Secretory lam ah protein thleidannak cu cell tthencheunak le homeostasis kilvennak caah a biapi tukmi a si. Shell-mediated thleidannak lengah, secretory phurhnak kalning ah kinesin thleidannak ah lipids riantuannak cu caan saupi a um cangmi a hrampi biahalnak a si i a phi a um rih lo. Hika ah, 3D caankhatte ah rong phunphun a sangmi a ttha ngaimi caan taktak hmanthlaknak kan tuah i, a thar in sermi glycosylphosphatidylinositol-immobilized proteins pawl cu a sau tukmi ceramide lipid moieties he an i fonh i specialized endoplasms Net exit site ah an i tthen, cucu transmembrane proteins nih an hmanmi he aa dang. Cun, endoplasmic reticulum membrane chung i ceramide hri saunak cu hi thleidannak thimnak caah a biapi tukmi a si ti kha kan langhter. Kan hlathlainak nih hin a hmasa bik direct in vivo tehte a kan pek i, lipid chain saunak cung ah hram bunh in protein thilri pawl kha secretory lamthluan ah thimcia mi chuahnak hmun ah tthen khawhnak ding caah a si.
Eukaryotic cells ah, endoplasmic reticulum (ER) ah sermi protein pawl cu an i tlakmi cell kalnak hmun ah kuat khawhnak ding caah secretory lam in kalpi lio ah an thleidan hna (1). Coat-mediated thleidannak lengah, lipid cheukhat cu a hleiin protein (2-5) hna kha a hleiin membrane domain ah an i fonh hna i thim khawhmi chuahnak hmun ah an hman khawh tiah caan saupi chung ruah a si. Sihmanhsehlaw, hi thil a cang khomi thau-hram bunhmi thil sining langhternak caah direct in vivo tehte a um rih lo. Hi a hrampi buainak hi tawlrel khawhnak ding caah, glycosylphosphatidylinositol (GPI) anchored proteins (GPI-APs) hna cu ER in aa dang tein zeitindah an chuah timi kha yeast ah kan hlathlai. GPI-APs cu lipid he aa pehtlaimi cell cunglei protein phunphun an si ( 6, 7). GPI-AP cu glycolipid moiety (GPI anchor) hmangin plasma membrane lenglei cahmai ah aa pehmi a chuakmi protein a si. GPI anchors cu ER lumen (8) ah a tlawm deuhmi lehnak hnu remhnak ah an cohlan. Peh hnu ah, GPI-AP cu ER in plasma membrane tiang Golgi thilri (5, 9) chungin a kal. GPI hri a ummi nih GPI-AP cu transmembrane chuahmi protein (plasma membrane protein dang aa telmi) he a dang tein chuahnak lam ah a kalter (5, 9, 10). Yeast cells ah, GPI-APs cu endoplasmic reticulum chung i a dang a chuakmi proteins hna he an i tthen, cun coat protein complex II (COPII) nih a khuhmi aa dangmi vesicles ah an khumh hna (6, 7). ER chuahnak kalning ah hi thleidannak kalning a thlentu thil hna cu a fiang lo, asinain hi thil kalning nih hin lipid a herh men lai tiah ruah a si, a hlei in GPI anchor (5, 8) i lipid tthen a sining remhnak. Yeast ah, GPI lipid remhnak cu GPI aa peh hnu ah aa thawk colh, cun thil tampi ah, ceramide kha 26-carbon long-chain saturated fatty acid (C26:0) (11, 12) he aa pehtlaihter. C26 ceramide cu a tu tiang ah yeast cells nih an chuahmi ceramide bik a si. ER ah sermi a si i a tam deuh cu COPII vesicles (13) hmangin Golgi thilri ah kuat a si. GPI-AP ER chuahnak nih a bik in a kal cuahmahmi ceramide sernak a herh (14, 15), cun a hnu ah, Golgi thilri chung i ceramide in inositol phosphate ceramide (IPC) ah thlennak cu GPI anchor sernak cungah aa hngat (16). Artificial membranes he biophysical hlathlainak nih a langhter mi cu a sau tukmi acyl chain ceramides pawl cu aa dangmi taksa sining he aa pehtlaimi domain pawl ser awkah an i fonh khawh (17, 18). Hi data nih hin C26 ceramide le GPI-AP cu C26 ceramide he an pumsa sining hna kha a ttha ngaimi hmun asiloah a ttha lomi ER membrane lipid pawngkam ah aa fonh khawhnak ding caah an hman timi ruahnak a chuahter. A bik in a tawimi le a tling lomi glycerolipids (C16:1 le C18:1) (19, 20) in sermi a si. Hi pengtlang hna cu ER chuahnak hmun (ERES) ah thim in an i hngat lai, cuka ahcun ceramide le ceramide-based GPI-AP cu COPII vesicle (5) ah Golgi ah an kalpi khawh hna lai.
Hi hlathlainak ah, hi lipid-based mechanism hi super-resolution confocal real-time imaging microscopy (SCLIM) hmangin direct in kan hneksak, cucu a thar in hmanmi microscopy thiamnak a si i, a ceumi min bunhmi protein pawl kha hmunkhat te ah a zoh khawh.
S. cerevisiae ah ER chuahtak hnu ah C26 ceramide phu he a ttha mi GPI-AP cu transmembrane chuahmi proteins in zeitindah thleidan a si timi fianternak caah SCLIM thiamnak kha kan hman hmasa. ER thleidannak zohfel awkah, ERES ah a lutmi thilri thar sermi kha direct in hmuh khawhnak genetic system kan hman (7, 23). Thilri ah, C26 ceramide-based GPI-AP Gas1 cu a senmi a ceumi protein (GFP) he aa ttialmi le transmembrane secreted protein Mid2 cu near-infrared a ceumi protein (iRFP) he aa ttialmi kan thim, cu hna pahnih nih cun plasma membrane kha an target (24–26). sec31-1 lum le kih a hngalh khomi thlennak ah, hi thilri pahnih hna hi galactose-inducible promoter le constitutive ERES marker tangah an langhter. A lin tukmi (37°C) ah, sec31-1 thlennak nih COPII hrinnak le ER chuahnak khamnak caah COPII coat component Sec31 riantuannak a hnorsuan caah, a thar in sermi thilri cu ER ah aa pum (23). A niam deuhmi lumnak (24°C) tiang a kik hnu ah, sec31-1 mutant cells cu a chuahnak hmun in an dam ṭhan, cun a khonmi thilri thar cu ER in chuah hram an thawk. CLIM hmuh khawhnak nih a langhter mi cu a thar in sermi Gas1-GFP le Mid2-iRFP tam deuh cu 37°C ah chiah hnu ah sec31-1 mutant cells ER ah an i khon rih i 24°C ah minutes 5 chung an chuah (Figure 1). Mid2-iRFP cu ER membrane dihlak ah aa phawt caah, cun Gas1-GFP cu aa pehtlaimi ER membrane hmun ah aa fonh i aa fonh caah, an i phawtzamhnak cu aa dang dih (Figure 1, A in C le Movie S1). Cun, Figure 1D ah langhter bantuk in, Gas1-GFP phu nih Mid2-iRFP a ngei lo. Hi hmuhmi nih hin GPI-AP le transmembrane protein pawl cu ER membrane hmun dangdang ah an i tthen hmasa ti a langhter. Gas1-GFP phu cu mCherry's COPII coat protein Sec13 (Figure 1, E le F, le movie S1) (23) he aa pehtlaimi ERES he aa naih.
