Kev cais cov protein hauv txoj kev tso zis yog qhov tseem ceeb rau kev tswj hwm kev faib cov cell thiab homeostasis. Ntxiv rau kev cais cov plhaub, lub luag haujlwm ntawm lipids hauv kinesin kev cais hauv cov txheej txheem ntawm kev thauj mus los ntawm secretory yog ib lo lus nug ntev uas tseem tsis tau teb. Ntawm no, peb ua 3D tib lub sijhawm ntau xim siab daws teeb meem tiag tiag los ua pov thawj hauv vivo tias cov glycosylphosphatidylinositol-immobilized proteins tshiab nrog cov ceramide lipid moieties ntev heev tau sib sau ua ke thiab faib ua cov endoplasms tshwj xeeb Net tawm qhov chaw, uas txawv ntawm qhov siv los ntawm cov protein transmembrane. Tsis tas li ntawd, peb qhia tias qhov ntev ntawm cov saw hlau ntawm ceramide hauv endoplasmic reticulum membrane yog qhov tseem ceeb rau qhov kev xaiv cais no. Peb txoj kev tshawb fawb muab thawj qhov pov thawj ncaj qha hauv vivo los faib cov protein cargoes raws li qhov ntev ntawm cov saw hlau mus rau hauv cov chaw xa khoom xaiv hauv txoj kev tso zis.
Hauv cov hlwb eukaryotic, cov protein uas tau tsim ua ke hauv endoplasmic reticulum (ER) ces raug txheeb xyuas thaum lub sijhawm thauj mus los ntawm txoj kev zais cia rau kev xa mus rau lawv qhov chaw tsim nyog ntawm tes (1). Ntxiv nrog rau kev cais tawm ntawm lub tsho tiv no, nws tau xav ntev tias qee cov lipids kuj tseem tuaj yeem ua haujlwm ua cov ntsiab lus xaiv tawm los ntawm kev sib sau ua ke rau hauv cov cheeb tsam membrane tshwj xeeb uas cov protein tshwj xeeb (2-5). Txawm li cas los xij, tseem tsis muaj pov thawj ncaj qha hauv vivo los ua pov thawj qhov ua tau lipid-raws li mechanism no. Txhawm rau daws qhov teeb meem yooj yim no, peb tau kawm hauv cov poov xab li cas glycosylphosphatidylinositol (GPI) anchored proteins (GPI-APs) raug xa tawm txawv ntawm ER. GPI-APs yog ntau yam lipid-txuas nrog cell nto proteins (6, 7). GPI-AP yog cov protein zais cia txuas nrog cov ntawv sab nraud ntawm cov plasma membrane los ntawm glycolipid moiety (GPI anchor). Lawv lees txais GPI anchors ua kev hloov kho tom qab kev txhais lus hauv ER lumen (8). Tom qab txuas, GPI-AP hla dhau Golgi apparatus (5, 9) los ntawm ER mus rau plasma membrane. Qhov muaj GPI anchors ua rau GPI-AP raug thauj mus sib cais los ntawm transmembrane secreted proteins (suav nrog lwm cov plasma membrane proteins) raws txoj kev secretory (5, 9, 10). Hauv cov hlwb poov xab, GPI-APs raug sib cais los ntawm lwm cov protein secreted hauv endoplasmic reticulum, thiab tom qab ntawd ntim rau hauv cov vesicles tshwj xeeb qhwv los ntawm txheej protein complex II (COPII) (6, 7). Cov kev txiav txim siab ntawm cov txheej txheem kev faib tawm no hauv ER export txheej txheem tsis meej, tab sis nws tau xav tias cov txheej txheem no yuav xav tau lipids, tshwj xeeb tshaj yog qhov kev hloov kho dua tshiab ntawm cov lipid ntawm GPI anchor (5, 8). Hauv poov xab, GPI lipid remodeling pib tam sim ntawd tom qab GPI txuas, thiab hauv ntau qhov xwm txheej, nws ua rau ceramide khi rau 26-carbon ntev-saw saturated fatty acid (C26: 0) (11, 12). C26 ceramide yog lub ceramide tseem ceeb uas tsim los ntawm cov hlwb poov xab txog tam sim no. Nws yog synthesized hauv ER thiab feem ntau ntawm nws yog exported mus rau Golgi apparatus los ntawm COPII vesicles (13). ER export ntawm GPI-AP tshwj xeeb xav tau kev txuas ntxiv ceramide synthesis (14, 15), thiab nyob rau hauv lem, kev hloov pauv ntawm ceramide mus rau inositol phosphate ceramide (IPC) hauv Golgi apparatus nyob ntawm GPI anchor synthesis (16). Kev tshawb fawb biophysical nrog cov membranes dag tau qhia tias cov acyl saw hlau ceramides ntev heev tuaj yeem sib sau ua ke los tsim cov npe sau nrog cov khoom tshwj xeeb (17, 18). Cov ntaub ntawv no ua rau muaj kev xav tias C26 ceramide thiab GPI-AP nrog C26 ceramide siv lawv cov khoom siv lub cev los sib sau ua ke rau hauv cov cheeb tsam lossis cov cheeb tsam hauv qhov chaw ER membrane lipid tsis zoo. Nws feem ntau yog tsim los ntawm luv luv thiab unsaturated glycerolipids (C16: 1 thiab C18: 1) (19, 20). Cov cheeb tsam no yuav raug xaiv los tsom mus rau cov chaw tawm ER tshwj xeeb (ERES), qhov twg ceramide thiab ceramide-based GPI-AP tuaj yeem thauj mus rau Golgi hauv tib lub COPII vesicle (5).
Hauv kev tshawb fawb no, peb tau sim ncaj qha rau cov txheej txheem lipid-based no los ntawm kev siv super-resolution confocal real-time imaging microscopy (SCLIM), uas yog ib txoj kev siv tshuab microscopy uas tuaj yeem soj ntsuam cov protein uas muaj fluorescently labeled tib lub sijhawm. Cov duab peb xim thiab peb-seem (3D) muaj kev daws teeb meem siab heev thiab ceev hauv cov hlwb muaj sia nyob (21, 22).
Peb thawj zaug siv SCLIM thev naus laus zis los txhais ntxiv txog li cas GPI-AP ib txwm muaj nrog C26 ceramide pawg tau raug tshuaj xyuas los ntawm cov protein transmembrane secreted tom qab tawm hauv ER hauv S. cerevisiae. Txhawm rau txheeb xyuas qhov kev faib tawm ntawm ER, peb tau siv lub kaw lus genetic uas tuaj yeem pom cov khoom thauj tshiab synthesized nkag mus rau ERES hauv vivo (7, 23). Raws li cov khoom thauj, peb tau xaiv C26 ceramide-based GPI-AP Gas1 labeled nrog cov protein fluorescent ntsuab (GFP) thiab transmembrane secreted protein Mid2 labeled nrog ze-infrared fluorescent protein (iRFP), ob qho tib si tsom mus rau plasma membrane (24–26). Hauv sec31-1 kub-sensitive mutant, ob qho khoom thauj no tau qhia nyob rau hauv galactose-inducible promoter thiab constitutive ERES marker. Thaum qhov kub heev (37°C), vim tias qhov kev hloov pauv sec31-1 cuam tshuam rau kev ua haujlwm ntawm COPII txheej Cheebtsam Sec31 kom tiv thaiv COPII germination thiab ER xa tawm, cov khoom thauj tshiab synthesized sib sau ua ke ntawm ER (23). Tom qab txias mus rau qhov kub qis (24°C), cov hlwb sec31-1 mutant rov qab los ntawm thaj chaw secretory, thiab cov khoom thauj tshiab synthetic tau pib xa tawm los ntawm ER. CLIM visualization qhia tau hais tias feem ntau ntawm cov Gas1-GFP thiab Mid2-iRFP tshiab synthesized tseem sib sau ua ke hauv ER ntawm cov hlwb sec31-1 mutant tom qab incubation ntawm 37°C thiab tom qab ntawd tso tawm ntawm 24°C rau 5 feeb (Daim Duab 1). Vim tias Mid2-iRFP tau faib rau ntawm tag nrho ER membrane, thiab Gas1-GFP tau concentrated thiab sau ua ke hauv thaj chaw discontinuous ER membrane, lawv qhov kev faib tawm txawv kiag li (Daim Duab 1, A rau C thiab Movie S1). Tsis tas li ntawd, raws li pom hauv Daim Duab 1D, pawg Gas1-GFP tsis muaj Mid2-iRFP. Cov txiaj ntsig no qhia tau tias GPI-AP thiab cov protein transmembrane tau raug cais mus rau hauv thaj chaw sib txawv ntawm ER membrane thaum ntxov. Pawg Gas1-GFP nyob ib sab ntawm ERES tshwj xeeb uas muaj mCherry's COPII coat protein Sec13 (Daim Duab 1, E thiab F, thiab zaj duab xis S1) (23).
