. cerevisiae. Kuti tubone kwaandaanya kwa ER, twakabelesya nzila yama gene iikonzya kubona zintu zipya zyakabambwa kazinjila mu ERES mumubili (7, 23). Mbuli mukuli, twakasala C26 ceramide-based GPI-AP Gas1 iilembedwe a green fluorescent protein (GFP) antoomwe a transmembrane secreted protein Mid2 iilembedwe a near-infrared fluorescent protein (iRFP), zyoonse zyobile zilanga plasma membrane (24–26). Mu sec31-1 mutant iikonzya kumvwa kupya, eezyi zintu zyobile zilatondezyegwa munsi lya galactose-inducible promoter antoomwe a constitutive ERES marker. Aawo kakupya kapati (37°C), nkaambo kakuti sec31-1 mutation ilajatikizya mulimo wa COPII coat component Sec31 kuti ilesye COPII kumena akutumwa kwa ER, zintu zipya zyakabambwa zilabungika ku ER (23). Nikwakamana kutontola kusika aawo mpokutontola (24°C), maselu aa sec31-1 akasanduka akazwa kubusena bwakuvwumbilila, mpawo zintu zipya zyakabungikwa zyakatalika kuzwa ku ER. Kulanga kwa CLIM kwakatondezya kuti bunji bwa Gas1-GFP a Mid2-iRFP zyakabambwa zipya zyakachili kuyoboloka mu ER ya sec31-1 mutant cells nikwakamana kubikkwa a 37°C mpawo zyakagwisigwa a 24°C kwamaminiti aali 5 (Chitondeezyo 1). Mbwaanga Mid2-iRFP ilamwaikide aa ER membrane yoonse, mpawo Gas1-GFP ilibungene akuyoboloka mubusena bwa ER membrane butazumanani, kwaabwa kwazyo kuliimpene (Chitondeezyo 1, A kusikila ku C a Movie S1). Mukuyungizya, mbuli mbokutondezyedwe achifanikisyo 1D, kabunga ka Gas1-GFP takajisi Mid2-iRFP pe. Eezyi mpindu zitondezya kuti GPI-AP antoomwe amapuloteni aazwa mumubili akaandaanizyigwa muzibeela zyaandeene zya ER kumatalikilo. Kabunga ka Gas1-GFP kali afwaafwi a ERES ilembedwe a mCherry's COPII coat protein Sec13 (Chitondeezyo 1, E a F, antoomwe a filimu S1) (23).
sec31-1 maselu atondezya galactose-induced secretions, acyl chain mulamfu (C26) ceramide GPI-AP Gas1-GFP (GPI-AP, green) antoomwe a transmembrane protein Mid2-iRFP (TMP, blue) mpawo eechi Constructive ERES labeling Sec13-mCherry (E) chakabikkwa mu RES 30 maminiti, yakatolwa ku 24°C, mpawo yakatolwa a SCLIM maminiti aali 5 musule. (A kusikila ku C) zitondezya mufanikisyo uusanganizidwe naakuti omwe wa 2D wa ndeke (A), mufanikisyo wa 2D wa 10 z-sections (B) naakuti mufanikisyo wa 3D cell hemisphere wa cargo a ERES markers (C). Chipimyo 1μm (A a B). Chipimyo chili 0.551μm (C). Gas1-GFP yakajanwa mumasena aandeene aa ER naakuti mutubunga, mpawo Mid2-iRFP yakajanwa mpawo yakamwaigwa mu ER membrane yoonse (C). (D) Igilafu itondezya nguzu zyakumweka kwa Gas1-GFP antoomwe a Mid2-iRFP mukabunga ka Gas1-GFP kweendelana amusemo uutuba (kulumwensyi). AU, chibeela chakulisalila. (E a F) ziiminina mufanikisyo wa 3D uusanganya zintu a ERES mark. Tubunga twa Gas1-GFP twakajanika afwaafwi a ERES. Chipimyo chili 0.551μm. (F) Musemu uutuba uusimide utondeezya Gas1-GFP cluster iiswaangene a ERES. Zibeela zyaakati azyakululyo zitondezya mufanikisyo wa 3D uusanganizidwe antoomwe akuzinguluka kwa Gas1-GFP cluster yakasalwa.
Kuswaangana kwabusena aakati ka Gas1-GFP cluster a ERES imwi kutondeezya kuti Gas1-GFP ilakonzya kunjila mu ERES iisalidwe, eeyo yiindene akusala kubelesyegwa a Mid2-iRFP kuzwa mu ER. Kuti tukonzye kulanga-langa eezi, twakapima mweelwe wa ERES wachintu chomwe naa zyobile luzutu (Chitondeezyo 2, A kusikila ku C). Twakajana kuti bunji bwa ERES (70%) ilijisi buyo musyobo omwe wazyintu zyakunyamuna. Cifwanikiso chili munsi cha Cifwanikiso 2C chitondezya zikozyanyo zyobile zya ERES zili a Gas1-GFP luzutu (Cifwanikiso 1) naa Mid2-iRFP luzutu (Cifwanikiso 2). Mukwiimpana, aali 20% ya ERES ilijisi zintu zyobile zivwumbilidwe mubusena bomwe. Kwakajanwa kuti zimwi ERES (10%) zyakalijisi misyobo yobile yamikuli, pesi zyakali kwaandaanizyidwe mumasena aasiyene. Aboobo, ooku kuvwuntauzya kwanamba kutondeezya kuti ER niyakagwisigwa, GPI-AP Gas1-GFP antoomwe a transmembrane cargo Mid2-iRFP zyaandaanizyigwa kuba ERES ziindene (Chitondeezyo 2D). Ooku kusalazya kweendelana loko akuvwuntauzya kwakacitwa musule (6) akuziba kwabupange (7). Tulakonzya kubona mbuzichita zintu zyakabikkwa mubusena bwakukkala mu ERES (Chitondeezyo 2E aMuvi S2). Cifwanikiso 2E chitondeezya kuti buyo kabeela kaniini ka Gas1-GFP (chibeela 3) naa Mid2-iRFP (chibeela 4) chinjila mu ERES kuzwa kulubazu lomwe mpawo chijalilidwe mubusena butazyibidwe. Chibeela 5 chachifanikisyo 2E chitondezya kuti Gas1-GFP antoomwe a Mid2-iRFP chimwi chiindi zilajanwa mu ERES yomwe, pesi zilanjila kuzwa kumabazu aasiyene mpawo zilajanika mumasena aasiyene aakonzya kwiiminina COPII vesicles zisiyene. Twakasinizya kuti kwaandaanya kwakabonwa akwaandaanya kwa C26 ceramide-based GPI-AP Gas1 mbuli ERES iisalidwe nchintu chilibedelede nkaambo chimwi chintu chakuzwa mumubili, GFP-tagged plasma membrane protein Axl2 (27 ), chitondezya kulilemeka kukozyenye a Mid2-iRFP. (Cifwanikiso S1 aMuvi S3). Axl2-GFP mpya yakapangwa ilamwaigwa kwiinda mu ER membrane mbuli Mid2-iRFP (Mufanikisyo S1, A a B), mpo ilajanika antoomwe a Mid2-iRFP mu ERES zinji (Mufanikisyo S1, B kusikila ku D). Zibeela 1 a 2 zyaChitondeezyo 1. S1C itondezya zikozyanyo zyobile zya ERES aawo zintu zyobile zya transmembrane nkoziswaangana. Muzyiimo ezi, zintu zyoonse zyobile zilanjila mu ERES antoomwe (Chitondeezyo S1E, Chitondeezyo 3 a Muvi S3).
