Say panag-sort na protina ed secretory pathway et importante pian napansiansia so cell compartmentalization tan homeostasis. Nilikud ed shell-mediated sorting, say papel na lipids ed kinesin sorting ed proseso na secretory transport et abayag lan manunan tepet ya agni naebatan. Dia, manggagawa kami na 3D simultaneous multicolor high-resolution real-time imaging pian napaneknekan ed vivo ya saray balon ginawan glycosylphosphatidylinositol-immobilized proteins ya walaan na andukey iran ceramide lipid moieties et mankakasakey tan niklasipika ed saray espisyal ya endoplasm Net exit site, ya duma ed saray protina ya uusaren na transmembrane. Niarum ni, ipapanengneng mi ya say karukey na kadena na ceramide ed endoplasmic reticulum membrane et importante ed sayan panag-sort selectivity. Say impanaral mi so mangiiter na sankaunaan ya direktan in vivo ya ebidensya pian naklasipika iray karga na protina base ed karukey na lipid chain diad saray pinilin export sites diad secretory pathway.
Diad saray selula na eukaryotic, saray protina ya ginawa ed endoplasmic reticulum (ER) et i-sort legan na transportasyon diad secretory pathway pian nasabi ed dugan destinasyon na selula (1). Nilikud ed coat-mediated sorting, abayag lan iisipen ya wala ray lipid ya nayarin manserbi met ya selective exit points diad pangitipon ed saratan ed espisipikon membrane domain ya espisipikon protina (2-5). Balet, wala ni so kakulangan na direktan in vivo ya ebidensya pian napaneknekan iyan posiblin mekanismo ya base ed lipid. Pian nasolbar iyan manunan problema, inaral mi ed lebadura no panon ya saray glycosylphosphatidylinositol (GPI) anchored proteins (GPI-APs) et mandumaan ya ipapaway manlapud ER. Saray GPI-APs et nanduruman klase na lipid-connected cell surface proteins ( 6, 7). Say GPI-AP et sakey a protina ya akapeket ed paway na plasma membrane panamegley na glycolipid moiety (GPI anchor). Aawaten da ray GPI anchors bilang konserbatibon post-translational ya pananguman ed ER lumen (8). Kayari na inkabit, say GPI-AP et ondalan ed Golgi apparatus (5, 9) manlapud ER ya anggad plasma membrane. Say kiwawala na GPI anchors so pansengegan na pangiyakar na GPI-AP ya nibiig ed saray transmembrane secreted proteins (kaiba lay arum nin plasma membrane proteins) diad dalan na panag-sekreto (5, 9, 10). Diad saray selula na lebadura, saray GPI-APs et nibiig ed arum ya nisekreto iran protina ed endoplasmic reticulum, insan ira i-pack ed nikadkaduman vesicles ya binalkot na coat protein complex II (COPII) (6, 7). Saray sengegan na sayan proseso na klasipikasyon ed proseso na ER export et agmalinew, balet ta iisipen ya sayan mekanismo et nayarin mankaukolan na lipids, lalo la ed structural remodeling na lipid portion na GPI anchor (5, 8). Diad lebadura, say GPI lipid remodeling et onggapo kayari na pangipeket na GPI, tan diad dakel a kaso, saya so pansengegan na pangipeket na ceramide ed 26-carbon long-chain saturated fatty acid (C26:0) (11, 12). Say C26 ceramide so manunan ceramide ya napapawala na saray selula na lebadura anggad natan. Saya so nagawa ed ER tan maslak ed saya et ipapaway ed Golgi apparatus panamegley na COPII vesicles (13). Say ER export na GPI-AP et espisipikon mankaukolan na tuloytuloy ya panaggawa na ceramide (14, 15), tan diad saginonor, say pananguman na ceramide ed inositol phosphate ceramide (IPC) diad Golgi apparatus et mandedependi ed GPI anchor synthesis (16). Saray biophysical ya impanaral ed artipisyal iran membrane so angipatnag ya saray andukey a tuloy ya acyl chain ceramides et nayarin mankasakey pian manporma na saray mauksoy a domain ya walaan na nikadkaduman pisikal iran kablian (17, 18). Sarayan datos so mangitonton ed hypothesis ya say C26 ceramide tan GPI-AP ya walay C26 ceramide et manguusar na pisikal iran kablian da pian mankakasakey ed mauksoy iran rehyon odino rehyon ed medyo magulon ER membrane lipid environment. Saya so manunan tugyopen na antikey tan unsaturated glycerolipids (C16:1 tan C18:1) (19, 20). Sarayan rehyon et napili ya manpokus ed espisipikon ER exit sites (ERES), ya ditan et say ceramide tan ceramide-based GPI-AP et nayarin i-co-transport ed Golgi diad parehon COPII vesicle (5).
Diad sayan impanaral, direktan sinubok mi iyan lipid-based mechanism diad impangusar na super-resolution confocal real-time imaging microscopy (SCLIM), ya sakey a cutting-edge microscopy technique ya makapan-obserba na saray protina ya walay fluorescently label da.
Inusar mi nin unona so teknolohiya na SCLIM pian lalon nadetermina no panon ya say normal ya GPI-AP ya walay C26 ceramide group et na-screen manlapud transmembrane secreted proteins kayari na impaway ed ER ed S. cerevisiae. Pian naseguro so klasipikasyon na ER, angusar kami na genetic system ya direktan makanengneng ed balon ginawan kargamento ya onloloob ed ERES diad vivo (7, 23). Bilang kargamento, pinili mi so C26 ceramide-based GPI-AP Gas1 ya walay marka ton green fluorescent protein (GFP) tan transmembrane secreted protein Mid2 ya walay marka ton near-infrared fluorescent protein (iRFP), ya parehon manpupuntirya ed plasma membrane (24-26). Diad sec31-1 temperature-sensitive mutant, sarayan duaran kargamento et nipatnag ed silong na galactose-inducible promoter tan sakey a constitutive ERES marker. Diad arawrawin temperatura (37°C), lapud say sec31-1 mutation et mangaapekta ed kimey na COPII coat component Sec31 pian nasebel so COPII germination tan ER export, say balon ginawan kargamento et mantitipon ed ER (23). Kayari na impamalemew ed abeban temperatura (24°C), saray sec31-1 mutant cells so akabawi manlapud secretory area, tan say natipon a balon sintetikon kargamento so ginmapon impaway manlapud ER. Impanengneng na CLIM ya maslak ed saray balon ginawan Gas1-GFP tan Mid2-iRFP et natipon ni ed ER na sec31-1 mutant cells kayari na inkubasyon ed 37°C insan nibulos ed 24°C ed loob na 5 minuto (Figure 1). Lapud say Mid2-iRFP et nibunog ed interon ER membrane, tan say Gas1-GFP et akasentro tan natipon ed ag-ontutumbok ya ER membrane area, say inkaapag-apag da et sigpot a duma (Figure 1, A anggad C tan Movie S1). Niarum ni, singa nanengneng ed Figure 1D, say Gas1-GFP cluster et anggapoy Mid2-iRFP to. Ipapanengneng na sarayan resulta ya say GPI-AP tan transmembrane proteins et insian ed nanduruman ER membrane regions nen masakbay. Say Gas1-GFP cluster et asingger ed sakey ya espisipikon ERES ya aka-label na mCherry's COPII coat protein Sec13 (Figure 1, E tan F, tan movie S1) (23).
