Secretory pathway-a protein sorting hi cell compartmentalization leh homeostasis vawng reng turin a pawimawh hle. Shell-mediated sorting bakah hian secretory transport kalpui danah kinesin sorting-a lipids te chanvo hi hun rei tak atanga lo awm tawh, chhanna la awm lo a ni. Hetah hian 3D simultaneous multicolor high-resolution real-time imaging kan ti a, in vivo-ah kan finfiah a, glycosylphosphatidylinositol-immobilized protein siam thar, ceramide lipid moieties sei tak tak nei te chu clustered leh classified in specialized endoplasms Net exit site, transmembrane proteins te hman dan nen a danglam a ni. Chu bakah, endoplasmic reticulum membrane-a ceramide chain sei zawng hi he sorting selectivity atan hian a pawimawh hle tih kan lantir bawk. Kan zirchianna hian lipid chain sei zawng atanga protein cargo te chu secretory pathway-a selective export site-a classified theihna tur direct in vivo evidence hmasa ber a pe a ni.
Eukaryotic cell-ah chuan endoplasmic reticulum (ER)-a protein siamte chu an cellular destination dik takah an thlen theih nan secretory pathway kaltlangin an kal laiin an thliar hrang a ni (1). Coat-mediated sorting bakah hian lipid thenkhat chu protein bikte membrane domain bik-ah cluster-in selective exit point atan pawh an thawk thei tih hi hun rei tak chhung ngaihtuah a ni tawh (2-5). Mahse, he lipid-based mechanism awm thei hi finfiah turin direct in vivo evidence a la awm lo. He harsatna bulpui chinfel nan hian yeast-ah glycosylphosphatidylinositol (GPI) anchored proteins (GPI-APs) te chu ER atanga differentially export dan kan zirchiang a. GPI-APs hi lipid nena inzawm cell surface protein chi hrang hrang an ni ( 6, 7). GPI-AP hi secreted protein a ni a, plasma membrane pawn lam leaflet-ah glycolipid moiety (GPI anchor) hmangin a inzawm a ni. GPI anchor te hi ER lumen-a conservative post-translational modification angin an pawm (8). Attachment hnuah GPI-AP chu Golgi apparatus (5, 9) kaltlangin ER atanga plasma membrane-ah a kal a. GPI anchor awmna hian GPI-AP chu transmembrane secreted proteins (plasma membrane protein dang pawh tel) atanga secretory pathway-ah a hranin a phur chhuak thin (5, 9, 10). Yeast cell-ah chuan GPI-APs te hi endoplasmic reticulum-a secreted protein dang atang hian an inthen a, chutah chuan coat protein complex II (COPII) hmanga khuh vesicles danglam takah an package a ni (6, 7). ER export process-a he classification process tichiangtute hi a chiang lo hle a, mahse he mechanism hian lipids a mamawh mai thei niin an sawi a, a bik takin GPI anchor-a lipid portion structural remodeling (5, 8). Yeast-ah chuan GPI lipid remodeling hi GPI a inzawm hnuah a intan nghal a, a tam zawkah chuan ceramide chu 26-carbon long-chain saturated fatty acid (C26:0) nen a inzawm tir thin (11, 12). Tun thlenga yeast cell-in ceramide an siam chhuah ber chu C26 ceramide a ni. ER-ah siam a ni a, a tam zawk chu COPII vesicles hmangin Golgi apparatus-ah thawn chhuah a ni (13). GPI-AP ER export atan hian a bik takin ceramide siam chhuah chhunzawm zel a ngai a ( 14 , 15 ), a lehlamah chuan Golgi apparatus-a ceramide chu inositol phosphate ceramide (IPC)-a chantirna chu GPI anchor synthesis-ah a innghat a ni (16). Artificial membrane hmanga biophysical study-ah chuan acyl chain sei tak tak ceramide te chu an inzawm khawm a, physical property danglam tak nei ordered domain an siam thei tih hmuhchhuah a ni (17, 18). Heng data te hian C26 ceramide leh C26 ceramide nei GPI-AP te hian an physical property hmangin ER membrane lipid environment buaithlak takah hian orderly region emaw region emaw ah an inzawm khawm thin tih hypothesis a siam a ni. Glycerolipids tawi leh unsaturated (C16:1 leh C18:1) atanga siam a ni ber (19, 20). Heng region te hi specific ER exit sites (ERES)-ah thlan bikin an dah ang a, chutah chuan ceramide leh ceramide-based GPI-AP te chu Golgi-ah co-transported a ni ang a, chu chu dedicated COPII vesicle khatah (5).
He zirchiannaah hian he lipid-based mechanism hi super-resolution confocal real-time imaging microscopy (SCLIM) hmangin direct-in kan test a, hei hi cutting-edge microscopy technique a ni a, fluorescently labeled proteins te chu a rualin a enfiah thei a ni Three-color leh three-dimensional (3D) images hian cell nungah resolution leh speed sang tak a nei a (21, 22).
S. cerevisiae-a ER chhuahsan hnua transmembrane secreted proteins atanga C26 ceramide group nei GPI-AP pangngai screen dan tur sawifiah belh turin SCLIM technology kan hmang hmasa a. ER classification enfiah nan hian genetic system kan hmang a, chu chuan in vivo-a ERES-a lut tur cargo siam tharte chu direct-in a hmu thei a ni (7, 23). Cargo atan C26 ceramide-based GPI-AP Gas1 green fluorescent protein (GFP) hmanga label leh transmembrane secreted protein Mid2 near-infrared fluorescent protein (iRFP) hmanga label kan thlang a, chungte chuan plasma membrane an target vek a ni (24–26). sec31-1 temperature-sensitive mutant-ah hian heng cargo pahnih hi galactose-inducible promoter leh constitutive ERES marker hnuaiah an express a ni. Extreme temperature (37°C)-ah chuan sec31-1 mutation hian COPII coat component Sec31 hnathawh a nghawng avangin COPII germination leh ER export a titawp a, ER-ah hian cargo siam thar a awm khawm thin (23). Temperature hniam (24°C)-a a lum hnuah sec31-1 mutant cells te chu secretory area atangin an lo harh chhuak a, synthetic cargo thar an khawlkhawm chu ER atang hian an thawn chhuak tan ta a ni. CLIM visualization atanga a lan dan chuan Gas1-GFP leh Mid2-iRFP siam thar tam zawk chu sec31-1 mutant cells ER-ah 37°C-a an incubation hnuah an la pungkhawm a, chutah chuan 24°C-ah minute 5 chhung an chhuah leh a ni (Figure 1). Mid2-iRFP hi ER membrane pumpuiah a insem darh a, Gas1-GFP chu discontinuous ER membrane area-ah a concentrate a, a khawl khawm a, chuvangin an distribution chu a danglam vek a ni (Figure 1, A to C leh Movie S1). Tin, Figure 1D-a kan hmuh angin Gas1-GFP cluster hian Mid2-iRFP a nei lo. Heng results te hian GPI-AP leh transmembrane protein te hi ER membrane region hrang hrangah an inthen hmasa tih a tarlang. Gas1-GFP cluster hi mCherry’s COPII coat protein Sec13 hmanga label ERES bik nen a inhnaih hle a (Figure 1, E leh F, leh movie S1) (23).