Maseelo aa sec31-1 aatondezya galactose inducible secretions, Gas1-GFP (GPI-AP, green) antoomwe a Mid2-iRFP (TMP, blue) antoomwe a constitutive ERES labeling Sec13-mCherry (ERES, magenta) akabikkwa a 37 Nikwakamana kuyobolwa kwamaminiti aali 30 a °C, 24°C, akugwisigwa ku ion a SCLIM musule lyamaminiti aali makumi abili. (A kusikila ku C) Zifwanikiso zya 2D zitondezya (A; chipimyo, 1μm) naakuti zifwanikiso zya 3D zya cell hemisphere (B a C; chipimyo, 0.456μm) zyakunyamuna antoomwe a 10 z-sections zilembedwe a ERES. Ibbazu lyamunsi mu (B) abbazu lyamu (C) litondezya zifwanikiso zyakabambwa kuti zitondezye buyo zintu zili mu ERES (magenta) [Gas1-GFP (mubala wadota) a Mid2-iRFP (mubala wa bbululu)]. (C) Musemo uujalukide: ERES inyamwide buyo chintu chomwe (1 kusikila ku 4). Musemo wamubala wadota: ERES ilijisi zintu zyaandeene (5). Musemo muyumu uutuba: ERES iijisi zintu zibikkwa antoomwe. Munsi: ERES yomwe yakasalwa ilijisi buyo Gas1-GFP (1) naa Mid2-iRFP (2). Chipimyo, 100 nm. (D) Kupima kwachifanikisyo chaambwa mu (C). Mweelwe waakati-kati wa ERES uulaa buyo chintu chomwe (Gas1-GFP naa Mid2-iRFP), zintu zyaandaanizyidwe antoomwe azintu zivwumbilidwe. Mumasunko otatwe aalikke, n=432 mumaseelo aali 54. Ibbaala lyakulubizya = SD. Musunko wa t-tailed unpaired. *** P = 0.0002. (E) Cifwanikiso cha 3D cha ERES yakasalwa yazintu zyakabikkwa ambali zyakalembwa a (C). Gas1-GFP (mubala wa sokwe) (3) naa Mid2-iRFP (mubala wa bbuluu) (4) ulanjila mu ERES (mubala wa bbululu) kuzwa kulubazu lumwi mpawo ulabikkwa buyo mubusena busyoonto mukati ka ERES. Chimwi chiindi, misyobo yoonse yobile yamikuli ilanjila mu ERES yomwe (5) kuzwa kulubazu lomwe mpawo ilaangidwe kubusena bulikke mukati ka ERES. Chipimyo, 100 nm.
Kuzwa waawo, twakasunka muzeezo wakuti acyl chain ceramide (C26) iili mu ER membrane njiyeendelezya kubunganya akusalazya kwa Gas1 mu ERES. Kusikila kumamanino, twakabelesya musyobo wa yisiti wakasandulwa GhLag1, oomo ma endogenous ceramide synthases Lag1 a Lac1 mobakanjililwa busena a GhLag1 (Lag1 homolog ya cotton), eezi zyakapa kuti kube musyobo wa yisiti uulaa musyobo wa cell membrane Ceramide mufwiifwi kwiinda wa wild type (Chitondeezyo 3A) (28). Kuvwuntauzya kwa mass spectrometry (MS) kwakatondeezya kuti mumisyobo yamusyobo wamusyokwe, 95% ya ceramide yoonse njilamfwu loko (C26) nketani ya ceramide, mpawo mu GhLag1, 85% ya ceramide njilamfwu loko (C18 a C16). ), 2% luzutu lwa ceramide ndulamfwu loko (C26) nketani ya ceramide. Nikuba kuti C18 a C16 ceramides ngama ceramides mapati aakajanika mu GhLag1 membrane kusikila lino, MS analysis yakasinizya kuti GPI anchor ya Gas1-GFP yakatondezyegwa mu GhLag1 strain ilijisi C26 ceramide, eeyo iikozyenye a wild-type lipids. Bubotu mbubonya (Fig. 3A) (26). Aboobo, eezi zyaamba kuti enzyme yakubambulula ceramide Cwh43 ilasala kapati C26 ceramide, mbuli mbokutondezyedwe muchifanikisyo 26, ilayandisya kubikkilizya GPI anchor kuzwa ku C26 ceramide nsyoonto mumusyobo wa GhLag1. S2 (29). Nikuliboobo, chikumba cha GhLag1 chilijisi buyo C18-C16 ceramide, mpawo Gas1-GFP ichijisi C26 ceramide. Kaambo aaka kapa kuti musyobo ooyu ube chibelesyo chibotu chakumanizya penzi lyabulamfwu bwa acyl chain ya membrane ceramide mu ER. Mulimu wakuyeeyela wachiinga akusalaula. Mpawo, twakasaanguna kwiiya nguzu zya C26 Gas1-GFP zyakuyoboloka muzibeela mu GhLag1 a allele iikonzya kuchincha ya sec31-1 kwiinda mukubelesya nzila ya fluorescence microscopy, oomo buyo nketani ndamfu (C18-C16) moili mu ER membrane Ceramide (Fig. 3). Twakabona kuti mu sec31-1, bunji bwa Gas1-GFP bwakali muzibeela, mpawo Gas1-GFP mu sec31-1 GhLag1 a (C18-C16) bulamfu bwa ceramide ER membrane tiiyakabungene pe mpawo yakamwaigwa mu ER membrane yoonse. Mubwini, nkaambo kakuti C26 ceramide-based clustering ilaswaangene a ERES (Mufanikisyo 1), twakalanga-langa kuti naa nzila eeyi ilakonzya kujatikizya mulimo wa ER export protein mechanism. GPI-AP ibelesya nzila yaalubazu ya COPII yakutumizya ER, eeyo yeendelezyegwa a Ted1′s kubamba bupange bwa glycan bwa GPI anchor (30, 31). I GPI-glycan iisanganizidwe mpawo ilazyibwa a transmembrane cargo receptor p24 complex, eeyo isalazya Lst1, eeyo iili isoform ya COPII cargo