Cov hlwb sec31-1 qhia txog cov kua qaub ncaug uas ua rau galactose, ib txoj saw acyl ntev (C26) ceramide GPI-AP Gas1-GFP (GPI-AP, ntsuab) thiab cov protein transmembrane Mid2-iRFP (TMP, xiav) thiab qhov Constructive ERES labeling Sec13-mCherry (ERES, magenta) no tau incubated ntawm 37°C rau 30 feeb, tsiv mus rau 24°C, thiab imageed los ntawm SCLIM 5 feeb tom qab. (A rau C) qhia txog ib daim duab 2D sib koom ua ke lossis ib daim duab ntawm lub dav hlau (A), ib daim duab projection 2D ntawm 10 z-sections (B) lossis ib daim duab 3D cell hemisphere ntawm cov khoom thauj thiab ERES markers (C). Scale bar 1μm (A thiab B). Lub scale unit yog 0.551μm (C). Gas1-GFP tau pom hauv cov cheeb tsam ER lossis pawg sib cais, thaum Mid2-iRFP tau pom thiab faib thoob plaws hauv ER membrane (C). (D) Daim duab qhia txog qhov sib piv ntawm qhov sib piv ntawm fluorescence ntawm Gas1-GFP thiab Mid2-iRFP hauv Gas1-GFP pawg raws li kab xub dawb (sab laug). AU, chav tsev tsis raws cai. (E thiab F) sawv cev rau daim duab 3D uas muab cov khoom lag luam thiab ERES cim ua ke. Cov pawg Gas1-GFP tau pom ze ntawm ERES tshwj xeeb. Lub ntsuas ntsuas yog 0.551μm. (F) Tus xub dawb khov kho cim cov pawg Gas1-GFP cuam tshuam nrog ERES. Cov vaj huam sib luag nruab nrab thiab sab xis qhia cov duab 3D uas tau sib koom ua ke thiab qhov pom tig ntawm pawg Gas1-GFP xaiv.
Qhov kev sib raug zoo ntawm qhov chaw ze ntawm pawg Gas1-GFP thiab ib qho ERES tshwj xeeb qhia tau hais tias Gas1-GFP tuaj yeem nkag mus rau hauv ERES xaiv, uas txawv ntawm qhov kev xaiv uas Mid2-iRFP siv los tawm hauv ER. Txhawm rau daws qhov ua tau no, peb tau ntsuas qhov piv ntawm ERES rau ib lossis ob yam khoom xwb (Daim Duab 2, A txog C). Peb pom tias feem ntau ERES (70%) tsuas muaj ib hom khoom thauj xwb. Daim duab hauv qab ntawm Daim Duab 2C qhia ob qho piv txwv ntawm ERES nrog tsuas yog Gas1-GFP (Daim Duab 1) lossis tsuas yog Mid2-iRFP (Daim Duab 2). Qhov sib txawv, kwv yees li 20% ntawm ERES muaj ob lub khoom thauj uas sib tshooj hauv tib thaj chaw. Nws tau pom tias qee qhov ERES (10%) muaj ob hom khoom thauj, tab sis lawv tau cais tawm hauv thaj chaw sib txawv. Yog li ntawd, qhov kev tshuaj xyuas txheeb cais no qhia tau hais tias tom qab ER raug xa tawm, GPI-AP Gas1-GFP thiab cov khoom thauj transmembrane Mid2-iRFP raug faib ua ERES sib txawv (Daim Duab 2D). Qhov kev ua haujlwm zoo ntawm kev cais tawm no zoo ib yam li kev tshuaj xyuas biochemical yav dhau los (6) thiab kev txiav txim siab morphological (7). Peb kuj tuaj yeem saib xyuas tus cwj pwm ntawm cov khoom thauj cais tawm nkag mus rau ERES (Daim Duab 2E thiab Movie S2). Daim Duab 2E qhia tau tias tsuas yog ib feem me me ntawm Gas1-GFP (vaj huam sib luag 3) lossis Mid2-iRFP (vaj huam sib luag 4) nkag mus rau ERES los ntawm ib sab thiab raug kaw hauv thaj chaw sib cais. Vaj Huam Sib Luag 5 ntawm Daim Duab 2E qhia tau tias Gas1-GFP thiab Mid2-iRFP qee zaum pom nyob rau hauv tib ERES, tab sis lawv nkag los ntawm ntau sab thiab tau sib sau ua ke hauv thaj chaw sib cais uas yuav sawv cev rau cov vesicles COPII sib txawv. Peb kuj tau lees paub tias qhov kev sib cais thiab kev faib tawm ntawm C26 ceramide-based GPI-AP Gas1 raws li kev xaiv ERES yog qhov tshwj xeeb vim tias lwm qhov transmembrane secretion cargo, GFP-tagged plasma membrane protein Axl2 (27), qhia txog tus cwj pwm zoo sib xws rau Mid2-iRFP. (Daim Duab S1 thiab Movie S3). Axl2-GFP tshiab tsim tau muab faib los ntawm ER membrane zoo li Mid2-iRFP (Daim Duab S1, A thiab B), thiab tau sib koom ua ke nrog Mid2-iRFP hauv feem ntau ERES (Daim Duab S1, B txog D). Cov Vaj Huam Sib Luag 1 thiab 2 ntawm Daim Duab 1. S1C qhia ob qho piv txwv ntawm ERES qhov twg ob lub khoom thauj transmembrane sib tshooj. Hauv cov xwm txheej no, ob qho khoom nkag mus rau ERES ua ke (Daim Duab S1E, Vaj Huam Sib Luag 3 thiab Movie S3).
Cov hlwb sec31-1 uas qhia txog cov galactose inducible secretions, Gas1-GFP (GPI-AP, ntsuab) thiab Mid2-iRFP (TMP, xiav) thiab cov cim ERES Sec13-mCherry (ERES, magenta) tau muab tso rau ntawm 37 Tom qab incubating rau 30 feeb ntawm °C, txav mus rau 24 °C kom tso tawm cov secretion block, thiab duab nrog SCLIM tom qab 20 feeb. (A rau C) Cov duab 2D projection sawv cev (A; scale bar, 1μm) lossis 3D cell hemisphere duab (B thiab C; scale unit, 0.456μm) ntawm cov khoom thauj thiab 10 z-sections cim los ntawm ERES. Lub vaj huam sib luag qis dua hauv (B) thiab lub vaj huam sib luag hauv (C) tso saib cov duab ua tiav los tso saib tsuas yog cov khoom muaj nyob hauv ERES (magenta) [Gas1-GFP (grey) thiab Mid2-iRFP (xiav daj)]. (C) Qhib xub: ERES tsuas yog nqa ib daim khoom thauj (1 rau 4). Xub grey: ERES muaj cov khoom thauj sib cais (5). Xub dawb khov kho: ERES muaj cov khoom thauj sib koom ua ke. Hauv qab no: Ib qho ERES uas xaiv tsuas muaj Gas1-GFP (1) lossis Mid2-iRFP (2). Scale bar, 100 nm. (D) Kev ntsuas ntawm daim duab thaij duab piav qhia hauv (C). Qhov feem pua ​​​​​​nruab nrab ntawm ERES uas muaj tsuas yog ib qho khoom thauj (Gas1-GFP lossis Mid2-iRFP), cov khoom thauj sib cais thiab cov khoom thauj sib tshooj. Hauv peb qhov kev sim ywj pheej, n = 432 hauv 54 lub hlwb. Yuam kev bar = SD. Ob-tailed unpaired t xeem. *** P = 0.0002. (E) 3D daim duab ntawm ERES xaiv ntawm cov khoom thauj cais tawm cim nrog (C). Gas1-GFP (ntsuab) (3) lossis Mid2-iRFP (xiav) (4) nkag mus rau ERES (magenta) los ntawm ib sab thiab txwv rau thaj chaw me me hauv ERES. Qee zaum, ob hom khoom thauj nkag mus rau tib ERES (5) los ntawm tib sab thiab raug kaw rau hauv thaj chaw cais tawm hauv ERES. Scale bar, 100 nm.