sec31-1 cells nih galactose-induced secretions an langhter, a sau mi acyl chain (C26) ceramide GPI-AP Gas1-GFP (GPI-AP, a sen) le transmembrane protein Mid2-iRFP (TMP, sen) le hi Constructive ERES labeling Sec13-mCherry (E) cu 30°C ah 37 °C ah an chiah minutes, 24°C ah ṭhial, cun minutes 5 hnu ah SCLIM nih hmanthlak a thlak. (A in C tiang) nih vanlawng pakhat i aa fonhmi asiloah pakhat lawng a simi 2D hmanthlak (A), 10 z-sections 2D langhternak hmanthlak (B) asiloah thilri le ERES hmelchunhnak (C) i 3D cell hemisphere hmanthlak a langhter. Scale bar 1μm (A le B). Tahnak unit cu 0.551μm (C) a si. Gas1-GFP cu a dang tein ER pengtlang asiloah bu ah hmuh a si, cu lio ah Mid2-iRFP cu hmuh a si i ER membrane (C) chung vialte ah phawtzamh a si. (D) Graph nih Gas1-GFP bu chung i Gas1-GFP le Mid2-iRFP i aa pehtlaimi ceunak tthawnnak kha thal rang line (orhlei) ah a langhter. AU, duh paoh in tuahmi unit. (E le F) nih thilri le ERES hmelchunhnak aa fonhmi 3D hmanthlak kha a aiawh. Gas1-GFP phu pawl cu a bik in ERES pawng ah hmuh an si. Tahnak unit cu 0.551μm a si. (F) A senmi thal nih ERES he aa pehtlaimi Gas1-GFP phu kha a langhter. A laifang le orhlei panel nih fonhmi ngantermi 3D hmanthlak le thimcia mi Gas1-GFP bu i aa merhmi hmuhnak a langhter.
Gas1-GFP bu le ERES pakhatkhat karlak i a naihmi hmunhma pehtlaihnak nih Gas1-GFP nih thim khawhmi ERES ah a luh khawh ti a langhter, cucu Mid2-iRFP nih ER chuahtak awkah a hmanmi thim khawhnak he aa dang. Hi thil cang khomi hi tawlrel awkah, thilri pakhat asiloah pahnih lawng caah ERES ratio kha kan tuak (Figure 2, A in C tiang). ERES tam deuh (70%) nih thilri phunkhat lawng an ngei ti kan hmuh. Figure 2C i a tanglei hmanthlak nih Gas1-GFP lawng (Figure 1) asiloah Mid2-iRFP lawng (Figure 2) he ERES tahchunhnak pahnih a langhter. Cu he aa dang in, ERES 20% hrawng nih hmunkhat ah aa tlukmi thilri pahnih a ngei. ERES cheukhat (10%) nih thilri phun hnih an ngei ti hmuh a si, asinain hmun dangdang ah an i ṭhen. Cucaah, hi zatlang hlathlainak nih ER chuah hnu ah, GPI-AP Gas1-GFP le transmembrane cargo Mid2-iRFP cu ERES dangdang ah an i ṭhen ti a langhter (Figure 2D). Hi thleidannak tthatnak cu a hlan i biochemical hlathlainak (6) le morphological tuaktannak (7) he aa tlak ngai. ERES chung a lutmi thilri chiahmi hna an ziaza zong kan zoh khawh (Figure 2E le Movie S2). Figure 2E nih a langhter mi cu Gas1-GFP (panel 3) asiloah Mid2-iRFP (panel 4) tlawmpal lawng nih ERES ah khatlei in a lut i a dang tein a ummi hmun ah a um. Figure 2E i Panel 5 nih a langhter mi cu Gas1-GFP le Mid2-iRFP cu a caan ah ERES pakhat ah hmuh an si, asinain kap dangdang in an lut i COPII vesicles dangdang aiawh khomi hmun dangdang ah an i pum. Cun, C26 ceramide-based GPI-AP Gas1 kha thimfung thlaknak ERES tiah hmuhmi thleidannak le thleidannak cu a hleiin a si ti zong kan fehter, zeicahtiah a dang transmembrane chuahnak thilri, GFP-tagged plasma membrane protein Axl2 (27 ), nih Mid2-iRFP he aa lo ngaimi ziaza a langhter. (Hmanthlak S1 le Movie S3). A thar in sermi Axl2-GFP cu Mid2-iRFP bantuk in ER membrane chungin a phawtzamh (Figure S1, A le B), cun ERES tam deuh ah Mid2-iRFP he hmunkhat ah a um (Figure S1, B in D tiang). Figure 1 i panel 1 le 2. S1C nih ERES tahchunhnak pahnih a langhter i cuka ahcun transmembrane thilri pahnih an i tluk. Hi bantuk thil sining ah, thilri pahnih cu ERES ah hmunkhat ah an lut (Figure S1E, Panel 3 le Movie S3).
Galactose inducible secretions a langhtermi sec31-1 cells, Gas1-GFP (GPI-AP, hring) le Mid2-iRFP (TMP, sen) le constitutive ERES labeling Sec13-mCherry (ERES, magenta) cu 37 ah chiah an si. minutes 20 hnu ah SCLIM he. (A in C) A aiawhtu 2D langhternak hmanthlak (A; tahfung bar, 1μm) asiloah 3D cell hemisphere hmanthlak (B le C; tahfung unit, 0.456μm) thilri le ERES in hmelchunhmi 10 z-sections. (B) chung i a tanglei panel le (C) chung i panel nih ERES (magenta) [Gas1-GFP (sen) le Mid2-iRFP (sen rang)] chung i a ummi thilri lawng langhter awkah hmanthlak suaimi an langhter. (C) A ongmi thal: ERES nih thilri pakhat lawng a phorh (1 in 4). A senmi thal: ERES nih aa ṭhenmi thilri (5) a ngei. A fekmi thal rang: ERES ah hmunkhat ah a ummi thilri aa tel. A tanglei: Thimmi ERES pakhat lawng nih Gas1-GFP (1) asiloah Mid2-iRFP (2) lawng a ngei. Tahfung, 100 nm. (D) (C) ah langhtermi hmanthlak hmete zat tahnak. ERES ah a tlangpi in thilri pakhat lawng aa telmi (Gas1-GFP asiloah Mid2-iRFP), thilri tthenmi le aa tlukmi thilri. Aa hngat lomi hneksaknak pathum ah, cell 54 ah n=432. Palhnak bar = SD. Two-tailed unpaired t test. *** P = 0.0002. (E) (C) in hmelchunhmi thleidanmi thilri thimcia mi ERES 3D hmanthlak. Gas1-GFP (a sen) (3) asiloah Mid2-iRFP (a sen) (4) cu khatlei in ERES (magenta) ah a lut i ERES chung hmun hmete ah a kham. Cheukhat ahcun, thilri phun hnih hna cu ERES (5) ah khatlei in an lut i ERES chung ah a dang tein a ummi hmun ah an i hren. Tahfung, 100 nm.