binding subunit Sec24, mpawo ipanga GPI-AP-rich COPII Vesicles are necessary (31-33). Aboobo, twakapanga mutant yobile yakaswaanganya kugwisigwa kwamapulotini aaya (p24 complex component Emp24, GPI-glycan remodeling enzyme Ted1 antoomwe a COPII subunit Lst1) antoomwe a sec31-1 mutant strain, mpawo twakaziiya. Sena zilakonzyeka kupanga Gas1-Gluster GFP (Figure 3). Twakabona kuti mu sec31-1emp24Δ antoomwe a sec31-1ted1Δ, Gas1-GFP tiili antoomwe mpawo ilamwaikide mu ER membrane, mbuli mbukwakabonwa mu sec31-1 GhLag1, mpawo mu sec31-1lst1Δ, Gas1-GFP mbuli sec31-1-1. Eezyi mpindu zitondezya kuti mukuyungizya kukubako kwa C26 ceramide mu ER membrane, kubunganya kwa Gas1-GFP akwalo kuyandika kuswaangana a p24 complex, mpawo takuyandiki Lst1. Mpawo, twakavwuntauzya kuti naa bulamfwu bweketani bwa ceramide mu ER membrane bulakonzya kweendelezya kuswaangana kwa Gas1-GFP ku p24. Nokuba boobo, twakajana kuti kubako kwa C18-C16 ceramide muchikumba takujatikizyi GPI-glycans yakayakululwa a p24 complex (Mifanikisyo S3 a S4, A a B) naa kuswaangana a GPI-AP akugwisya GPI-AP. kukonzya. Amubweze COPII subtype Lst1 (Chitondeezyo S4C). Aboobo, C26 ceramide-dependent clustering takuyandiki kuswaangana kwama protein anzila zyaandeene zya ER export protein, pesi zigwasyilizya nzila zimbi zyakusalazya zyeendelezyegwa abulamfwu bwamafuta. Mpawo, twakalanga-langa naa bulamfwu bwa nketani ya ceramide acyl mu ER membrane bulayandika kapati mukusalazya Gas1-GFP kuti ibe ERES iisalidwe. Mbwaanga Gas1 mumusyobo wa GhLag1 uujisi nketani mfwaafwi ya ceramide ulazwa mu ER akunjila mu membrane ya plasma (Mufanikisyo S5), tusyoma kuti ikuti kusala kweendelezyegwa abulamfwu bwa nketani ya ceramide acyl, Gas1 mumusyobo wa GhLag1 ilakonzya kujokolosyegwa akuzabuka. ERES zintu zili a membrane yomwe.
(A) Chikwabilizyo chamaseelo cha GhLag1 chilijisi ma C18-C16 ceramides mafwaafwi, mpawo GPI anchor ya Gas1-GFP ichijisi C26 IPC njiyonya mbuli maseelo aamusyobo wamusyokwe. Atala: kulanga bulamfwu bweketani ya acyl ya ceramide mumaseelo aamusyobo wa wild-type (Wt) antoomwe a GhLag1p strains kwiinda mukubelesya nzila ya mass spectrometry (MS). Data itondezya mweelwe wa ceramide yoonse. Mweelwe waakati-kati wamasunko otatwe aalikke. Ibbaala lyakulubizya = SD. Musunko wa t-tailed unpaired. **** P ＜0.0001. Chitondeezyo chaansi: MS buvwuntauzi bwa acyl chain length ya IPC iili mu Gas1-GFP (GPI-IPC) GPI anchor yakatondezyegwa mu wild-type a GhLag1p strains. Data itondezya mweelwe wa IPC yoonse. Mweelwe waakati-kati wamasunko aali musanu aalikke. Ibbaala lyakulubizya = SD. Musunko wa t-tailed unpaired. ns, taziyandiki pe. P = 0.9134. (B) Ma micrographs aa sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ antoomwe a sec31-1lst1Δ maselu aatondezya galactose-induced Gas1-GFP akabikkwa a 37°C kwamaminiti aali 30 mpawo akuchita kabotu chitondeezyo chipati nikwakainda 24°C. Musemo uutuba: ER Gas1-GFP kabunga. Musemo uujalukide: Gas1-GFP iitakwe mbungano ilamwaigwa a ER membrane yoonse, kutondezya ER bube bwa nuclear ring staining. Chipimyo, 5μm. (C) Kupima kwachifanikisyo chaambwa mu (B). Mweelwe waakati aakati wamaseelo ajisi bupange bwa Gas1-GFP. Mumasunko otatwe aalikke, n≥300 maselu. Ibbaala lyakulubizya = SD. Musunko wa t-tailed unpaired. **** P ＜0.0001.
Kuti tumanizye penzi eeli, twakachita SCLIM kutondezya Gas1-GFP antoomwe a Mid2-iRFP mu GhLag1 antoomwe a sec31-1 temperature-sensitive mutant allele (Chitondeezyo 4 a Muvi S4). Nikwakainda ER niyakabambwa a 37°C mpawo yakaangununwa a 24°C, bunji bwa Gas1-GFP mpya yakapangwa tiiyakabungene akumwaigwa mu ER membrane yoonse, mbuli mbokwakabonwa ama microscopes (Chitondeezyo 4, A a B). Mukuyungizya, mweelwe mupati wa ERES (67%) ubikkilizya misyobo yobile yazintu zyakunyamuna zili antoomwe mulinjiyo (Chitondeezyo 4D). Zibeela 1 a 2 zya Cifwanikiso 4C zitondezya zikozyanyo zyobile zya ERES zili a Gas1-GFP antoomwe a Mid2-GFP. Mukuyungizya, zintu zyoonse zyobile zyakabikkwa mu ERES yomwe (Chitondeezyo 4E, chibeela 3 antoomwe afilimu S4). Aboobo, mpindu zyesu zitondezya kuti bulamfu bwa nketani ya ceramide acyl mu ER membrane nchintu chipati loko mukubunganya ma protein aa ER akubikkwa muzibeela.