sec31-1 cells hian galactose-induced secretions an express a, acyl chain sei tak (C26) ceramide GPI-AP Gas1-GFP (GPI-AP, green) leh transmembrane protein Mid2-iRFP (TMP, blue) te chu an express a, he Constructive ERES labeling Sec13-mCherry (ERES, magenta) hi 37°C-ah minute 30 chhung incubate a ni a, sawn a ni 24°C, leh minute 5 hnuah SCLIM hmangin imaged a ni. (A to C) hian plane (A) pakhat aiawhtu merged emaw single 2D image emaw, z-section 10 (B) 2D projection image emaw, cargo leh ERES marker (C) 3D cell hemisphere image emaw a tarlang a ni. Scale bar 1μm (A leh B) a ni. Scale unit chu 0.551μm (C) a ni. Gas1-GFP chu discrete ER region emaw cluster emaw-ah hmuhchhuah a ni a, Mid2-iRFP erawh chu hmuhchhuah a ni a, ER membrane (C) pumpuiah a insem darh thung. (D) Graph-ah hian Gas1-GFP cluster-a Gas1-GFP leh Mid2-iRFP te relative fluorescence intensity chu white arrow line (khawi lamah nge) a ni. AU, duhthlanna unit. (E leh F) hian thil leh ERES mark inzawmkhawmna 3D image a entir a ni. ERES bik bulah hian Gas1-GFP cluster hmuhchhuah a ni. Scale unit chu 0.551μm a ni. (F) Solid arrow dum hian ERES nena inzawm Gas1-GFP cluster a tarlang a. Middle leh right panel-ah hian merged enlarged 3D image leh Gas1-GFP cluster thlan chhuah rotated view a lang a ni.
Gas1-GFP cluster leh ERES bik inkara spatial relationship hnai tak hian Gas1-GFP hi selective ERES-ah a lut thei tih a tilang a, hei hi Mid2-iRFP-in ER chhuahsan tura a hman selectivity nen a danglam a ni. Hetiang thil thleng thei hi sutkian nan hian thil pakhat emaw pahnih emaw chauh ERES ratio chu kan quantified a (Figure 2, A to C). ERES tam zawk (70%) hian bungrua chi khat chauh a nei tih kan hmuchhuak. Figure 2C hnuai lam thlalak hian ERES entirnan Gas1-GFP chauh (Figure 1) emaw Mid2-iRFP chauh (Figure 2) emaw a entir a. Chumi danglamna chu ERES 20% vel hian hmun khata inzawm khawm cargo pahnih a nei a ni. ERES thenkhat (10%) ah hian bungraw chi hnih a awm tih hmuhchhuah a ni a, mahse hmun hrang hrang chiang takah an inthen a ni. Chuvangin, he statistical analysis hian ER hi export a nih hnuah GPI-AP Gas1-GFP leh transmembrane cargo Mid2-iRFP te chu ERES hrang hrangah an inthen tih a tarlang (Figure 2D). He sorting efficiency hi a hmaa biochemical analysis (6) leh morphological determination (7) nen a inmil hle. ERES-a lut tur quarantine cargo-te awm dan pawh kan hmu thei bawk (Figure 2E leh Movie S2). Figure 2E atang hian Gas1-GFP (panel 3) emaw Mid2-iRFP (panel 4) emaw a tlem ber chauh chu a sir khat atanga ERES chhungah a lut a, hmun hrang hrangah a inkhung tlat a ni. Figure 2E-a Panel 5-ah hian Gas1-GFP leh Mid2-iRFP te hi ERES khatah hmuh tur a awm fo a, mahse a sir hrang hrang atanga lut an ni a, COPII vesicles hrang hrang aiawh thei tur hmun hrang hrangah an inzawm khawm a ni. Tin, C26 ceramide-based GPI-AP Gas1 chu selective ERES anga inthen leh classification hmuhchhuah chu a chiang hle tih kan nemnghet bawk a, a chhan chu transmembrane secretion cargo dang, GFP-tagged plasma membrane protein Axl2 (27 ), Mid2-iRFP nena inmil thiltih lantir vang a ni. (Thlalak S1 leh Movie S3 te). Axl2-GFP siam thar hi Mid2-iRFP angin ER membrane kaltlangin a insem darh a (Figure S1, A leh B), ERES tam zawkah chuan Mid2-iRFP nen co-localized a ni (Figure S1, B to D). Figure 1-a panel 1 leh 2-ah hian S1C-ah hian ERES entirnan langsar tak pahnih a lang a, chutah chuan transmembrane cargo pahnih a inzawm tlat a ni. Heng hunah hian bungrua pahnih hi ERES-ah an lut dun thin (Figure S1E, Panel 3 leh Movie S3).
Sec31-1 cells expressing galactose inducible secretions, Gas1-GFP (GPI-AP, green) leh Mid2-iRFP (TMP, blue) leh constitutive ERES labeling Sec13-mCherry (ERES, magenta) te chu °C-a minute 30 chhung incubate hnuah 37-ah dah a ni a, 24 °C-ah sawn a, secretion block chhuah tir a ni a, chumi hnuah SCLIM hmangin image a ni Minute 20 chhung a ni. (A atanga C) Cargo leh z-section 10 ERES hmanga chhinchhiah 2D projection image (A; scale bar, 1μm) emaw 3D cell hemisphere image (B leh C; scale unit, 0.456μm) aiawhtu. (B)-a panel hnuai lam leh (C)-a panel-ah chuan ERES (magenta) [Gas1-GFP (gray) leh Mid2-iRFP (light blue)]-a thil awmte chauh lantir turin processed images a lang a. (C) Open arrow: ERES hian bungrua pakhat (1 atanga 4) chauh a phur a. Gray arrow: ERES ah hian segregated cargo (5) a awm a. White solid arrow: ERES-ah hian co-located cargo a awm. A hnuaia mi hi: ERES pakhat thlan chhuah ah hian Gas1-GFP (1) emaw Mid2-iRFP (2) emaw chauh a awm. Scale bar, 100 nm a ni. (D) (C)-a tarlan photomicrograph quantification. ERES-a cargo pakhat chauh (Gas1-GFP emaw Mid2-iRFP), segregated cargo leh overlapping cargo awmna percentage average. Independent experiment pathum-ah chuan cell 54-ah n=432 a awm. Error bar = SD a awm. T test pahnih nei, unpaired t test. *** P = 0.0002 a ni. (E) Quarantine cargo ERES thlan chhuah 3D image (C) hmanga chhinchhiah. Gas1-GFP (green) (3) emaw Mid2-iRFP (blue) (4) emaw chu a sir khat atangin ERES (magenta) ah a lut a, ERES chhunga hmun tlemteah chauh tihkhawtlai a ni. A châng chuan, bungraw chi hnih hi a sir khat aṭangin ERES (5) khatah an lut a, ERES chhunga hmun hla takah an inkhung a ni. Scale bar, 100 nm a ni.