saray sec31-1 ya selula et mangipapaway na galactose-induced secretions, sakey ya andukey ya acyl chain (C26) ceramide GPI-AP Gas1-GFP (GPI-AP, berde) tan say transmembrane protein ya Mid2-iRFP (TMP, asul) tan sayan Constructive ERES labeling ya Sec13-mCherry (E) et inmage ed 30°C minuto, inyalis ed 24°C, tan in-image na SCLIM 5 minutos ed saginonor. Say (A anggad C) et mangipapanengneng na representative ya nankasakey odino saksakey ya 2D ya imahen na sakey ya eroplano (A), sakey ya 2D ya imahen na projection na 10 z-sections (B) odino sakey ya 3D ya imahen na cell hemisphere na kargamento tan ERES markers (C). Scale bar 1μm (A tan B). Say sukat et 0.551μm (C). Say Gas1-GFP et anengneng ed nanduruman ER regions o clusters, bangta say Mid2-iRFP et anengneng tan nibunog ed interon ER membrane (C). (D) Ipapanengneng na graph so relatibon intensidad na fluorescence na Gas1-GFP tan Mid2-iRFP ed Gas1-GFP cluster diad gilig na amputin linya na pana (kawanan). AU, arbitrary unit. (E tan F) so mangirerepresenta ed 3D ya imahen ya mankokombina na saray produkto tan ERES mark. Saray Gas1-GFP clusters et anengneng ya asingger ed espisipikon ERES. Say sukat et 0.551μm. (F) Say amputin solidon pana so mamaneknek ed Gas1-GFP cluster ya asosasyon na ERES. Say pegley tan kawanan ya panel et mangipapanengneng na pinagkasakey ya pinabaleg ya 3D ya imahen tan say inpaliber ya nengnengen na pinilin Gas1-GFP cluster.
Say maapit ya siglaotan na Gas1-GFP cluster tan sakey ya espisipikon ERES so mangipapanengneng ya say Gas1-GFP et nayarin onloob ed selective ERES, ya duma ed selectivity ya uusaren na Mid2-iRFP pian ompaway ed ER. Pian naresolbe iyan posibilidad, kinuenta mi so ERES ratio parad sakey odino duaran produkto labat (Figure 2, A anggad C). Nalmoan mi ya maslak ed ERES (70%) et walaan labat na sakey a klase na kargamento. Say litrato ed leksab na Figure 2C et mangipapanengneng na duaran tipikal ya alimbawa na ERES ya walaan labat na Gas1-GFP (Figure 1) odino Mid2-iRFP labat (Figure 2). Diad pidumaan, ngalngalin 20% na ERES so walaan na duaran kargamento ya mansasalet ed parehon lugar. Nalmoan ya wala ray ERES (10%) ya walaan na duaran klase na kargamento, balet ta nibiig iratan ed malinew ya nanduruman lugar. Kanian, ipapanengneng na sayan statistical analysis ya kayari na pangipaway na ER, say GPI-AP Gas1-GFP tan say transmembrane cargo Mid2-iRFP et naapag ed nanduruman ERES (Figure 2D). Sayan inkaepektibo na panag-sort et mipara ed akaunan biochemical analysis (6) tan morphological determination (7). Naobserbaan tayo met so ugali na saray aka-quarantine ya kargamento ya onloloob ed ERES (Figure 2E tan Movie S2). Ipapanengneng na Figure 2E ya melag labat a kabiangan na Gas1-GFP (panel 3) odino Mid2-iRFP (panel 4) so ​​onloloob ed ERES manlapud sakey a diking tan akakulong ed sakey a nikadkaduman lugar. Ipapanengneng na Panel 5 na Figure 2E ya say Gas1-GFP tan Mid2-iRFP et no maminsan et naromog ed parehon ERES, balet ta onloloob iratan manlapud nanduruman dapag tan mankakasakey ira ed nanduruman rehyon ya nayarin mangirerepresenta ed nanduruman COPII vesicles. Akumpirma mi met ya say naobserbaan ya inkaapag tan inkaklasipika na C26 ceramide-based GPI-AP Gas1 bilang selective ERES et espisipiko lapud sananey nin transmembrane secretion cargo, say GFP-tagged plasma membrane protein Axl2 (27 ), ya mangipapanengneng na mipadparan ugali ed Mid2-iRFP. (Litrato S1 tan Pelikula S3). Say balon ginawa ya Axl2-GFP et nibubunog ed ER membrane a singa say Mid2-iRFP (Figure S1, A tan B), tan kaiba toy Mid2-iRFP ed maslak ya ERES (Figure S1, B anggad D). Say panel 1 tan 2 na Figure 1. Ipapanengneng na S1C so duaran tipikal ya alimbawa na ERES ya ditan et mansasalet so duaran transmembrane cargoes. Diad sarayan kaso, parehon onloob ed ERES so duaran produkto (Figure S1E, Panel 3 tan Movie S3).
Saray sec31-1 cells ya mangipapatnag na galactose inducible secretions, Gas1-GFP (GPI-AP, berde) tan Mid2-iRFP (TMP, asul) tan constitutive ERES labeling Sec13-mCherry (ERES, magenta) so inyan ed 37 Kayari na inkubasyon ed loob na 30 minuto ed °C 24, pian nibulos so ion pian nibulos so ion diad SCLIM kayari na 20 minutos. (A anggad C) Representative 2D projection images (A; scale bar, 1μm) odino 3D cell hemisphere images (B tan C; scale unit, 0.456μm) na karga tan 10 z-sections ya minarkaan na ERES. Say abeban panel ed (B) tan say panel ed (C) et mangipapanengneng na saray naproseso lan imahen pian nipanengneng labat iray produkto ya walad ERES (magenta) [Gas1-GFP (gray) tan Mid2-iRFP (light blue)]. (C) Open arrow: Say ERES et walaan labat na sakey a piraso na kargamento (1 anggad 4). Gray arrow: Say ERES et walaan na nibiig a kargamento (5). Amputin solidon pana: ERES ya walaan na kargamento ya nankakasakey. Diad leksab: Say pinilin saksakey ya ERES et walaan labat na Gas1-GFP (1) odino Mid2-iRFP (2). Scale bar, 100 nm. (D) Say karakel na photomicrograph ya adeskribe ed (C). Say average ya porsiento na ERES ya walaan labat na sakey a kargamento (Gas1-GFP o Mid2-iRFP), saray kargamenton nibiig tan saray mankakasakey a kargamento. Diad taloran independentin eksperimento, n=432 ed 54 a selula. Error bar = SD. Two-tailed unpaired t test. *** P = 0.0002. (E) 3D ya imahen na pinilin ERES na saray aka-quarantine ya kargamento ya walay marka ton (C). Say Gas1-GFP (berde) (3) odino Mid2-iRFP (asul) (4) et onloob ed ERES (magenta) manlapud sakey a diking tan limitado ed melag a lugar ed loob na ERES. No maminsan, parehon onloob so duaran klase na kargamento ed parehon ERES (5) manlapud parehon dapag tan mankukulong ira ed nisulinek a pasen ed loob na ERES. Scale bar, 100 nm.