Sec31-1 GhLag1 maselu aatondezya galactose-induced secretions, Gas1-GFP (GPI-AP, green) antoomwe a Mid2-iRFP (TMP, blue) antoomwe a constitutive ERES-labeled Sec13-mCherry (ERES, magenta) Zibikkidwe a 37°C. Kozumanana kwa maminiti aali 30, mpawo uselemuke kusika ku 24°C kuti ugwisye zyintu zizwa mumubili, mpawo ufwefweme a SCLIM musule lya maminiti aali 20. (A kusikila ku C) Zifwanikiso zya 2D zitondezya (A; chipimyo, 1μm) naakuti zifwanikiso zya 3D zya cell hemisphere (B a C; chipimyo, 0.45μm) zya 10 z-sections zilembedwe a cargo a ERES. Ibbazu lyamunsi mu (B) abbazu lyamu (C) litondezya zifwanikiso zyakabambwa kuti zitondezye buyo zintu zili mu ERES (magenta) [Gas1-GFP (mubala wadota) a Mid2-iRFP (mubala wa bbululu)]. (C) Musemu uuzwide mubala uutuba: ERES, zintu zilavwumbilila. Jula musemo: ERES ilijisi buyo chintu chomwe. Chibeela chaansi: ERES yakasalwa ilijisi zintu zikozyenye (1 a 2) zilembedwe mu (C). Chipimyo, 100 nm. (D) Kupima kwachifanikisyo chaambwa mu (C). Muzibeela zya sec31-1 antoomwe a sec31-1 GhLag1, buyo chintu chomwe (Gas1-GFP naa Mid2-iRFP) mpachibikkilizyidwe, antoomwe amweelwe waakati-kati wa ERES wazyintu zili alikke azintu zizwide. Mumasunko otatwe aalikke, n = 432 mumaseelo aali 54 (sec31-1) antoomwe a n = 430 mumaseelo aali 47 (sec31-1 GhLag1). Ibbaala lyakulubizya = SD. Musunko wa t-tailed unpaired. *** P = 0.0002 (sec31-1) a ** P = 0.0031 (sec31-1 GhLag1). (E) Cifwanikiso cha 3D cha ERES yakasalwa antoomwe azintu zivwumbilidwe (3) zilembedwe mu (C). Gas1-GFP (mubala ukozyene asokwe) antoomwe a Mid2-iRFP (uusalala pyu) balaswena ku ERES (mubala wa bbululu) kuzwa kulubazu lomwe mpawo bakkala mubusena bomwe bwa ERES. Chipimyo, 100 nm.
Ooku kuvwuntauzya kupa bumboni bwini bwakuti ma protein aajisi mafuta abikkwa mumasena aasalidwe munzila yakuzwa, mpawo kutondezya mbociyandika bulamfwu bweketani lya acyl mukusalazya. Kubelesya nzila yanguzu alimwi yakusaanguna ya microscopy iitwa kuti SCLIM, twakatondezya Gas1-GFP (cibeela cipati ca plasma membrane GPI-AP cijisi acyl chain ndamfu kapati (C26) ceramide lipid portion) mu yeast ) Masena aabungene mu ERs aandeene-andeene alaswaangene a ERE, kakuli ma protein aazwa mu membrane ERRES alamwaigwa kwiinda mu membrane (Chitondeezyo 1). Mukuyungizya, misyobo yobile yazintu ilanjila mu ERES zisiyene (Chitondeezyo 2). Bulamfu bwa nketani ya acyl ya cellular ceramide mu membrane bwacheya kuzwa ku C26 kusika ku C18-C16, Gas1-GFP cluster yakanyonganizyigwa kuya kubusena bwa ER, mpawo Gas1-GFP yakacincwa kuti izwe mu ER antoomwe a transmembrane protein kwiinda mu ERES njiyonya (Mufanikisyo 3 a Cifwanikiso 3). 4).
Nikuba kuti GPI-AP ibelesya nzila yaalubazu yama protein kuzwa mu ER, twakajana kuti C26 ceramide-dependent separation tayeeme akusiyana kwama protein aakonzya kusololela ku ERES (Mifanikisyo S4 a S5). Muciindi, nzyotwajana zigwasyilizya nzila imbi yakusalazya iisololelwa akubunganya ma protein azwa kumafuta mpawo akugwisya zimwi zintu. Nzyotwakabona zitondezya kuti busena bwa Gas1-GFP naakuti nkamu iiswaangene a ERES imwi taikwe ma protein aazwa mumubili Mid2-iRFP, eezi zitondezya kuti nkamu ya C26 ceramide-dependent GPI-AP ilakonzya kugwasya kunjila mu ERES yeelede, mpawo aawo, akugwisya transmembrane. Mukwiimpana, kujanika kwa C18-C16 ceramides mu ER membrane takupi kuti GPI-AP ipange zibeela naakuti tubunga, aboobo tazigwisyi naakuti kuchincha ma protein azwa mu membrane mu ERES yomwe (Mifanikisyo 3 a 4). . Aboobo, twasala kuti C26 ceramide isololela kwaandaanya akwaandaanya kwiinda mukugwasya kubunganya ma protein aajatikizyidwe ku ERES imwi.
Ino mbuti mbotukonzya kuzuzikizya C26 ceramide-dependent clustering mubusena bumwi bwa ER? Kuyandisya kwa membrane ceramide kwaandaana kumbali kulakonzya kupa kuti GPI-AP a C26 ceramide zipange mafuta masyoonto alimwi aabikkidwe kabotu-kabotu mubusena bwamafuta butaluleme bwa ER membrane bujisi ma glycerolipids mafwaafwi alimwi aatakwe busani. Tubunga twabubotu (17, 18). Tubunga ootu tuniini twachiindi chifwiifwi tulakonzya kusanganyigwa kuba tubunga tupati, tutazungaani nikwakamana kuswaangana a p24 complex (34). Kweendelana aawa, twakatondezya kuti C26 Gas1-GFP yeelede kuswaangana a p24 kuti ipange tubunga tupati tulibonya (Chitondeezyo 3). I p24 complex ni heterozygous oligomer iijisi p24 transmembrane proteins zyone mu yeast (35), eeyo iipa kuti kube kujatana kwa multivalent, ooko kukonzya kusololela kukujatana kwatubunga tusyoonto twa GPI-AP, aboobo kupa kuti kube Stable cluster mpati (34). Kuswaangana akataa ma protein ectodomains aa GPI-APs akwalo kulakonzya kugwasya mukubunganya kwawo, mbuli mbokutondezyedwe muchiindi chakweendela kwa Golgi mumaseelo aanyama (36). Nokuba boobo, eelyo C18-C16 ceramide noiliwo mu ER membrane, eelyo p24 complex niyaswaangana a Gas1-GFP, tubunga tupati twaandeene tatukonzyi kupangwa pe. Nzila ibelesyegwa ilakonzya kweendelana abube bwamubili abwamakemikalu bwa acyl chain ceramide. Ziiyo zya biophysical zya membrane zyakupanga zitondezya kuti nikuba kuti zyoonse zyobile zilamfu (C24) azifwiifwi (C18-C16) acyl chain ceramides zilakonzya kupa kuti kube kwaandaana, acyl chain ceramides zilamfwu (C24) zilikke zilakonzya kusumpula kugonka kupati akukotama kwafilimu kuti ichite bupange bupya bwafilimu. Kwiinda mukulangana (17, 37, 38). Kwakatondezyegwa kuti transmembrane helix ya TMED2, imuntu uukozyenye a Emp24, ilaswaangana a C18 ceramide-based sphingomyelin mu cytoplasmic lobules (39). Kubelesya kweezyeezya kwa molecule dynamics (MD), twakajana kuti zyoonse zyobile C18 a C26 ceramides zilabungana kuzyunguluka cytoplasmic lobules ya Emp24 transmembrane helix, mpo zili azintu zikozyenye (Chitondeezyo S6). Nchintu chibotu kuzyiba kuti eezi zitondezya kuti transmembrane helix ya Emp24 ilakonzya kusololela kukumwaika kutali kabotu kwamafuta mu membrane. Eezi nzintu zyakajanwa ino-ino kuzwa kumaseelo aabanyama. Zikozyanyo zya MD zikozyenye azyalo zitondezya kubako kwamafuta aa ether (40) . Aboobo, twayeyela kuti C26 ceramide muzibeela zyobile zya ER26 ilivwubide. Eelyo GPI-AP mu luminal lobules naiswaanganya a multivalent p24 akuyoboloka kwa C26 ceramide kuzyunguluka p24 mu cytoplasmic lobules, ilakonzya kusumpula kujatana kwama Protein akuvwunguluka kwa membrane kulapangwa kwiinda muminwe (41), kupa kuti GPI-AP iandaane mumasena aaindene-indene aali afwaafwi a ERES, aalo aagwasya kuvwunguluka kwa RES ER membrane (42). Malipooti aakaindi akagwasyilizya nzila yakayeeyelwa (43, 44). Kuswaangana kwa oligolectins, ma pathogens naakuti ma antibodies ku ceramide-based glycosphingolipids (GSL) aa plasma membrane kupa kuti GSL ibungane, kuyungizya kwaandaana kwa phase mpawo kupa kuti membrane ichinche akunjila mukati (44). Iwabuchi azimwi. (43) Kwakajanika kuti kakuli nketani zya acyl zilamfu (C24) pesi kutali zifwiifwi (C16), ligand ya multivalent iijatikizyidwe ku GSL lactosylceramide yakapa kuti kube kupanga tubunga tupati akunjilila kwa membrane, mpawo cytoplasm Lyn-mediated signal transduction on the leaflets kweendelezyegwa a neutrophils mu couple.