Tom ntej no, peb tau sim ib qho kev xav tias cov acyl saw ceramide ntev (C26) uas muaj nyob rau hauv ER membrane tsav cov clustering thiab kev cais ntawm Gas1 mus rau hauv kev xaiv ERES. Txog qhov kawg no, peb tau siv cov poov xab hloov kho GhLag1, uas ob qho endogenous ceramide synthases Lag1 thiab Lac1 tau hloov los ntawm GhLag1 (Lag1 homolog ntawm paj rwb), ua rau cov poov xab nrog lub cell membrane Ceramide hom luv dua li hom qus (Daim Duab 3A) (28). Kev tshuaj xyuas pawg spectrometry (MS) qhia tau hais tias hauv cov kab mob qus, 95% ntawm tag nrho cov ceramide yog ntev heev (C26) saw ceramide, thaum nyob rau hauv GhLag1, 85% ntawm ceramide yog ntev heev (C18 thiab C16). ), tsuas yog 2% ntawm ceramide yog ntev heev (C26) saw ceramide. Txawm hais tias C18 thiab C16 ceramides yog cov ceramides tseem ceeb uas pom hauv GhLag1 daim nyias nyias txog tam sim no, MS kev tshuaj xyuas kuj tau lees paub tias GPI anchor ntawm Gas1-GFP qhia hauv GhLag1 hom muaj C26 ceramide, uas sib piv rau cov lipids qus. Qhov zoo yog tib yam (Daim Duab 3A) (26). Yog li ntawd, qhov no txhais tau tias ceramide remodeling enzyme Cwh43 yog xaiv heev rau C26 ceramide, raws li pom hauv Daim Duab 26, nws nyiam suav nrog GPI anchor los ntawm me me ntawm C26 ceramide hauv GhLag1 hom. S2 (29). Txawm li cas los xij, lub cell membrane ntawm GhLag1 tsuas yog muaj C18-C16 ceramide, thaum Gas1-GFP tseem muaj C26 ceramide. Qhov tseeb no ua rau hom no yog ib qho cuab yeej zoo tshaj plaws los daws qhov teeb meem ntawm acyl saw ntev ntawm daim nyias nyias ceramide hauv ER. Lub luag haujlwm hypothetical ntawm chav kawm thiab kev cais. Tom qab ntawd, peb thawj zaug kawm txog lub peev xwm ntawm C26 Gas1-GFP kom sib sau ua ke hauv cov pawg hauv GhLag1 nrog cov allele hloov pauv kub ntawm sec31-1 los ntawm kev siv lub tshuab fluorescence microscopy, qhov twg tsuas yog cov saw ntev (C18-C16) muaj nyob hauv ER membrane Ceramide (Daim Duab 3). Peb tau pom tias hauv sec31-1, feem ntau ntawm Gas1-GFP tau sib sau ua ke hauv cov pawg, thaum Gas1-GFP hauv sec31-1 GhLag1 nrog cov ceramide ER membrane ntev (C18-C16) feem ntau tsis tau sib sau ua ke thiab faib rau hauv tag nrho ER membrane. Yuav kom meej meej, vim tias C26 ceramide-based clustering muaj feem cuam tshuam nrog ERES tshwj xeeb (Daim Duab 1), peb tom ntej no tshawb xyuas seb cov txheej txheem no puas tseem yuav cuam tshuam nrog kev ua haujlwm ntawm ER export protein mechanism. GPI-AP siv lub kaw lus COPII tshwj xeeb rau ER export, uas tau tswj hwm los ntawm Ted1's structural remodeling ntawm glycan feem ntawm GPI anchor (30, 31). Tus recombinant GPI-glycan ces raug lees paub los ntawm transmembrane cargo receptor p24 complex, uas tig mus xaiv Lst1, uas yog ib qho isoform tshwj xeeb ntawm COPII cargo binding subunit Sec24, tsim GPI-AP-rich COPII Vesicles yog qhov tsim nyog (31-33). Yog li ntawd, peb tau tsim ob chav mutant uas ua ke kev rho tawm ntawm cov protein no (p24 complex component Emp24, GPI-glycan remodeling enzyme Ted1 thiab COPII subunit Lst1 tshwj xeeb) nrog sec31-1 mutant strain, thiab kawm lawv Puas yog nws ua tau los tsim Gas1-cluster GFP (Daim Duab 3). Peb tau pom tias hauv sec31-1emp24Δ thiab sec31-1ted1Δ, Gas1-GFP feem ntau yog unclustered thiab faib thoob plaws hauv ER membrane, raws li tau pom yav dhau los hauv sec31-1 GhLag1, thaum nyob hauv sec31-1lst1Δ, Gas1-GFP Zoo li sec31-1. Cov txiaj ntsig no qhia tau tias ntxiv rau qhov muaj C26 ceramide hauv ER membrane, kev sib sau ua ke ntawm Gas1-GFP kuj tseem yuav tsum khi rau p24 complex, thiab tsis tas yuav tsum muaj kev nrhiav neeg tshwj xeeb Lst1. Tom qab ntawd, peb tau tshawb nrhiav qhov ua tau tias qhov ntev ntawm ceramide hauv ER membrane tuaj yeem tswj hwm qhov kev khi ntawm Gas1-GFP rau p24. Txawm li cas los xij, peb pom tias qhov muaj C18-C16 ceramide hauv daim nyias nyias tsis cuam tshuam rau GPI-glycans rov tsim dua los ntawm p24 complex (Daim Duab S3 thiab S4, A thiab B) lossis khi rau GPI-AP thiab xa tawm GPI-AP. Muaj peev xwm. Nrhiav COPII subtype Lst1 (Daim Duab S4C). Yog li ntawd, C26 ceramide-dependent clustering tsis xav tau kev sib cuam tshuam ntawm cov protein nrog cov txheej txheem xa tawm ER sib txawv, tab sis txhawb nqa lwm txoj hauv kev xaiv uas tsav los ntawm qhov ntev lipid. Tom qab ntawd, peb tau tshuaj xyuas seb qhov ntev ntawm ceramide acyl saw hauv ER membrane puas tseem ceeb rau kev faib tawm zoo ntawm Gas1-GFP ua ERES xaiv. Txij li thaum Gas1 hauv GhLag1 hom nrog cov ceramide luv luv tawm hauv ER thiab nkag mus rau hauv plasma membrane (Daim Duab S5), peb ntseeg tias yog tias qhov kev cais tawm yog tsav los ntawm qhov ntev ntawm ceramide acyl saw, Gas1 hauv GhLag1 hom tuaj yeem raug xa mus thiab hla. ERES cov khoom nrog tib daim nyias nyias.
(A) Lub cell membrane ntawm GhLag1 feem ntau muaj cov C18-C16 ceramides luv dua, thaum GPI anchor ntawm Gas1-GFP tseem muaj tib C26 IPC li cov cell qus-hom. Saum toj no: acyl saw ntev tsom xam ntawm ceramide hauv lub cell membrane ntawm qus-hom (Wt) thiab GhLag1p hom los ntawm pawg spectrometry (MS). Cov ntaub ntawv sawv cev rau feem pua ​​ntawm tag nrho ceramide. Qhov nruab nrab ntawm peb qhov kev sim ywj pheej. Yuam kev bar = SD. Ob-tailed unpaired t xeem. **** P <0.0001. Hauv qab vaj huam sib luag: MS tsom xam ntawm acyl saw ntev ntawm IPC uas muaj nyob rau hauv Gas1-GFP (GPI-IPC) GPI anchor qhia nyob rau hauv cov qus-hom thiab GhLag1p hom. Cov ntaub ntawv sawv cev rau feem pua ​​ntawm tag nrho IPC teeb liab. Qhov nruab nrab ntawm tsib qhov kev sim ywj pheej. Yuam kev bar = SD. Ob-tailed unpaired t xeem. ns, tsis tseem ceeb. P = 0.9134. (B) Cov duab fluorescence micrographs ntawm sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ thiab sec31-1lst1Δ hlwb uas qhia txog galactose-induced Gas1-GFP tau incubated ntawm 37°C rau 30 feeb thiab dhau mus rau Ua qhov kev kuaj fluorescence microscopy tom qab 24°C. Xub dawb: ER Gas1-GFP pawg. Xub qhib: Unclustered Gas1-GFP tau faib rau ntawm tag nrho ER membrane, qhia txog ER tus yam ntxwv ntawm lub nplhaib nuclear staining. Scale bar, 5μm. (C) Kev ntsuas ntawm photomicrograph piav qhia hauv (B). Qhov feem pua ​​​​​​nruab nrab ntawm cov hlwb nrog punctate Gas1-GFP qauv. Hauv peb qhov kev sim ywj pheej, n≥300 hlwb. Yuam kev bar = SD. Ob-tailed unpaired t xeem. **** P <0.0001.