A hnu ah, ER membrane chung i a ummi acyl chain ceramide (C26) sau nih Gas1 kha thimfung thlakmi ERES ah a tthencheunak le thleidannak a hruai timi ruahnak cheuhnak kha kan hneksak. Cu caah, remhmi changvut phun GhLag1 kan hman, cu ah cun endogenous ceramide synthases pahnih Lag1 le Lac1 cu GhLag1 (cotton i Lag1 homolog) nih a thlen hna, cu nih cun changvut phun cu ramlak phun nakin a tawi deuhmi cell membrane Ceramide phun a chuahter (Figure 3A) (28). Mass spectrometry (MS) hlathlainak nih a langhter mi cu ramlak phun ah, ceramide dihlak 95% cu a sau tukmi (C26) chain ceramide a si, GhLag1 ahcun, ceramide 85% cu a sau tuk (C18 le C16). ), ceramide 2% lawng hi a sau tukmi (C26) hri ceramide a si. C18 le C16 ceramides cu atu tiang GhLag1 membrane ah hmuhmi ceramides bik an si ko nain, MS hlathlainak nih GhLag1 phun ah langhtermi Gas1-GFP GPI anchor ah C26 ceramide aa tel ti zong a fehter, cucu wild-type lipids he tahchunh khawh a si. A tthatnak cu aa khat (Fig. 3A) (26). Cucaah, a sullam cu ceramide remhthannak enzyme Cwh43 cu C26 ceramide caah a ttha tukmi a si tinak a si, Figure 26 ah hmuh khawhmi bantuk in, GhLag1 phun chung i C26 ceramide tlawmte in GPI anchor kha a duh deuh. S2 (29). Sihmanhsehlaw, GhLag1 cell membrane nih a hrampi in C18-C16 ceramide lawng a ngei, Gas1-GFP nih C26 ceramide a ngei rih. Hi thil nih hin hi phun hi ER chung i membrane ceramide acyl chain saunak kong he pehtlai in a bik in tawlrelnak caah thilri tha taktak ah a ser. Class le thleidannak riantuannak ruahnak. Cun, C26 Gas1-GFP nih GhLag1 ah sec31-1 i lum le kih a tuar khomi aa thlengmi allele he bu in a i fonh khawhnak kha a hmasa bik ah kan hlathlai hmasa, cuka ahcun ER membrane Ceramide ah a saumi (C18-C16) hri lawng a um (Fig. 3). Sec31-1 ah, Gas1-GFP tam deuh cu bu ah aa fonh ti kan hmuh, cu lio ah sec31-1 GhLag1 chung i Gas1-GFP cu a sau (C18-C16) sau ceramide ER membrane he a tlangpi in aa fonh lo i ER membrane dihlak ah aa ṭhen. A dikthliar in chim ahcun, C26 ceramide-based clustering cu ERES (Figure 1) he aa naih caah, hi thil kalning nih hin ER chuahnak protein mechanism riantuannak zong aa tel kho men maw ti kha kan hlathlai tthan. GPI-AP nih ER chuahnak caah a hleiin COPII system a hman, cucu Ted1′s nih GPI anchor (30, 31) i glycan tthen a sining remhnak nih a uk. Recombinant GPI-glycan cu transmembrane cargo receptor p24 complex nih a hngalh, cu nih cun Lst1 kha a thim in a lak, cucu COPII cargo binding subunit Sec24 i a hleiin a ummi isoform a si, GPI-AP-rich COPII Vesicles a ser (31-33). Cucaah, hi protein pakhat lawng (p24 complex component Emp24, GPI-glycan remhthannak enzyme Ted1 le a hleiin COPII subunit Lst1) hna kha sec31-1 mutant strain he fonh in double mutant kan ser i, Gas1-cluster ser khawh a si maw (Figure 3FP) kan hlathlai. Sec31-1emp24Δ le sec31-1ted1Δ ah, Gas1-GFP cu a tlangpi in aa fonh lomi le ER membrane chung vialte ah aa ṭhenmi a si ti kha kan hmuh, sec31-1 GhLag1 ah a hlan ah hmuh cangmi bantuk in, sec31-1lst1Δ ah, Gas1-GFP Like sec31-1-1Δ ah. Hi hmuhmi nih hin ER membrane ah C26 ceramide a um pin ah, Gas1-GFP i fonhnak zong nih p24 complex he aa pehtlaih a herh, cun Lst1 laknak a herh lo ti a langhter. Cun, ER membrane chung i ceramide i a hri saunak nih Gas1-GFP le p24 pehtlaihnak a uk khawhnak ding lam kha kan hlathlai. Sihmanhsehlaw, membrane chungah C18-C16 ceramide a ummi nih p24 complex nih a ser ṭhanmi GPI-glycans (Figure S3 le S4, A le B) asiloah GPI-AP he aa pehtlaimi le GPI-AP a chuahtermi kha a hnorsuan lo ti kha kan hmuh. thil ti khawhnak. COPII subtype Lst1 (Figure S4C) kha lak. Cucaah, C26 ceramide-dependent clustering nih hin ER chuahnak protein mechanism phunphun he protein pehtlaihnak a herh lo, asinain lipid saunak nih a hruaimi thleidannak mechanism dang a dirpi. Cun, ER membrane chung i ceramide acyl chain saunak cu Gas1-GFP kha thimfung thlaknak ERES tiah ttha tein thleidan khawhnak caah a biapi maw ti kha kan hlathlai. GhLag1 phun chung i Gas1 cu short-chain ceramide he ER in a chuak i plasma membrane chungah a lut caah (Figure S5), thleidannak cu ceramide acyl chain saunak nih a hruai ahcun, GhLag1 phun chung i Gas1 cu a thlen khawh i a tan khawh tiah kan zumh. ERES thilri hna cu aa khatmi membrane he.
