di protεin sכt insay di sekret path na implεnt fכ mεnten di sεl kכmpartimεntayzεshכn εn homכstasis. apat frכm di sכt we di shεl mεdiet, di rol we di lipid dεm de ple fכ sכt di kinesin insay di prכsεs fכ sekret transpכt na wan bεsik kכwεshכn we de fכ lכng tεm we dεn nכ ansa yet. Hia, wi de du 3D simultaneous multicolor high-resolution rial-time imaging fכ pruv in vivo se nyu sεntez glycosylphosphatidylinositol-immobilized protin dεm wit vεri lכng sεramid lipid moieties dεn kכlכs εn klas insay spεshal εndoplasms Net εkzit sayt, we difrεnt frכm di wan we transmembran protin dεm de yuz. apat frכm dat, wi sho se di chen lεngth fכ sεramid insay di εndoplasmik rεtikul mεmbran na krichכl fכ dis sכt sεlektiviti. wi stכdi de gi di fכs dayrekt in vivo pruf fכ klas protin kכgo dεm bays pan lipid chen lεngth insay sεlektiv εkspכt sayt dεm na di sekret path.
insay yukariכtik sεl dεm, di protin dεm we dεn sεntez insay di εndoplasmik rεtikul (ER) dεn de sכt dεm we dεn de transpכt tru di sekret path fכ dεlivεri to dεn aprכpriet sεlyul dεstinεshכn (1). apat frכm di kot-mεdiet sכt, i bin lכng spεkul se sכm lipid dεm kin sεv bak as sεlektiv εkzit pכynt bay we dεn de kכlכs dεm insay spεsifi k mεmbran domεn dεm we spεsifi k protin dεm (2-5). כltu, i stil lכk fכ dayrekt in vivo pruf fכ pruv dis posεbul lipid-bεys mεkanism. fכ sכlv dis bεsik prכblεm, wi stכdi insay yist aw glycosylphosphatidylinositol (GPI) anchored protin dεm (GPI-APs) de difrεntli εkspכt frכm di ER. GPI-AP dεm na difrεn lipid-kכnekt sεl sεf protin dεm ( 6, 7). GPI-AP na sekret protin we de atak di כta liflet dεm na di plasma mεmbran tru di glycolipid moiety (GPI anchor). dεn aksept GPI ankכr dεm as kכnsyuv pכst-transleshכnal modifyushכn dεm na di ER lumεn (8). afta di atachmεnt, GPI-AP de pas tru di Golgi apεrat (5, 9) frכm di ER to di plasma mεmbran. di prεsεns fכ GPI ankכr dεm de mek GPI-AP de transpכt sεparat frכm transmεmbran sekret protin dεm (inklud כda plasma mεmbran protin dεm) along di sekret path (5, 9, 10). insay yist sεl dεm, di GPI-AP dεm de separet frכm כda sekret protin dεm na di εndoplasmik rεtikul, εn afta dat dεn de pak dεm insay yכnik vεsikul dεm we dεn rap bay kot protin kכmpleks II (COPII) (6, 7). di ditarminant dεm fכ dis klasification prכsεs in di ER εkspכt prכsεs nכ klia, bכt dεn spεkul se dis mεkanism kin nid lipid dεm, spεshal wan di strכkchכral rimכdelin fכ di lipid pat pan di GPI ankכ ( 5 , 8 ). insay yist, di GPI lipid rimכdelin de bigin wantεm afta di GPI atak, εn insay bכku kes dεm, i de mek sεramid de biεn di 26-kabכn lכng chen satεrayt fεt asid (C26:0) (11, 12). C26 sεramid na di men sεramid we yist sεl dεm de prodyuz te naw. i de sεntez insay di εr εn mכst pan am de εkspכt to di Golgi apεrat tru COPII vεsikul dεm (13). di ER εkspכt fכ GPI-AP spεshal nid fכ כngoing sεramid sεntez ( 14 , 15 ), εn in tכn, di kכnvכshכn fכ sεramid to inositol fכsfεt sεramid (IPC) insay di Golgi apεrat dipכnt pan GPI ankכr sεntesis ( 16 ). bayofizikal stכdi wit atifishal mεmbran dεn sho se vεri lכng asil chen sεramid dεm kin kכales fכ fכm כda domεn wit yunik fכshal prכpati dεm ( 17 , 18 ). dis data de lid to di haypothεsis se C26 sεramid εn GPI-AP wit C26 sεramid de yuz dεn fכs prכpati fכ kכales insay כda rijyכn כ rijyכn dεm na di rili mεsi εr mεmbran lipid invayroment. i mεnli kכmכp fכ sכt εn כnsatyuret glisεrolipid dεm (C16:1 εn C18:1) (19, 20). dis rijyכn dεm go sεlektiv fכs pan spεsifi k ER εkzit sayt dεm (ERES), usay sεramid εn sεramid-bεys GPI-AP kin kכ-transpכt to di Golgi insay di sem dεdiket COPII vεsikul (5).
insay dis stכdi, wi dεn tεst dis lipid-bεys mεkanism dεn dεn wan bay we wi yuz supεr-rεsכlushכn kכnfכkal rial-taym imej maykroskכpi (SCLIM), we na kכt-εj maykroskכpi tεknik we kin simultaneously obsכv fכrosεnt lεbul protin dεm Di tri-kכla εn tri-dimεnshכnal (3D) imej dεm gεt ekstrim hכy rεsכlushכn εn spid insay liv sεl dεm (21, 22).
Wi fכs aplay SCLIM tεknכlכji fכ fכs difayn aw nכmal GPI-AP wit C26 sεramid grup bin skrεn frכm transmεmbran sekret protin dεm afta i kכmכt na di ER insay S. cerevisiae. In orda fכ chεk di klasifikasiכn fכ ER, wi yuz wan jεnεtik sistεm we kin dayrekt vishכnal nyu sεntez kכgo we de enta ERES in vivo ( 7 , 23 ). As kago, wi pik C26 seramid-bεys GPI-AP Gas1 we dεn lεbul wit grεn fכrosεnt protin (GFP) εn transmεmbran sekret protin Mid2 we lεbul wit nia-infrared fכrosεnt protin (iRFP), we dεn tu de tכk to di plasma mεmbran ( 24–26 ). insay di sec31-1 tεmprachכ-sεnsitiv mutant, dεn tu kכgo dεm ya de εksprεs כnda wan galaktכz-inducible prכmotεr εn wan kכnstityushכn ERES mak. na di ekstrim tεmprachכ (37°C), biכs di sec31-1 mכtεshכn de afekt di fכnshכn fכ COPII kot kכmכpכnt Sec31 fכ inhεbit COPII jεmεnshכn εn ER εkspכt, nyu sεntez kכgo de akumulet na di ER (23). afta dεn kol to lכw tεmprachכ (24°C), di sec31-1 mutant sεl dεm rεkכv frכm di sekret εria, εn di akumulet nyu sεntetik kכgo bigin fכ εkspכt frכm di ER. CLIM visualization sho se mכst pan di nyu sεntez Gas1-GFP εn Mid2-iRFP stil akכmyuleyt in di ER fכ di sec31-1 mutant sεl dεm afta inkכbeshכn na 37°C εn afta dat rilis na 24°C fכ 5 minit (Figure 1). sכm Mid2-iRFP de distribyut pan di כl ER mεmbran, εn Gas1-GFP de kכnsantreyt εn gεt insay di diskontinyu ER mεmbran εria, dεn distribushכn de kכmplit difrεnt (Figure 1, A to C εn Movie S1). apat frכm dat, lεk aw dεn sho na fכg 1D, di Gas1-GFP kכlכsta nכ gεt Mid2-iRFP. dis rizulεt dεm sho se GPI-AP εn transmembran protin dεm bin separet insay difrεn ER membran rijyכn dεm ali. di Gas1-GFP klasta de nia wan spεsifi k ERES we dεn lεbul wit mCherry in COPII kot protin Sec13 (Figure 1, E εn F, εn muvi S1) (23).