Insan, sinubok mi so sakey a hypothesis ya say andukey ya acyl chain ceramide (C26) ya walad ER membrane so mamapaarap ed espisipikon panagtipon tan panag-sort na Gas1 ed selective ERES. Diad sayan gagala, inusar mi so inuman ya yeast strain GhLag1, ya say duaran endogenous ceramide synthases Lag1 tan Lac1 et sinalatan na GhLag1 (say Lag1 homolog na cotton), ya nanresulta ed yeast strain ya walay cell membrane Ceramide strain ya mas antikey nen say wild type (Figure 3A) (28). Say mass spectrometry (MS) analysis et angipatnag ya diad wild-type strains, 95% na dagup na ceramide et andukey a tuloy (C26) chain ceramide, bangbalet diad GhLag1, 85% na ceramide et andukey a tuloy (C18 tan C16). ), 2% labat na ceramide so andukey a tuloy (C26) a kadena na ceramide. Anggaman say C18 tan C16 ceramides so manunan ceramides ya anengneng ed GhLag1 membrane anggad natan, akumpirma met na MS analysis ya say GPI anchor na Gas1-GFP ya nipatnag ed GhLag1 strain et walaan na C26 ceramide, ya mipara ed wild-type lipids. Pareho so kalidad (Fig. 3A) (26). Kanian, kabaliksan na saya ya say ceramide remodeling enzyme Cwh43 et mapilin maong parad C26 ceramide, singa nanengneng ed Figure 26, mas labay ton ilaktip so GPI anchor manlapud daiset ya C26 ceramide ed GhLag1 strain. S2 (29). Anggaman ontan, say cell membrane na GhLag1 et walaan labat na C18-C16 ceramide, bangta say Gas1-GFP et walaan ni na C26 ceramide. Sayan katuaan so manggagawa ed sayan strain ya sakey ya sankaabigan ya kagawaan pian espisipikon nasolbar so problema na karukey na acyl chain na membrane ceramide ed ER. Say hypothetical ya papel na klase tan panag-sort. Insan, inaral mi nin unona so abilidad na C26 Gas1-GFP ya mantipon ed saray grupo ed GhLag1 ya walay temperature-sensitive mutant allele na sec31-1 panamegley na conventional fluorescence microscopy, ya say andukey (C18-C16) ya kadena labat so walad ER membrane Ceramide (Fig. 3). Naobserbaan mi ya diad sec31-1, maslak ed Gas1-GFP et akatipon ed saray grupo, bangta say Gas1-GFP ed sec31-1 GhLag1 ya walay andukey (C18-C16) ya andukey ya ceramide ER membrane et ag-akatipon tan nibunog ed interon ER membrane. Pian eksakto, lapud say C26 ceramide-based clustering et maapit a misiglaotan ed espisipikon ERES (Figure 1), inusisa mi no kasin sayan proseso et nayarin mangilaktip met ed kimey na ER export protein mechanism. Say GPI-AP et manguusar na espisyal a COPII system parad ER export, ya aktibon ireregulate na Ted1′s structural remodeling na glycan portion na GPI anchor (30, 31). Say recombinant GPI-glycan et nabidbir na transmembrane cargo receptor p24 complex, ya diad saginonor et manpipili na Lst1, ya sakey ya espisipikon isoform na manunan COPII cargo binding subunit Sec24, ya manporma na GPI-AP-rich COPII Vesicles are necessary (31-33). Kanian, nanggawa kami na doble ya mutant ya intipon so inkaandi na sarayan single proteins (say p24 complex component Emp24, GPI-glycan remodeling enzyme Ted1 tan say specific COPII subunit Lst1) ed sec31-1 mutant strain, tan inaral mi iratan. Naobserbaan mi ya diad sec31-1emp24Δ tan sec31-1ted1Δ, say Gas1-GFP et manunan ag-akagrupo tan nibunog ed interon ER membrane, a singa datin anengneng ed sec31-1 GhLag1, bangta diad sec31-1lst1Δ, say Gas1-GFP et singa sec31-1-11. Ipapanengneng na sarayan resulta ya nilikud ed kiwawala na C26 ceramide ed ER membrane, say pankakasakey na Gas1-GFP et kaukolan met ya man-bind ed p24 complex, tan agto kaukolan so espisipikon Lst1 recruitment. Insan, ineksplor mi so posibilidad ya say karukey na kadena na ceramide ed ER membrane et makapangiregula ed pangibiig na Gas1-GFP ed p24. Balet, aromog mi ya say kiwawala na C18-C16 ceramide ed membrane et ag makaapekto ed GPI-glycans ya ginawa lamet na p24 complex (Figures S3 tan S4, A tan B) odino say pangibiig ed GPI-AP tan pangipaway na GPI-AP. abilidad. I-recruit so COPII subtype Lst1 (Figure S4C). Kanian, say C26 ceramide-dependent clustering et agmankaukolan na pidedeneng na protina ed nanduruman mekanismo na ER export protein, balet susuportaan toy alternatibon mekanismo na panag-sort ya ipapaarap na karukey na lipid. Insan, inaral mi no kasin say karukey na ceramide acyl chain ed ER membrane et importante parad epektibon klasipikasyon na Gas1-GFP bilang selective ERES. Lapud say Gas1 ed GhLag1 strain ya walay short-chain ceramide to et ontaynan ed ER tan onloob ed plasma membrane (Figure S5), manisia kami ya no say panag-sort et igiya na karukey na ceramide acyl chain, say Gas1 ed GhLag1 strain et nayarin i-redirect tan i-cross. ERES ya produkto ya parehoy membrane to.