Yuav kom daws tau qhov teeb meem no ncaj qha, peb tau ua SCLIM visualization ntawm Gas1-GFP thiab Mid2-iRFP hauv GhLag1 nrog rau sec31-1 qhov kub thiab txias-sensitive mutant allele (Daim Duab 4 thiab Movie S4). Tom qab ER khaws cia ntawm 37 ° C thiab tom qab ntawd tso tawm ntawm 24 ° C, feem ntau ntawm cov Gas1-GFP tshiab tsim tsis tau clustered thiab faib thoob plaws hauv ER membrane, raws li pom los ntawm cov microscopes ib txwm muaj (Daim Duab 4, A thiab B). Tsis tas li ntawd, feem pua ​​​​​​ntawm ERES (67%) suav nrog ob hom khoom thauj sib koom ua ke hauv nws (Daim Duab 4D). Cov Vaj Huam Sib Luag 1 thiab 2 ntawm Daim Duab 4C qhia ob qho piv txwv ntawm ERES nrog kev sib tshooj Gas1-GFP thiab Mid2-GFP. Tsis tas li ntawd, ob qho khoom tau raug xaiv rau hauv tib ERES (Daim Duab 4E, vaj huam sib luag 3 thiab movie S4). Yog li ntawd, peb cov txiaj ntsig qhia tau tias qhov ntev ntawm cov saw hlau ceramide acyl hauv ER membrane yog qhov tseem ceeb ntawm kev sib sau ua ke thiab kev faib tawm ntawm ER protein.
Cov qe ntshav Sec31-1 GhLag1 uas qhia txog cov kua qaub ncaug uas ua rau galactose, Gas1-GFP (GPI-AP, ntsuab) thiab Mid2-iRFP (TMP, xiav) thiab cov khoom siv ERES-labeled Sec13-mCherry (ERES, magenta) Incubate ntawm 37°C. Txuas ntxiv mus rau 30 feeb, poob rau 24°C kom tso cov kua qaub ncaug tawm, thiab thaij duab nrog SCLIM tom qab 20 feeb. (A txog C) Cov duab 2D projection sawv cev (A; scale bar, 1μm) lossis 3D cell hemisphere duab (B thiab C; scale unit, 0.45μm) ntawm 10 z-sections cim los ntawm cargo thiab ERES. Lub vaj huam sib luag qis dua hauv (B) thiab lub vaj huam sib luag hauv (C) tso saib cov duab ua tiav los tso saib tsuas yog cov khoom muaj nyob hauv ERES (magenta) [Gas1-GFP (grey) thiab Mid2-iRFP (xiav daj)]. (C) Xub dawb puv: ERES, cov khoom sib tshooj. Qhib xub: ERES tsuas muaj ib yam khoom xwb. Vaj huam sib luag hauv qab: ERES xaiv muaj cov khoom sib tshooj (1 thiab 2) cim rau hauv (C). Scale bar, 100 nm. (D) Kev ntsuas ntawm photomicrograph piav qhia rau hauv (C). Hauv sec31-1 thiab sec31-1 GhLag1 units, tsuas yog ib qho khoom thauj (Gas1-GFP lossis Mid2-iRFP) suav nrog, thiab qhov feem pua ​​​​​​nruab nrab ntawm ERES rau cov khoom thauj cais thiab cov khoom thauj sib tshooj. Hauv peb qhov kev sim ywj pheej, n = 432 hauv 54 lub hlwb (sec31-1) thiab n = 430 hauv 47 lub hlwb (sec31-1 GhLag1). Yuam kev bar = SD. Ob-tailed unpaired t xeem. *** P = 0.0002 (sec31-1) thiab ** P = 0.0031 (sec31-1 GhLag1). (E) 3D duab ntawm ERES xaiv nrog cov khoom thauj sib tshooj (3) cim rau hauv (C). Gas1-GFP (ntsuab) thiab Mid2-iRFP (xiav) mus txog ERES (magenta) los ntawm tib sab thiab nyob hauv tib thaj chaw txwv ERES. Scale bar, 100 nm.
Txoj kev tshawb fawb no muab cov pov thawj ncaj qha hauv vivo tias cov khoom thauj protein lipid tau muab faib ua cov chaw xa khoom xaiv hauv txoj kev zais cia, thiab qhia txog qhov tseem ceeb ntawm qhov ntev ntawm acyl saw rau kev xaiv kev faib tawm. Siv cov txheej txheem microscopy muaj zog thiab txiav-ntug hu ua SCLIM, peb tau ua pov thawj Gas1-GFP tshiab tsim (lub plasma membrane loj GPI-AP nrog lub acyl saw ntev heev (C26) ceramide lipid feem) hauv cov poov xab) Cov cheeb tsam sib sau ua ke hauv discrete ERs yog txuam nrog ERES tshwj xeeb, thaum cov protein transmembrane secreted tau faib thoob plaws hauv ER membrane (Daim Duab 1). Tsis tas li ntawd, ob hom khoom no nkag mus rau ERES sib txawv xaiv (Daim Duab 2). Qhov ntev ntawm acyl saw ntawm cellular ceramide hauv daim nyias nyias raug txo los ntawm C26 mus rau C18-C16, Gas1-GFP pawg raug cuam tshuam rau hauv thaj chaw ER sib cais, thiab Gas1-GFP raug xa rov qab mus rau ER nrog cov protein transmembrane los ntawm tib ERES (Daim Duab 3 thiab Daim Duab 3). 4).
Txawm hais tias GPI-AP siv cov txheej txheem protein tshwj xeeb los tawm ntawm ER, peb pom tias C26 ceramide-dependent kev sib cais tsis vam khom rau kev sib cuam tshuam ntawm cov protein sib txawv uas yuav ua rau ERES tshwj xeeb (Cov Duab S4 thiab S5). Hloov chaw, peb cov kev tshawb pom txhawb nqa lwm txoj hauv kev faib tawm uas tsav los ntawm lipid-based protein clustering thiab tom qab ntawd tshem tawm lwm cov khoom thauj. Peb cov kev soj ntsuam qhia tau hais tias thaj tsam Gas1-GFP lossis pawg uas cuam tshuam nrog ib qho ERES tshwj xeeb tsis muaj cov protein transmembrane secreted Mid2-iRFP, uas qhia tau hais tias C26 ceramide-dependent GPI-AP pawg yuav pab txhawb lawv nkag mus rau hauv ERES, thiab tib lub sijhawm, tshem tawm cov secretions nkag mus rau hauv ERES tshwj xeeb no (Cov Duab 1 thiab 2). Qhov sib piv, qhov muaj C18-C16 ceramides hauv ER membrane tsis ua rau GPI-AP tsim cov cheeb tsam lossis pawg, yog li lawv tsis tshem tawm lossis hloov cov protein transmembrane secreted rau hauv tib ERES (Cov Duab 3 thiab 4). . Yog li ntawd, peb xav tias C26 ceramide tsav kev sib cais thiab kev faib tawm los ntawm kev pab txhawb kev sib sau ua ke ntawm cov protein txuas nrog cov ERES tshwj xeeb.