sec31-1 sel dεm de εksprεs galaktכz-indyus sekreshכn dεm, wan lכng asil chen (C26) sεramid GPI-AP Gas1-GFP (GPI-AP, grεn) εn di transmεmbran protin Mid2-iRFP (TMP, blu) εn dis Kכnstrכktiv ERES lεbul Sec13-mCherry (ERES, magenta) bin inkכba na 37°C fכ 30 minit, muv to 24°C, ɛn dɛn tek am pikchɔ bay SCLIM 5 minit afta dat. (A to C) sho wan riprizentiv mεrj כ singl 2D imej fכ wan plen (A), wan 2D projεkshכn imej fכ 10 z-sεkshכn (B) כ wan 3D sel hεmisfya imej fכ kago εn ERES mak dεm (C). Skel bar 1μm (A ɛn B). Di skel yunit na 0.551μm (C). Gas1-GFP wεn dεtekt in diskrεt ER rijyכn כ kכlכsta, we Mid2-iRFP bin dεtekt εn distribyut tru di ER mεmbran (C). (D) Di grafik sho di rilitiv fluorescence intensiti fɔ Gas1-GFP ɛn Mid2-iRFP insay di Gas1-GFP klasta along di wayt aro layn (lɛft). AU, yunit we dɛn want. (E ɛn F) de sho di 3D pikchɔ we de jɔyn di guds ɛn ERES mak. Gas1-GFP klasta dɛn bin detekt nia di spɛsifi k ERES. Di skel yunit na 0.551μm. (F) Di wayt sɔlid aro de mak di Gas1-GFP klasta we gɛt fɔ du wit ERES. Di midul ɛn rayt panɛl dɛn sho di 3D imej we dɛn dɔn jɔyn ɛn wan rɔta we fɔ si di Gas1-GFP klasta we dɛn dɔn pik.
di klos spatial rilayshכn bitwin di Gas1-GFP klasta εn wan spεsifi k ERES de sho se Gas1-GFP kin enta sεlektiv ERES, we difrεnt frכm di sεlektiviti we Mid2-iRFP de yuz fכ kכmכt na di ER. Fɔ adrɛs dis pɔsibul, wi kwantifay di ERES rɛsɛshɔn fɔ wan ɔ tu guds nɔmɔ (Figure 2, A to C). Wi fain se bɔku pan di ERES (70%) gɛt wan kayn kago nɔmɔ. di bכtכm imej na fכg 2C sho tu tכpik εgzampl dεm fכ ERES wit כnli Gas1-GFP (Figכ 1) כ כnli Mid2-iRFP (Figכ 2). Difrɛn frɔm dat, lɛk 20% pan ERES gɛt tu kago dɛn we de ɔvalap na di sem eria. Dɛn bin si se sɔm ERES (10%) bin gɛt tu kayn kago, bɔt dɛn bin de ayd dɛn na difrɛn say dɛn we klia wan. כl dis mek dis stεdi analisis sho se afta dεn εkspכt di ER, dεn divayd GPI-AP Gas1-GFP εn di transmembran kכgo Mid2-iRFP insay difrεn ERES (Figure 2D). Dis sכt efyushכn rili kכnsist wit di fכs bayokεmik analisis (6) εn mכfכlכjik ditarminεshכn (7). Wi kin wach bak di bihayvya fɔ di kwarantin kago we de go insay ERES (Figure 2E ɛn Movie S2). fכg 2E sho se na כnli sכm pat pan Gas1-GFP (panεl 3) כ Mid2-iRFP (panεl 4) de enta di ERES frכm wan say εn i de kכnfyus insay wan diskrεt εria. panεl 5 na fכg 2E sho se sכmtεm dεn kin fכnshכn Gas1-GFP εn Mid2-iRFP insay di sem ERES, bכt dεn kin enta frכm difrεn sayd dεm εn dεn kin kכnsantreyt insay sεparat rijyכn dεm we kin riprizent difrεn COPII vεsikul dεm. We konfam bak se di obsεv separeshכn εn klasifikasiכn כf C26 sεramid-bεys GPI-AP Gas1 as sεlektiv ERES na spεsifi k biכs wan כda transmεmbran sekreshכn kago, di GFP-tag plasma mεmbran protin Axl2 (27 ), we sho di sem bihayvya to Mid2-iRFP. (Pikchɔ S1 ɛn Fim S3). di nyu sεntez Axl2-GFP de distribyut tru di ER mεmbran lεk Mid2-iRFP (Figure S1, A εn B), εn i de kכ-lכkal wit Mid2-iRFP in mכst ERES (Figure S1, B to D). Panɛl 1 ɛn 2 na Fig 1. S1C sho tu tipik ɛgzampul dɛn fɔ ERES usay tu transmɛmbran kago dɛn de ɔvalap. Insay dɛn kayn tin ya, ɔl tu di guds dɛn kin go insay ERES togɛda (Figure S1E, Panel 3 ɛn Movie S3).
di sec31-1 sel dεm we de εksprεs galaktכs indusibl sekreshכn, Gas1-GFP (GPI-AP, grεn) εn Mid2-iRFP (TMP, blu) εn kכnstityushכn ERES lεbul Sec13-mCherry (ERES, magenta) bin put na 37 Afta dεn inkכba fכ 30 minit na °C, muv to 24 °C fכ rilis di sekreshכn blכk, εn imej wit SCLIM afta 20 minit. (A to C) Riprεzεntεtiv 2D projεkshכn imej dεm (A; skel bar, 1μm) כ 3D sεl hεmisfya imej dεm (B εn C; skel yunit, 0.456μm) fכ di kago εn 10 z-sεkshכn dεm we dεn mak wit ERES. Di panɛl we de dɔŋ na (B) ɛn di panɛl we de na (C) de sho di pikchɔ dɛn we dɛn dɔn prosɛs fɔ sho ɔl di guds dɛn we de insay ERES (magenta) [Gas1-GFP (grey) ɛn Mid2-iRFP (layt blu)]. (C) Opin aro: ERES de kɛr wan pat pan kago nɔmɔ (1 to 4). Grey aro: ERES gɛt kago we dɛn dɔn sheb (5). Wait sɔlid aro: ERES we gɛt kago we de togɛda. dכn dכn: di singl ERES we dεn sεlekt kכntεn כnli Gas1-GFP (1) כ Mid2-iRFP (2). Skel bar, 100 nm. (D) Kwantifikeshɔn fɔ di fotomaykrograf we dɛn diskrayb na (C). Di avrej pasɛnt fɔ ERES we gɛt wan kago nɔmɔ (Gas1-GFP ɔ Mid2-iRFP), segregated kago ɛn ɔvalapin kago. In tri indipεndεnt εkspεriεns, n=432 in 54 sεl dεm. Bar we gɛt mistek = SD. Tu-tɛl unpaired t tɛst. *** P = 0,0002. Di wan dɛn we de wok. (E) 3D imej fɔ sɛlɛkɛt ERES fɔ kwarantin kago we dɛn mak wit (C). Gas1-GFP (grin) (3) כ Mid2-iRFP (blu) (4) de enta ERES (magenta) frכm wan sayd εn i de rεstrikt to sכm εria insay ERES. Sɔntɛnde, ɔl tu di kayn kago dɛn kin go insay di sem ERES (5) frɔm di sem say ɛn dɛn kin put dɛn na wan say we nɔ de insay di ERES. Skel bar, 100 nm.