A dawtah chuan ER membrane-a long acyl chain ceramide (C26) awm hian Gas1 specific clustering leh sorting chu selective ERES-ah a hruai lut tih hypothesis kan test leh a. Chumi atan chuan modified yeast strain GhLag1 kan hmang a, chutah chuan endogenous ceramide synthases pahnih Lag1 leh Lac1 te chu GhLag1 (cotton-a Lag1 homolog) hmangin kan thlak a, chu chuan yeast strain cell membrane Ceramide strain wild type aiin a tawi zawk a siam ta a ni (Figure 3A) (28). Mass spectrometry (MS) hmanga an zirchiannaah chuan wild-type strain-ah chuan ceramide zawng zawng zinga 95% chu chain ceramide sei tak (C26) a ni a, GhLag1-ah chuan ceramide 85% chu a sei hle (C18 leh C16) a ni thung. ), ceramide 2% chauh hi chain sei tak (C26) ceramide a ni. Tun thlenga GhLag1 membrane-a C18 leh C16 ceramide hmuhchhuah zingah hian ceramide hmuhchhuah ber ni mah se, MS analysis hmangin GhLag1 strain-a Gas1-GFP expressed GPI anchor-ah hian C26 ceramide a awm tih a nemnghet bawk a, hei hi wild-type lipids nen tehkhin theih a ni. Quality pawh a inang vek (Fig. 3A) (26). Chuvangin, hei hian ceramide remodeling enzyme Cwh43 hian C26 ceramide tan a thlang thiam hle tihna a ni a, Figure 26-a kan hmuh angin, GhLag1 strain-a C26 ceramide tlemte atanga GPI anchor chu a dah tel duh zawk a ni. S2 (29) a ni. Chuti chung pawh chuan GhLag1 cell membrane-ah hian a bul berah chuan C18-C16 ceramide chauh a awm a, Gas1-GFP erawh chuan C26 ceramide a la nei thung. He thudik hian he strain hi ER-a membrane ceramide acyl chain sei zawng chungchanga buaina chiang taka chinfelna hmanraw tha tak a ni. Class leh sorting-in a chanvo (hypothetical role) a neih dan. Tichuan, C26 Gas1-GFP chu GhLag1-a temperature-sensitive mutant allele of sec31-1 hmangin cluster-a a khawlkhawm theihna chu conventional fluorescence microscopy hmangin kan zir hmasa a, chutah chuan ER membrane Ceramide-ah hian chain sei tak (C18-C16) chauh a awm a (Fig. 3). Sec31-1-ah hian Gas1-GFP tam zawk chu cluster-ah a concentrate tih kan hmu a, sec31-1 GhLag1-ah chuan Gas1-GFP chu sec31-1 GhLag1-a sei (C18-C16) sei ceramide ER membrane neiah chuan a bulpui berah chuan In the entire ER membrane-ah clustered lo leh distributed a ni. A dik tak chuan C26 ceramide-based clustering hi ERES bik nen a inzawm tlat avangin (Figure 1), a dawtah chuan he process hian ER export protein mechanism hnathawh pawh a huam thei em tih kan zirchiang leh a. GPI-AP hian ER export atan COPII system bik a hmang a, chu chu Ted1′s structural remodeling of the glycan portion of the GPI anchor (30, 31) hmangin active takin a regulate a ni. Chumi hnuah recombinant GPI-glycan chu transmembrane cargo receptor p24 complex chuan a hrechhuak a, chu chuan Lst1 chu a thlangin a recruit a, chu chu major COPII cargo binding subunit Sec24 isoform bik a ni a, GPI-AP-rich COPII siam turin Vesicles a ngai (31-33). Chuvangin, heng protein pakhat chauh (p24 complex component Emp24, GPI-glycan remodeling enzyme Ted1 leh specific COPII subunit Lst1) deletion chu sec31-1 mutant strain nen inzawmkhawmtu double mutant kan siam a, Gas1-cluster GFP siam theih a ni em tih kan zirchiang a (Figure 3). Sec31-1emp24Δ leh sec31-1ted1Δ-ah te hian Gas1-GFP hi a tam zawk chu unclustered niin ER membrane pumpuiah a insem darh tih kan hmu a, hei hi a hmaa sec31-1 GhLag1-a kan hmuh tawh angin, sec31-1lst1Δ-ah erawh chuan Gas1-GFP Like sec31-1. Heng results te hian ER membrane-a C26 ceramide awmna bakah hian Gas1-GFP clustering hian p24 complex-ah pawh a binding a ngai tih a tilang a, Lst1 recruitment bik a ngai lo. Tichuan, ER membrane-a ceramide chain sei zawng hian Gas1-GFP leh p24 inzawmna a tidanglam thei tih kan zirchiang a. Mahse, membrane chhunga C18-C16 ceramide awm hian p24 complex-in GPI-glycans a siam thar (Figure S3 leh S4, A leh B) emaw, GPI-AP nena inzawmna leh GPI-AP export emaw a nghawng lo tih kan hmuchhuak. theihna. COPII subtype Lst1 chu recruit rawh (Figure S4C). Chuvangin, C26 ceramide-dependent clustering hian ER export protein mechanism hrang hrang nena protein inzawmna a mamawh lo va, mahse lipid sei zawngin a kaihhruai sorting mechanism dang a thlawp thung. Tichuan, ER membrane-a ceramide acyl chain sei zawng chu Gas1-GFP chu selective ERES anga classification \ha tak atan a pawimawh em tih kan zirchiang a. Short-chain ceramide nei GhLag1 strain-a Gas1 hian ER a chhuahsan a, plasma membrane-ah a luh avangin (Figure S5), sorting hi ceramide acyl chain sei zawnga kalpui a nih chuan GhLag1 strain-a Gas1 chu redirect leh cross theih a ni tih kan ring. ERES bungrua chu membrane inang nei a ni.