Mumaseelo aabanyama, bunji bwa anti-Golgi network (TGN) kusikila aawo mpaali plasma membrane yeendelezya kwaandaanya akusalazya GPI-AP (10, 45). Ooku kuswaangana kweendelezyegwa a GPI-AP oligomerization (36), asi kulakonzya kweendelana abulamfwu bwa nketani ya ceramide njotujana mu yisiti. Nikuba kuti GPI-AP yabanyama bavwubwa ilijisi nsinga zya ether lipid, alimwi bupange bwayo buliindene kapati a acyl chain ceramide, buvwuntauzi bwakacitwa ino-ino bwakajana kuti ma lipid oonse obile alijisi bube bukozyenye bwamubili alimwi amulimo (40). Aboobo, cibeela cha ether lipid mumaseelo aabanyama chilakonzya kukozyanya a C26 ceramide mu yeast, mpawo mulimo wayo ngwakuswaangana a ceramide iijisi nketani ndamfu mu membrane kuchitila kuti GPI-AP ibungane akusalazya. Nikuba kuti eezi zilakonzya kuchitwa kuti zikonzye kusunkwa, eezyo zyakajanwa musule zitondezya kuti kweenzya kwa acyl chain ceramide kuya kumubili wa Golgi takuchitiki ama protein aazwa ku cytoplasmic, pesi kuyeeme akupanga GPI anchors mbuli yeast. Aboobo, nzila yakukwabilila yakusanduka ilibonya kuti ilakonzya kweenzya antoomwe acyl chain ceramide a GPI-AP (13, 16, 20, 46, 47) munzila yomwe.
Mumaseelo aajisi bumena amunyama, kuswaangana kwa GPI-AP akwaandaana kuzwa kuma puloteni aambi aa plasma membrane zyoonse zilachitika kaatanasika atala aamaseelo. Paladino abamwi. (48) bakajana kuti a TGN yamaseelo aanyama, kuswaangana kwa GPI-AP takuyandiki buyo mukusala kwa GPI-APs ku apical plasma membrane, pele alimwi kulalanganya mbungano ya GPI-APs alimwi amulimo wayo wabuumi. Busena bwaseelo. Mubumena, chiiyo eechi chakatondezya kuti C26 ceramide-dependent GPI-AP cluster a ER ilakonzya kweendelezya bubambe bwa cluster akubeleka kwa GPI-AP a plasma membrane (24, 49). Kweendelana achikozyano eechi, maselu aa GhLag1 alakonzya kuchisa GPI inhibitors naakuti misamu iijatikizya kusyomeka kwabwaanda bwamaselu (28), mpawo kuyandika kwa Gas1-GFP clusters zibeleka (49) zya tip ceramide zilangilwa mukujatana kwa maselu aa yeast zitondezya G� zikonzya kuchitika akaambo kamaseelo aa hLag1. GPI-AP kulubizya. Nokuba boobo, kusunka kunembo kuti naa bubambe bwakubeleka bwamaseelo bwakabambilwa kuzwa ku ER anzila yakusalazya kweendelanya abulamfwu bwamafuta kuyooba makani aakuvwuntauzya kwesu kwakumbele.
Misyobo ya Saccharomyces cerevisiae yakabelesegwa mumulimo ooyu ililembedwe mu Table S1. Misyobo ya MMY1583 a MMY1635 ya SCLIM yakutondezya maselu apona yakapangwa musule lya W303. Misyobo eeyi iitondezya Sec13-mCherry a fluorescent protein tag yakabambwa kubelesya nzila ya polymerase chain reaction (PCR)-based method a pFA6a plasmid mbuli chikozyano (23). Musyobo uutondezya Mid2-iRFP uulembedwe ama protein aamweka-mweka kweendelezyegwa a GAL1 promoter wakapangwa mbuli mbokutondezyedwe. Kuyungizya kwa PCR kwa iRFP-KanMx kuzwa ku pKTiRFP-KAN vector (chipego cha E. O'Shea, Addgene plasmid number 64687; http://n2t.net/addgene: 64687; research resource identifier (RRID): Addgene_64687) Mpawo kwakabikkwa mu Cendous-termind ya Midgeneus. Nikwakainda Mid2-iRFP genome sequence yakayungizigwa akubikkwa mu GAL1 promoter, yakabikkwa mu Not I-Sac I site ya integration plasmid pRS306. I plasmid pRGS7 yakazwa yakabikkwa antoomwe a Pst I kuti injile mu URA3 locus.
I Gas1-GFP fusion gene ilajanika kweendelezyegwa a GAL1 promoter mu centromere (CEN) plasmid, eeyo yakabambwa boobu. Mulongo wa Gas1-GFP wakayungizigwa a PCR kuzwa ku pRS416-GAS1-GFP plasmid (24) (chipego cha L. Popolo) mpawo wakabikkwa mubusena bwa Xma I-Xho I bwa CEN plasmid pBEVY-GL LEU2 (chipego cha C). Miller; Addgene plasmid namba 51225; http://n2t.net/addgene: 51225; RRID: Addgene_51225). I plasmid yakazwa yakaitwa kuti pRGS6. Axl2-GFP fusion gene ayalo ilajanika mubweendelezi bwa GAL1 promoter ya pBEVY-GL LEU2 vector, mpawo bupange bwayo buli boobu. Axl2-GFP yakayungizigwa kuzwa ku pRS304-p2HSE-Axl2-GFP plasmid (23) a PCR, mpawo yakabikkwa mu Bam HI-Pst I site ya pBEVY-GL LEU2 vector. I plasmid yakazwa yakaitwa kuti pRGS12. Mulongo wama oligonucleotides akabelesegwa mukwiiya ooku ulilembedwe mu Table S2.