nεks, wi tεst wan haypothεsis se di lכng asil chen sεramid (C26) we de insay di εr mεmbran de drεb di spεsifi k kכlכsta εn sכt fכ Gas1 insay sεlektiv ERES. Fכ dis, wi yuz wan modifyed yist strayn GhLag1, we di tu εndojεnik sεramid sεntaz Lag1 εn Lac1 bin riples bay GhLag1 (di Lag1 homolog fכ kכtכn), we rεsult in wan yist strayn wit wan sεl mεmbran Ceramide strayn sכt pas wayl tכp (Figure 3A) (28). Mas spɛktrometri (MS) analisis sho se insay wayl-tayp strayn, 95% pan di tɔtal sɛramid na rili lɔng (C26) chen sɛramid, we insay GhLag1, 85% pan di sɛramid rili lɔng (C18 ɛn C16). ), na 2% nכmכ pan di sεramid na vεri lכng (C26) chen sεramid. pan tap we C18 εn C16 sεramid na di men sεramid dεm we dεn dεtekt insay di GhLag1 mεmbran so fa, MS analisis kכnfכm bak se di GPI ankכ fכ Gas1-GFP we dεn εksprεs insay di GhLag1 strayn gεt C26 sεramid, we kכmparabl wit wayl-tayp lipid dεm. Di kwaliti na di sem (Fig. 3A) (26). כl dis min se di sεramid rimכdelin εnzym Cwh43 na hεli sεlektiv fכ C26 sεramid, lεk aw dεn sho na fכg 26, i fכ inkכrporεshכn di GPI ankכ frכm sכm sכm C26 sεramid insay di GhLag1 strayn. S2 (29) we dɛn kɔl. כl dis de mek di sεl mεmbran fכ GhLag1 bεsכs כnli kכntεn C18-C16 sεramid, we Gas1-GFP stil gεt C26 sεramid. dis fכt de mek dis strayn bi wan ideal tul fכ spεshal sכlv di prכblεm fכ di asil chen lεngth fכ di mεmbran sεramid na di ER. Di hypothetical rol we klas ɛn sɔt de ple. afta dat, wi fכs stכdi di abiliti fכ C26 Gas1-GFP fכ akumulet in klasta in GhLag1 wit wan tεmprachכ-sεnsitiv mutant alεl fכ sec31-1 tru kכvεshכnal fכrosεns maykroskכpi, usay nכmכ di lכng (C18-C16) chen de insay di ER mεmbran Ceramide (Fig. 3). We observ se in sec31-1, mכst pan Gas1-GFP bin kכnsantreyt in klasta, we Gas1-GFP in sec31-1 GhLag1 wit lכng (C18-C16) lכng sεramid ER mεmbran bin mεnli nכ kכlכsta εn distribyut in In di כl ER mεmbran. fכ bi prεsis, biכs C26 sεramid-bεys kכlכsta de kכlכs rilet to spεsifi k ERES (Figure 1), wi nεks invεstigat if dis prכsεs kin involv bak di fכnshכn fכ di ER εkspכt protin mεkanism. GPI-AP de yuz wan spεshal COPII sistεm fכ ER εkspכt, we de aktivli rεgulεt bay Ted1′s strכkchכral rimכdelin fכ di glycan pat pan di GPI ankכr ( 30 , 31 ). di rεkombinεnt GPI-glycan de dכn rεkכgnεz bay di transmεmbran kago rεsεptכr p24 kכmpleks, we in tכn sεlektivli rikrut Lst1, we na spεsifi k isoform fכ di mεjכr COPII kago binding sכbyunit Sec24, fכm wan GPI-AP-rich COPII Vεsikul dεm nid (31-33). כl dis mek wi kכnstrכkt wan dכbl mutant we kכmbayn di dilit fכ dεn singl protin dεm (di p24 kכmpleks kכmכpכnt Emp24, GPI-glycan rimodelin εnzym Ted1 εn di spεsifi k COPII sabyunit Lst1) wit di sec31-1 mutant strayn, εn stכdi dεm Is i posibul fכ fכm Gas1-klכsta GFP (Figure 3). Wi obsεv se in sec31-1emp24Δ εn sec31-1ted1Δ, Gas1-GFP na mεnli unclustered εn distributed truutout di ER membran, lεk aw wi bin si bifo in sec31-1 GhLag1, we in sec31-1lst1Δ, Gas1-GFP Lεk sec31-1. dis rizulεt dεm sho se apat frכm di prεsεns fכ C26 sεramid insay di ER mεmbran, di kכlכsta fכ Gas1-GFP nid bak fכ biεn di p24 kכmpleks, εn i nכ nid spεsifi k Lst1 rikrutmεnt. afta dat, wi εksplכr di posisibul se di chen lεngth fכ sεramid insay di ER mεmbran kin rεgεl di binding fכ Gas1-GFP to p24. כltu, wi fכnd se di prεsεns fכ C18-C16 sεramid insay di mεmbran nכ de afekt di GPI-glycans we di p24 kכmpleks rεkכnstrכkt (Figure S3 εn S4, A εn B) כ binding to GPI-AP εn εkspכt GPI-AP. ebul fɔ yuz. Rikrut COPII sabtayp Lst1 (Figure S4C). fכ dat, C26 sεramid-dipεndεnt kכlכsta nכ nid protin intarakshכn wit difrεn ER εkspכt protin mεkanism dεm, bכt i de sכpכt כda sכt mεkanism we di lipid lεngth de drεb. afta dat, wi analכz if di sεramid asil chen lεngth in di ER mεmbran imכtant fכ di ifektiv klasifikasiכn fכ Gas1-GFP as sεlektiv ERES. sins Gas1 in di GhLag1 strain wit sכt-chen sεramid de kכmכt na di ER εn enta di plasma membran (Figure S5), wi biliv se if di sכt de drεb bay di lεngth fכ di sεramid asil chen, di Gas1 in di GhLag1 strayn kin ridayrekt εn kכros. ERES guds wit di sem membran.
(A) di sel mεmbran fכ GhLag1 mεnli kכntεn sכt C18-C16 sεramid dεm, we di GPI ankכ fכ Gas1-GFP stil gεt di sem C26 IPC lεk wayl-tayp sεl dεm. Abov: asil chen lεngth analisis fכ sεramid in di sεl mεmbran fכ wayl-tayp (Wt) εn GhLag1p strayn bay mas spεktrכmetri (MS). Di data riprizent di pasɛnt fɔ di tɔtal sɛramid. Di avrej fɔ tri indipɛndɛnt ɛkspiriɛns dɛn. Bar we gɛt mistek = SD. Tu-tɛl unpaired t tɛst. **** P <0.0001. Di wan dɛn we de wok. Bɔt panɛl: MS analisis fɔ di asil chen lɔng we di IPC we de insay di Gas1-GFP (GPI-IPC) GPI ankɔ we dɛn ɛksprɛs insay di wayl-tayp ɛn GhLag1p strayn dɛn. Di data riprizent di pasɛnt fɔ di totɛl IPC signal. Avrej fɔ fayv indipɛndɛnt ɛkspiriɛns dɛn. Bar we gɛt mistek = SD. Tu-tɛl unpaired t tɛst. ns, nɔ impɔtant. P = 0,9134. Di wan dɛn we de wok. (B) Fluorescence micrographs of sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ ɛn sec31-1lst1Δ sɛl dɛn we de ɛksprɛs galaktoz-indyuz Gas1-GFP bin inkubayt na 37°C fɔ 30 minit ɛn pas dɔŋ fɔ Pɛrfɔm rutin fluorescence maykroskɔpi afta 24°C na di ples. Wait aro: ER Gas1-GFP klasta. opin aro: Unclustered Gas1-GFP de distribut pan di entire ER membran, we de sho di ER kכntribyushכn nyuklia ring stεyning. Skel bar, 5μm. (C) Kwantifikeshɔn fɔ di fotomaykrograf we dɛn tɔk bɔt na (B). di avrej pasεnshכn fכ sεl dεm wit punktat Gas1-GFP strכkchכ. In tri indipεndεnt εkspεriεns, n≥300 sεl dεm. Bar we gɛt mistek = SD. Tu-tɛl unpaired t tɛst. **** P <0.0001. Di wan dɛn we de wok.