Yuav ua li cas thiaj ua tiav qhov kev sib sau ua ke ntawm C26 ceramide no rau hauv ib cheeb tsam ER tshwj xeeb? Qhov kev nyiam ntawm daim nyias nyias ceramide kom sib cais sab nraud yuav ua rau GPI-AP thiab C26 ceramide tsim cov lipids me me thiab txiav txim siab tam sim ntawd hauv qhov chaw lipid tsis sib xws ntawm daim nyias nyias ER uas muaj cov glycerolipids luv dua thiab tsis muaj zog. Cov pawg zoo (17, 18). Cov pawg me me ib ntus no tuaj yeem sib xyaw ua ke rau hauv cov pawg loj dua, ruaj khov dua tom qab khi rau p24 complex (34). Sib xws nrog qhov no, peb tau qhia tias C26 Gas1-GFP xav tau kev sib cuam tshuam nrog p24 complex los tsim cov pawg pom loj dua (Daim Duab 3). Lub p24 complex yog ib qho heterozygous oligomer uas muaj plaub qhov sib txawv p24 transmembrane proteins hauv cov poov xab (35), uas muab ntau yam kev sib txuas, uas tuaj yeem ua rau kev sib txuas ntawm cov pawg me me GPI-AP, yog li tsim cov pawg ruaj khov loj dua (34). Qhov kev sib cuam tshuam ntawm cov protein ectodomains ntawm GPI-APs kuj tseem yuav pab txhawb rau lawv cov kev sib sau ua ke, raws li pom thaum lub sijhawm lawv Golgi thauj mus los hauv cov hlwb epithelial polarized mammalian (36). Txawm li cas los xij, thaum C18-C16 ceramide muaj nyob rau hauv ER membrane, thaum p24 complex khi rau Gas1-GFP, cov pawg loj sib cais yuav tsis tsim. Lub hauv paus mechanism yuav nyob ntawm cov khoom siv lub cev thiab tshuaj ntawm cov acyl saw ntev ceramide. Kev tshawb fawb biophysical ntawm cov membranes dag qhia tau tias txawm hais tias ob qho tib si ntev (C24) thiab luv (C18-C16) acyl saw ceramides tuaj yeem ua rau muaj kev sib cais theem, tsuas yog ntev acyl saw ceramides (C24) tuaj yeem txhawb nqa siab Curvature thiab zaj duab xis khoov kom rov ua dua zaj duab xis. Los ntawm kev sib txuas lus (17, 37, 38). Nws tau pom tias lub transmembrane helix ntawm TMED2, tib neeg homologue ntawm Emp24, xaiv sib cuam tshuam nrog C18 ceramide-based sphingomyelin hauv cytoplasmic lobules (39). Siv cov molecular dynamics (MD) simulations, peb pom tias ob qho tib si C18 thiab C26 ceramides sib sau ua ke nyob ib puag ncig cytoplasmic lobules ntawm Emp24 transmembrane helix, thiab lawv muaj kev nyiam zoo sib xws (Daim Duab S6). Nws tsim nyog sau cia tias qhov no qhia tau tias lub transmembrane helix ntawm Emp24 tuaj yeem ua rau muaj kev faib tawm asymmetric ntawm lipids hauv daim nyias nyias. Qhov no yog qhov tshwm sim tsis ntev los no raws li cov hlwb mammalian. Cov kev sim MD zoo sib xws kuj qhia txog qhov muaj ether lipids (40). Yog li ntawd, peb xav tias C26 ceramide hauv ob lub lobules ntawm ER26 yog qhov nplua nuj hauv zos. Thaum GPI-AP hauv cov luminal lobules ncaj qha khi rau ntau tus p24 thiab kev sib sau ua ke ntawm C26 ceramide nyob ib puag ncig p24 hauv cov cytoplasmic lobules, nws tuaj yeem txhawb nqa cov protein sib sau ua ke thiab cov curvature membrane tau tsim los ntawm cov ntiv tes (41), ua rau GPI-AP sib cais mus rau hauv thaj chaw sib cais uas nyob ib sab ntawm ERES, uas kuj nyiam cov cheeb tsam nkhaus heev ntawm ER membrane (42). Cov ntawv ceeb toom yav dhau los txhawb nqa cov txheej txheem tau hais tseg (43, 44). Kev sib txuas ntau tus ntawm oligolectins, cov kab mob lossis cov tshuaj tiv thaiv rau ceramide-based glycosphingolipids (GSL) ntawm cov plasma membrane ua rau muaj kev sib sau ua ke GSL loj, txhim kho kev sib cais theem thiab ua rau cov membrane deformation thiab internalization (44). Iwabuchi thiab lwm yam (43) Nws tau pom tias nyob rau hauv qhov muaj cov saw hlau acyl ntev (C24) tab sis tsis luv (C16), cov ligand ntau yam khi rau GSL lactosylceramide ua rau muaj cov pawg loj thiab cov membrane invagination, thiab cytoplasm Lyn-mediated signal transduction ntawm cov ntawv yog interdigitated los ntawm acyl saw hlau hauv cov neutrophils txuas.
Hauv cov hlwb epithelial polarized ntawm cov tsiaj txhu, qhov kev sib sau ua ke ntawm lub network anti-Golgi (TGN) mus rau theem ntawm daim nyias nyias apical plasma tswj kev sib cais thiab kev cais ntawm GPI-AP (10, 45). Qhov kev sib sau ua ke no yog tsav los ntawm GPI-AP oligomerization (36), tab sis nws kuj tseem yuav nyob ntawm qhov ntev ntawm cov saw hlau ceramide uas peb pom hauv cov poov xab. Txawm hais tias cov tsiaj txhu GPI-AP muaj lub ether lipid-based anchor, thiab nws cov qauv tshuaj lom neeg txawv ntawm cov acyl saw hlau ceramide ntev heev, kev tshawb fawb tsis ntev los no pom tias ob qho tib si lipids muaj cov khoom siv lub cev thiab tshuaj lom neeg zoo sib xws thiab kev ua haujlwm (40). Yog li ntawd, qhov ether lipid ib feem hauv cov hlwb tsiaj txhu yuav zoo ib yam li C26 ceramide hauv cov poov xab, thiab nws lub luag haujlwm yog koom nrog cov saw hlau ceramide ntev hauv daim nyias nyias los txhawb GPI-AP kev sib sau ua ke thiab kev cais. Txawm hais tias qhov ua tau no tseem yuav tsum tau sim ncaj qha, cov kev tshawb pom yav dhau los txhawb nqa tias kev thauj mus los ntawm cov acyl saw ceramide ntev mus rau Golgi lub cev tsis yog nqa tawm los ntawm cytoplasmic hloov pauv protein, tab sis nyob ntawm kev tsim cov GPI anchors zoo li poov xab. Yog li ntawd, lub tshuab kev hloov pauv zoo li muaj peev xwm xaiv tau co-transport ntev heev acyl saw ceramide thiab GPI-AP (13, 16, 20, 46, 47) hauv tib lub vesicle thauj mus los.
Hauv cov poov xab thiab cov tsiaj txhu uas muaj cov hlwb epithelial polarized, GPI-AP kev sib sau ua ke thiab kev sib cais los ntawm lwm cov protein plasma membrane txhua yam tshwm sim ua ntej mus txog qhov chaw ntawm lub hlwb. Paladino et al. (48) pom tias ntawm TGN ntawm cov tsiaj txhu uas muaj cov hlwb epithelial polarized, GPI-AP clustering tsis yog tsuas yog tsim nyog rau kev xaiv pawg ntawm GPI-APs rau apical plasma membrane, tab sis kuj tswj hwm kev sib sau ua ke ntawm GPI-APs thiab nws cov haujlwm biological. Qhov chaw ntawm lub hlwb. Hauv poov xab, kev tshawb fawb no tau qhia tias C26 ceramide-dependent GPI-AP cluster ntawm ER tuaj yeem tswj hwm kev sib sau ua ke thiab kev ua haujlwm ntawm GPI-AP ntawm plasma membrane (24, 49). Sib xws nrog tus qauv no, GhLag1 hlwb ua xua rau GPI inhibitors lossis cov tshuaj uas cuam tshuam rau kev ncaj ncees ntawm phab ntsa hlwb (28), thiab qhov xav tau rau kev ua haujlwm Gas1-GFP clusters (49) ntawm lub taub hau ceramide projected hauv mating ntawm cov hlwb poov xab qhia G​​​​ Tej zaum yuav muaj cov txiaj ntsig ntawm lub cev ntawm hLag1 hlwb. GPI-AP yuam kev. Txawm li cas los xij, kev sim ntxiv seb qhov kev ua haujlwm ntawm lub cell nto puas tau raug programmed los ntawm ER los ntawm txoj kev cais raws li lipid ntev yuav yog lub ntsiab lus ntawm peb txoj kev tshawb fawb yav tom ntej.