Musyobo ooyu wakayungizyigwa a 0.2% adenine a 2% glucose [YP-dextrose (YPD)], 2% raffinose [YP-raffinose] rich yeast extract protein p (YP) medium (1 % Yeast extract a 2% protein ept). (YPR)] naa 2% galactose [YP-galactose (YPG)] mbuli nzila yakujana kaboni, naa munzila yakupanga (0.15% yeast nitrogen base a 0.5% ammonium sulfate) kuti iyungizye ma amino acid aama base ayelede kuyandika mukulya, Alimwi a 2% glucose (2% glucose glucose) naa minimal glucose galactose (galactose yakapangwa asyoonto) mbuli nzila yakujana kaboni.
Mukutondezya muchiindi cheenzu, maselu aa sec31-1 aakonzya kupya aatondezya bupange bwa GAL1 promoter akakomezyegwa mu YPR medium a 24°C masiku woonse kusikila akati-kati ka log phase. Musule akunjizigwa mu YPG a 24°C kwawoola lyomwe, maselu akabikkwa mu SG a 37°C kwamaminiti aali 30, mpawo akagwisigwa ku 24°C kuti azwe muchibaka chakuzwa. Concanavalin A yakabelesegwa kubamba maselu aagilazi mpawo yakatolwa a SCLIM. SCLIM nchintu chisanganizidwe a Olympus IX-71 inverted fluorescence microscope antoomwe a UPlanSApo 100×1.4 numerical aperture oil lens (Olympus), high-speed and high-signal-to-noise ratio rotating disc confocal scanner (Yokogawa Electric), custom spectrometer, intenseing systems and the intensiveing ​​systems (Hamamatsu Photonics) ilakonzya kupa nzila yakukomezya lensi iijisi kukomezya kwamamanino kwa ×266.7 antoomwe akkamera iijisi magesi iiyungizya ma electron (Hamamatsu Photonics) (21). Kubweza zifwanikiso kuchitwa apulogilamu iipangidwe (Yokogawa Electric). Kumifanikisyo ya 3D, twakabelesya chintu chakapangwa a piezoelectric actuator kuzungaanya lensi yakulanga, mpawo twakayobolola zibeela zyamumuni 100 nm kuzwa kuli umwi. Cifwanikiso cha Z-stack chasandulwa kuba 3D voxel data, mpawo nzila yakumwaya mapointi iibelesyegwa ku rotating disc confocal microscope ilabelesyegwa mu deconvolution processing a Volocity software (PerkinElmer). Kwiinda mukubelesya pulogilamu ya Volocity kuti ichite zyintu zyoonse zyeelede kuchitwa, ERES kubikkilizya azintu zyakali kubelesegwa zyakali kupimwa. Kuvwuntauzya kwakachitwa kubelesya pulogilamu ya MetaMorph (Molecular Devices).
Belesya pulogilamu ya GraphPad Prism kuti ubone bupati bwakubala. Ku t-test ya Student's two-tailed antoomwe a ordinary one-way analysis of variance (ANOVA) test, kwiimpana aakati kankamu kulabonwa kuti kuli ampindu mpati a P <0.05 (*).
Mukulanga ma microscope aa Gas1-GFP, maselu aa log phase akakomezyegwa masiku woonse mu YPD mpawo akayobolwa a centrifugation, akasanzyigwa tubili a phosphate buffered saline, mpawo akabikkwa mu ice kwamaminiti aatasiki ku 15, mpawo akazumanana aama microscope mbuli mbukwakapandululwa musule Check (24). I Leica DMi8 microscope (HCX PL APO 1003/1.40 oil PH3 CS) iijisi lensi yakulanga, L5 (GFP) filter, Hamamatsu camera antoomwe a Application Suite X (LAS X) software zyakabelesegwa mukujana. .
Isampulu zyakasandululwa a SDS sample buffer a 65°C kwamaminiti aali kkumi, mpawo zyakaandaanizyigwa a SDS-polyacrylamide gel electrophoresis (PAGE). Mukuvwuntauzya kwa immunoblotting, 10 μl yasampulu yakabikkwa mumugwagwa omwe. Antibody yakusaanguna: Belesya rabbit polyclonal anti-Gas1 a dilution ya 1:3000, rabbit polyclonal anti-Emp24 a dilution ya 1:500, antoomwe a rabbit polyclonal anti-GFP (chipego kuzwa kuli H. Riezman) a dilution ya 1:30000. I mbeba ya monoclonal anti-Pgk1 antibody yakabelesegwa a dilution ya 1:5000 (chipego kuzwa kuli J. de la Cruz). I antibody yabili: Horseradish peroxidase (HRP) conjugated goat anti-rabbit immunoglobulin G (IgG) yakabelesyegwa a dilution ya 1:3000 (Pierce). HRP-conjugated goat anti-mouse IgG yakabelesegwa a dilution ya 1:3000 (Pierce). Busena bwakukwabilila mubili bwakabonwa anzila ya chemiluminescence ya SuperSignal West Pico reagent (Thermo Fisher Scientific).
Mbuli mbokwaambidwe mu (31), kusunka kwakukwabilila mubili kwakachitwa a ER fraction yakavwubidwe. Mubufwiifwi, sanzya maselu aa yisiti a TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride antoomwe a protease inhibitor mixture) a 600 nm ( OD600) a 100 optical density tubili. Lyakatyolwa aamabwe aagilazi, mpawo maselu aamabwe aagilazi zyakagwisigwa kwiinda mukubelesya nzila ya centrifugation. Maanzi aakasyaala mpawo akabikkwa mu 17,000 g kwamaminiti ali kkumi amusanu a 4°C. Ipellet yakabikkwa alimwi mu TNE mpawo digitalis saponin yakayungizigwa kusika kumamanino aa 1%. Ikuyuminina kwakabikkwa mubusena bwawoola lyomwe kakuzungaanisigwa a 4°C, mpawo zibeela zitakonzyi kunywika zyakagwisigwa kwiinda mukuzisofwaazya a 13,000 g a 4°C kwamaminiti aali 60. Kutegwa Gas1-GFP ikwabilile mubili, sanguna kubikka musamu ooyu amabwe aatakwe chintu (ChromoTek) a 4°C kwawoola lyomwe, mpawo ubikke a GFP-Trap_A (ChromoTek) a 4°C kwamawoola otatwe. Insinga zyakasanzyigwa ziindi zyosanwe a TNE iijisi 0.2% digoxigenin, zyakagwisigwa a SDS sample buffer, zyakaandaanizyigwa a SDS-PAGE, mpawo zyakavwuntauzyigwa a immunoblotting.