Fɔ sɔlv dis prɔblɛm dairekt wan, wi bin du SCLIM vishɔnalizeshɔn fɔ Gas1-GFP ɛn Mid2-iRFP insay GhLag1 wit di sec31-1 tɛmpracha-sɛnsitiv mutant alɛl ( Fig 4 ɛn Muvi S4 ). afta di ER bin rεtεn na 37°C εn afta dat i rilis na 24°C, mכst pan di nyu sεntez Gas1-GFP nכ bin kכlכsta εn distribyut tru di ER mεmbran, lεk aw dεn obsεv bay kכvεshכnal maykroskכp (Figure 4, A εn B ). Apat frɔm dat, wan big pasɛnt pan ERES (67%) inklud tu kayn kago we de kɔ-lɔk insay am (Figure 4D). Panɛl 1 ɛn 2 na Fig 4C sho tu tipik ɛgzampul dɛn fɔ ERES wit ɔvalap Gas1-GFP ɛn Mid2-GFP. Apat frɔm dat, dɛn bin rikrut ɔl tu di guds dɛn na di sem ERES (Figure 4E, panel 3 ɛn movie S4). כl dis mek wi rizulεt dεn sho se di lεngth fכ di sεramid asil chen na di εr mεmbran na imכtant ditarminant fכ di εr protin agregεshכn εn klasifikasiכn.
Sec31-1 GhLag1 sel dεm we de εksprεs galaktכz-indyus sekreshכn, Gas1-GFP (GPI-AP, grεn) εn Mid2-iRFP (TMP, blu) εn kכnstityushכn ERES-lεbul Sec13-mCherry (ERES, magenta) Inkubayt na 37°C. kכntinyu fכ 30 minit, dכp to 24°C fכ rilis sekreshכn, εn imej wit SCLIM afta 20 minit. (A to C) Riprεzεntεtiv 2D projεkshכn imej dεm (A; skel bar, 1μm) כ 3D sεl hεmisfya imej dεm (B εn C; skel yunit, 0.45μm) fכ di 10 z-sεkshכn dεm we dεn mak wit kago εn ERES. Di panɛl we de dɔŋ na (B) ɛn di panɛl we de na (C) de sho di pikchɔ dɛn we dɛn dɔn prosɛs fɔ sho ɔl di guds dɛn we de insay ERES (magenta) [Gas1-GFP (grey) ɛn Mid2-iRFP (layt blu)]. (C) Wait ful-ɔp aro: ERES, guds ɔvalap. Opin aro: ERES gɛt wan tin nɔmɔ. Lɔwa panɛl: Di ERES we yu dɔn pik gɛt ɔvalap guds (1 ɛn 2) we dɛn mak insay (C). Skel bar, 100 nm. (D) Kwantifikeshɔn fɔ di fotomaykrograf we dɛn diskrayb na (C). Insay di sec31-1 ɛn sec31-1 GhLag1 yunit dɛn, na wan kago nɔmɔ (Gas1-GFP ɔ Mid2-iRFP) de insay, ɛn di avɛrej pasɛnt ERES fɔ isol kago ɛn ɔvalap kago. In tri indipεndεnt εkspεriεns, n = 432 in 54 sεl dεm (sec31-1) εn n = 430 in 47 sεl dεm (sec31-1 GhLag1). Bar we gɛt mistek = SD. Tu-tɛl unpaired t tɛst. *** P = 0.0002 (sek31-1) ɛn ** P = 0.0031 (sek31-1 GhLag1). (E) 3D imej fɔ sɛlɛkɛt ERES wit ɔvalap kago (3) we dɛn mak insay (C). Gas1-GFP (grin) εn Mid2-iRFP (blu) de aproch ERES (magenta) frכm di sem sayd εn de na di sem ERES rεstrikt εria. Skel bar, 100 nm.
dis stכdi de gi dayrekt in vivo pruf se lipid-bεys protin kago dεm de klas insay sεlektiv εkspכt sayt dεm na di sekret path, εn i de rivεl di imכpכtants fכ asil chen lεngth fכ klasification sεlektiviti. yuz wan pawaful εn kכt-εj maykroskכpi tεknik we dεn kכl SCLIM, wi sho di nyu sεntez Gas1-GFP (wan mεjכr plasma mεmbran GPI-AP wit wan vεri lכng asil chen (C26) sεramid lipid pat) insay yist ) Di rijyכn dεm we kכlכs insay diskrεt ER dεm de כsכsiayt wit spεsifi k ERES, we transmεmbran sekret protin dεm de distribyut tru di ER mεmbran (Figure 1). Apat frɔm dat, dɛn tu kayn guds ya kin ɛnta difrɛn ERES dɛn we dɛn kin pik (Figure 2). di asil chen lεngth fכ di sεlyul sεramid insay di mεmbran de ridyus frכm C26 to C18-C16, di Gas1-GFP kכlכsta de disrupt insay di diskrεt ER rijyכn, εn Gas1-GFP de rirout fכ kכmכt na di ER wit di transmembran protin tru di sem ERES (Figure 3 εn Figure 3). 4).
pan tap we GPI-AP de yuz spεshal protin mεkanism fכ kכmכt na ER, wi fכnd se C26 sεramid-dipεndεnt sεparashכn nכ de dip pan difrεnt protin intarakshכn dεm we kin lid to ERES spεshalization (Figures S4 εn S5). insted, di tin dεm we wi fכnshכn sכpכt wan כda klasification mεkanism we dεn drεb bay lipid-bεys protin kכlכsta εn sכbsεkvεnt εksklushכn כf כda kago dεm. di obsכbishכn dεm we wi obsכv sho se di Gas1-GFP rijyכn כ kכlכsta we de aכsכsiayt wit wan spεsifi k ERES nכ gεt di transmεmbran sekret protin Mid2-iRFP, we sho se di C26 sεramid-dipεndεnt GPI-AP kכlכsta go fεsilitεt dεn εntri insay di rilevεnt ERES, εn di sem tεm, kכl transmembran Di sekreshכn dεm de enta dis patikyula ERES (Figure 1 εn 2). difrεnt frכm dat, di prεsεns fכ C18-C16 sεramid dεm na di ER mεmbran nכ de mek GPI-AP fכm rijyכn כ kכlכsta, so dεn nכ de kכl כ riples transmembran sekret protin dεm insay di sem ERES (Figure 3 εn 4). . כl dis mek wi prכpos se C26 sεramid de drεb separeshכn εn klasification bay we i de fεsilit di kכlכsta fכ protin dεm we lεnk to spεsifi k ERES.