(A) GhLag1 cell membrane ah a tawi deuhmi C18-C16 ceramides aa tel, cu lio ah Gas1-GFP GPI anchor nih cun wild-type cells he aa khatmi C26 IPC a ngei rih. A cunglei: mass spectrometry (MS) hmangin wild-type (Wt) le GhLag1p phun hna i cell membrane chung i ceramide acyl chain saunak hlathlainak. Data nih hin ceramide dihlak zatuak a langhter. Aa hngat lomi hneksaknak pathum i a tlangpi. Palhnak bar = SD. Two-tailed unpaired t test. **** P ＜0.0001. A tanglei hmanthlak: Gas1-GFP (GPI-IPC) GPI anchor ah a ummi IPC i acyl chain saunak MS hlathlainak cu wild-type le GhLag1p phun ah langhtermi a si. Data nih IPC signal dihlak zatuak a aiawh. Aa hngat lomi hneksaknak panga karlak. Palhnak bar = SD. Two-tailed unpaired t test. ns, a biapi lo. P = 0.9134. (B) Galactose-induced Gas1-GFP a langhtermi sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ le sec31-1lst1Δ cells hna i an ceunak micrographs cu 37°C ah minutes 30 chung an chiah hna i an riantuannak kha an tlawmter 24°C hnu ah microscopy. Thla rang: ER Gas1-GFP bu. A ongmi thal: Aa fonh lomi Gas1-GFP cu ER membrane dihlak ah aa ṭhen, ER sining nuclear ring rong a langhter. Tahnak hri, 5μm. (C) (B) ah langhtermi hmanthlak hmete zat tahnak. Gas1-GFP sining a ngeimi cells zatuak tlawmpal. Aa hngat lomi hneksaknak pathum ah, n≥300 cells. Palhnak bar = SD. Two-tailed unpaired t test. **** P ＜0.0001.
Hi buainak hi direct in tawlrel khawhnak ding caah, GhLag1 chung i Gas1-GFP le Mid2-iRFP kha sec31-1 lum le lum lo aa thlengmi allele (Figure 4 le Movie S4) he SCLIM hmuhsaknak kan tuah. ER cu 37°C ah chiah a si hnu ah 24°C ah chuah a si hnu ah, a thar in sermi Gas1-GFP tam deuh cu ER membrane chung vialte ah aa fonh lo i a karh lo, a tlangpi in hmanmi microscope in hmuh khawh a si (Figure 4, A le B ). Cun, ERES zatuak tam deuh (67%) nih a chung ah hmunkhat ah a ummi thilri phun hnih aa tel (Figure 4D). Figure 4C i panel 1 le 2 nih Gas1-GFP le Mid2-GFP aa tlukmi ERES tahchunhnak pahnih an langhter. Cun, thilri pahnih cu ERES pakhat ah an lak hna (Figure 4E, panel 3 le movie S4). Cucaah, kan hmuhmi nih ER membrane chung i ceramide acyl chain saunak cu ER protein fonhnak le thleidannak ah a biapi mi thil pakhat a si ti a langhter.
Sec31-1 GhLag1 cells galactose-induced secretions, Gas1-GFP (GPI-AP, green) le Mid2-iRFP (TMP, blue) le constitutive ERES-labeled Sec13-mCherry (ERES, magenta) a langhtermi 37°C ah chiah. Minute 30 chung pehzulh, 24°C ah tthumh law a chuakmi chuah, cun minutes 20 hnu ah SCLIM in hmanthlak. (A in C) 2D langhternak hmanthlak (A; tahfung bar, 1μm) asiloah 3D cell hemisphere hmanthlak (B le C; tahfung unit, 0.45μm) cargo le ERES in hmelchunhmi z-section 10 chungin. (B) chung i a tanglei panel le (C) chung i panel nih ERES (magenta) [Gas1-GFP (sen) le Mid2-iRFP (sen rang)] chung i a ummi thilri lawng langhter awkah hmanthlak suaimi an langhter. (C) A sen in a khatmi thal: ERES, thilri aa tluk. Onmi thal: ERES nih thil pakhat lawng a ngei. A tanglei tthen: Thimmi ERES nih (C) ah hmelchunhmi thilri (1 le 2) aa tlukmi a ngei. Tahfung, 100 nm. (D) (C) ah langhtermi hmanthlak hmete zat tahnak. Sec31-1 le sec31-1 GhLag1 unit ah, thilri pakhat lawng (Gas1-GFP asiloah Mid2-iRFP) aa tel, cun a dang tein a ummi thilri le aa tlukmi thilri caah ERES zatuak tlawmpal aa tel. Aa hngat lomi hneksaknak pathum ah, n = 432 in 54 cells (sec31-1) le n = 430 in 47 cells (sec31-1 GhLag1). Palhnak bar = SD. Two-tailed unpaired t test. *** P = 0.0002 (sec31-1) le ** P = 0.0031 (sec31-1 GhLag1). (E) (C) ah hmelchunhmi aa tlukmi thilri (3) he thimmi ERES 3D hmanthlak. Gas1-GFP (a sen) le Mid2-iRFP (a sen) nih ERES (a sen) kha khatlei in an naih i ERES khamnak hmun pakhat ah an um. Tahfung, 100 nm.
Hi hlathlainak nih hin direct in vivo tehte a pek i, lipid-based protein cargoes cu secretory lamthluan ah thimfung thlaknak hmun ah tthen an si, cun thleidannak thim khawhnak caah acyl chain saunak a biapitnak a langhter. SCLIM timi a tthawngmi le a thar in sermi microscopy thiamnak hmangin, a thar in sermi Gas1-GFP (plasma membrane GPI-AP nganpi cu a sau tukmi acyl chain (C26) ceramide lipid tthen a ngeimi) kha yeast ah kan langhter) A dang tein ERs ah aa fonhmi hmun hna cu a hleiin ERE he aa pehtlaimi an si, cu lio ah transmembrane proteins cu membrane chungin an i phawt (Hmanthlak 1). Cun, hi thilri phun hnih hna nih hin ERES dangdang ah thim tein an lut (Figure 2). Membrane chung i cellular ceramide i acyl chain saunak cu C26 in C18-C16 tiang a zor, Gas1-GFP bu cu aa thleidangmi ER peng ah aa thlen, cun Gas1-GFP cu ERES pakhat chungin transmembrane protein he ER chuahtak dingin lam a thlen (Figure 3 le Figure 3). 4).
GPI-AP nih ER chuahtak awkah a hleiin sermi protein mechanism a hman ko nain, C26 ceramide-a hngatmi tthennak cu ERES thiamnak a chuahter khomi aa dangmi protein pehtlaihnak cungah aa hngat lo ti kha kan hmuh (Figures S4 le S5). Cu nakin, kan hmuhmi nih hin lipid-based protein clustering le a hnu ah a dang thilri pawl hrialnak nih a hruaimi thleidannak lam dang kha a dirpi. Kan hmuhmi nih a langhter mi cu Gas1-GFP peng asiloah ERES he aa pehtlaimi bu nih hin transmembrane chuahmi protein Mid2-iRFP a ngei lo, cucu C26 ceramide-a hngatchanmi GPI-AP bu nih aa pehtlaimi ERES ah an luh khawhnak ding caah a bawmh hna lai, cun cu caan te ah cun, transmembrane kha a chuahter lai. Cu he aa dang in, ER membrane chung i C18-C16 ceramides a ummi nih GPI-AP kha pengtlang asiloah bu a serter lo, cucaah annih nih transmembrane chuahmi protein pawl kha ERES pakhat ah an chuah hna lo asiloah an thlen hna lo (Figure 3 le 4). . Cucaah, C26 ceramide nih ERES he aa pehtlaimi protein pawl i fonhnak a bawmh hna i tthennak le thleidannak a hruai tiah ruahnak kan cheuh.