Mbuli mbokwaambidwe mu (31), kusalazya kwakachitwa aa ER fraction yakavwubidwe. Mubufwiifwi, ER fraction yakayungizigwa yakabikkwa a 0.5 mM dithiobis(succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, USA; 20°C, 20 min). Ikujatana kwakazima kwiinda mukuyungizya glycine (50 mM kumamanino, maminiti aali 5, 20°C).
Mbuli mbukwakapandululwa musule (50), kuvwuntauzya kwa MS kwa ceramide mumusyobo wa wild-type antoomwe a GhLag1 kwakachitwa. Mubufwiifwi, maselu akakomezyegwa kusikila achiindi chakuyungizya (3 kusikila ku 4 OD600 yuniti/ml) mu YPD a 30°C, mpawo maselu aali 25×107 akatebulwa. Kusanduka kwabo kwamubili kulazimwa a trichloroacetic acid. Belesya musamu wakugusya [ethanol, maanzi, ether, pyridine a 4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] antoomwe a 1.2 nmol ya internal standard C17 ceramide (860517, Avanti polar lipid) quality). Belesya monomethylamine reagent [methanol, maanzi, n-butanol antoomwe a methylamine solution (4:3:1:5 v/v)] kuchita mild alkaline hydrolysis ya extract, mpawo kobelesya n-butanol iizwide maanzi kugwisya munyo. Kumamanino, chakagwisigwa chakabikkwa muchintu chibotu [chloroform/methanol/maanzi (2:7:1) + 5 mM ammonium acetate] mpawo chakabikkwa mu mass spectrometer. Kulanga-langa zintu zyiingi (MRM) kwakachitwa kuti kuzyibwe akubalwa kwama molecule aa sphingolipid. TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) ilijisi robotic nanoflow ion source Nanomate HD (Advion Biosciences, Ithaca, NY) yakuvwuntauzya mafuta. Nguzu zyakudonkolana zili kabotu kuli umwi awumwi musyobo wa ceramide. Data ya MS yakajanwa munzila iili kabotu. Kuli chimwi achimwi chikozyano chabuumi, chitondezyo chamafuta nchintu chili akati-akati kazipimyo zyotatwe zitazulide.
Mbuli mbokwaambidwe mu (31), maselu (800×107) aatondezya Gas1-GFP akabikkwa munzila yakukwabilila mubili. Gas1-GFP yakasalala yakaandaanizyigwa a SDS-PAGE mpawo yakabikkwa mu polyvinylidene fluoride (PVDF) membrane. Ma protein akabonwa kwiinda mukupenta PVDF a amide black. Ibbanti lya Gas1-GFP lyakakosolwa kuzwa ku PVDF mpawo lyakasanzyigwa ziindi zili musanu a methanol mpawo aawo a liquid chromatography-MS (LC-MS) grade water. Kwiinda mukubikka 500μl 0.3 M NaOAc (pH 4.0), buffer a 500μl 1 M sodium nitrite mixture a 37°C kwamahola otatwe, cibeela cha lipid chilazwa ku Gas1-GFP mpawo chilazwa kuzwa mu Gas1-GFP mpawo chilazwa mu inophoosphamiophaosi akati ka ceramiode. (51). Kuzwa waawo, bbazu lya membrane lyakasanzigwa ziindi zyone amaanzi aa LC-MS, lyakayuminizyigwa aawo mpaakali kupya, mpawo lyakabikkwa mumulengalenga wa nitrogen a -80°C kusikila lyakavwuntauzyigwa. Mbuli nzila yakulanga, musamu uutakwe chintu wa PVDF membrane wakabelesegwa kuli umwi awumwi musunko. Mafuta aakagwisigwa kuzwa ku Gas1-GFP mpawo akavwuntauzyigwa a MS mbuli mbokwaambidwe (50). Mubufwiifwi, zibeela zya PVDF zijisi GPI-lipid zyakabikkwa alimwi mu 75μl negative mold solvent [chloroform/methanol (1:2) + 5 mM ammonium acetate] mpawo zyakainda electrospray ionization (ESI)-MRM/MS Analysis of sphingolipid species (TSQ Vantage). Muchiimo eechi, data ya MS yakajanwa munzila ya negative ion.
Mbuli mbukwaambidwe kale, cibeela cha GPI anchor chakaandaanizyigwa kuzwa ku [3H]-inositol-labeled GPI-AP (16). Mafuta akaandaanizyigwa akubelesya nzila ya solvent (55:45:10 chloroform-methanol-0.25% KCl) mpawo akabonwa kubelesya FLA-7000 (Fujifilm).
Maseelo aatondezya Gas1-GFP (600×107) akasanzyigwa ziindi zyobile a TNE buffer antoomwe a TNE buffer, mpawo atyolwa amagilazi, mpawo akabikkwa mu centrifuge kuti agwisye tombe lyamaseelo amagilazi. Maanzi aakasyaala mpawo akabikkwa mu 17,000 g kwawoola lyomwe a 4°C. Ipellet yakasanzigwa mu TNE mpawo yakabikkwa a 1 U PI-PLC (Invitrogen) mu TNE iijisi 0.2% digitalis saponin kwawoola lyomwe a 37°C. Nikwakamana kusilikwa a enzyme, chikumba chakagwisigwa a 17,000 g a 4°C kwawoola lyomwe. Kuti Gas1-GFP ichite kabotu, maanzi aakasyaala akabikkwa a GFP-Trap_A (ChromoTek) a 4°C masiku woonse. I Gas1-GFP iisalazyidwe yakaandaanizyigwa a SDS-PAGE yakabikkwa mubala wa Coomassie uusalala pyu. Ibbanti lyakupenta lya Gas1-GFP lyakakosolwa kuzwa kumubala wadota uuzingulukide mugwagwa wamaanzi, mpawo nikwakamana kusanzyigwa a iodoacetamide akucheya a dithiothreitol, kugaya kwa in-gel a trypsin kwakachitwa. Gwisya akuyuminizya ma peptide aama peptide a GPI-glycans. I peptide yakayumide yakanyonyoonwa mu 20 μl yamaanzi. Bika chibeela (8μl) mu LC. Octadecylsilane (ODS) column (Develosil 300ODS-HG-5; inner diameter 150 mm×1.0 mm; Nomura Chemical, Aichi Prefecture, Japan) yakabelesegwa kwaandaanya ma peptides mubukkale bumwi. Chibeela chakweendeenda nchintu chiyumu A (0.08% asidi yafoomu) antoomwe aasidi B (0.15% asidi yafoomu mu 80% acetonitrile). Inzila ya Accela HPLC (Thermo Fisher Scientific, Boston, Massachusetts) yakabelesegwa kugwisya musamu A mukati ka maminiti aali 55 amweelwe wa 50 μl min-1 kwamaminiti aali 5, mpawo bunji bwa musamu B bwakayungizigwa kusika ku 40%. , Amelika). I eluate yakazumanana kunjila mu ESI ion source, mpawo ma peptide aa tryptic ama peptide a GPI-glycans akavwuntauzyigwa a LTQ Orbitrap XL (hybrid linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific). Mububambe bwa MS, nguzu zya magesi zya capillary zyakabikkwa a 4.5 kV, mpawo kupya kwa capillary yakabikkwa a 300°C. Inguzu zya capillary azya tube lens zyakabikkwa a 15 V a 50 V, kweendelana. Data ya MS yakajanwa munzila ya ion iili kabotu (kusalala kwa 60,000; kulondoka kwa 10 zibeela muli miliyoni) mumweelwe wa 300/m/z mass/charge ratio (m/z) 3000. Data ya MS/MS yakajanwa kwiinda mu ion trap mu LTQ Orbit [ XL data yakusaanguna, eeyo iiyeeme a data kutyompokelwa kwakapa kuti kube kwaandaana (CID)].