aw fכ achy dis C26 sεramid-dipεndεnt kכlכsta insay wan spεsifi k ER εria? di tεndens fכ di mεmbran sεramid fכ sεparayt lateral wan kin mek GPI-AP εn C26 sεramid fכ fכm sכm sכm εn instantaneously כda lipid dεm na di mכr irεgul lipid envayroment fכ di ER mεmbran we gεt sכt εn כnsatyuret glycerolipid dεm. Kwaliti klasta dɛn (17, 18). dis sכm sכm tεmporari kכlכsta dεm kin fכs fכs insay big, mכr stebul kכlכsta dεm afta dεn biεn to di p24 kכmpleks ( 34 ). kכnsistεnt wit dis, wi sho se C26 Gas1-GFP nid fכ intarakt wit di p24 kכmpleks fכ fכm big visible kכlכsta dεm (Figure 3). di p24 komplex na hεterozaygכs כligomεr we kכmכp fכ difrεnt p24 transmεmbran protin dεm insay yist ( 35 ), we de gi mכltivalεnt binding, we kin lid to kכros-link fכ sכm GPI-AP kכlכsta dεm, we de jεnarεt big Stebul kכlכsta ( 34 ). di intarakshכn bitwin di protin εktodomεn dεm fכ GPI-AP dεm kin kכntribyut bak to dεn agregεshכn, lεk aw dεn sho di tεm we dεn Golgi transpכt insay mamal polarized εpitεlial sεl dεm ( 36 ). כltu, we C18-C16 sεramid de insay di εr mεmbran, we di p24 kכmpleks de biεn to Gas1-GFP, big sεparayt kכlכsta dεm nכ go fכm. di כndalayn mεkanism kin dipכnt pan di spεsifi k fכs εn kεmikכl prכpati dεm fכ di lכng asil chen sεramid. bayofizikal stכdi dεm pan atifishal mεmbran dεm sho se pan כl we כl tu di lכng (C24) εn sכt (C18-C16) asil chen sεramid dεm kin mek fεz separeshכn, na כnli lכng asil chen sεramid dεm (C24) kin promuot hכy Kכvεr εn film bεnd fכ rishεp di film. Tru mutual rεfrεns (17, 37, 38). i sho se di transmembran hεliks fכ TMED2, di hכman homolog fכ Emp24, de sεlektivli intarakt wit C18 sεramid-bεys sphingomyelin insay di saytoplasmik lכbul dεm ( 39 ). yuz mכlikul dynamiks (MD) simulεshכn, wi fכnd se כl tu di C18 εn C26 sεramid dεm de akumulet rawnd di saytoplasmik lכbul dεm na di Emp24 transmεmbran hεliks, εn dεn gεt di sem kayn prεfrεns (Figure S6). i fayn fכ no se dis de sho se di transmεmbran hεliks fכ Emp24 kin lid to asimεtrik distribushכn fכ lipid dεm na di mεmbran. dis na rizulyt we dεn jכs du we dεn bays pan mami dεm sεl dεm. di sem kayn MD simulεshכn dεm de sho bak di prεsεns fכ εta lipid dεm (40) . כl dis mek wi spεkul se C26 sεramid insay di tu lכbul dεm na ER26 na lokal εnrich. we GPI-AP insay di luminal lכbul dεm de biεn dayrekt to mכltivalεnt p24 εn di akyumyuleshכn fכ C26 sεramid arawnd p24 insay di saytoplasmik lכbul dεm, i kin protεkt di akkompayn Protein agregεshכn εn mεmbran kכvεshכn de jεnarεt tru di finga dεm ( 41 ), we de mek GPI-AP separet insay diskrεt rijyכn dεm we de nia ERES, we de fכvכr bak di hכy kכv rijyכn dεm na di εr mεmbran ( 42 ). Ripɔt dɛn we bin dɔn de bifo bin sɔpɔt di mɛkanism we dɛn bin dɔn pruv ( 43 , 44 ). di mכltivalεnt binding fכ oligolectins, pathogens כ antibodies to sεramid-bεys glycosphingolipids (GSL) pan di plasma mεmbran de trigεr big GSL agregεshכn, εnhans fεz sεparashכn εn mek mεmbran difכmeshכn εn internalization (44). Iwabuchi εtk nyutrofil dɛn.
insay mammalian polarized epithelial sel dεm, di kכnsantreshכn fכ di anti-Golgi nεtwכk (TGN) to di lεvεl fכ di apikal plasma mεmbran de kכntro di separeshכn εn sכt fכ GPI-AP ( 10 , 45 ). dis agregεshכn de drεb bay GPI-AP oligomerization ( 36 ), bכt i kin dipכnt bak pan di sεramid chen lεngth we wi de fכn insay yist. pan tap we mamal GPI-AP gεt εta lipid-bεys ankכ, εn in kεmikכl strכkchכ rili difrεnt frכm di vεri lכng asil chen sεramid, wan stכdi we dεn jכs du sho se di tu lipid dεm gεt evolushכnal sεm kayn fכs εn kεmikכl prכpati εn fכnshכn ( 40 ). כl dis mek di εta lipid pat na di mama sεl dεm kin sכm kayn we lεk di C26 sεramid insay yist, εn in rol na fכ asosiet wit di lכng chen sεramid insay di mεmbran fכ protεkt GPI-AP agregεshכn εn sכt. pan ɔl we dis posisibul stil nid fɔ tɛst dairekt wan, di tin dɛn we dɛn bin dɔn fɛn bifo dis tɛm de sɔpɔt se di transpɔt fɔ di lɔng asil chen sɛramid to di Golgi bɔdi nɔ de apin bay saytoplasmik transfyushɔn protin dɛn, bɔt i dipen pan di sεntesis fכ GPI ankכ dεm lεk yist. fכ dat, i lεk se di evolushכnal kכnsyuv mεkanism ebul fכ sεlektivli kכ-transpכt vεri lכng asil chen sεramid εn GPI-AP (13, 16, 20, 46, 47) insay di sem transpכt vεsikul.
insay yist εn mamal polarized epithelial sel sistεm dεm, GPI-AP agregεshכn εn separeshכn frכm כda plasma mεmbran protin dεm כl de apin bifo i rich di sεl sεf. Paladino ɛn ɔda pipul dɛn. . Sεl surface. in yist, dis stכdi sho se di C26 sεramid-dipεndεnt GPI-AP kכlכsta pan di ER kin rεgεl di kכlכsta כganayzeshn εn fכnshכnal aktiviti fכ GPI-AP pan di plasma mεmbran ( 24 , 49 ). kכnsistεnt wit dis mכdel, GhLag1 sεl dεm de alerjik to GPI inhibito כ drog dεm we de afekt sεl wכl integriti ( 28 ), εn di nid fכ fכnshכnal Gas1-GFP kכlכsta dεm ( 49 ) fכ di tip sεramid we dεn projεkt insay di mating fכ yist sεl dεm indikεt G Posisibul fysiolojikal kכnsekvεns fכ hLag1 sεl dεm. GPI-AP mistek we dɛn mek. כltu, fכ tεst if di fכnshכnal כganayzeshn fכ di sεl sεf dεn bin progrεm frכm di εr bay wan sכt mεtכd bays pan lipid lεngth go bi di sכbjek fכ wi fכs risεch.
di Saccharomyces cerevisiae strayn dεm we dεn yuz fכ dis wok dεn list dεm na Tεbul S1. Di MMY1583 ɛn MMY1635 strayn dɛn fɔ SCLIM fɔ layf sɛl imej bin kɔnstrɔk insay di bakgrɔn fɔ W303. Dis strayn dεm we de εksprεs Sec13-mCherry wit wan fluorescent protin tεg bin kכnstrכkt yuz wan polimεrayz chen riakshכn (PCR)-bεys mεtכd wit pFA6a plasmid as tεmplat ( 23 ). di strain we de εksprεs Mid2-iRFP we dεn lεbul wit fכrosεnt protin כnda di kכntrכl fכ di GAL1 prכmotεr bin kכnstrכkt lεk dis. PCR amplifyushכn fכ iRFP-KanMx sikεns frכm pKTiRFP-KAN vεktכr (gift fכ E. O'Shea, Addgene plasmid nכmba 64687; http://n2t.net/addgene: 64687; risεch risכs aydentifaya (RRID): Addgene_64687) εn insay di C-tεrminכs fכ εndojεnik Mid2. Afta di Mid2-iRFP jεnom sikεns bin amplify εn klon insay di GAL1 prכmotεr, i bin intagret insay di Not I-Sac I sayt fכ di intagrεshכn plasmid pRS306. Di rizultant plasmid pRGS7 bin linearized wit Pst I fɔ intagret insay di URA3 lɔkus.
di Gas1-GFP fכs jin de εksprεs כnda di kכntrכl fכ di GAL1 prכmotεr insay di sεntromεr (CEN) plasmid, we dεn kכnstrכkt lεk dis. Di Gas1-GFP sikεns bin amplify bay PCR frכm di pRS416-GAS1-GFP plasmid (24) (gift fכ L. Popolo) εn klon insay di Xma I–Xho I sayt fכ di CEN plasmid pBEVY-GL LEU2 (gift fכ C) . Miller; Addjin plasmid nɔmba 51225; http://n2t.net/addgene: 51225, di wan dɛn we de stɔdi bɔt di Baybul; RRID: Addjin_51225) we de na di wɔl. Di plasmid we kɔmɔt bin gɛt di nem pRGS6. di Axl2-GFP fכs jin de εksprεs bak כnda di kכntrכl fכ di GAL1 prכmotεr fכ di pBEVY-GL LEU2 vεktכr, εn in kכnstrכkshכn na lεk dis. Di Axl2-GFP sikεns bin amplify frכm pRS304-p2HSE-Axl2-GFP plasmid (23) bay PCR, εn klon insay di Bam HI-Pst I sayt כf pBEVY-GL LEU2 vεktכr. Di plasmid we kɔmɔt bin gɛt di nem pRGS12. di sikεns fכ di oligonuklεotayd dεm we dεn yuz na dis stכdi de na di list na Tεbul S2.