(A) GhLag1 cell membrane-ah hian C18-C16 ceramide tawi zawk a awm ber a, Gas1-GFP-a GPI anchor-ah chuan wild-type cell ang chiah C26 IPC a la nei thung. A chungah hian: wild-type (Wt) leh GhLag1p chi hrang hrangte cell membrane-a ceramide awm zat mass spectrometry (MS) hmanga acyl chain sei zawng tehna. Data hian ceramide zawng zawng percentage a entir a ni. Independent experiment pathum atanga a vaiin. Error bar = SD a awm. T test pahnih nei, unpaired t test. **** P <0.0001 a ni. Bottom panel: Wild-type leh GhLag1p strain-a Gas1-GFP (GPI-IPC) GPI anchor-a IPC awm acyl chain sei zawng MS hmanga zirchianna. Data hian IPC signal zawng zawng percentage a entir a ni. Independent experiment panga a vaiin. Error bar = SD a awm. T test pahnih nei, unpaired t test. ns, a pawimawh lo. P = 0.9134 a ni. (B) Sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ leh sec31-1lst1Δ cells expressing galactose-induced Gas1-GFP te fluorescence micrograph te chu 37°C ah minute 30 chhung incubate a ni a, a hnuah Perform routine fluorescence microscopy ah pass down a ni 24°C a ni. Arrow dum: ER Gas1-GFP cluster a ni. Open arrow: Unclustered Gas1-GFP hi ER membrane pumpuiah a insem darh a, ER characteristic nuclear ring staining a lantir a ni. Scale bar, 5μm a ni. (C) (B)-a tarlan photomicrograph quantification. Punctate Gas1-GFP structure nei cell percentage zatve. Independent experiment pathum-ah chuan n≥300 cells. Error bar = SD a awm. T test pahnih nei, unpaired t test. **** P <0.0001 a ni.
He harsatna hi direct-a chinfel nan hian sec31-1 temperature-sensitive mutant allele hmangin GhLag1-a Gas1-GFP leh Mid2-iRFP te SCLIM visualization kan ti a (Figure 4 leh Movie S4). ER chu 37°C-a dah a nih hnuah, a hnuah 24°C-a tihchhuah a nih hnuah, Gas1-GFP siam thar tam zawk chu a clustered lo va, ER membrane pumpuiah a insem darh lo va, hei hi microscope pangngai hmanga hmuh a ni (Figure 4, A leh B ). Chu bakah, ERES za zela tam tak (67%) hian a chhunga awm cargo chi hnih a huam tel bawk (Figure 4D). Figure 4C-a panel 1 leh 2-ah hian ERES entirnan langsar tak pahnih, Gas1-GFP leh Mid2-GFP inzawmkhawm te kan hmu a. Tin, bungrua pahnih hi ERES khatah an recruit vek bawk (Figure 4E, panel 3 leh movie S4). Chuvangin, kan result atang hian ER membrane chhunga ceramide acyl chain sei zawng hi ER protein aggregation leh classification atana tichiangtu pawimawh tak a ni tih a tarlang.
Sec31-1 GhLag1 cells expressing galactose-induced secretions, Gas1-GFP (GPI-AP, green) leh Mid2-iRFP (TMP, blue) leh constitutive ERES-labeled Sec13-mCherry (ERES, magenta) te chu 37°C-ah incubate rawh. Minute 30 chhung chhunzawm la, 24°C-ah dah la, secretions chhuah tir la, minute 20 hnuah SCLIM hmangin image rawh. (A atanga C) Cargo leh ERES hmanga chhinchhiah z-section 10 zinga 2D projection image (A; scale bar, 1μm) emaw 3D cell hemisphere image (B leh C; scale unit, 0.45μm) emaw aiawhtu. (B)-a panel hnuai lam leh (C)-a panel-ah chuan ERES (magenta) [Gas1-GFP (gray) leh Mid2-iRFP (light blue)]-a thil awmte chauh lantir turin processed images a lang a. (C) White filled arrow: ERES, bungrua inzawmkhawm. Open arrow: ERES ah hian item pakhat chauh a awm. Lower panel: ERES thlan chhuah hian (C)-a chhinchhiah thil (1 leh 2) inzawmkhawm (overlapping goods) a nei a. Scale bar, 100 nm a ni. (D) (C)-a tarlan photomicrograph quantification. sec31-1 leh sec31-1 GhLag1 unit-ah hian cargo pakhat (Gas1-GFP emaw Mid2-iRFP) chauh a tel a, chu chu isolated cargo leh overlapping cargo-a ERES percentage average a ni. Independent experiment pathum-ah chuan cell 54 (sec31-1)-ah n = 432 leh cell 47 (sec31-1 GhLag1)-ah n = 430 a ni. Error bar = SD a awm. T test pahnih nei, unpaired t test. *** P = 0.0002 (sec31-1) leh ** P = 0.0031 (sec31-1 GhLag1) a ni. (E) ERES thlan bikte 3D image, (C)-a chhinchhiah cargo (3) inzawm khawm. Gas1-GFP (green) leh Mid2-iRFP (blue) te hian ERES (magenta) chu a sir khatah an hnaih a, ERES khapna hmun khatah an awm reng a ni. Scale bar, 100 nm a ni.
He zirchianna hian lipid-based protein cargo te chu secretory pathway-ah selective export site-ah an classified tih direct in vivo evidence a pe a, classification selectivity atana acyl chain length pawimawhzia a tilang bawk. Microscopy technique chak tak leh hmasawn tak SCLIM tih hmangin, yeast-a Gas1-GFP (a major plasma membrane GPI-AP with a very long acyl chain (C26) ceramide lipid portion) kan siam thar chu kan entir a (Figure 1) a ni. Tin, heng thil chi hnih te hi ERES hrang hrangah thlan bik takin an lut thin (Figure 2). Membrane chhunga cellular ceramide acyl chain sei zawng chu C26 atanga C18-C16 ah a tlahniam a, Gas1-GFP cluster chu discrete ER region ah a disrupted a, Gas1-GFP chu ERES ang chiah hmangin transmembrane protein nen ER chhuahsan turin rerout a ni bawk (Figure 3 leh Figure 3). 4).
GPI-AP hian ER chhuahsan nan hian specialized protein mechanism hmang mahse, C26 ceramide-a innghat separation hian ERES specialization thlen thei tur differential protein interaction-ah a innghat lo tih kan hmuchhuak (Figure S4 leh S5). Chu ai chuan kan thil hmuhchhuahte hian lipid-based protein clustering leh a hnu lama bungraw dangte paih chhuahna hmanga kalpui classification mechanism dang a thlawp a ni. Kan thil hmuhchhuahte chuan ERES bik nena inzawm Gas1-GFP region emaw cluster emaw hian transmembrane secreted protein Mid2-iRFP a tlachham tih a tilang a, hei hian C26 ceramide-dependent GPI-AP cluster hian ERES kaihhnawih-a an luh theihna tur a siamsak dawn tih a tilang a, chutih rual chuan transmembrane The secretions enter this particular ERES-ah pawh a tel lo (Figure 1 leh 2). Chumi danglamna chu ER membrane-a C18-C16 ceramides awm hian GPI-AP hian region emaw cluster emaw a siam lo va, chuvangin transmembrane secreted proteins te chu ERES khatah an paih chhuak lo va, an thlak lo bawk (Figure 3 leh 4). . Chuvangin, C26 ceramide hian ERES bik nena inzawm proteins clustering awlsam zawka siamin separation leh classification a tichak tih kan rawt a ni.