Cov kab mob Saccharomyces cerevisiae siv rau hauv txoj haujlwm no tau teev nyob rau hauv Rooj S1. Cov kab mob MMY1583 thiab MMY1635 ntawm SCLIM rau kev thaij duab ntawm lub hlwb tau tsim nyob rau hauv keeb kwm yav dhau ntawm W303. Cov kab mob no qhia Sec13-mCherry nrog lub cim protein fluorescent tau tsim los ntawm kev siv txoj kev polymerase chain reaction (PCR)-raws li nrog pFA6a plasmid ua tus qauv (23). Cov kab mob qhia Mid2-iRFP uas muaj cov protein fluorescent nyob rau hauv kev tswj hwm ntawm GAL1 promoter tau tsim raws li hauv qab no. PCR amplification ntawm iRFP-KanMx kab lus los ntawm pKTiRFP-KAN vector (khoom plig ntawm E. O'Shea, Addgene plasmid tus lej 64687; http://n2t.net/addgene: 64687; kev tshawb fawb cov peev txheej txheeb xyuas (RRID): Addgene_64687) Thiab ntxig rau hauv C-terminus ntawm endogenous Mid2. Tom qab Mid2-iRFP genome sequence tau raug amplified thiab cloned rau hauv GAL1 promoter, nws tau integrated rau hauv Not I-Sac I site ntawm integration plasmid pRS306. Lub plasmid pRGS7 uas tshwm sim tau linearized nrog Pst I kom integrate rau hauv URA3 locus.
Tus Gas1-GFP fusion gene tau qhia nyob rau hauv kev tswj hwm ntawm GAL1 promoter hauv centromere (CEN) plasmid, uas tau tsim raws li hauv qab no. Cov kab ke Gas1-GFP tau amplified los ntawm PCR los ntawm pRS416-GAS1-GFP plasmid (24) (khoom plig ntawm L. Popolo) thiab cloned rau hauv Xma I-Xho I qhov chaw ntawm CEN plasmid pBEVY-GL LEU2 (khoom plig ntawm C). Miller; Addgene plasmid tus lej 51225; http://n2t.net/addgene: 51225; RRID: Addgene_51225). Tus plasmid tshwm sim tau hu ua pRGS6. Tus Axl2-GFP fusion gene kuj tau qhia nyob rau hauv kev tswj hwm ntawm GAL1 promoter ntawm pBEVY-GL LEU2 vector, thiab nws cov kev tsim kho yog raws li hauv qab no. Cov kab ke Axl2-GFP tau raug nthuav dav los ntawm pRS304-p2HSE-Axl2-GFP plasmid (23) los ntawm PCR, thiab cloned rau hauv Bam HI-Pst I qhov chaw ntawm pBEVY-GL LEU2 vector. Cov plasmid tshwm sim tau hu ua pRGS12. Cov kab ke ntawm oligonucleotides siv hauv kev tshawb fawb no tau teev nyob rau hauv Rooj S2.
Cov kab mob tau ntxiv nrog 0.2% adenine thiab 2% glucose [YP-dextrose (YPD)], 2% raffinose [YP-raffinose] nplua nuj poov xab extract protein p (YP) nruab nrab (1% poov xab extract thiab 2% protein ept). (YPR)] lossis 2% galactose [YP-galactose (YPG)] ua qhov chaw carbon, lossis hauv cov khoom siv tsawg kawg nkaus (0.15% poov xab nitrogen puag thiab 0.5% ammonium sulfate) los ntxiv cov amino acids thiab cov hauv paus tsim nyog rau kev noj haus, Thiab muaj 2% glucose (synthetic glucose tsawg kawg nkaus nruab nrab) lossis 2% galactose (synthetic galactose tsawg kawg nkaus nruab nrab) ua qhov chaw carbon.
Rau kev thaij duab tiag tiag, cov hlwb hloov pauv sec31-1 uas rhiab heev rau qhov kub thiab txias uas qhia txog cov qauv hauv qab GAL1 promoter tau loj hlob hauv YPR nruab nrab ntawm 24 ° C thaum hmo ntuj mus rau theem nruab nrab. Tom qab induction hauv YPG ntawm 24 ° C rau 1 teev, cov hlwb tau incubated hauv SG ntawm 37 ° C rau 30 feeb, thiab tom qab ntawd hloov mus rau 24 ° C kom tso tawm ntawm lub thaiv secretion. Concanavalin A tau siv los kho cov hlwb ntawm daim iav swb thiab thaij duab los ntawm SCLIM. SCLIM yog kev sib xyaw ua ke ntawm Olympus IX-71 inverted fluorescence microscope thiab UPlanSApo 100 × 1.4 numerical aperture roj lens (Olympus), high-speed thiab high-signal-to-noise ratio rotating disc confocal scanner (Yokogawa Electric), custom spectrometer, thiab custom cooling Lub system's image intensifier (Hamamatsu Photonics) tuaj yeem muab lub magnifying lens system nrog qhov kawg magnification ntawm × 266.7 thiab lub koob yees duab charge-coupled device uas multiplies electrons (Hamamatsu Photonics) (21). Kev tau txais duab yog ua los ntawm kev cai software (Yokogawa Electric). Rau cov duab 3D, peb siv lub piezoelectric actuator ua los ntawm kev cai los co lub lens lub hom phiaj vertically, thiab sau cov khoom optical 100 nm sib nrug hauv ib pawg. Daim duab Z-stack yog hloov mus rau hauv 3D voxel cov ntaub ntawv, thiab lub theoretical point spread function siv rau lub rotating disc confocal microscope yog siv rau deconvolution processing los ntawm Volocity software (PerkinElmer). Los ntawm kev siv Volocity software los ntsuas qhov chaw sib koom ua ke, ERES suav nrog cov khoom thauj tau ntsuas. Kev tshuaj xyuas kab tau ua tiav los ntawm kev siv MetaMorph software (Molecular Devices).
Siv GraphPad Prism software los txiav txim siab qhov tseem ceeb ntawm kev suav lej. Rau qhov kev xeem t ob-tailed Student's t thiab qhov kev xeem ib txoj kev ntsuas qhov sib txawv (ANOVA), qhov sib txawv ntawm cov pab pawg raug suav hais tias muaj kev cuam tshuam loj rau P <0.05 (*).
Rau kev siv lub tshuab tsom iav fluorescence ntawm Gas1-GFP, cov hlwb theem log tau loj hlob ib hmos hauv YPD thiab sau los ntawm centrifugation, ntxuav ob zaug nrog phosphate buffered saline, thiab incubated rau ntawm dej khov tsawg kawg 15 feeb, thiab tom qab ntawd mus rau hauv qab lub tshuab tsom iav raws li tau piav qhia ua ntej Check (24). Lub tshuab tsom iav Leica DMi8 (HCX PL APO 1003/1.40 roj PH3 CS) nruab nrog lub lens hom phiaj, L5 (GFP) lim, Hamamatsu lub koob yees duab thiab Application Suite X (LAS X) software tau siv rau kev tau txais.
Cov qauv kuaj tau denatured nrog SDS qauv buffer ntawm 65 ° C rau 10 feeb, thiab tom qab ntawd cais los ntawm SDS-polyacrylamide gel electrophoresis (PAGE). Rau kev tshuaj ntsuam immunoblotting, 10 μl ntawm cov qauv kuaj tau thauj khoom rau ib txoj kab. Primary antibody: Siv rabbit polyclonal anti-Gas1 ntawm qhov dilution ntawm 1: 3000, rabbit polyclonal anti-Emp24 ntawm qhov dilution ntawm 1: 500, thiab rabbit polyclonal anti-GFP (khoom plig los ntawm H. Riezman) ntawm qhov dilution ntawm 1: 3000. Tus nas monoclonal anti-Pgk1 antibody tau siv ntawm qhov dilution ntawm 1: 5000 (khoom plig los ntawm J. de la Cruz). Secondary antibody: Horseradish peroxidase (HRP) conjugated tshis anti-rabbit immunoglobulin G (IgG) siv ntawm qhov dilution ntawm 1: 3000 (Pierce). HRP-conjugated tshis tiv thaiv nas IgG tau siv ntawm qhov dilution ntawm 1: 3000 (Pierce). Lub cheeb tsam tiv thaiv kab mob tau pom los ntawm txoj kev chemiluminescence ntawm SuperSignal West Pico reagent (Thermo Fisher Scientific).