Di strayn bin supliment wit 0.2% adenin ɛn 2% glukɔs [YP-dɛkstroz (YPD)], 2% rafinɔs [YP-rafinɔs] rich yist ɛkstrakt protin p (YP) mɛdiɔm (1 % Yist ɛkstrakt ɛn 2% protin ept). (YPR)] ɔ 2% galaktɔs [YP-galaktoz (YPG)] as kabɔn sɔs, ɔ insay sɛntetik minim mɛdiɔm (0.15% yist naytrɔjen bays ɛn 0.5% amɔniɔm sɔlfɛt) fɔ sɔpɔt di rayt amino asid ɛn bays dɛn we dɛn nid fɔ it , Ɛn we gɛt 2% glukɔs (sintetik glukɔs minimal mɛdiɔm) ɔ 2% galaktɔs (sintetik galaktכz minim mεdiכm) as kכbכn sכs.
Fכ rial-taym imej, tεmprachכ-sεnsitiv sec31-1 mutant sεl dεm we de εksprεs di kכnstrכkt כnda di GAL1 prכmotεr bin gro insay YPR mεdiכm na 24°C כvnayt to mid-lכg fεz. Afta indukshכn in YPG na 24°C fכ 1 awa, dεn inkכba di sεl dεm insay SG na 37°C fכ 30 minit, εn afta dat dεn transfכr to 24°C fכ rilis frכm di sekreshכn blכk. Concanavalin A wεn yuz fכ fiks di sεl dεm pan wan glas slayd εn imej bay SCLIM. SCLIM na wan kɔmbaynshɔn fɔ Olympus IX-71 invɛst fluorescence maykroskɔp ɛn UPlanSApo 100×1.4 nyumɛrik aperchɔ ɔyl lens (Olympus), ay-spid ɛn ay-saynal-to-nɔys rɛtɛshɔn rɔtin disk kɔnfɔkal skan (Yokogawa Ilɛktrik), kɔstɔm spɛktromita, ɛn kɔstɔm kol Di sistɛm in imej intensifaya (Hamamatsu Fɔtoniks) kin gi wan magnifying lens sistem wit wan fayn magnifyeshɔn we na ×266.7 ɛn wan chaj-kɔpl divays kamɛra we de multiply ilɛktron dɛn (Hamamatsu Photonics) (21). Na kɔstɔm sɔftwɛl (Yokogawa Ilɛktrik) de du di pikchɔ akwyeshɔn. Fɔ 3D imej, wi yuz wan kɔstɔm-mɛd piezoɛlektrik aktyuator fɔ vaybrɛt di ɔbjɛktiv lens vertikal, ɛn gɛda di optik pat dɛn 100 nm apat insay wan stak. Di Z-stak imej de kכnvכlt to 3D vכksεl data, εn di tiori pכynt spεd fכnshכn we dεn yuz fכ di rotating disk kכnfכkal maykroskכp dεn yuz fכ dikonvכlushכn prכsεsin bay Volocity softwεa (PerkinElmer). Bay we dɛn yuz Volocity softwe fɔ ɔtomɛtik threshold fɔ kɔ-lɔkeshɔn analisis, dɛn mɛzhɔ ERES inklud kago. Layn skan analisis bin de yuz MetaMorph softwe (Molecular Devices).
Yuz GraphPad Prism softwe fɔ no di statystik siginifikɛns. Fɔ di tu-tɛl Student’s t-tɛst ɛn di ɔdinari wan-way analisis ɔf varyans (ANOVA) tɛst, dɛn tek difrɛns bitwin grup dɛn fɔ gɛt signifyant impak pan P <0.05 (*).
Fכ fluorescence maykroskכpi fכ Gas1-GFP, di log fεz sεl dεm bin gro כvnayt insay YPD εn kכlekt bay sεntrifugayshכn, was tu tεm wit fכsfεt bכfa salin, εn inkכba pan ays fכ at le 15 minit, εn afta dat dεn bin go כnda di maykroskכp lεk aw dεn bin dεskrεb bifo Chεk (24). Di Leica DMi8 maykroskɔp (HCX PL APO 1003/1.40 ɔyl PH3 CS) we gɛt ɔbjɛktiv lens, L5 (GFP) filta, Hamamatsu kamɛra ɛn Aplikeshɔn Suit X (LAS X) softwea bin yuz fɔ akwyeshɔn. .
Di samplε dεm bin denature wit SDS sεmpl bכfa na 65°C fכ 10 minit, εn afta dat dεn bin separet bay SDS-polyacrylamide jel εlektrofכresis (PAGE). Fכ immunoblotting analisis, 10 μl sεmpl bin lod pan wan len. Praymari antibodi: Yuz rabit polyclonal anti-Gas1 we di dilution 1:3000, rabit polyclonal anti-Emp24 we dilution 1:500, ɛn rabit polyclonal anti-GFP (wan gift frɔm H. Riezman) we dilution 1:3000. Di maws monoklonal anti-Pgk1 antibodi bin yuz pan dilushon we na 1:5000 (wan gift frɔm J. de la Cruz). Sεkɔndari antibodi: Hכsεradish pεrכksidayz (HRP) kכnjugεt got anti-rεbεt imyunoglobulin G (IgG) we dεn yuz pan dilushכn we na 1:3000 (Pierce). HRP-konjugεt got anti-maus IgG bin yuz pan dilushכn 1:3000 (Pierce). Di imyun rεspכns zon bin obsεv bay di kεmiluminesεns mεtכd כf SuperSignal Wεst Piko riεjεnt (Tεrmo Fisher Scientific).
As dεn dεskrεb in (31), wan nεchכral immunoprεsipitεshכn εkspεriεns bin de pan di εnrich εr frakshכn. in sכt, was yist sεl dεm wit TNE bכfa [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM fεnylmethylsulfonyl fluoride εn protease inhibitor miksכp) na 600 nm ( OD600) na 100 optik dεnsiti tu tεm. Dɛn bin de brok am wit glas bidz, dɔn dɛn bin de pul di dɔti we de na di sɛl ɛn di glas bidz bay we dɛn yuz sɛntrifugayshɔn. Afta dat, dɛn sɛntrifug di supamatant na 17,000 g fɔ 15 minit na 4°C. Di pellet bin risuspɛnd insay TNE ɛn dɛn ad digitalis saponin to wan fayn kɔnsɛntreshɔn we na 1%. Di suspension bin inkubayt fɔ 1 awa wit roteshɔn na 4°C, ɛn afta dat dɛn pul di insɔlubul kɔmpɔnɛnt dɛn bay sɛntrifugayshɔn na 13,000 g na 4°C fɔ 60 minit. Fכ Gas1-GFP immunoprecipitation, fכs prε-inkubayt di sεmpl wit εmpti agarose bidz (ChromoTek) na 4°C fכ 1 awa, εn afta dat inkכba wit GFP-Trap_A (ChromoTek) na 4°C fכ 3 awa. Di immunoprecipitated bidz dɛn was fayv tɛm wit TNE we gɛt 0.2% digoxigenin, eluted wit SDS sampul bafa, separet pan SDS-PAGE, ɛn analayz bay immunoblotting.