(A) Say cell membrane na GhLag1 et walaan na mas antikey iran C18-C16 ceramides, bangta say GPI anchor na Gas1-GFP et walaan ni na parehon C26 IPC a singa saray wild-type cells. Diad tagey: say acyl chain length analysis na ceramide ed cell membrane na wild-type (Wt) tan GhLag1p strains panamegley na mass spectrometry (MS). Say datos et mangirerepresenta ed porsiento na dagup na ceramide. Say average na taloran independentin eksperimento. Error bar = SD. Two-tailed unpaired t test. **** P ＜0.0001. Diad leksab a panel: MS analysis na acyl chain length na IPC ya walad Gas1-GFP (GPI-IPC) GPI anchor ya nipatnag ed wild-type tan GhLag1p strains. Say data et mangirerepresenta ed porsiento na dagup ya signal na IPC. Average na limaran independentin eksperimento. Error bar = SD. Two-tailed unpaired t test. ns, aliwan importante. P = 0.9134. (B) Saray fluorescence micrographs na sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ tan sec31-1lst1Δ ya selula ya mangipapatnag na galactose-induced Gas1-GFP et in-incubate ed 37°C ed loob na 30 minuto tan linmabas so fluoresce mikroskopyo kayari na 24°C. Amputin pana: ER Gas1-GFP cluster. Open arrow: Say ag-akatipon ya Gas1-GFP et nibunog ed interon ER membrane, ya mangipapanengneng na ER ya ugali na nuclear ring staining. Scale bar, 5μm. (C) Say karakel na photomicrograph ya adeskribe ed (B). Say average ya porsiento na saray selula ya walay punctate Gas1-GFP ya istruktura. Diad taloran independentin eksperimento, n≥300 ya selula. Error bar = SD. Two-tailed unpaired t test. **** P ＜0.0001.
Pian direktan nasolbar iyan problema, nanggawa kami na SCLIM visualization na Gas1-GFP tan Mid2-iRFP ed GhLag1 ya walay sec31-1 temperature-sensitive mutant allele (Figure 4 tan Movie S4). Kayari na impansiansia na ER ed 37°C insan nibulos ed 24°C, maslak ed saray balon ginawan Gas1-GFP et ag-akatipon tan ag-akabunog ed interon ER membrane, a singa naobserbaan na saray kombensional a mikroskopyo (Figure 4, A tan B ). Niarum ni, say baleg a porsiento na ERES (67%) et walaan na duaran klase na kargamento ya walad loob to (Figure 4D). Say panel 1 tan 2 na Figure 4C et mangipapanengneng na duaran tipikal ya alimbawa na ERES ya mansasalat so Gas1-GFP tan Mid2-GFP. Niarum ni, parehon saray produkto so inala ed parehon ERES (Figure 4E, panel 3 tan movie S4). Kanian, ipapanengneng na saray resulta mi ya say karukey na ceramide acyl chain ed ER membrane et importantin determinante na ER protein aggregation tan classification.
Sec31-1 GhLag1 cells ya mangipapaway na galactose-induced secretions, Gas1-GFP (GPI-AP, berde) tan Mid2-iRFP (TMP, asul) tan constitutive ERES-labeled Sec13-mCherry (ERES, magenta) I-incubate ed 37°C. Ituloy ed loob na 30 minutos, ipelag ed 24°C pian ompaway iray sekreto, tan i-imadyin panamegley na SCLIM kayari na 20 minutos. (A anggad C) Representative 2D projection images (A; scale bar, 1μm) odino 3D cell hemisphere images (B tan C; scale unit, 0.45μm) na 10 z-sections ya minarkaan na kargamento tan ERES. Say abeban panel ed (B) tan say panel ed (C) et mangipapanengneng na saray naproseso lan imahen pian nipanengneng labat iray produkto ya walad ERES (magenta) [Gas1-GFP (gray) tan Mid2-iRFP (light blue)]. (C) Amputin napno na pana: ERES, saray produkto so mansasagpot. Bukas ya pana: Say ERES et walaan labat na sakey ya aytem. Diad leksab a panel: Say pinilin ERES et walaan na saray mankakasakey a produkto (1 tan 2) ya akamarka ed (C). Scale bar, 100 nm. (D) Say karakel na photomicrograph ya adeskribe ed (C). Diad sec31-1 tan sec31-1 GhLag1 units, saksakey labat so kargamento (Gas1-GFP o Mid2-iRFP) ya inlaktip, tan say average ya porsiento na ERES parad nisulinek ya kargamento tan mansasalet ya kargamento. Diad taloran independentin eksperimento, n = 432 ed 54 a selula (sec31-1) tan n = 430 ed 47 a selula (sec31-1 GhLag1). Error bar = SD. Two-tailed unpaired t test. *** P = 0.0002 (sec31-1) tan ** P = 0.0031 (sec31-1 GhLag1). (E) 3D ya imahen na pinilin ERES ya walay manpapaspas ya kargamento (3) ya akamarka ed (C). Say Gas1-GFP (berde) tan Mid2-iRFP (asul) et onaasingger ed ERES (magenta) manlapud parehon diking tan mansiansia ed parehon ERES restricted area. Scale bar, 100 nm.
Sayan impanaral so mangiiter na direktan in vivo ya ebidensya ya saray lipid-based protein cargoes et niklasipika ed selective export sites diad secretory pathway, tan ipaparungtal to so kaimportantian na acyl chain length parad classification selectivity. Diad pangusar na mabiskeg tan sankabaloan a teknik na mikroskopyo ya tatawagen ya SCLIM, impanengneng mi so balon ginawan Gas1-GFP (sakey a manunan plasma membrane GPI-AP ya walaan na andukey a acyl chain (C26) ceramide lipid portion) ed lebadura) Saray rehyon ya akatipon ed nanduruman ERs et misiglaotan ed espisipikon ERES bangta saray protina ya transmembrane et nibubunog ed membrane ERRES (Figure 1). Niarum ni, sarayan duaran klase na produkto et onloob ed nanduruman ERES ya mapili (Figure 2). Say acyl chain length na cellular ceramide ed membrane et nabawasan manlapud C26 ya anggad C18-C16, say Gas1-GFP cluster et naderal ed discrete ER region, tan say Gas1-GFP et na-reroute pian taynanan so ER a kaiba so transmembrane protein diad parehon ERES (Figure 3 tan Figure 3). 4).
Anggaman say GPI-AP et manguusar na espisyal ya mekanismo na protina pian makapaway ed ER, aromog mi ya say C26 ceramide-dependent separation et agmandedependi ed saray nanduruman protina ya nayarin manresulta ed ERES specialization (Figures S4 tan S5). Imbes, saray adiskobre mi et manusuporta ed alternatibon mekanismo na panagklasipika ya ipapaarap na lipid-based protein clustering tan say ontumbok ya pangipulisay ed arum nin kargamento. Ipapanengneng na saray naobserbaan mi ya say Gas1-GFP ya rehyon odino cluster ya misiglaotan ed sakey ya espisipikon ERES et anggapoy transmembrane secreted protein ya Mid2-iRFP, ya mangipapanengneng ya say C26 ceramide-dependent GPI-AP cluster so mamagaan ed iloob da ed matukoy ya ERES, tan diad saman met a panaon, et ag to ilaktip so transmembrane. Diad pidumaan, say kiwawala na C18-C16 ceramides ed ER membrane et agto pansengegan na GPI-AP ya manporma na saray rehyon odino grupo, kanian ag da ipulisay odino isalet iray transmembrane secreted proteins ed parehon ERES (Figures 3 tan 4). . Kanian, iproproponin mi ya say C26 ceramide so mangitonton ed panagsian tan panagklasipika diad panamegley na pangipasimpli ed panagtipon na saray protina ya nisiglaot ed espisipikon ERES.