Raws li tau piav qhia hauv (31), kev sim tshuaj tiv thaiv kab mob ntuj tau ua tiav rau ntawm cov feem ER nplua nuj. Hauv ntej, ntxuav cov hlwb poov xab nrog TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride thiab protease inhibitor sib tov) ntawm 600 nm (OD600) ntawm 100 optical density ob zaug. Nws tau tawg nrog cov hlaws iav, thiab tom qab ntawd cov khib nyiab ntawm tes thiab cov hlaws iav tau raug tshem tawm los ntawm centrifugation. Lub supernatant tom qab ntawd tau centrifuged ntawm 17,000 g rau 15 feeb ntawm 4 ° C. Lub pellet tau resuspended hauv TNE thiab digitalis saponin tau ntxiv rau qhov kawg concentration ntawm 1%. Cov kev ncua tau incubated rau 1 teev nrog kev tig ntawm 4 ° C, thiab tom qab ntawd cov khoom tsis yaj tau raug tshem tawm los ntawm centrifugation ntawm 13,000 g ntawm 4 ° C rau 60 feeb. Rau Gas1-GFP immunoprecipitation, ua ntej tshaj plaws ua ntej muab cov qauv nrog cov hlaws agarose khoob (ChromoTek) ntawm 4°C rau 1 teev, thiab tom qab ntawd muab GFP-Trap_A (ChromoTek) ntawm 4°C rau 3 teev. Cov hlaws immunoprecipitation tau ntxuav tsib zaug nrog TNE uas muaj 0.2% digoxigenin, eluted nrog SDS sample buffer, sib cais ntawm SDS-PAGE, thiab tshuaj xyuas los ntawm immunoblotting.
Raws li tau piav qhia hauv (31), kev txiav txim siab cross-linking tau ua tiav rau ntawm cov feem ER nplua nuj. Hauv luv luv, cov feem ER nplua nuj tau incubated nrog 0.5 mM dithiobis (succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, USA; 20 ° C, 20 feeb). Qhov kev sib txuas lus crosslinking tau quenched los ntawm kev ntxiv glycine (50 mM kawg concentration, 5 feeb, 20 ° C).
Raws li tau piav qhia ua ntej (50), MS kev tshuaj xyuas ntawm ceramide hauv cov kab mob qus thiab GhLag1 tau ua tiav. Hauv ntej, cov hlwb tau loj hlob mus rau theem exponential (3 txog 4 OD600 units / ml) hauv YPD ntawm 30 ° C, thiab 25 × 107 hlwb tau sau. Lawv cov metabolism yog quenched nrog trichloroacetic acid. Siv cov tshuaj extraction [ethanol, dej, ether, pyridine thiab 4.2 N ammonium hydroxide (15:15:5:1:0.018 v / v)] thiab 1.2 nmol ntawm tus qauv sab hauv C17 ceramide (860517, Avanti polar lipid) zoo). Siv monomethylamine reagent [methanol, dej, n-butanol thiab methylamine kua (4:3:1:5 v / v)] los ua cov alkaline hydrolysis me me ntawm cov extract, thiab tom qab ntawd siv dej-saturated n-butanol kom desalt. Thaum kawg, cov extract tau resuspended hauv cov tshuaj yaj hom zoo [chloroform/methanol/dej (2:7:1) + 5 mM ammonium acetate] thiab txhaj rau hauv lub mass spectrometer. Kev soj ntsuam ntau yam (MRM) tau ua rau kev txheeb xyuas thiab kev ntsuas ntawm cov sphingolipid molecules. Lub TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) tau nruab nrog lub tshuab robotic nanoflow ion source Nanomate HD (Advion Biosciences, Ithaca, NY) rau kev tshuaj xyuas lipid. Lub zog sib tsoo tau zoo rau txhua pawg ceramide. Cov ntaub ntawv MS tau txais hauv hom zoo. Rau txhua qhov kev rov ua dua ntawm lub cev, lub teeb liab lipid yog qhov nruab nrab ntawm peb qhov kev ntsuas ywj pheej.
Raws li tau piav qhia hauv (31), cov hlwb (800 × 107) uas qhia Gas1-GFP tau raug rau kev tiv thaiv kab mob ntuj. Cov Gas1-GFP uas tau lim tau raug cais los ntawm SDS-PAGE thiab hloov mus rau polyvinylidene fluoride (PVDF) daim nyias nyias. Cov protein tau pom los ntawm kev pleev xim PVDF nrog amide dub. Cov Gas1-GFP band tau txiav los ntawm PVDF thiab ntxuav 5 zaug nrog methanol thiab ib zaug nrog kua chromatography-MS (LC-MS) qib dej. Los ntawm kev ua kom cov daim nyias nyias nrog 500μl 0.3 M NaOAc (pH 4.0), buffer thiab 500μl tshiab yaj 1 M sodium nitrite sib tov ntawm 37 ° C rau 3 teev, cov roj lipid tau tso tawm los ntawm Gas1-GFP thiab lysed Tso tawm ntawm inosine phosphate ceramide ntawm glucosamine thiab inositol (51). Tom qab ntawd, daim kab xev membrane tau ntxuav plaub zaug nrog dej LC-MS qib, ziab ntawm chav tsev kub, thiab khaws cia rau hauv huab cua nitrogen ntawm -80 ° C kom txog thaum tshuaj xyuas. Ua tus tswj, ib qho qauv dawb ntawm PVDF membrane tau siv rau txhua qhov kev sim. Cov lipid rho tawm los ntawm Gas1-GFP tom qab ntawd tau tshuaj xyuas los ntawm MS raws li tau piav qhia (50). Hauv ntej, PVDF daim kab xev uas muaj GPI-lipid tau rov ua dua hauv 75μl cov tshuaj pwm tsis zoo [chloroform / methanol (1: 2) + 5 mM ammonium acetate] thiab dhau electrospray ionization (ESI) -MRM / MS Kev Tshuaj Xyuas ntawm cov hom sphingolipid (TSQ Vantage). Hauv qhov no, cov ntaub ntawv MS tau txais hauv hom ion tsis zoo.
Raws li tau hais ua ntej lawm, qhov lipid ntawm GPI anchor tau sib cais los ntawm [3H]-inositol-labeled GPI-AP (16). Cov lipids tau sib cais los ntawm cov txheej nyias nyias chromatography siv lub tshuab solvent (55:45:10 chloroform-methanol-0.25% KCl) thiab pom siv FLA-7000 (Fujifilm).
Cov hlwb uas qhia Gas1-GFP (600 × 107) tau ntxuav ob zaug nrog TNE buffer nrog TNE buffer, thiab tawg nrog cov hlaws iav, thiab tom qab ntawd centrifuged kom tshem tawm cov khib nyiab ntawm tes thiab cov hlaws iav. Lub supernatant ces centrifuged ntawm 17,000 g rau 1 teev ntawm 4 ° C. Lub pellet tau ntxuav hauv TNE thiab incubated nrog 1 U PI-PLC (Invitrogen) hauv TNE uas muaj 0.2% digitalis saponin rau 1 teev ntawm 37 ° C. Tom qab kev kho mob enzyme, daim nyias nyias tau tshem tawm los ntawm centrifugation ntawm 17,000 g ntawm 4 ° C rau 1 teev. Txhawm rau immunoprecipitate Gas1-GFP, lub supernatant tau incubated nrog GFP-Trap_A (ChromoTek) ntawm 4 ° C ib hmos. Lub Gas1-GFP purified sib cais los ntawm SDS-PAGE tau stained nrog Coomassie brilliant xiav. Cov kab xim Gas1-GFP tau txiav tawm ntawm cov xim grey uas nyob ib puag ncig cov kav dej, thiab tom qab ntawd tom qab alkylation nrog iodoacetamide thiab txo nrog dithiothreitol, kev zom zaub mov hauv-gel nrog trypsin tau ua tiav. Extract thiab qhuav tryptic peptides thiab peptides nrog GPI-glycans. Cov peptide qhuav tau yaj rau hauv 20 μl ntawm dej. Txhaj ib feem (8μl) rau hauv LC. Ib kem octadecylsilane (ODS) (Develosil 300ODS-HG-5; sab hauv txoj kab uas hla 150 hli × 1.0 hli; Nomura Chemical, Aichi Prefecture, Nyiv) tau siv los cais cov peptides nyob rau hauv cov xwm txheej gradient tshwj xeeb. Lub theem mobile yog cov kuab tshuaj A (0.08% formic acid) thiab cov kuab tshuaj B (0.15% formic acid hauv 80% acetonitrile). Ib lub tshuab Accela HPLC (Thermo Fisher Scientific, Boston, Massachusetts) tau siv los elute lub kem nrog cov kuab tshuaj A hauv 55 feeb ntawm tus nqi ntws ntawm 50 μl min-1 rau 5 feeb, thiab tom qab ntawd qhov concentration ntawm cov kuab tshuaj B tau nce mus txog 40%. , Tebchaws Meskas). Cov eluate tau txuas ntxiv mus rau hauv ESI ion qhov chaw, thiab cov tryptic peptides thiab peptides nrog GPI-glycans tau soj ntsuam los ntawm LTQ Orbitrap XL (hybrid linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific). Hauv MS teeb tsa, qhov hluav taws xob ntawm qhov chaw capillary tau teeb tsa rau 4.5 kV, thiab qhov kub ntawm cov capillary hloov pauv tau khaws cia ntawm 300 ° C. Qhov hluav taws xob capillary thiab lub raj lens voltage tau teeb tsa rau 15 V thiab 50 V, raws li. Cov ntaub ntawv MS tau txais hauv hom ion zoo (kev daws teeb meem ntawm 60,000; qhov tseeb ntawm qhov hnyav ntawm 10 feem ib lab) hauv qhov ntau ntawm qhov hnyav ntawm 300/m/z qhov hnyav/nqi (m/z) 3000. Cov ntaub ntawv MS/MS tau txais los ntawm lub cuab yeej ntes ion hauv LTQ Orbitrap XL [3 tus lej thawj zaug uas cov ntaub ntawv nyob ntawm, kev sib tsoo ua rau muaj kev sib cais (CID)].