Zeitindah hi C26 ceramide-a hngatmi ER hmun pakhatkhat ah fonh khawh a si lai? Membrane ceramide nih a kam lei in aa ṭhen duhnak nih GPI-AP le C26 ceramide kha a tawi deuhmi le a tling lomi glycerolipids aa telmi ER membrane i a tlarimi lipid pawngkam ah a hme deuhmi le a rannak in a tlarimi lipid a chuahter khawh. Quality clusters (17, 18). Hi caan karlak bu hmete hna cu p24 complex (34) he an i pehtlaih hnu ah a ngan deuhmi, a fek deuhmi bu ah an i fonh khawh. Cu he aa tlak in, C26 Gas1-GFP nih hmuh khawhmi bu ngan deuh ser awkah p24 complex he pehtlaihnak a herh ti kha kan langhter (Figure 3). p24 complex cu heterozygous oligomer a si i yeast (35) chung i p24 transmembrane protein aa dangmi pali in sermi a si, mah nih hin multivalent binding a pek, cu nih cun GPI-AP clusters hmete pawl i pehtlaihnak a chuahter khawh, cucaah Stable cluster ngan deuh a chuahter khawh (34). GPI-APs i protein ectodomains karlak pehtlaihnak zong nih an i fonhnak ah a bawmh khawh hna, cucu saram polarized epithelial cells (36) ah an Golgi phorh lio ah a lang. Sihmanhsehlaw, C18-C16 ceramide cu ER membrane ah a um tikah, p24 complex nih Gas1-GFP he aa pehtlaih tikah, a dang tein a ummi bu ngan pipi an chuak lai lo. A tanglei thil kalning cu a saumi acyl chain ceramide i a hleiin a ummi thilri le chemical sining cungah aa hngat kho men. Artificial membranes kong hlathlainak nih a langhter mi cu a sau (C24) le a tawi (C18-C16) acyl chain ceramides nih tthencheunak a chuahter ko nain, a saumi acyl chain ceramides (C24) lawng nih film a sersiam khawhnak ding caah a sangmi a tliknak le film bending a chuahter khawh. Pakhat le pakhat i pehtlaihnak thawngin (17, 37, 38). TMED2 i transmembrane helix, Emp24 i minung homologue nih, cytoplasmic lobules chung i C18 ceramide-based sphingomyelin he thim tein pehtlaihnak a tuah ti a si (39). Molecular dynamics (MD) simulations hmangin, C18 le C26 ceramides pahnih cu Emp24 transmembrane helix i cytoplasmic lobules pawng ah an i pum ti kan hmuh, cun an duhnak aa khat (Figure S6). Emp24 transmembrane helix nih membrane chung i lipids aa tluk lomi tthennak a chuahter khawh ti a langhter ti kha hngalh awk tlak a si. Mah cu saram cell cung ah hram bunh in naite ah hmuhmi a si. Cu bantuk MD zohchunhnak nih ether lipids (40) a um ti zong a langhter. Cucaah, ER26 lobule pahnih chung i C26 ceramide cu hmunkhat te ah a rum tiah kan ruah. Luminal lobules chung i GPI-AP nih multivalent p24 he direct in aa pehtlaih tikah le cytoplasmic lobules chung i p24 pawngkam ah C26 ceramide a khon tikah, a zulmi Protein fonhnak le membrane curvature cu kutdong in a chuak (41), cu nih cun GPI-AP cu E he aa pehtlaimi hmun dangdang ah a ṭhenter khawh, cu nih cun a sangmi RES zong a bawmh ER membrane (42). A hlan report nih ruahnak cheuhnak mechanism kha a dirpi (43, 44). Plasma membrane cung i ceramide-based glycosphingolipids (GSL) he oligolectins, zawtnak rungrul asiloah antibodies hna i an i pehtlaihnak nih GSL fonhnak nganpi a chuahter, tthencheunak a ṭhanchoter i membrane thlennak le chunglei luhternak a chuahter (44). Iwabuchi tbk. (43) A sau (C24) asinain a tawi lo (C16) acyl hri a um tikah, GSL lactosylceramide he aa pehtlaimi multivalent ligand nih bu nganpi sernak le membrane luhhnawhnak a chuahter, cun cytoplasm Lyn-mediated signal transduction cu leaflets cung i neutrophils chains in aa pehtlaimi a si ti kha hmuh a si.
Mammalian polarized epithelial cells ah, anti-Golgi network (TGN) a tlawmtam ning cu apical plasma membrane tiang a si i GPI-AP tthennak le thleidannak kha a uk (10, 45). Hi fonhnak cu GPI-AP oligomerization (36) nih a hruai, asinain thilnu ah kan hmuhmi ceramide hri saunak cungah zong aa hngat kho. Saram GPI-AP nih ether lipid-based anchor a ngeih ko nain, a chemical sining cu a sau tukmi acyl chain ceramide he aa dang tuk ko nain, naite ah tuahmi hlathlainak nih lipid pahnih hna cu thlennawnnak lei in aa khatmi pumsa le chemical sining le riantuannak an ngei ti a hmuh (40). Cucaah, saram cell chung i ether lipid tthen cu changvut chung i C26 ceramide he aa lo kho, cun a rian cu GPI-AP fonhnak le thleidannak thanchoternak caah membrane chung i long-chain ceramide he pehtlaihnak tuah a si. Hi thil cang khomi hi direct in hneksak a herh rih ko nain, a hlan i hmuhmi nih a sau mi acyl chain ceramide cu Golgi pum ah kalpi cu cytoplasmic thlennak proteins nih a tuah lo, asinain yeast bantuk GPI anchors sernak cungah aa hngat ti kha a dirpi. Cucaah, thlennawnnak kilvennak mechanism nih a sau tukmi acyl chain ceramide le GPI-AP (13, 16, 20, 46, 47) kha phurhnak vesicle pakhat ah thim tein a kalpi khawh hna.
Yeast le mammalian polarized epithelial cell systems ah, GPI-AP i fonhnak le a dang plasma membrane proteins he i tthennak cu cell cunglei a phanh hlanah a cang dih. Paladino le a dangdang. (48) nih saram polarized epithelial cells TGN cung ah, GPI-AP fonhnak cu GPI-APs kha apical plasma membrane ah thimfung thlaknak caah a herh lawng si loin, GPI-APs fonhnak bu le a nunnak cawlcanghnak zong a uk ti kha a hmuh. Cell cunglei. Yeast ah, hi hlathlainak nih ER cung i C26 ceramide-dependent GPI-AP bu nih plasma membrane cung i GPI-AP bu dirhmun le riantuannak cawlcanghnak kha a uk khawh ti a langhter (24, 49). Hi model he aa tlak in, GhLag1 cells cu GPI khamnak sii asiloah cell vampang tthatnak a hnorsuanmi sii (28) cungah an lung a tling lo, cun yeast cells i tthennak ah langhtermi tip ceramide (49) riantuannak Gas1-GFP phu (49) a herhnak nih G hLag1 cells nih a chuahpi khawhmi pumsa lei thil sining a langhter. GPI-AP palhnak. Sihmanhsehlaw, cell cunglei riantuannak dirhmun cu ER in lipid saunak cung ah hram bunh in thleidannak lam in program tuah a si maw ti hneksaknak tuah chap cu kan hmailei hlathlainak kong a si lai.