Zikozyanyo zya MD zyakachitilwa kubelesya GROMACS (52) pulogilamu antoomwe a MARTINI 2 force field (53-55). CHARMM GUI Membrane Builder (56, 57) mpawo yakabelesegwa kuyaka bbazu lyabili lijisi dioleoylphosphatidylcholine (DOPC) a Cer C18 naa DOPC a Cer C26. Bupange abwaanda bwa Cer C26 bwakajanwa kuzwa ku DXCE kwiinda mukugwisya nsinga zimbi kuzwa kumusela wa sphingosine. Belesya nzila iipanduludwe munsi aawa kuti ubambe zibeela zyobile mpawo uchichite, mpawo ubelesye ma coordinates aamamanino aanzila kuyaka nzila iijisi Emp24. Busena bwa transmembrane bwa yeast Emp24 (zisyaala 173 kusikila ku 193) bwakayakwa mbuli α-helix kubelesya nzila ya visual MD (VMD) molecular structure (58). Mpawo, nikwakamana kugwisigwa mafuta aakali kuvwumbilila, ma protein akabikkwa muzibeela zyobile kubelesya CHARMM GUI. Inzila yamamanino ilijisi 1202 DOPC a 302 Cer C26 naa 1197 DOPC antoomwe a 295 Cer C18 a Emp24. Bikka ion system kusika ku 0.150M. Zikozyano zyone zitazulide zyakachitilwa zibeela zyobile zya bilayer.
I lipid bilayer ilabikkwa kabotu kubelesya nzila ya CHARMM GUI, eeyo iijatikizya kucesya mpawo akubamba ntaamu zili 405,000, ooko zyeelelo zyabusena nkozicheya asyoonto-syoonto akugwisigwa, mpawo ntaamu yachiindi ilayungizigwa kuzwa ku 0.005 ps kusika ku 0.02 ps. Musule akweezyanisya, yakazyala 6 μs achiindi cha 0.02 ps. Mwamana kubikka Emp24, amubelesye nzila njiyonya ya CHARMM GUI kutegwa mucesye akubamba kabotu nzila, mpoonya akubeleka kwamasekondi aali 8 mukupanga.
Kuzibelesyo zyoonse, muchiindi chakweezyanisya, kusinikizyigwa kweendelezyegwa a Berendsen barostat (59), mpawo muchiindi chakupanga, kusinikizyigwa kweendelezyegwa a Parrinello-Rahman barostat (60). Muzyiimo zyoonse, mweelwe waakati aakati wa 1 bar mpawo nzila ya semi-isotropic pressure coupling scheme ilabelesyegwa. Mukweezyanisya akupanga, chintu chakutontola (61) chilafwambaana kubelesegwa kuswanizya kupya kwamapulotini, mafuta aamafuta. Muchiindi choonse chakubeleka, kupya kwachiyandisyo nku 310K. Kuswaangana kutali kwakujatana kulabalilwa akupanga mulongo wazyintu zyobile kubelesya nzila ya Verlet a 0.005 buffer tolerance. Ibbala lya Coulomb lilabalilwa kubelesya nzila yakusanduka antoomwe abulemu bwakukosola bwa 1.1 nm. Ibbala lya Vander Waals libelesya nzila yakugela abulemu bwa 1.1 nm, mpawo nzila yakugela ya Verlet ibelesyegwa kukukonzya kweenda (62).
Kubelesya VMD, mweelwe wa wavelength akati ka DOPC phosphate beads naa ceramide AM1 beads a protein ngu 0.7 nm, mpawo mweelwe wama lipids aajatikizyidwe a protein ulabalilwa. Kweelana anzila itobela, bala mweelwe wakugwisya-kuyungizya (DE) mbuli mu (63): DE factor = (mweelwe wamafuta woonse mupulotiini 0.7) mupulotiini 0.7 (mweelwe waCer mumafuta woonse)
Mpindu yakaambwa yakajanwa mbuli mweelwe waakati-kati, mpawo tumpenda twakulubizya tuli makkopi one aatali aalikke aa SE. Bupati bwa DE factor bulabalilwa a t test [(averageDE-factor-1)/SE]. Bala mweelwe wa P kuzwa kukumwaigwa kwamusela omwe.
Chibelesyo cha GROMACS chakabelesegwa kubala 2D lateral density map ya system iijisi Emp24 mukati ka 250 ns yamamanino ya trace. Kuti tujane maapu yakuvwuzya/kumana kwa ceramide, maapu yabunji bwa Cer ilaandaanizyigwa amweelwe wamaapu ya Cer a DOPC, mpawo yaabwa amweelwe wa Cer mumubili. Chipimyo chamaapu yamubala nguwenya chibelesegwa.
Kuti ujane zimwi zilembedwe muchibalo eechi, langa http://advances.sciencemag.org/cgi/content/full/6/50/eaba8237/DC1
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Sofia Rodriguez-Gallardo, Kazuo Kurokawa, Susana Sabido-Bozo, Alejandro Cortez · Gomez (Alejandro Cortes-Gomez), Atsuko Ikeda (Atsuko Ikeda), Valeria Zoni (Valeria Zoni), Auxiliadora Aguilera-Romero, Ana Maria Perez -Lingio Serero), Wazero Lopez (Serhopez), (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Zifwanikiso zya 3D ziboneka kabotu muchiindi cheenzu zitondezya mbociyandika bulamfwu bwa nketani ya ceramide mukusalazya ma protein mumasena aasalidwe.
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 -Lingio Serero), Wazero Lopez (Serhopez), (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Zifwanikiso zya 3D ziboneka kabotu muchiindi cheenzu zitondezya mbociyandika bulamfwu bwa nketani ya ceramide mukusalazya ma protein mumasena aasalidwe.
©2020 Kabunga ka Amelika kakusumpula sayensi. zyeelelo zyoonse zilikwabilidwe. AAAS mweenzinyina wa HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef a COUNTER. ScienceAdvances ISSN 2375-2548.