Panon ya nagawaan iyan C26 ceramide-dependent clustering ed sakey ya espisipikon ER area? Say tendensia na membrane ceramide ya mansian ed gilig et nayarin pansengegan na GPI-AP tan C26 ceramide ya manporma na melag tan tampol ya akauksoy iran lipid diad mas ag-regular ya lipid ya kaliberliber na ER membrane ya walaan na mas antikey tan ag-inka-saturated ya glycerolipids. Saray kalidad na grupo (17, 18). Sarayan melag a temporaryon grupo et nayarin lalon mankasakey ed angkakabaleg tan mas malet iran grupo kayari na pan-bind ed p24 complex (34). Mitunosan ed saya, impanengneng mi ya say C26 Gas1-GFP et kaukolan ya mitongtong ed p24 complex pian makaporma na mas angkakabaleg iran nanengneng ya grupo (Figure 3). Say p24 complex et sakey ya heterozygous oligomer ya tugyopen na apatiran nanduruman p24 transmembrane proteins ed lebadura (35), ya mangiiter na multivalent binding, ya nayarin manresulta ed cross-linking na melag iran GPI-AP clusters, kanian makapawala na mas baleg ya Stable cluster (34). Say pidedeneng na saray protina ya ectodomain na GPI-APs et nayarin makatulong met ed pankakasakey da, a singa nanengneng legan na Golgi transport da ed saray mammalian polarized epithelial cells (36). Balet, no say C18-C16 ceramide et walad ER membrane, no say p24 complex et onsisiglaot ed Gas1-GFP, ag naporma iray angkakabaleg ya nanduruman grupo. Say manunan mekanismo et nayarin mandedepende ed espisipikon pisikal tan kemikal iran kablian na andukey ya acyl chain ceramide. Ipapanengneng na saray biophysical ya impanaral ed artipisyal iran membrane ya anggaman parehon andukey (C24) tan antikey (C18-C16) ya acyl chain ceramides et nayarin pansengegan na phase separation, saray andukey ya acyl chain ceramides (C24) labat so makapangiter na atagey ya Curvature tan film bending pian nauman so itsura na pelikula. Diad panamegley na pankakasakey (17, 37, 38). Napatnagan ya say transmembrane helix na TMED2, say human homologue na Emp24, et mapilin mibabali ed C18 ceramide-based sphingomyelin diad cytoplasmic lobules (39). Diad pangusar na molecular dynamics (MD) simulations, aromog mi ya parehon mantitipon iray C18 tan C26 ceramides ed kaliberliber na cytoplasmic lobules na Emp24 transmembrane helix, tan pareho so labay da (Figure S6). Nepeg ya imanoen ya saya so mangipapanengneng ya say transmembrane helix na Emp24 et nayarin manresulta ed ag-inkayadyari na lipids ed membrane. Saya so agano nin resulta base ed saray selula na mammal. Saray mipadparan MD simulation et mangipapanengneng met na kiwawala na ether lipids (40) . Kanian, iisipen mi ya say C26 ceramide ed duaran lobule na ER26 et lokal ya pinayaman. No say GPI-AP ed luminal lobules et direktan onsisiglaot ed multivalent p24 tan say idakel na C26 ceramide ed kaliberliber na p24 diad cytoplasmic lobules, nayarin napaaligwas so kaiba ton pantipon na protina tan say kurba na membrane et napapawala panamegley na saray gamet (41), ya pansengegan na pansian na GPI-AP ed nanduruman rehyon ya asingger ed ERES, ya pabor met ed atagey a kurba na rehyon say ER membrane (42). Saray akaunan report so manuporta ed iproproponin mekanismo (43, 44). Say multivalent binding na oligolectins, pathogens o antibodies ed ceramide-based glycosphingolipids (GSL) ed plasma membrane et pansengegan na baleg ya GSL aggregation, mamaaligwas na phase separation tan pansengegan na deformation tan internalization na membrane (44). Iwabuchi etc. (43) Nalmoan ya diad kiwawala na andukey (C24) balet aliwan antikey (C16) ya acyl chains, say multivalent ligand ya akapeket ed GSL lactosylceramide so sengegan na inkaporma na angkakabaleg iran clusters tan membrane invagination, tan say cytoplasm Lyn-mediated signal transduction ed saray leaflets et ipapasen na saray neutrophil chains ya mankakasakey.
Diad saray mammalian polarized epithelial cells, say konsentrasyon na anti-Golgi network (TGN) anggad lebel na apical plasma membrane so mangokontrol ed inkaapag tan inkaapag-apag na GPI-AP (10, 45). Sayan pankakasakey et ipapaarap na GPI-AP oligomerization (36), balet nayarin mandedepende met itan ed karukey na kadena na ceramide ya naromog tayo ed lebadura. Anggaman say GPI-AP na mammalian et walaan na ether lipid-based anchor, tan say kemikal ya istruktura to et midumaan a tuloy ed andukey a tuloy ya acyl chain ceramide, diad sakey ya agano nin impanaral et adiskobre ya parehon walaan na parehon pisikal tan kemikal iran kablian tan kimey so ebolusyon (40). Kanian, say ether lipid part ed saray selula na mammal et nayarin mipara ed C26 ceramide ed lebadura, tan say papel to et mikonektaan ed long-chain ceramide ed membrane pian napaaligwas so GPI-AP aggregation tan sorting. Anggaman kaukolan nin direktan suboken iyan posibilidad, susuportaan na saray akaunan adiskobre ya say pangiyakar ed andukey ya acyl chain ceramide ed laman na Golgi et ag-agawaan na saray cytoplasmic transfer proteins, noagta mandedependi ed panaggawa na GPI anchors a singa say lebadura. Kanian, say ebolusyonaryon konserbatibon mekanismo et singa sarag ton pilien so man-co-transport na andukey ya acyl chain ceramide tan GPI-AP (13, 16, 20, 46, 47) diad parehon transport vesicle.
Diad yeast tan mammalian polarized epithelial cell systems, say GPI-AP aggregation tan separation manlapud arum ya plasma membrane proteins et nagagawa antis ya onsabi ed tapew na selula. Paladino et al. (48) aromog da ya diad TGN na mammalian polarized epithelial cells, say GPI-AP clustering et aglabat nakaukolan parad selective classification na GPI-APs ed apical plasma membrane, noagta manreregulate met ed clustering organization na GPI-APs tan say biological activity to. Say tapew na selula. Diad lebadura, impanengneng na sayan impanaral ya say C26 ceramide-dependent GPI-AP cluster ed ER et makapangiregula ed organisasyon na cluster tan say aktibidad na GPI-AP ed plasma membrane (24, 49). Mitunosan ed sayan modelo, saray GhLag1 cells et alerdyik ed GPI inhibitors odino saray tambal ya mangaapekta ed integridad na cell wall (28), tan say pankaukolan parad functional Gas1-GFP clusters (49) na tip ceramide ya iproproyekto ed pan-mating na saray selula na lebadura so mangipapanengneng na G Possible physiological consequences na hLag1 cells. GPI-AP error. Balet, say kaaruman a panagsukimat no kasin say functional ya organisasyon na tapew na selula et naprograma manlapud ER panamegley na paraan na panag-sort base ed karukey na lipid so tema na arapen ya panagsukimat tayo.