As dεn dεskrεb in (31), kכros-linkin ditarminεshכn bin de pan di εnrich εr frakshכn. Brifly, di enrich ER frakshɔn bin inkubayt wit 0.5 mM dithiobis(succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rɔkfɔd, IL, Amɛrika; 20°C, 20 min). Di kroslinkin riakshכn bin kεnch bay we dεn ad glycine (50 mM fainal kכnsantreshכn, 5 minit, 20°C).
As wi bin dεskrεb bifo (50), MS analisis כf sεramid in wayl-tayp εn GhLag1 strayn dεm bin du. In shot, sεl dεm bin gro to εkspכnεnshal fεz (3 to 4 OD600 yunit/ml) insay YPD na 30°C, εn 25×107 sεl dεm bin avεst. di mεtabolism dεm de kεnch wit trikloroasetεk asid. Yuz ɛkstrakshɔn sɔlvɛnt [ɛtɛnol, wata, ɛtɛr, piridin ɛn 4.2 N amɔniɔm haydroksayd (15:15:5:1:0.018 v/v)] ɛn 1.2 nmol intanɛnt standad C17 sɛramid (860517, Avanti polar lipid) kwaliti). Yuz monomethylamine reagent [methanol, wata, n-butanol ɛn methylamine solution (4:3:1:5 v/v)] fɔ du mild alkaline hydrolysis fɔ di ekstrakt, ɛn afta dat yuz wata-satyureted n-butanol fɔ desalt. Fכ finish, di εkstrakt bin rεsuspεnd insay wan positifu mכd sכlvεnt [klorofכm/mεtanכl/wata (2:7:1) + 5 mM amoniכm asetεt] εn injεkt insay di mas spεktrכmita. Multi-reaction monitoring (MRM) wεn pεrform fכ di idεntifikεshכn εn kwantifikεshכn כf sphingolipid mכlikul dεm. Di TSQ Vantage teshari kwadrupol mas spɛktromita (Thermo Fisher Scientific) gɛt wan rɔbɔt nanoflɔ ayɔn sɔs Nanomate HD (Advion Biosciences, Ithaca, NY) fɔ lipid analisis. Di kolishon enaji na optimiz fɔ ɛni sɛramid kategori. MS data wεn כbtain in positifu mכd. fכ εvri bayolojikal rεplikεt, di lipid signal na di mεdian fכ tri indipεndεnt mεzhɔmεnt dεm.
As dεn dεskrεb in (31), di sεl dεm (800×107) we de εksprεs Gas1-GFP bin sכbjεkt to nεchכral immunoprecipitation. Di purifyed Gas1-GFP bin separet bay SDS-PAGE ɛn transfa to wan polyvinylidene fluoride (PVDF) mɛmbran. Di protin bin visualiz bay we dɛn stɛyn PVDF wit amid blak. Di Gas1-GFP band bin kot frɔm di PVDF ɛn was 5 tɛm wit mɛtanɔl ɛn wan tɛm wit likwid kromatografi-MS (LC-MS) gred wata. bay we dεn inkכba di membran strip wit 500μl 0.3 M NaOAc (pH 4.0), bכfa εn 500μl fכs sכlv 1 M sכdiכm naytrayt miksכp na 37°C fכ 3 awa, di lipid frakshכn de kכmכt frכm Gas1-GFP εn layz Rilis fכ inosine fכsfεt sεramid bitwin glukosamin εn inositol (51). Afta dat, dɛn was di mɛmbran strip 4 tɛm wit LC-MS gred wata, drɛy am na rum tɛmpracha, ɛn kip am na naytrɔjen atmosfɛs na -80°C te dɛn analayz am. As a kontrכl, wan blank sεmpl כf PVDF mεmbran bin yuz fכ εvri εkspεriεns. Di lipid we dɛn pul frɔm Gas1-GFP bin afta dat analayz bay MS lɛk aw dɛn diskrayb am ( 50 ). In shot, PVDF strip dɛn we gɛt GPI-lipid bin risuspɛnd insay 75μl nɛgitiv mold sɔlvɛnt [klorofɔm/mɛtanɔl (1:2) + 5 mM amɔniɔm asɛtat] ɛn pas ilɛktrɔsprɛy ayɔnizashɔn (ESI)-MRM/MS Analysis of sphingolipid spisis (TSQ Vantage). In dis kes, MS data bin ɔbtain in nɛgitiv ayɔn mɔd.
as wi bin dכn mεnshכn, di lipid pat pan di GPI ankכr bin separet frכm di [3H]-inositol-lεbul GPI-AP ( 16 ). Di lipid dεm bin separet bay tin-layer kromatografi yuz wan sכlvεnt sistεm (55:45:10 kloroform-mεtanכl-0.25% KCl) εn visualiz yuz FLA-7000 (Fujifilm).
Di sel dεm we de εksprεs Gas1-GFP (600×107) dεn was tu tεm wit TNE bכfa wit TNE bכfa, εn brok wit glas bidz, εn afta dat dεn sεntrifug fכ pul sεl dεbris εn glas bidz. Afta dat, dɛn bin sɛntrifug di supamatant na 17,000 g fɔ 1 awa na 4°C. Di pellet bin was insay TNE ɛn inkubayt wit 1 U PI-PLC (Invitrogen) insay TNE we gɛt 0.2% digitalis saponin fɔ 1 awa na 37°C. Afta di εnzym tritmεnt, dεn pul di mεmbran bay sεntrifugayshכn na 17,000 g na 4°C fכ 1 awa. Fכ immunoprecipitate Gas1-GFP, di supεrnatant bin inkכba wit GFP-Trap_A (ChromoTek) na 4°C ovanayt. Di purifyed Gas1-GFP we dɛn separet bay SDS-PAGE bin stɛyn wit Coomassie briliant blu. Di Gas1-GFP steyning band bin kot ɔf frɔm di grey we de rawnd di akwedakt, ɛn afta dat afta alkylation wit iodoacetamide ɛn ridyushɔn wit dithiothreitol, in-gel digestion wit trypsin bin du. Ekstrakt ɛn dray triptik pεpti dεm εn pεptida dεm wit GPI-glycans. Di dray pεptida bin sכlv insay 20 μl wata. Injekt wan pat (8μl) insay di LC. Wan oktadecylsilane (ODS) kɔlɔm (Develosil 300ODS-HG-5; inana dayamita 150 mm×1.0 mm; Nomura Kεmikכl, Aichi Prεfεkshכn, Japan) bin yuz fכ separet pεptida dεm כnda spεsifi k grεdiεnt kכndishכn dεm. Di mobayl fεz na sכlvεnt A (0.08% fכmik asid) εn sכlvεnt B (0.15% fכmik asid insay 80% asetonitril). Wan Accela HPLC sistem (Thermo Fisher Scientific, Boston, Massachusetts) bin yuz fɔ elute di kɔlɔm wit sɔlvɛnt A insay 55 minit pan wan flɔ rit we na 50 μl min-1 fɔ 5 minit, ɛn afta dat dɛn inkrisayz di kɔnsɛntreshɔn fɔ sɔlvɛnt B to 40%. , Amɛrika). Di eluate bin kɔntinyu fɔ introduks insay di ESI ayɔn sɔs, ɛn di triptik peptida ɛn peptida dɛn wit GPI-glycans bin analayz bay LTQ Orbitrap XL (haybrid linya ayɔn trap-ɔrbitrap mas spɛktromita; Thermo Fisher Scientific). insay di MS setup, dεn bin set di vכltεj fכ di kapilari sכs to 4.5 kV, εn di tεmprachכ fכ di tכnfכs kapilari bin kip na 300°C. Di kapilari vכltεj εn tiub lεns vכltεj bin set to 15 V εn 50 V, rispεktivli. MS data bin gɛt insay di pɔzitiv ayɔn mɔd (rɛzolushɔn fɔ 60,000; mas akkuracy fɔ 10 pat pan wan milyɔn) insay wan mas rɛnj we na 300/m/z mas/chaj rɛtɛshɔn (m/z) 3000. Dɛn bin gɛt di MS/MS data tru di ayɔn trap insay di LTQ Ɔbitrap XL [di fɔs 3 dijit dɛn we di data dipen pan, kɔlishin indyuz dissosieshɔn (CID)) we dɛn kɔl].