Cov kev sim ua MD tau ua tiav siv GROMACS (52) software thiab MARTINI 2 lub zog teb (53-55). Tom qab ntawd CHARMM GUI Membrane Builder (56, 57) tau siv los tsim ib txheej bilayer uas muaj dioleoylphosphatidylcholine (DOPC) thiab Cer C18 lossis DOPC thiab Cer C26. Lub topology thiab kev sib koom ua ke ntawm Cer C26 yog los ntawm DXCE los ntawm kev tshem tawm cov hlaws ntxiv los ntawm tus Tsov tus tw sphingosine. Siv cov txheej txheem piav qhia hauv qab no kom sib npaug ob txheej thiab khiav nws, tom qab ntawd siv cov kev sib koom ua ke kawg ntawm lub kaw lus los tsim ib lub kaw lus uas muaj Emp24. Lub transmembrane domain ntawm cov poov xab Emp24 (cov seem 173 txog 193) tau tsim ua α-helix siv cov cuab yeej pom MD (VMD) cov qauv molecular (58). Tom qab ntawd, tom qab tshem tawm cov lipids overlapping, cov protein tau coarsely granulated thiab ntxig rau hauv bilayer siv CHARMM GUI. Lub kaw lus kawg muaj 1202 DOPC thiab 302 Cer C26 lossis 1197 DOPC thiab 295 Cer C18 thiab Emp24. Ionize lub kaw lus rau qhov concentration ntawm 0.150 M. Plaub qhov kev rov ua dua ywj pheej tau ua rau ob lub bilayer compositions.
Cov lipid bilayer raug sib npaug siv cov txheej txheem CHARMM GUI, uas cuam tshuam nrog kev txo qis thiab tom qab ntawd sib npaug 405,000 kauj ruam, qhov twg cov kev txwv txoj haujlwm maj mam txo qis thiab tshem tawm, thiab cov kauj ruam sijhawm nce ntxiv los ntawm 0.005 ps mus rau 0.02 ps. Tom qab sib npaug, nws tsim 6 µs nrog lub sijhawm kauj ruam ntawm 0.02 ps. Tom qab ntxig Emp24, siv tib cov txheej txheem CHARMM GUI los txo qis thiab sib npaug lub kaw lus, thiab tom qab ntawd khiav rau 8 s hauv kev tsim khoom.
Rau txhua lub tshuab, thaum lub sijhawm ua kom sib npaug, lub siab raug tswj los ntawm Berendsen barostat (59), thiab thaum lub sijhawm tsim khoom, lub siab raug tswj los ntawm Parrinello-Rahman barostat (60). Hauv txhua qhov xwm txheej, qhov nruab nrab siab yog 1 bar thiab siv cov txheej txheem sib txuas siab semi-isotropic. Hauv kev sib npaug thiab cov txheej txheem tsim khoom, lub thermostat (61) nrog kev rov kho dua qhov ceev yog siv los sib txuas qhov kub ntawm cov protein, lipid thiab cov khoom siv yaj. Thaum lub sijhawm ua haujlwm tag nrho, qhov kub thiab txias yog 310K. Kev sib cuam tshuam tsis sib txuas yog suav los ntawm kev tsim cov npe sib txuas siv cov txheej txheem Verlet nrog 0.005 buffer kam rau siab. Lub sijhawm Coulomb yog suav siv lub tshav pob tshuaj tiv thaiv thiab qhov deb ntawm 1.1 nm. Lub sijhawm Vander Waals siv cov txheej txheem txiav nrog qhov deb ntawm 1.1 nm, thiab cov txheej txheem txiav Verlet yog siv rau qhov muaj peev xwm hloov pauv (62).
Siv VMD, qhov cutoff wavelength ntawm DOPC phosphate hlaws lossis ceramide AM1 hlaws thiab cov protein yog 0.7 nm, thiab tus lej ntawm cov lipids uas cuam tshuam nrog cov protein raug suav. Raws li cov mis hauv qab no, xam qhov depletion-enrichment (DE) factor raws li hauv (63): DE factor = (qhov ntau ntawm tag nrho cov lipids hauv cov protein 0.7) hauv cov protein 0.7 (qhov ntau ntawm Cer hauv tag nrho cov lipids)
Tus nqi tshaj tawm tau txais los ntawm qhov nruab nrab, thiab cov kab yuam kev yog plaub daim ntawv theej ywj pheej ntawm SE. Qhov tseem ceeb ntawm DE factor yog xam los ntawm t xeem [(averageDE-factor-1)/SE]. Xam tus nqi P los ntawm kev faib tawm ib-tailed.
Cov cuab yeej GROMACS tau siv los xam daim ntawv qhia 2D lateral density ntawm lub cev uas muaj Emp24 nyob rau hauv 250 ns kawg ntawm qhov ntsuas. Yuav kom tau txais daim ntawv qhia enrichment/depletion ntawm ceramide, daim ntawv qhia density ntawm Cer raug faib los ntawm qhov sib ntxiv ntawm daim ntawv qhia ntawm Cer thiab DOPC, thiab tom qab ntawd faib los ntawm qhov concentration ntawm Cer hauv lub cev. Tib daim ntawv qhia xim siv.
Yog xav paub ntxiv txog tsab xov xwm no, thov mus saib http://advances.sciencemag.org/cgi/content/full/6/50/eaba8237/DC1
Qhov no yog ib tsab xov xwm qhib rau kev nkag mus tau faib raws li cov nqe lus ntawm Creative Commons Attribution-Non-Commercial License, uas tso cai rau kev siv, kev faib tawm thiab kev tsim dua tshiab hauv txhua qhov chaw, tsuav yog qhov kev siv zaum kawg tsis yog rau kev lag luam thiab lub hauv paus yog tias cov haujlwm thawj yog qhov tseeb. Kev siv.
Lus Cim: Peb tsuas thov kom koj muab koj qhov chaw nyob email kom tus neeg uas koj pom zoo rau nplooj ntawv paub tias koj xav kom lawv pom email thiab nws tsis yog spam. Peb yuav tsis ntes cov chaw nyob email twg li.
Lo lus nug no yog siv los sim seb koj puas yog tus qhua thiab tiv thaiv kev xa tawm spam tsis siv neeg.
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, AnaroSergio (-Lópezio Maria Perez), Waga (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Cov duab 3D siab daws teeb meem tiag tiag qhia txog qhov tseem ceeb ntawm qhov ntev ntawm cov saw hlau ceramide rau kev cais cov protein hauv cov chaw tso zis xaiv.
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, AnaroSergio (-Lópezio Maria Perez), Waga (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Cov duab 3D siab daws teeb meem tiag tiag qhia txog qhov tseem ceeb ntawm qhov ntev ntawm cov saw hlau ceramide rau kev cais cov protein hauv cov chaw tso zis xaiv.
© 2020 American Association rau Advancement of Science. tag nrho cov cai. AAAS yog tus khub ntawm HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef thiab COUNTER. ScienceAdvances ISSN 2375-2548.