Saray strain na Saccharomyces cerevisiae ya inusar ed sayan kimey et akalista ed Table S1. Say MMY1583 tan MMY1635 ya strain na SCLIM parad live cell imaging et ginawa ed benegan na W303. Sarayan strain ya mangipapatnag na Sec13-mCherry ya walay fluorescent protein tag et ginawa ya inusar so polymerase chain reaction (PCR)-based a paraan ya walay pFA6a plasmid bilang template (23). Say strain ya mangipapatnag na Mid2-iRFP ya walay marka ton fluorescent protein ya kontrolado na GAL1 promoter et ginawa unong ed onggendan. PCR amplification na iRFP-KanMx sequence manlapud pKTiRFP-KAN vector (regalo nen E. O'Shea, Addgene plasmid number 64687; http://n2t.net/addgene: 64687; research resource identifier (RRID): Addgene_64687) Tan inloob ed endogenous ya C.2. Kayari na impan-amplify tan in-clone na Mid2-iRFP genome sequence ed GAL1 promoter, in-integrate itan ed Not I-Sac I site na integration plasmid pRS306. Say resulta na plasmid pRGS7 et inlinearize ed Pst I pian makaloob ed URA3 locus.
Say Gas1-GFP fusion gene et nipatnag ed silong na kontrol na GAL1 promoter diad centromere (CEN) plasmid, ya ginawa unong ed onggendan. Say Gas1-GFP sequence et pinalaknab na PCR manlapud pRS416-GAS1-GFP plasmid (24) (regalo nen L. Popolo) tan in-clone ed Xma I-Xho I site na CEN plasmid pBEVY-GL LEU2 (regalo nen C). Miller; Addgene plasmid number 51225; http://n2t.net/addgene: 51225; RRID: Addgene_51225). Say resulta na plasmid et tinawag ya pRGS6. Say Axl2-GFP fusion gene et nipatnag met ed silong na kontrol na GAL1 promoter na pBEVY-GL LEU2 vector, tan say inkagawa to et singa onggendan. Say Axl2-GFP sequence et pinalaknab manlapud pRS304-p2HSE-Axl2-GFP plasmid (23) panamegley na PCR, tan in-clone ed Bam HI-Pst I site na pBEVY-GL LEU2 vector. Say resulta na plasmid et inngaran ya pRGS12. Say nantutumbokan ya oligonucleotides ya inusar ed sayan impanaral et akalista ed Table S2.
Say strain et indagdag na 0.2% adenine tan 2% glucose [YP-dextrose (YPD)], 2% raffinose [YP-raffinose] ya mayaman ed yeast extract protein p (YP) medium (1 % Yeast extract tan 2% protein ept). (YPR)] odino 2% galactose [YP-galactose (YPG)] bilang lapuan na carbon, odino diad sintetikon minimal a medium (0.15% yeast nitrogen base tan 0.5% ammonium sulfate) pian nadagdag iray matukoy ya amino acid tan base ya nakaukolan parad nutrisyon, Tan walaan na 2% glucose glucose (2% glucose medium) odino minimal galactose (synthetic galactose minimal medium) bilang lapuan na carbon.
Parad real-time imaging, saray sec31-1 mutant cells ya sensitibo ed temperatura ya mangipapatnag na construct ed silong na GAL1 promoter et pinabaleg ed YPR medium ed 24°C ed sanlabi anggad mid-log phase. Kayari na induction ed YPG ed 24°C ed loob na 1 oras, saray selula so in-incubate ed SG ed 37°C ed loob na 30 minutos, insan inyalis ed 24°C pian nibulos ed secretion block. Say Concanavalin A so inusar pian naayos iray selula ed sakey a glass slide tan in-image na SCLIM. Say SCLIM et kombinasyon na Olympus IX-71 inverted fluorescence microscope tan UPlanSApo 100×1.4 numerical aperture oil lens (Olympus), high-speed tan high-signal-to-noise ratio rotating disc confocal scanner (Yokogawa Electric), custom spectrometer, intensive systems tan saray imahen na sistema Say (Hamamatsu Photonics) et makapangiter na magnifying lens system ya walay pinal ya magnification ya ×266.7 tan sakey ya charge-coupled device camera ya mamaparakel na electrons (Hamamatsu Photonics) (21). Say pakagamor na litrato et gagawaen na custom software (Yokogawa Electric). Parad 3D ya imahen, angusar kami na custom-made piezoelectric actuator pian na-vibrate so objective lens ya patayo, tan tinipon mi ray optical parts ya 100 nm so kaarawi ra ed sakey a stack. Say Z-stack ya imahen et nauman ed 3D voxel data, tan say theoretical point spread function ya inusar parad rotating disc confocal microscope et inusar parad deconvolution processing na Volocity software (PerkinElmer). Diad impangusar na Volocity software pian automatikon nawalaan na threshold parad co-location analysis, say ERES kaiba lay kargamento so nasukat. Say line scan analysis et ginawa ya inusar so MetaMorph software (Molecular Devices).
Usaren so GraphPad Prism software pian naamtaan so kabaliksan na estadistika. Parad two-tailed Student's t-test tan say ordinaryon one-way analysis of variance (ANOVA) test, saray pandumaan na grupo et ipapasen ya walay baleg ya epekto to ed P <0.05 (*).
Parad fluorescence microscopy na Gas1-GFP, saray log phase cells so pinabaleg ed sanlabi ed YPD tan inala panamegley na centrifugation, inurasan na mamidua na phosphate buffered saline, tan in-incubate ed yelo ed loob na ag-onkulang ed 15 minutos, insan intuloy ed silong na mikroskopyo unong ed akauna lan indeskribe Check (24). Say Leica DMi8 microscope (HCX PL APO 1003/1.40 oil PH3 CS) ya walay objective lens, L5 (GFP) filter, Hamamatsu camera tan Application Suite X (LAS X) software so inusar parad panag-ala. .
Saray sample et in-denatured panamegley na SDS sample buffer ed 65°C ed loob na 10 minutos, insan insian panamegley na SDS-polyacrylamide gel electrophoresis (PAGE). Parad immunoblotting analysis, 10 μl na sample so inkarga ed kada lane. Manunan antibody: Usaren so rabbit polyclonal anti-Gas1 ed dilution ya 1:3000, rabbit polyclonal anti-Emp24 ed dilution ya 1:500, tan rabbit polyclonal anti-GFP (regalo nen H. Riezman) ed dilution ya 1:3000. Say mouse monoclonal anti-Pgk1 antibody et inusar ed dilution ya 1:5000 (regalo nen J. de la Cruz). Segundaryon antibody: Horseradish peroxidase (HRP) conjugated goat anti-rabbit immunoglobulin G (IgG) ya inusar ed dilution ya 1:3000 (Pierce). Say HRP-conjugated goat anti-mouse IgG et inusar ed dilution ya 1:3000 (Pierce). Say immune response zone et naobserbaan panamegley na chemiluminescence method na SuperSignal West Pico reagent (Thermo Fisher Scientific).