Hi rian ah hmanmi Saccharomyces cerevisiae phun pawl cu Table S1 ah an langhter. A nungmi cell hmanthlaknak caah SCLIM phun MMY1583 le MMY1635 cu W303 hnulei ah sermi an si. Sec13-mCherry a langhtermi hi phun pawl cu a ceumi protein tag he polymerase chain reaction (PCR)-based method hmangin pFA6a plasmid kha template ah an ser (23). GAL1 promoter uknak tangah a ceumi protein in min bunhmi Mid2-iRFP a langhtermi phun cu a tanglei bantuk in ser a si. pKTiRFP-KAN vector (E. O'Shea laksawng, Addgene plasmid number 64687; http://n2t.net/addgene: 64687; research resource identifier (RRID): Addgene_64687) chungin iRFP-KanMx sequence PCR amplification le Midgenous Cend endous chungah khumh. Mid2-iRFP genome sequence cu a nganter i GAL1 promoter ah a thlen hnu ah, aa fonhmi plasmid pRS306 i Not I-Sac I hmun ah a fonh. A chuakmi plasmid pRGS7 cu Pst I he URA3 locus ah fonh dingin linearized a si.
Gas1-GFP fonhmi gene cu centromere (CEN) plasmid chung i GAL1 promoter uknak tangah langhter a si, cucu a tanglei bantuk in sermi a si. Gas1-GFP sequence cu pRS416-GAS1-GFP plasmid (24) (L. Popolo laksawng) in PCR in a karhter i CEN plasmid pBEVY-GL LEU2 (C laksawng) i Xma I–Xho I hmun ah clone tuah a si. Miller; Addgene plasmid nambar 51225; http://n2t.net/addgene: 51225; RRID: Addgene_51225). A chuakmi plasmid cu pRGS6 tiah min an sak. Axl2-GFP fonhmi gene cu pBEVY-GL LEU2 vector i GAL1 promoter uknak tangah zong langhter a si, cun a serning cu a tanglei bantuk in a si. Axl2-GFP sequence cu pRS304-p2HSE-Axl2-GFP plasmid (23) in PCR in an karhter, cun pBEVY-GL LEU2 vector i Bam HI-Pst I hmun ah an thlen. A chuakmi plasmid cu pRGS12 tiah min an sak. Hi hlathlainak ah hmanmi oligonucleotides pawl an i zulhnak cu Table S2 ah langhter a si.
Cu phun cu 0.2% adenine le 2% glucose [YP-dextrose (YPD)], 2% raffinose [YP-raffinose] rich yeast extract protein p (YP) medium (1 % Yeast extract le 2% protein ept) he an chap. (YPR)] asiloah 2% galactose [YP-galactose (YPG)] cu carbon hrampi ah, asiloah sermi a tlawm bik thilri (0.15% yeast nitrogen base le 0.5% ammonium sulfate) ah rawl eidin caah a herhmi aa tlakmi amino acid le base pawl chap awkah, Cun 2% glucose (2% glucose glucose) aa telmi (2% glucose medium) asiloah a tlawmbik galactose (synthetic galactose minimal medium) cu carbon hrampi ah hman a si.
Caan hmaan tein hmanthlaknak caah, GAL1 promoter tangah a ummi thilri a langhtermi lum le kih a hngalh khomi sec31-1 mutant cells pawl cu YPR medium ah 24°C ah zankhat chung mid-log phase tiang an ṭhanter hna. YPG ah 24°C ah suimilam 1 chung an luhter hnu ah, cells pawl cu SG ah 37°C ah minutes 30 chung an chiah hna, cun 24°C ah an thlen hna i secretion block chungin an chuah hna. Concanavalin A cu thlalang cung i cell pawl remhnak caah hman a si i SCLIM nih hmanthlak a thlak. SCLIM cu Olympus IX-71 inverted fluorescence microscope le UPlanSApo 100×1.4 numerical aperture oil lens (Olympus), a rangmi le a sangmi signal-to-noise ratio aa mer khomi disc confocal scanner (Yokogawa Electric), custom spectrometer, cotomofier hmanthlak le a tthawngmi hmanthlak pawl fonhmi a si (Hamamatsu Photonics) nih a donghnak ah ×266.7 a ngantermi le electrons a karhtertu charge-coupled thilri camera (Hamamatsu Photonics) (21) a pek khawh. Hmanthlak laknak cu mah tein sermi software (Yokogawa Electric) nih a tuah. 3D hmanthlak caah, kanmah tein sermi piezoelectric actuator hmangin kan hmanmi lens kha a dirhmun in kan hninh, cun 100 nm aa hlatnak ah a ummi optical parts pawl kha stack ah kan khawmh hna. Z-stack hmanthlak cu 3D voxel data ah thlen a si, cun aa mer lengmangmi disc confocal microscope caah hmanmi theoretical point spread function cu Volocity software (PerkinElmer) nih deconvolution tuahnak caah hman a si. Volocity software hmangin hmunkhat ah umttinak hlathlainak caah amah tein threshold tuah in, thilri telh in ERES kha tah a si. Line scan hlathlainak cu MetaMorph software (Molecular Devices) hmangin tuah a si.
Statistical significance hngalh khawhnak dingah GraphPad Prism software kha hmang. Two-tailed Student's t-test le a tlangpi in one-way analysis of variance (ANOVA) test caah, phu karlak i dannak cu P <0.05 (*) ah tthatnak a chuahpi tiah ruah a si.
Gas1-GFP i a ceunak microscopy caah, log phase cells cu YPD ah zankhuadei an ṭhanter hna i centrifugation in an lak hna, phosphate buffered saline in voihnih an tawl hna, cun a tlawmbik minutes 15 chung tikhor cungah an chiah hna, cun a hlan ah langhtermi bantukin microscope tangah an kalpi hna Check (24). Leica DMi8 microscope (HCX PL APO 1003/1.40 oil PH3 CS) cu objective lens, L5 (GFP) filter, Hamamatsu camera le Application Suite X (LAS X) software he aa pehtlaimi kha laknak caah hman a si. .