Engtin nge he C26 ceramide-a innghat clustering hi ER area bik pakhatah kan tih theih ang? Membrane ceramide te hi laterally a inthen duhna hian GPI-AP leh C26 ceramide te chu ER membrane lipid environment mumal lo zawk, glycerolipids tawi zawk leh unsaturated te awmnaah lipid te tak te leh instantaneously ordered a siam thei a ni. Quality cluster hrang hrang (17, 18) te a ni. Heng temporary cluster te te hi p24 complex nena an binding hnuah cluster lian zawk, stable zawkah an fuse leh thei a ni ( 34 ). Chumi nena inmil takin C26 Gas1-GFP hian p24 complex nen inzawmna neiin visible cluster lian zawk siam a ngai tih kan hmuchhuak a (Figure 3). p24 complex hi heterozygous oligomer a ni a, yeast-a p24 transmembrane protein hrang hrang pali atanga siam a ni (35), hei hian multivalent binding a pe a, hei hian GPI-AP cluster te te cross-linking a thlen thei a, chu chuan Stable cluster lian zawk a siam thei a ni (34). GPI-APs-a protein ectodomains inkara inzawmna hian an aggregation-ah pawh a pui thei a, hei hi mammalian polarized epithelial cells-a Golgi transport an neih laiin a lang (36). Mahse, ER membrane-a C18-C16 ceramide a awm chuan, p24 complex chu Gas1-GFP nena a inzawm chuan cluster hrang hrang lian tak tak a lo awm dawn lo. A hnuaia mechanism chu long acyl chain ceramide physical leh chemical property bik ah a innghat thei. Artificial membrane biophysical study-ah chuan acyl chain ceramide sei (C24) leh tawi (C18-C16) te hian phase separation thlen thei mahse, acyl chain ceramide sei (C24) chauh hian Curvature sang tak leh film bending a tichak thei a, film chu a siam danglam thei a ni. Inhriatthiam tawnna hmangin (17, 37, 38). Emp24 mihring homologue TMED2 transmembrane helix hian cytoplasmic lobules-a C18 ceramide-based sphingomyelin nen thlan bik takin a inzawm tih hmuhchhuah a ni ( 39 ). Molecular dynamics (MD) simulations hmangin C18 leh C26 ceramide pahnih hi Emp24 transmembrane helix-a cytoplasmic lobules vel ah an pung khawm tih kan hmu a, an duh dan inang an nei (Figure S6). Hriat tur chu hei hian Emp24-a transmembrane helix hian membrane chhunga lipids te asymmetric distribution a thlen thei tih a tilang a ni. Hei hi tun hnaia mammalian cells atanga result chhuak a ni. MD simulation ang chiah hian ether lipids a awm tih a lang bawk (40) . Chuvangin ER26 lobule pahnih a C26 ceramide hi locally enriched a ni tih kan ring. Luminal lobules-a GPI-AP hian multivalent p24-ah direct-a a binding a, cytoplasmic lobules-a p24 vel a C26 ceramide a punkhawm chuan a kaihhnawih Protein aggregation a tichak thei a, membrane curvature chu fingers (41) hmangin a siam thei a, chu chuan GPI-AP chu ERES bula discrete region-ah a inthen tir thei a, hei hian highly curved region-ah a inthen thei bawk ER membrane-a bial hrang hrang (42). Tun hmaa report-te chuan he mechanism rawt hi an thlawp a ni (43, 44). Plasma membrane-a oligolectins, pathogens emaw antibodies te chu ceramide-based glycosphingolipids (GSL) nena multivalent binding hian GSL aggregation lian tak a tichhuak a, phase separation a tichak a, membrane deformation leh internalization a thlen bawk (44). Iwabuchi etc. (43) Acyl chain sei (C24) mahse tawi lo (C16) awmnaah chuan GSL lactosylceramide nena inzawm multivalent ligand chuan cluster lian tak tak leh membrane invagination a siam a, leaflets-a cytoplasm Lyn-mediated signal transduction chu acyl chain in coupled hmangin interdigitated a ni tih hmuhchhuah a ni neutrophils a ni.
Mammalian polarized epithelial cells-ah chuan anti-Golgi network (TGN) concentration chu apical plasma membrane level thlenga a awm chuan GPI-AP inthen leh sorting a control a ni (10, 45). He aggregation hi GPI-AP oligomerization (36) vang a ni a, mahse yeast-a kan hmuh ceramide chain sei zawngah pawh a innghat thei bawk. Mammalian GPI-AP hian ether lipid-based anchor nei mahse, a chemical structure chu acyl chain sei tak ceramide nen a danglam hle a, tun hnaia zirchianna pakhatah chuan lipid pahnih hian evolutionarily physical leh chemical property And function inang an nei tih hmuhchhuah a ni (40). Chuvangin, mammalian cell-a ether lipid part hi yeast-a C26 ceramide nen a inang thei a, a hna chu GPI-AP aggregation leh sorting tichak turin membrane chhunga long-chain ceramide nena inzawm a ni. Hetiang thil thleng thei hi direct-a test a la ngai a, mahse a hmaa thil hmuhchhuahte chuan Golgi taksaa long acyl chain ceramide phurh chhuahna hi cytoplasmic transfer proteins hmanga kalpui a ni lo va, yeast ang chi GPI anchor siamnaah a innghat tih a nemnghet a ni. Chuvangin, evolutionary conservative mechanism hian acyl chain sei tak tak ceramide leh GPI-AP (13, 16, 20, 46, 47) te chu transport vesicle khatah selective-in a co-transport thei niin a lang.
Yeast leh mammalian polarized epithelial cell system-ah chuan GPI-AP aggregation leh plasma membrane protein dang atanga inthen hi cell chunglam a thlen hmain a thleng vek a ni. Paladino leh a thawhpuiten an sawi. (48) te chuan mammalian polarized epithelial cells TGN-ah GPI-AP clustering hi GPI-APs te apical plasma membrane-a selective classification atan chauh ni lovin, GPI-APs te clustering organization leh Its biological activity te pawh a tidanglam tih an hmuchhuak bawk. Cell chunglam a ni. Yeast-ah chuan he zirchianna hian ER-a C26 ceramide-dependent GPI-AP cluster hian plasma membrane-a GPI-AP cluster organization leh functional activity a tidanglam thei tih a tarlang ( 24 , 49 ). He model nen hian a inmil hle a, GhLag1 cells te hian GPI inhibitor emaw cell wall integrity tichhe thei damdawi emaw an allergy a ( 28 ), yeast cells inpawlnaah projected tip ceramide functional Gas1-GFP clusters ( 49 ) mamawhna hian G hLag1 cells te physiological consequences awm thei te a tilang a ni. GPI-AP tihsual a awm. Mahse, cell surface functional organization chu ER atanga lipid sei zawng atanga sorting method hmanga programme a nih leh nih loh test belh leh chu kan nakin lawka kan zirchianna tur a ni ang.