Unong ed adeskribe ed (31), sakey a natural ya eksperimento ed immunoprecipitation so ginawa ed pinayaman ya ER fraction. Diad antikey, urasan iray selula na lebadura na TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride tan protease inhibitor mixture) ed 600 nm (OD600) ed 100 optical dens ya mamidua. Pinuter itan panamegley na saray glass beads, insan inekal iray debris na selula tan saray glass beads panamegley na centrifugation. Say supernatant et in-centrifuge ed 17,000 g ed loob na 15 minutos ed 4°C. Say pellet et insuspinde lamet ed TNE tan say digitalis saponin et inyarum ed sampot ya konsentrasyon ya 1%. Say suspension et in-incubate ed loob na 1 oras ya in-rotate ed 4°C, insan saray ag-ontutunaw ya kabiangan et inekal panamegley na centrifugation ed 13,000 g ed 4°C ed loob na 60 minutos. Parad Gas1-GFP immunoprecipitation, unaan ya i-pre-incubate so sample ya walay empty agarose beads (ChromoTek) ed 4°C ed loob na 1 oras, insan i-incubate ed GFP-Trap_A (ChromoTek) ed 4°C ed loob na 3 oras. Saray immunoprecipitated beads et inurasan na maminlima na TNE ya walay 0.2% digoxigenin, in-elute na SDS sample buffer, insian ed SDS-PAGE, tan in-analisa panamegley na immunoblotting.
Unong ed adeskribe ed (31), say cross-linking determination et ginawa ed pinayaman ya ER fraction. Diad antikey, say pinayaman ya ER fraction et in-incubate ed 0.5 mM dithiobis(succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, USA; 20°C, 20 min). Say crosslinking reaction et naerep diad pangiyarum na glycine (50 mM ya sampot ya konsentrasyon, 5 minutos, 20°C).
Unong ed akaunan adeskribe (50), ginawa so MS analysis na ceramide ed wild-type tan GhLag1 strains. Diad antikey, saray selula so pinabaleg ed exponential phase (3 anggad 4 OD600 units/ml) ed YPD ed 30°C, tan 25×107 a selula so inani. Say metabolismo ra et naekal na trichloroacetic acid. Mangusar na extraction solvent [ethanol, danum, ether, pyridine tan 4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] tan 1.2 nmol na internal standard C17 ceramide (860517, Avanti polar lipid) ya kalidad). Mangusar na monomethylamine reagent [methanol, danum, n-butanol tan methylamine solution (4:3:1:5 v/v)] pian nagawaan so mild alkaline hydrolysis na extract, insan usaren so water-saturated n-butanol pian naekal so asin. Diad kaunoran, say extract et insuspinde lamet ed positibon paraan na solvent [chloroform/methanol/danum (2:7:1) + 5 mM ammonium acetate] tan inyan ed mass spectrometer. Say multi-reaction monitoring (MRM) so ginawa parad pakabidbiran tan pakabilang na saray molekula na sphingolipid. Say TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) et walaan na robotic nanoflow ion source Nanomate HD (Advion Biosciences, Ithaca, NY) parad lipid analysis. Say enerhiya na panbabanggaan et na-optimize parad kada kategorya na ceramide. Say datos na MS et naala ed positibon paraan. Parad kada biological replicate, say lipid signal et say median na taloran independentin sukat.
Unong ed adeskribe ed (31), saray selula (800×107) ya mangipapaway na Gas1-GFP et inpaarap ed natural ya immunoprecipitation. Say pinalinis ya Gas1-GFP et insian panamegley na SDS-PAGE tan inyalis ed polyvinylidene fluoride (PVDF) ya membrane. Say protina et anengneng diad pangi-stain ed PVDF na amide black. Say Gas1-GFP band et pinuter manlapud PVDF tan inurasan na 5 beses na methanol tan maminsan ya inurasan na liquid chromatography-MS (LC-MS) grade water. Diad pangi-incubate ed membrane strip ya walay 500μl 0.3 M NaOAc (pH 4.0), buffer tan 500μl ya balon tinunaw ya 1 M sodium nitrite mixture ed 37°C ed loob na 3 oras, say lipid fraction et nibulos manlapud Gas1-GFP tan na-lysed Release of ininphoosine phoosphamine ed baetan na ceramiosfaodeit tan ceramiosdeit (51). Kayari to, say membrane strip et inurasan na apatiran beses na LC-MS grade water, pinamaga ed temperatura na kuarto, tan insimpen ed nitrogen atmosphere ed -80°C anggad naanalisa. Bilang kontrol, sakey a blangko ya sample na PVDF membrane so inusar parad kada eksperimento. Say lipid ya inala ed Gas1-GFP et inaral na MS unong ed nideskribe (50). Diad antikey, saray PVDF strips ya walay GPI-lipid to et insuspinde lamet ed 75μl ya negatibon mold solvent [chloroform/methanol (1:2) + 5 mM ammonium acetate] tan inpasa so electrospray ionization (ESI)-MRM/MS Analysis of sphingolipid species (TSQ Vantage). Diad sayan kaso, say datos na MS et naala ed negatibon ion mode.
Unong ya asalambit la, say lipid portion na GPI anchor et insian ed [3H]-inositol-labeled GPI-AP (16). Saray lipid et insian panamegley na thin-layer chromatography ya inusar so solvent system (55:45:10 chloroform-methanol-0.25% KCl) tan in-visualize ya inusar so FLA-7000 (Fujifilm).