Sample pawl cu SDS sample buffer in 65°C ah minutes 10 chung an thlen hna, cun SDS-polyacrylamide gel electrophoresis (PAGE) in an tthen hna. Immunoblotting hlathlainak caah, lam pakhat ah 10 μl sample an chiah. A hrampi antibody: 1:3000 tlawmternak ah vok polyclonal anti-Gas1, 1:500 tlawmternak ah vok polyclonal anti-Emp24, le 1:3000 tlawmternak ah vok polyclonal anti-GFP (H. Riezman laksawng) hmang. Mouse monoclonal anti-Pgk1 antibody cu 1:5000 (J. de la Cruz laksawng) tlawmternak in hman a si. Secondary antibody: Horseradish peroxidase (HRP) conjugated goat anti-rabbit immunoglobulin G (IgG) 1:3000 (Pierce) tlawmternak ah hmanmi. HRP-conjugated goat anti-mouse IgG cu 1:3000 (Pierce) tlawmternak ah hman a si. SuperSignal West Pico reagent (Thermo Fisher Scientific) i chemiluminescence phunglam in pumsa ralvennak hmun cu hmuh a si.
(31) ah langhtermi bantukin, a tthawnmi ER tthencheunak cungah a chuakmi immunoprecipitation hneksaknak tuah a si. A tawinak in, TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride le protease inhibitor cawhmi) in 600 nm ( OD600) ah 100 optical density voihnih in yeast cells kha tawl hna. Thlalang hri in an hrawh, cun cell rawhnak le thlalang hri pawl cu centrifugation in an lak hna. Cu hnu ah a cunglei ti cu 17,000 g ah 4°C ah minutes 15 chung an cawh. Pellet cu TNE ah an hnim ṭhan i digitalis saponin cu a donghnak 1% tiang an cawh. Suspension cu 4°C ah suimilam 1 chung thlennak he an chiah, cun a cawh kho lomi thilri pawl cu 13,000 g ah 4°C ah minutes 60 chung centrifugation in an lak hna. Gas1-GFP immunoprecipitation caah, a hmasa bik ah sample kha a thiang lomi agarose beads (ChromoTek) he 4°C ah suimilam 1 chung chiah hmasa, cun GFP-Trap_A (ChromoTek) he 4°C ah suimilam 3 chung chiah. Immunoprecipitated beads cu 0.2% digoxigenin aa telmi TNE in voi nga an tawl hna, SDS sample buffer in an thlet hna, SDS-PAGE ah an ṭhen hna, cun immunoblotting in an hlathlai hna.
(31) ah langhtermi bantukin, a tthawntermi ER tthencheunak ah cross-linking biakhiahnak tuah a si. A tawinak in, a rummi ER tthen cu 0.5 mM dithiobis(succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, USA; 20°C, 20 min) he an cawh. Crosslinking reaction cu glycine (50 mM a donghnak tlawmtam, minutes 5, 20°C) chapnak in a dai.
A hlan ah langhtermi (50) bantuk in, wild-type le GhLag1 phun ah ceramide MS hlathlainak tuah a si. A tawinak in, cells cu YPD ah 30°C ah exponential phase (3 in 4 OD600 units/ml) tiang an ṭhanter hna i, 25×107 cells an lak hna. An pumsa thlennak cu trichloroacetic acid in a hmih. Extraction solvent [ethanol, ti, ether, pyridine le 4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] le 1.2 nmol chunglei standard C17 ceramide (860517, Avanti polar lipid) tthatnak kha hmang. Monomethylamine reagent [methanol, ti, n-butanol le methylamine cawhmi (4:3:1:5 v/v)] hmangin chuahmi thilri kha a nemmi alkaline hydrolysis tuah law, cun ti-a khatmi n-butanol hmangin chiti hlonh. A donghnak ah, chuahmi cu a ttha mi thlennak [chloroform/methanol/ti (2:7:1) + 5 mM ammonium acetate] ah an hnim tthan i mass spectrometer chungah an thun. Sphingolipid molecule pawl hngalh khawhnak le zat tuak khawhnak caah multi-reaction zohkhenhnak (MRM) tuah a si. TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) cu lipid hlathlainak caah robot nanoflow ion source Nanomate HD (Advion Biosciences, Ithaca, NY) he aa thuam. I pahnak thazaang cu ceramide phun pakhat cio caah a ttha bikmi a si. MS data cu a ttha lei in hmuh a si. Biological replicate pakhat cio caah, lipid signal cu aa hngat lomi tahnak pathum karlak a si.
(31) ah langhtermi bantukin, Gas1-GFP a langhtermi cells (800×107) cu a chuakmi immunoprecipitation ah an chiah hna. A thiangmi Gas1-GFP cu SDS-PAGE in tthen a si i polyvinylidene fluoride (PVDF) membrane ah thlen a si. Protein cu PVDF kha amide sen in rong in hmuh khawh a si. Gas1-GFP band cu PVDF chungin an tan i methanol in voi 5 le liquid chromatography-MS (LC-MS) grade ti in voikhat an tawl. Membrane strip cu 500μl 0.3 M NaOAc (pH 4.0), buffer le 500μl a thar in a cawhmi 1 M sodium nitrite cawhmi he 37°C ah suimilam 3 chung chiah in, lipid fraction cu Gas1-GFP in a chuak i lysed Release of infoosines of inphoosphatecomosphate in gluecomide le ceraeamin (51). Cu hnu ah, membrane strip cu LC-MS grade ti in voi li an tawl, khaan lumternak ah an roter, cun hlathlainak tuah hlan tiang -80°C ah nitrogen thli ah an chiah. Control ah, hneksaknak pakhat cio caah PVDF membrane a thiangmi sample hman a si. Gas1-GFP chungin chuahmi lipid cu MS nih a langhtermi (50) bantuk in hlathlainak tuah a si. A tawinak in, GPI-lipid aa telmi PVDF strips cu 75μl negative mold solvent [chloroform/methanol (1:2) + 5 mM ammonium acetate] ah an hren ṭhan hna i electrospray ionization (ESI)-MRM/MS Analysis of sphingolipid species (TSQ Vantage) an palh. Hi kongah, MS data cu negative ion mode in hmuh a si.
A hlan deuh ah kan chim cang bantuk in, GPI anchor i lipid tthen cu [3H]-inositol-labeled GPI-AP (16) he an i tthen. Lipid pawl cu a tlawmmi chromatography in solvent system (55:45:10 chloroform-methanol-0.25% KCl) hmangin an tthen hna i FLA-7000 (Fujifilm) hmangin an hmuh khawh hna.