MD simulshɔn dɛn bin du yuz GROMACS (52) softwe ɛn MARTINI 2 fɔs fil (53-55). Afta dat, dɛn yuz di CHARMM GUI Mɛmbran Bilda (56, 57) fɔ kɔnstrɔk wan bilayer we gɛt dioleoylphosphatidylcholine (DOPC) ɛn Cer C18 ɔ DOPC ɛn Cer C26. di tכpכlכji εn kכdכnayt dεm fכ Cer C26 kכmכt frכm DXCE bay we dεn de pul di ekstra bidz dεm frכm di sphingosine tel. Yuz di prɔses we dɛn tɔk bɔt dɔŋ ya fɔ balans di dabl layt ɛn rɔn am, dɔn yuz di las kɔdinɛt dɛn na di sistɛm fɔ bil wan sistɛm we gɛt Emp24. di transmembran domεn fכ yist Emp24 (rεsidεns 173 to 193) bin kכnstrכkt as α-hεliks yuz di vishכnal MD (VMD) tul mכlikul strכkchכ ( 58 ). afta dat, afta dεn pul di lipid dεm we de כvalap, dεn kכs granul di protin εn insay di bilayer yuz CHARMM GUI. Di fainal sistem gɛt 1202 DOPC ɛn 302 Cer C26 ɔ 1197 DOPC ɛn 295 Cer C18 ɛn Emp24. Ayoniz di sistem to wan kɔnsɛntreshɔn we na 0.150M. Fo indipendent rεplikεt dεm bin mek fכ tu bilayer kכmכshכn dεm.
di lipid bilayer de balans yuz di CHARMM GUI prכsεs, we involv fכ minimiz εn afta dat bεlε 405,000 stεp dεm, usay di posishכn kכnstrכkshכn dεm de rεdכks sכmtεm εn εliminet, εn di tεm stεp de inkrεs frכm 0.005 ps to 0.02 ps. afta ikwilεbrεshכn, i de prodyuz 6 μs wit tεm stεp we na 0.02 ps. Afta yu put Emp24, yuz di sem CHARMM GUI prɔses fɔ minimiz ɛn balans di sistɛm, ɛn afta dat rɔn fɔ 8 s insay prodakshɔn.
Fɔ ɔl di sistɛm dɛn, di tɛm we dɛn de balans, di prɛshɔn na di Berendsen barostat (59) de kɔntrol am, ɛn di tɛm we dɛn de prodyuz, di prɛshɔn na di Parrinello-Rahman barostat (60) de kɔntrol am. In ɔl di kes dɛm, di avɛrej prɛshɔn na 1 bar ɛn dɛn yuz sɛmi-isotropik prɛshɔn kɔpl skim. insay di bεlε εn prodakshכn prכsεs, dεn de yuz tεmכstat (61) wit spid rεkalibrayshכn fכ kכpl di tεmprachכ fכ protin, lipid εn sכlvεnt patikyula dεm rispεktivli. Durin di entire opareshon, di target temperecha na 310K. Di nכn-bכnding intarakshכn dεn kכl am bay we dεn jεnarεt wan pairing list yuz di Verlet skim wit 0.005 bafa tכlerεns. di Kulomb tεm dεn kכl am yuz di riakshכn fil εn wan kכt-כf distans we na 1.1 nm. Di Vander Waals tεm de yuz wan kכt-כf skim wit wan kכt-כf distans we na 1.1 nm, εn di Verlet kכt-כf skim de yuz fכ pכtεnshal drift (62).
we yu yuz VMD, di kכtכf wevlεngth bitwin DOPC fכsfεt bidz כ sεramid AM1 bidz εn di protin na 0.7 nm, εn dεn kכl di nכmba fכ di lipid dεm we de intarakt wit di protin. akɔdin to di fכmula we de kam biεn, kכl di diplεshכn-εnrichmεnt (DE) fכktכ lεk na (63): DE fכktכ = (di amoun fכ di tכtal lipid dεm na di protin 0.7) insay di protin 0.7 (di amoun fכ Cer insay di tכtal lipid dεm) .
Di valyu we dɛn ripɔt, dɛn kin gɛt am as avɛrej, ɛn di mistek bar dɛn na 4 indipɛndɛnt kɔpi dɛn fɔ SE. Di stεdi sכgεns fכ DE fכktכr dεn kכl am bay t tεst [(avεrejDE-faktכr-1)/SE]. Kalkul di P valyu frɔm di wan-tɛl distribyushɔn.
Di GROMACS tul bin yuz fɔ kɔl di 2D lateral density map fɔ di sistɛm we gɛt Emp24 insay di las 250 ns fɔ di trays. fכ mek dεn gεt di enrichmεnt/diplεshכn map fכ sεramid, dεn divayd di dεnsiti map fכ Cer wit di sכm fכ di map fכ Cer εn DOPC, εn afta dat dεn divayd am bay di kכnsantreshכn fכ Cer na di bכdi. Dɛn kin yuz di sem kɔlɔ map skel.
Fɔ sɔpɔt ɔda tin dɛn fɔ dis atikul, duya luk http://advances.sciencemag.org/cgi/content/full/6/50/eaba8237/DC1
Dis na opin akses atikul we dɛn sheb ɔnda di tin dɛn we de na di Krio Kɔmɔns Atribiushɔn-Nɔn-Kɔmɛshɔn Laysens, we de alaw fɔ yuz, sheb ɛn riprodyuz insay ɛni mɛdiɔm, as lɔng as di las yus nɔto fɔ kɔmɛshɔn bɛnifit ɛn di prɛmis na se di ɔrijinal wok kɔrɛkt. Tɔk bɔt.
Notis: Wi jɔs de aks yu fɔ gi yu imel adrɛs so dat di pɔsin we yu rɛkɔmɛnd to di pej go no se yu want dɛn fɔ si di imel ɛn i nɔto spam. Wi nɔ go kech ɛni imel adrɛs.
Dɛn kin yuz dis kwɛstyɔn fɔ tɛst if yu na visitɔ ɛn fɔ mek dɛn nɔ sɛn spam ɔtomɛtik wan.
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 Pɛriz -Linero), Sergio Lopez (Sergio Lopez), Miho Waga (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stɛfano Fani, Akihiko Nakano, Manuɛl Muniz
3D hεy-rεsכlushכn rial-taym imej de sho di imכpכtants fכ sεramid chen lεngth fכ protin sכt in sεlektiv כtput sayt dεm.
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 Pɛriz -Linero), Sergio Lopez (Sergio Lopez), Miho Waga (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stɛfano Fani, Akihiko Nakano, Manuɛl Muniz
3D hεy-rεsכlushכn rial-taym imej de sho di imכpכtants fכ sεramid chen lεngth fכ protin sכt in sεlektiv כtput sayt dεm.
©2020 Amɛrikan Asɛmbli fɔ di Advansmɛnt fɔ Sayns. ɔl di rayt dɛn de fɔ am. AAAS na patna fɔ HINARI, AGORA, OARE, CHORUS, KLOKSS, KrosRɛf ɛn KƆNTA. SaynsAdvans ISSN 2375-2548.