He hnathawhna atana hman Saccharomyces cerevisiae chi hrang hrangte chu Table S1-ah hian tarlan a ni. Live cell imaging atana SCLIM MMY1583 leh MMY1635 strain te hi W303 hnung lamah siam a ni. Heng strain te hi fluorescent protein tag hmanga Sec13-mCherry express tu te hi polymerase chain reaction (PCR)-based method hmanga siam niin pFA6a plasmid chu template atan hman a ni ( 23 ). GAL1 promoter control hnuaia fluorescent protein hmanga label Mid2-iRFP expressing strain chu a hnuaia mi ang hian siam a ni. pKTiRFP-KAN vector atanga iRFP-KanMx sequence PCR amplification (E. O’Shea thilpek, Addgene plasmid number 64687; http://n2t.net/addgene: 64687; research resource identifier (RRID): Addgene_64687) Tin, endogenous Mid2 C-terminus-ah dah a ni bawk. Mid2-iRFP genome sequence chu amplified a nih hnuah GAL1 promoter-ah clone a nih hnuah integration plasmid pRS306-a Not I-Sac I site-ah integrate a ni. Chuta chhuak plasmid pRGS7 chu Pst I hmangin linearized niin URA3 locus-ah a inzawm khawm a ni.
Gas1-GFP fusion gene hi centromere (CEN) plasmid-ah GAL1 promoter control hnuaiah express a ni a, a hnuaia mi ang hian siam a ni. Gas1-GFP sequence chu pRS416-GAS1-GFP plasmid (24) (L. Popolo thilpek) atanga PCR hmangin amplified a ni a, CEN plasmid pBEVY-GL LEU2 (gift of C) Xma I–Xho I site-ah clone a ni. Miller-a chuan; Addgene plasmid number chu 51225 a ni a; A rilru a hah lutuk chuan a rilru a buai em em a. 51225; RRID: Addgene_51225 tih a ni. Plasmid lo chhuak chu pRGS6 tih a ni. Axl2-GFP fusion gene pawh hi pBEVY-GL LEU2 vector GAL1 promoter control hnuaiah express a ni a, a siam dan chu hetiang hi a ni. Axl2-GFP sequence chu pRS304-p2HSE-Axl2-GFP plasmid (23) atanga PCR hmangin amplified a ni a, pBEVY-GL LEU2 vector Bam HI-Pst I site-ah clone a ni. Plasmid lo chhuak chu pRGS12 tih a ni. He zirchiannaa oligonucleotide hmante sequence chu Table S2-ah hian tarlan a ni.
He strain hi 0.2% adenine leh 2% glucose [YP-dextrose (YPD)], 2% raffinose [YP-raffinose] rich yeast extract protein p (YP) medium (1 % Yeast extract leh 2% protein ept) hmanga tihpun a ni. (YPR)] emaw 2% galactose [YP-galactose (YPG)] carbon source atan emaw, synthetic minimal medium (0.15% yeast nitrogen base leh 0.5% ammonium sulfate)-ah emaw ei tur atana mamawh amino acid leh base dik tak tihpun nan , Tin, 2% glucose (synthetic glucose minimal medium) emaw 2% galactose emaw a awm bawk (synthetic galactose minimal medium) chu carbon source atan hman a ni.
Real-time imaging atan chuan GAL1 promoter hnuaia construct express tu temperature-sensitive sec31-1 mutant cells te chu YPR medium-ah 24°C-ah zan khat chhung mid-log phase thlengin an tipung a. YPG-a 24°C-a darkar 1 chhung induction hnuah, cell-te chu SG-ah 37°C-ah minute 30 chhung incubate a ni a, chutah chuan 24°C-ah dahin secretion block atanga chhuah a ni. Concanavalin A hmangin glass slide-ah cell te chu fix a ni a, SCLIM hmangin imaged a ni. SCLIM hi Olympus IX-71 inverted fluorescence microscope leh UPlanSApo 100×1.4 numerical aperture oil lens (Olympus), high-speed leh high-signal-to-noise ratio rotating disc confocal scanner (Yokogawa Electric), custom spectrometer, leh custom cooling hmanga siam a ni a, The system’s image intensifier (Hamamatsu Photonics) hian magnifying lens system a pe thei a, a tawp ber chu ×266.7 a ni a, electron tipung thei charge-coupled device camera a pe thei bawk (Hamamatsu Photonics) (21). Image lakkhawm hi custom software (Yokogawa Electric) hmanga tih a ni. 3D images atan chuan custom-made piezoelectric actuator hmangin objective lens chu vertical-in kan vibrate a, optical parts te chu 100 nm a inhlat takin stack-ah kan khawlkhawm a. Z-stack image hi 3D voxel data-ah a chantir a, rotating disc confocal microscope atana hman theoretical point spread function chu Volocity software (PerkinElmer) hmanga deconvolution processing atan hman a ni. Volocity software hmangin co-location analysis atan automatic-a threshold siamin, cargo telin ERES chu teh a ni. Line scan analysis chu MetaMorph software (Molecular Devices) hmangin an ti a.
Statistical significance hriat nan GraphPad Prism software hmang ang che. Two-tailed Student’s t-test leh ordinary one-way analysis of variance (ANOVA) test atan chuan group hrang hrangte inthlauhna hian P <0.05 (*)-ah nghawng lian tak a nei nia ngaih a ni.
Gas1-GFP fluorescence microscopy atan chuan log phase cell te chu YPD-ah zan khat chhung an tipung a, centrifugation hmangin an la khawm a, phosphate buffered saline hmangin vawi hnih an silfai a, ice-ah minute 15 tal an incubate a, chutah chuan a hmaa kan sawi tawh angin microscope hmangin an kalpui leh a ni Check (24). Leica DMi8 microscope (HCX PL APO 1003/1.40 oil PH3 CS) chu objective lens, L5 (GFP) filter, Hamamatsu camera leh Application Suite X (LAS X) software hmanga thuam a ni. .