Gas1-GFP (600×107) a langhtermi cells pawl cu TNE buffer he TNE buffer in voihnih an tawl hna, cun thlalang hri in an hrawh hna, cun cell rawhnak le thlalang hri pawl hloh awkah centrifuge an tuah hna. Cu hnu ah a cunglei ti cu 17,000 g ah suimilam 1 chung 4°C ah an cawh. Pellet cu TNE ah an tawl i 0.2% digitalis saponin aa telmi TNE ah 1 U PI-PLC (Invitrogen) he 37°C ah suimilam 1 chung an chiah. Enzyme thlopnak hnu ah, membrane cu 17,000 g ah 4°C ah suimilam 1 chung centrifugation in chuah a si. Gas1-GFP khamnak caah, a cunglei ti cu GFP-Trap_A (ChromoTek) he 4°C ah zankhuadei an chiah. SDS-PAGE in tthenmi thianh cangmi Gas1-GFP cu Coomassie a ceumi sen in an thuh. Gas1-GFP rong cu ti chuahnak pawngkam a senmi in tan a si, cun iodoacetamide he alkylation tuah hnu le dithiothreitol he tlawmter hnu ah, trypsin he in-gel rawhnak tuah a si. GPI-glycans he tryptic peptides le peptides pawl kha chuah law roter. A ro cangmi peptide cu ti 20 μl ah cawh a si. LC chungah tlawmpal (8μl) thun. Octadecylsilane (ODS) column (Develosil 300ODS-HG-5; chunglei diameter 150 mm×1.0 mm; Nomura Chemical, Aichi Prefecture, Japan) cu a hleiin gradient dirhmun tangah peptide pawl thleidannak caah hman a si. A kal khomi tthen cu solvent A (0.08% formic acid) le solvent B (0.15% formic acid in 80% acetonitrile) an si. Accela HPLC system (Thermo Fisher Scientific, Boston, Massachusetts) cu 55 minutes chung ah 50 μl min-1 in minutes 5 chung a luannak rate in solvent A he column kha elute tuahnak caah hman a si, cun solvent B a tlawmtam kha 40% tiang a karhter. , United States). Eluate cu ESI ion source chungah pehzulh tein luhter a si, cun tryptic peptides le GPI-glycans he peptides cu LTQ Orbitrap XL (hybrid linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific) in an hlathlai. MS timhtuahnak ah, thihri hram i a tthawnnak cu 4.5 kV ah chiah a si, cun thlennak thirpi lumnak cu 300°C ah chiah a si. Capillary voltage le tube lens voltage cu 15 V le 50 V ah chiah an si. MS data cu positive ion mode (resolution 60,000; mass accuracy of 10 parts per million) ah 300/m/z mass/charge ratio (m/z) 3000 karlak ah hmuh a si. MS/MS data cu LTQ Orbit chung i ion trap hmangin hmuh a si i pahnak nih a chuahtermi tthencheunak (CID)].
MD lemsoinak cu GROMACS (52) software le MARTINI 2 force field (53-55) hmangin tuah a si. CHARMM GUI Membrane Builder (56, 57) cu dioleoylphosphatidylcholine (DOPC) le Cer C18 asiloah DOPC le Cer C26 aa telmi bilayer sernak caah hman a si. Cer C26 i a cunglei le a tthencheunak hna cu DXCE in sphingosine hngawng chungin a dang hri pawl chuah in lakmi an si. A tanglei ah langhtermi kalning kha hmangin tthen hnih kha tlukruannak tuah law kalpi, cun Emp24 aa telmi system sernak caah system a donghnak bik coordinate kha hmang. Yeast Emp24 (residues 173 in 193) i a transmembrane domain cu hmuh khawhmi MD (VMD) thilri molecular structure (58) hmangin α-helix in ser a si. Cun, aa tlukmi thau pawl kha hloh dih hnu ah, protein cu a tlawmte in an cawh i CHARMM GUI hmangin bilayer chungah an khumh. A donghnak system ah 1202 DOPC le 302 Cer C26 asiloah 1197 DOPC le 295 Cer C18 le Emp24 aa tel. System kha 0.150M tiang ionize tuah. Bilayer composition pahnih caah aa hngat lomi tlukruannak pali an tuah.
Lipid bilayer cu CHARMM GUI kalning hmangin aa tluk tein chiah a si, cu ah cun 405,000 step tlawmter le tlukruannak aa tel, cuka ahcun dirhmun khamnak cu zaangfah tein zorter le hloh a si, cun caan step cu 0.005 ps in 0.02 ps tiang karhter a si. Aa tluk hnu ah, 0.02 ps caan step he 6 μs a chuahter. Emp24 na khumh hnu ah, CHARMM GUI kalning kha hmang law system kha tlawmter le tlukruannak tuah, cun 8 s chung chuahnak ah kalpi.
System vialte caah, tlukruannak tuah lio ah, hneknak cu Berendsen barostat (59) nih a uk, cun chuah lio ah, hneknak cu Parrinello-Rahman barostat (60) nih a uk. Caan vialte ah, a tlangpi in hneknak cu 1 bar a si i semi-isotropic hneknak pehtlaihnak timhtuahnak hman a si. Tlukruannak le chuahnak kalning ah, thermostat (61) cu rang tukin thlennak he protein, lipid le solvent particles hna i an lumnak kha fonh awkah hman a si. Riantuannak chung vialte ah, tinhmi lumnak cu 310K a si. Aa pehtlaimi a si lomi pehtlaihnak cu 0.005 buffer tuar khawhnak he Verlet scheme hmangin aa pehtlaimi cazin sernak in tuak a si. Coulomb term cu lehrulhnak hmun le 1.1 nm tluknak hlatnak hmangin tuak a si. Vander Waals biafang nih 1.1 nm tthennak hlatnak he tthennak timhtuahnak a hman, cun Verlet tthennak timhtuahnak cu a cang khomi tliknak caah hman a si (62).
VMD hmangin, DOPC phosphate beads asiloah ceramide AM1 beads le protein karlak i cutoff wavelength cu 0.7 nm a si, cun protein he aa pehtlaimi lipids zat kha tuak a si. A tanglei formula ningin, (63) bantuk in depletion-enrichment (DE) factor kha tuak: DE factor = (protein chung i lipid dihlak zat 0.7) protein 0.7 chung i (lipids dihlak ah Cer zat)
Report tuahmi man cu a tlangpi in hmuh a si, cun palhnak hna cu SE i aa hngatmi copy pali an si. DE factor i a sullam cu t test [(averageDE-factor-1)/SE] in tuak a si. P man kha pakhat-tthen phawtzamhnak in tuak.
GROMACS thilri cu hnuzulhnak a donghnak 250 ns chung ah Emp24 aa telmi system i 2D lateral density map tuaktannak caah hman a si. Ceramide a tthanchonak/a zornak map hmuh khawhnak ding caah, Cer a tlawmtam map cu Cer le DOPC map fonhmi in tthen a si, cun pum chung i Cer a tlawm le tam in tthen a si. A rong aa khatmi map tahfung kha hman a si.
Hi capar caah bawmtu thilri caah, http://advances.sciencemag.org/cgi/content/full/6/50/eaba8237/DC1 zoh
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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, Lopez (Serho), (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
3D a sangmi a fiangmi caan hman tein hmanthlaknak nih thimmi chuahnak hmun ah protein thleidannak caah ceramide hri saunak a biapitnak a langhter.
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, Lopez (Serho), (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
3D a sangmi a fiangmi caan hman tein hmanthlaknak nih thimmi chuahnak hmun ah protein thleidannak caah ceramide hri saunak a biapitnak a langhter.
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