Saray selula ya walay Gas1-GFP (600×107) et amiduan inurasan na TNE buffer ya walay TNE buffer, tan pinuter na glass beads, insan in-centrifuge pian naekal so cell debris tan glass beads. Say supernatant et in-centrifuge ed 17,000 g ed loob na 1 oras ed 4°C. Say pellet et inurasan ed TNE tan in-incubate ed 1 U PI-PLC (Invitrogen) ed TNE ya walay 0.2% digitalis saponin ed loob na 1 oras ed 37°C. Kayari na enzyme treatment, say membrane et inekal panamegley na centrifugation ed 17,000 g ed 4°C ed loob na 1 oras. Pian na-immunoprecipitate so Gas1-GFP, say supernatant et in-incubate ed GFP-Trap_A (ChromoTek) ed 4°C ed sanlabi. Say pinalinis ya Gas1-GFP ya insian na SDS-PAGE et inkoloran na Coomassie brilliant blue. Say Gas1-GFP staining band et pinuter ed gray ya akapaliber ed aqueduct, insan kayari na alkylation ya walay iodoacetamide tan reduction ya walay dithiothreitol, ginawa so in-gel digestion ya walay trypsin. I-extract tan i-dry iray tryptic peptides tan peptides ya walay GPI-glycans. Say pinamaga ya peptide et tinunaw ed 20 μl na danum. I-inject so sakey a porsyon (8μl) ed LC. Say octadecylsilane (ODS) column (Develosil 300ODS-HG-5; inner diameter 150 mm×1.0 mm; Nomura Chemical, Aichi Prefecture, Japan) so inusar pian ibiig iray peptide diad silong na espisipikon kondisyon na gradient. Say mobile phase et say solvent A (0.08% formic acid) tan solvent B (0.15% formic acid ed 80% acetonitrile). Say Accela HPLC system (Thermo Fisher Scientific, Boston, Massachusetts) so inusar pian na-elute so column ya walay solvent A ed loob na 55 minutos diad flow rate ya 50 μl min-1 ed loob na 5 minutos, insan say konsentrasyon na solvent B et inmatagey ed 40%. , Estados Unidos). Say eluate et tuloytuloy ya inloob ed ESI ion source, tan saray tryptic peptides tan peptides ya walay GPI-glycans et inanalisa panamegley na LTQ Orbitrap XL (hybrid linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific). Diad MS setup, say boltahe na capillary source et inset ed 4.5 kV, tan say temperatura na transfer capillary et nansiansia ed 300°C. Say boltahe na capillary tan boltahe na tube lens et inset ed 15 V tan 50 V, unong ed inkauksoy da. Say MS data et naala ed positibon ion mode (resolusyon ya 60,000; mass accuracy ya 10 parts kada milyon) diad mass range ya 300/m/z mass/charge ratio (m/z) 3000. Say MS/MS data et naala panamegley na ion trap ed LTQ Orbit [XL XL) ya unonan mandedependi ed data collision induced dissociation (CID)].
Saray MD simulation et ginawa ya inusar so GROMACS (52) software tan MARTINI 2 force field (53-55). Say CHARMM GUI Membrane Builder (56, 57) so inusar pian manggawa na bilayer ya walay dioleoylphosphatidylcholine (DOPC) tan Cer C18 odino DOPC tan Cer C26. Say topology tan coordinates na Cer C26 et nanlapud DXCE diad pangekal ed saray ekstran beads ed ikol na sphingosine. Usaren so proseso ya adeskribe ed leksab pian nabalanse so doble a layer tan paandaren itan, insan usaren so unor iran coordinate na sistema pian makapanggawa na sistema ya walaan na Emp24. Say transmembrane domain na yeast Emp24 (residues 173 anggad 193) et ginawa bilang α-helix ya inusar so visual MD (VMD) tool molecular structure (58). Insan, kayari na pangekal ed saray manpapaspas ya lipid, say protina et magaspang ya granulate tan inloob ed bilayer ya inusar so CHARMM GUI. Say unor a sistema et walaan na 1202 DOPC tan 302 Cer C26 odino 1197 DOPC tan 295 Cer C18 tan Emp24. Ionize so sistema ed konsentrasyon ya 0.150M. Apatiran independentin replika so ginawa parad duaran bilayer a komposisyon.
Say lipid bilayer et nabalanse ya uusaren so proseso na CHARMM GUI, ya mangilalanor na pambawas insan pambalanse na 405,000 a kundang, ya ditan et kalkalnan nabawasan tan naekal iray limitasyon ed posisyon, tan say time step et ondarakel manlapud 0.005 ps ya anggad 0.02 ps. Kayari na equilibration, makapawala itan na 6 μs ya walay time step ya 0.02 ps. Kayari na pangiloob ed Emp24, usaren so parehon proseso na CHARMM GUI pian nabawasan tan nabalanse so sistema, insan manpaandar na 8 s ed produksyon.
Parad amin ya sistema, legan na proseso na panagbalanse, say pressure et kokontrolen na Berendsen barostat (59), tan legan na proseso na produksyon, say pressure et kokontrolen na Parrinello-Rahman barostat (60). Diad amin a kaso, say average ya presyon et 1 bar tan say semi-isotropic pressure coupling scheme so uusaren. Diad proseso na panagbalanse tan produksyon, sakey a thermostat (61) ya walay speed recalibration so uusaren pian na-couple so temperatura na protina, lipid tan solvent particles. Legan na interon operasyon, say target ya temperatura et 310K. Say non-bonding interaction et nakalkula diad panggawa na pairing list ya uusaren so Verlet scheme ya walay 0.005 buffer tolerance. Say Coulomb term et nakalkula ya uusaren so reaction field tan say cut-off distance ya 1.1 nm. Say terminon Vander Waals et manguusar na cut-off scheme ya walay cut-off distance ya 1.1 nm, tan say Verlet cut-off scheme et uusaren parad posiblin drift (62).
Diad pangusar na VMD, say cutoff wavelength ed baetan na DOPC phosphate beads odino ceramide AM1 beads tan say protina et 0.7 nm, tan say bilang na lipids ya mibabali ed protina so nakalkula. Unong ed onggendan a pormula, kalkulaen so depletion-enrichment (DE) factor a singa ed (63): DE factor = (say karakel na amin a lipid ed protina 0.7) ed protina 0.7 (say karakel na Cer ed amin a lipid)
Say nireport ya balor et naala bilang average, tan saray error bar et apatiran independentin kopya na SE. Say statistical significance na DE factor et nakalkula panamegley na t test [(averageDE-factor-1)/SE]. Kalkulaen so P value manlapud one-tailed distribution.
Say GROMACS tool so inusar pian nakalkula so 2D lateral density map na sistema ya walaan na Emp24 ed loob na unor a 250 ns na trace. Pian naala so enrichment/depletion map na ceramide, say density map na Cer et naapag ed dagup na mapa na Cer tan DOPC, insan naapag ed konsentrasyon na Cer ed laman. Parehon kolor na mapa so inusar.
Parad kaaruman iran materyales parad sayan artikulo, nengnengen so http://advances.sciencemag.org/cgi/content/full/6/50/eaba8237/DC1
Saya so open access ya artikulo ya ibubunog unong ed saray kondisyon na Creative Commons Attribution-Non-Commercial License, ya mangiiter na pankanawnawan mangusar, mangibunog tan manpawala lamet ed antokaman a medium, basta say unor ya pangusar et aliwan parad komersyal a gunggona tan say premis et duga so orihinal ya kimey. Reperensya.
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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), Wagazhope Lopez (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Say 3D high-resolution real-time imaging so mangipapatnag na kaimportantian na ceramide chain length parad panag-sort na protina ed saray pinilin output sites.
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), Wagazhope Lopez (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Say 3D high-resolution real-time imaging so mangipapatnag na kaimportantian na ceramide chain length parad panag-sort na protina ed saray pinilin output sites.
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