Sample te chu SDS sample buffer hmangin 65°C ah minute 10 chhung an denature a, chutah chuan SDS-polyacrylamide gel electrophoresis (PAGE) hmangin an inthen a ni. Immunoblotting analysis atan chuan lane khatah sample 10 μl load a ni. Primary antibody: Rabbit polyclonal anti-Gas1 chu dilution 1:3000-ah, rabbit polyclonal anti-Emp24 chu dilution 1:500-ah, leh rabbit polyclonal anti-GFP (H. Riezman hnen atanga thilpek) chu dilution 1:3000-ah hman tur a ni. Mouse monoclonal anti-Pgk1 antibody hi dilution 1:5000 (J. de la Cruz hnen atanga thilpek)-ah hman a ni. Secondary antibody: Horseradish peroxidase (HRP) conjugated kêl anti-rabbit immunoglobulin G (IgG) chu dilution 1:3000-a hman a ni (Pierce). HRP-conjugated goat anti-mouse IgG chu dilution 1:3000-ah hman a ni (Pierce). Immune response zone hi SuperSignal West Pico reagent (Thermo Fisher Scientific) hmanga chemiluminescence hmanga enfiah a ni.
(31)-a kan sawi tawh angin, enriched ER fraction-ah hian natural immunoprecipitation experiment an nei a. A tawi zawngin, yeast cell te chu TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride leh protease inhibitor mixture) hmangin 600 nm ( OD600) ah 100 optical density ah vawi hnih silfai tur a ni. Glass beads hmangin an tikehsawm a, chutah chuan cell debris leh glass beads te chu centrifugation hmangin an paih chhuak ta a ni. Chumi hnuah supernatant chu 17,000 g-ah minute 15 chhung 4°C-ah centrifuged a ni. Pellet chu TNE-ah resuspended a ni a, digitalis saponin chu final concentration 1%-ah dah a ni. Suspension chu darkar 1 chhung 4°C-ah rotation-in an incubate a, chutah chuan insoluble components chu 13,000 g-ah 4°C-ah minute 60 chhung centrifugation hmangin an paih chhuak a ni. Gas1-GFP immunoprecipitation atan chuan sample chu empty agarose beads (ChromoTek) nen 4°C-ah darkar 1 chhung pre-incubate hmasa la, chutah chuan GFP-Trap_A (ChromoTek) nen 4°C-ah darkar 3 chhung incubate hmasa phawt ang che. Immunoprecipitated beads te chu 0.2% digoxigenin awmna TNE hmangin vawi nga silfai a ni a, SDS sample buffer hmangin eluted a ni a, SDS-PAGE-ah then a ni a, immunoblotting hmangin an zirchiang a ni.
(31)-a kan sawi tawh angin, enriched ER fraction-ah cross-linking determination an ti a. A tawi zawngin, enriched ER fraction chu 0.5 mM dithiobis(succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, USA; 20°C, 20 min) nen incubate a ni. Crosslinking reaction chu glycine (50 mM final concentration, minute 5, 20°C) dahin a titawp a ni.
A hmaa kan sawi tawh ang khan (50) wild-type leh GhLag1 strain-a ceramide awm zat MS analysis an ti a. A tawi zawngin, YPD-ah 30°C-ah cell te chu exponential phase (3 to 4 OD600 units/ml) thlengin an tipung a, 25×107 cells an la khawm a ni. An metabolism chu trichloroacetic acid hmangin a titawp a ni. Extraction solvent [ethanol, tui, ether, pyridine leh 4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] leh internal standard C17 ceramide (860517, Avanti polar lipid) quality 1.2 nmol hmang rawh. Monomethylamine reagent [methanol, tui, n-butanol leh methylamine solution (4:3:1:5 v/v)] hmangin extract chu mild alkaline hydrolysis ti la, chutah chuan water-saturated n-butanol hmangin desalt rawh. A tawpah chuan extract chu positive mode solvent [chloroform/methanol/water (2:7:1) + 5 mM ammonium acetate]-ah resuspended lehin mass spectrometer-ah inject a ni. Sphingolipid molecule te hriatchhuah leh quantification atan multi-reaction monitoring (MRM) an nei a. TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) hi lipid analysis atan robotic nanoflow ion source Nanomate HD (Advion Biosciences, Ithaca, NY) hmanga siam a ni. Ceramide category tin tan collision energy hi optimized a ni. MS data chu positive mode-a lak a ni. Biological replicate tin tan lipid signal chu independent measurement pathum median a ni.
(31)-a kan sawi tawh ang khan Gas1-GFP express tu cell (800×107) te chu natural immunoprecipitation an nei a. Gas1-GFP tihthianghlim chu SDS-PAGE hmangin a then a, polyvinylidene fluoride (PVDF) membrane-ah dah a ni. Protein chu PVDF chu amide black hmanga stain hmangin hmuh theih a ni. Gas1-GFP band chu PVDF atang hian tan a ni a, methanol hmangin vawi 5 leh liquid chromatography-MS (LC-MS) grade water hmangin vawi khat silfai a ni. Membrane strip chu 500μl 0.3 M NaOAc (pH 4.0), buffer leh 500μl 1 M sodium nitrite mixture hmin thar nen 37°C-ah darkar 3 chhung incubate-in, lipid fraction chu Gas1-GFP atangin a chhuak a, lysed Release of inosine phosphate ceramide between glucosamine and inositol (51). Chumi hnuah chuan membrane strip chu LC-MS grade tui hmangin vawi li silfai a ni a, room temperature-ah a vawt a, nitrogen boruak -80°C-ah analysis thlengin dah a ni. Control atan chuan experiment tin atan PVDF membrane sample blank hman a ni. Chumi hnuah Gas1-GFP atanga lipid lakchhuah chu a sawi angin MS hmangin an zirchiang a (50). A tawi zawngin, GPI-lipid awmna PVDF strip te chu 75μl negative mold solvent [chloroform/methanol (1:2) + 5 mM ammonium acetate]-ah resuspended lehin electrospray ionization (ESI)-MRM/MS Analysis of sphingolipid species (TSQ Vantage) passed a ni. Hetiang a nih chuan MS data chu negative ion mode-ah hmuh a ni.
Sawi tawh ang khan GPI anchor-a lipid portion chu [3H]-inositol-labeled GPI-AP (16) atang hian a inthen a ni. Lipids te hi thin-layer chromatography hmangin solvent system (55:45:10 chloroform-methanol-0.25% KCl) hmangin an then a, FLA-7000 (Fujifilm) hmangin visualized an ni.
Gas1-GFP (600×107) expressing cell te chu TNE buffer hmanga TNE buffer hmangin vawi hnih silfai a ni a, glass beads hmangin tihchhiat a ni a, chutah chuan centrifuge hmangin cell debris leh glass beads te chu paih chhuah a ni. Chumi hnuah supernatant chu 17,000 g-ah darkar 1 chhung 4°C-ah centrifuged a ni. Pellet chu TNE-ah silfai a ni a, 0.2% digitalis saponin awmna TNE-ah 1 U PI-PLC (Invitrogen) nen darkar 1 chhung 37°C-ah incubate a ni. Enzyme hmanga enkawl hnuah membrane chu 17,000 g-ah 4°C-ah darkar 1 chhung centrifugation hmangin lakchhuah a ni. Gas1-GFP immunoprecipitate turin supernatant chu GFP-Trap_A (ChromoTek) nen 4°C-ah zan khat chhung an incubate a. SDS-PAGE hmanga then hran Gas1-GFP thianghlim chu Coomassie brilliant blue hmangin an stain a. Gas1-GFP staining band chu aqueduct chhehvel gray atanga tan a ni a, chutah chuan iodoacetamide hmanga alkylation leh dithiothreitol hmanga reduction hnuah trypsin hmanga in-gel digestion tih a ni. GPI-glycans hmangin tryptic peptide leh peptide te chu extract leh dry rawh. Peptide vawt chu tui 20 μl-ah a hmin a. LC chhungah hian a then (8μl) inject rawh. Octadecylsilane (ODS) column (Develosil 300ODS-HG-5; chhung lam diameter 150 mm×1.0 mm; Nomura Chemical, Aichi Prefecture, Japan) hmangin peptide te chu gradient condition bik hnuaiah an thliar hrang a. Mobile phase chu solvent A (0.08% formic acid) leh solvent B (0.15% formic acid 80% acetonitrile-a siam) te an ni. Accela HPLC system (Thermo Fisher Scientific, Boston, Massachusetts) hmangin column chu solvent A hmangin minute 55 chhungin flow rate 50 μl min-1-ah minute 5 chhung elute a ni a, chutah chuan solvent B concentration chu 40%-ah tihsan a ni. , United States-ah a awm). Eluate chu ESI ion source-ah dah chhunzawm zel a ni a, tryptic peptide leh GPI-glycans nei peptide te chu LTQ Orbitrap XL (hybrid linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific) hmangin an zirchiang a. MS setup-ah chuan capillary source voltage chu 4.5 kV-ah dah a ni a, transfer capillary temperature chu 300°C-ah dah a ni. Capillary voltage leh tube lens voltage chu 15 V leh 50 V ah dah a ni. MS data chu positive ion mode (resolution 60,000; mass accuracy part 10 per million)-ah mass range 300/m/z mass/charge ratio (m/z) 3000-ah hmuh a ni a, MS/MS data hi LTQ Orbitrap XL-a ion trap hmanga hmuh a ni [data innghahna digit 3 hmasa ber, collision induced dissociation (CID) tih a ni].
MD simulations hi GROMACS (52) software leh MARTINI 2 force field (53-55) hmangin an ti a. Chumi hnuah CHARMM GUI Membrane Builder (56, 57) hmangin dioleoylphosphatidylcholine (DOPC) leh Cer C18 emaw DOPC leh Cer C26 emaw awmna bilayer siam a ni. Cer C26 topology leh coordinate te hi DXCE atanga lak chhuah niin sphingosine tail atanga extra beads te chu lakchhuah a ni. A hnuaia tarlan process hmang hian double layer balance la, run la, chutah chuan system coordinate hnuhnung ber hmangin Emp24 awmna system siam rawh. Yeast Emp24 (residue 173 atanga 193) transmembrane domain chu visual MD (VMD) tool molecular structure hmangin α-helix angin siam a ni (58). Tichuan, lipids inzawmkhawmte chu an paih chhuah hnuah protein chu coarse granulated niin CHARMM GUI hmangin bilayer-ah dah a ni. Final system-ah hian DOPC 1202 leh Cer C26 302 emaw DOPC 1197 leh Cer C18 leh Emp24 295 emaw a awm a ni. System chu 0.150M concentration ah ionize rawh. Bilayer composition pahnih atan independent replicate pali siam a ni.
Lipid bilayer hi CHARMM GUI process hmangin balance a ni a, chutah chuan step 405,000 tih tlem a, chutah chuan balance a ngai a, chutah chuan position constraints chu zawi zawiin tihhniam leh tihbo a ni a, time step chu 0.005 ps atanga 0.02 ps ah tihsan a ni. Equilibration hnuah 6 μs a siam chhuak a, time step chu 0.02 ps a ni. Emp24 i dah hnuah CHARMM GUI process ang chiah kha hmang la, system chu minimize leh balance la, chutah chuan production-ah 8 s chhung run rawh.
System zawng zawng tan chuan balancing process chhung hian Berendsen barostat (59) hian pressure a control a, production process chhung hian Parrinello-Rahman barostat (60) hmangin pressure a control bawk. A zawng zawngah chuan pressure average chu 1 bar a ni a, semi-isotropic pressure coupling scheme hman a ni. Balance leh production process-ah chuan speed recalibration nei thermostat (61) hmangin protein, lipid leh solvent particle te temperature chu couple a ni. Operation chhung zawng hian target temperature chu 310K a ni. Non-bonding interaction hi Verlet scheme hmanga pairing list siamin 0.005 buffer tolerance neiin chhut a ni. Coulomb term hi reaction field leh cut-off distance 1.1 nm hmanga chhut a ni. Vander Waals term hian cut-off scheme hmangin cut-off distance 1.1 nm a hmang a, Verlet cut-off scheme hi potential drift atan hman a ni bawk (62).
VMD hmang hian DOPC phosphate beads emaw ceramide AM1 beads emaw leh protein inkar cutoff wavelength chu 0.7 nm a ni a, protein nena inzawm lipids zat chu chhut a ni. A hnuaia formula ang hian (63)-a kan hmuh angin depletion-enrichment (DE) factor chu chhut la: DE factor = (protein chhunga lipid zawng zawng 0.7) protein 0.7-a Cer awm zat (lipid zawng zawnga Cer awm zat)
Reported value chu average anga hmuh a ni a, error bars te hi SE independent copy pali a ni. DE factor statistical significance chu t test [(averageDE-factor-1)/SE] hmanga chhut a ni. One-tailed distribution atang hian P value chu chhut rawh.
GROMACS tool hmang hian trace hnuhnung ber 250 ns chhunga Emp24 awmna system 2D lateral density map chu chhut a ni. Ceramide enrichment/depletion map hmuh theih nan Cer density map chu Cer leh DOPC map sum nen kan zat a, chutah chuan taksaa Cer awm zat nen kan zat a ni. Color map scale ang chiah hi hman a ni.
He thuziak atana supplementary materials hriat duh chuan http://advances.sciencemag.org/cgi/content/full/6/50/eaba8237/DC1 ah hian en theih a ni
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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 -Linero), Sergio Lopez (Sergio Lopez), Miho Waga (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz te an ni
3D high-resolution real-time imaging hmangin selective output site-a protein sorting atan ceramide chain sei zawng a pawimawhzia a lang.
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 -Linero), Sergio Lopez (Sergio Lopez), Miho Waga (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz te an ni
3D high-resolution real-time imaging hmangin selective output site-a protein sorting atan ceramide chain sei zawng a pawimawhzia a lang.
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