Ing pamag-sort kareng protina keng secretory pathway importanti ya para keng pamag-maintain ning cell compartmentalization ampo homeostasis. Dagdag pa keng shell-mediated sorting, ing papil da reng lipids keng kinesin sorting keng prosesu ning secretory transport metung yang makabang kutang a e pa mekibat. keni, gagawan mi ing 3D simultaneous multicolor high-resolution real-time imaging para patunayan keng vivo na deng bayung me-synthesized a glycosylphosphatidylinositol-immobilized proteins a maki makabang ceramide lipid moieties maka-cluster la ampo maka-classify la kareng specialized endoplasms Net exit site, na miyayaliwa kareng transmembrane proteins a gagamitan da. Dagdag pa, papakit mi na ing chain length ning ceramide keng endoplasmic reticulum membrane kritikal ya para keng sorting selectivity. Ing kekaming pamagaral mamye yang mumunang direktang in vivo a ebidensya para a-classify la reng protein cargoes base keng lipid chain length kareng selective export sites keng secretory pathway.
kareng eukaryotic cells, deng protina a me-synthesize keng endoplasmic reticulum (ER) mi-sort la kabang magtransport la keng secretory pathway para ideliver la keng karelang cellular destination (1). Dagdag pa keng coat-mediated sorting, makaba na ing pamag-isip na deng mapilan a lipids malyari la namang magsilbing selective exit points kapamilatan ning pamag-cluster kareti kareng specific membrane domains na specific proteins (2-5). Makanyan man, kulang pa ing direktang in vivo a ebidensya para patunayan ing posibling lipid-based a mekanismo. Para asolve ining basic a problema, inaral mi keng yeast nung makananu la mipapalual deng glycosylphosphatidylinositol (GPI) anchored proteins (GPI-APs) ibat keng ER. Deng GPI-APs ila reng miyayaliwang lipid-connected cell surface proteins ( 6, 7). Ing GPI-AP metung yang secreted protein a makadikit kareng outer leaflets ning plasma membrane kapamilatan ning glycolipid moiety (GPI anchor). Tatanggapan da la reng GPI anchors bilang conservative post-translational modifications keng ER lumen (8). Kaybat ning pamag-attach, ing GPI-AP dumalan ya keng Golgi apparatus (5, 9) manibat keng ER papunta keng plasma membrane. Ing presensya da reng GPI anchors ing sangkan nung bakit ing GPI-AP midala yang miyayaliwa kareng transmembrane secreted proteins (kayabe nala reng aliwang plasma membrane proteins) keng secretory pathway (5, 9, 10). kareng yeast cells, deng GPI-APs makabukud la kareng aliwang secreted proteins keng endoplasmic reticulum, at kaybat mibabalut la kareng unique vesicles a makabalut keng coat protein complex II (COPII) (6, 7). E malino ing determinants na niting prosesu ning pamagklasipika king prosesu ning pamaglual ning ER, dapot mipaisip a ing mekanismo a ini malyari yang mangailangan lipid, lalu na ing pamagbayu king istruktura ning lipid portion ning GPI anchor (5, 8). keng yeast, ing GPI lipid remodeling magumpisa ya agad kaybat ning GPI makadikit ya, at keng keraklan a kasu, iti ing sangkan nung bakit ing ceramide makatali ya keng 26-carbon long-chain saturated fatty acid (C26:0) (11, 12). Ing C26 ceramide ing peka ceramide a gagawan da reng yeast cells angga ngeni. Iti me-synthesize ya keng ER at keraklan kaniti mi-export ya keng Golgi apparatus kapamilatan da reng COPII vesicles (13). Ing ER export ning GPI-AP partikular yang mangailangan keng patuloy a ceramide synthesis (14, 15), at ing pamagbayu ning ceramide keng inositol phosphate ceramide (IPC) keng Golgi apparatus makadependi ya keng GPI anchor synthesis (16). Deng biophysical a pamagaral kareng artipisyal a membrane pepakit da na deng makabang acyl chain ceramides malyari lang misasanmetung para gawang ordered domains a maki makatagulaling a physical properties (17, 18). Deting data magdala la keng hypothesis na ing C26 ceramide ampo ing GPI-AP a maki C26 ceramide gagamitan do reng karelang physical properties para misasanmetung la kareng maayus a rehiyon o rehiyon keng medyu magulu a ER membrane lipid a kapaligiran. Iti keraklan bibilugan de reng makuyad ampo e saturated a glycerolipids (C16:1 ampo C18:1) (19, 20). Deng rehiyon a reni ila reng mapiling magfocus kareng specific ER exit sites (ERES), nung nukarin ing ceramide ampo ing ceramide-based GPI-AP malyari lang miyabe keng Golgi keng pareung dedicated COPII vesicle (5).
Kening pamagaral ayni, direkta mi yang sinubukan ining lipid-based mechanism kapamilatan ning pamangamit super-resolution confocal real-time imaging microscopy (SCLIM), na metung yang cutting-edge microscopy technique na malyaring sabay-sabay maka-obserba kareng fluorescently labeled a protina Deng atlung kule ampo atlung-dimensional (3D) a larawan atin lang sobrang matas a resolution ampo bilis kareng mabie cell (21, 21).
Mumuna ming ginamit ing SCLIM technology para mas abalu nung makananu ya ing normal a GPI-AP a maki C26 ceramide group a me-screen ibat kareng transmembrane secreted proteins kaybat dang likuan ing ER keng S. cerevisiae. Para asuri ing klasipikasyun ning ER, ginamit kaming genetic system na direktang maka-visualize kareng bayung synthesized a karga na lulub keng ERES in vivo (7, 23). Bilang karga, pinili mi ing C26 ceramide-based GPI-AP Gas1 a maka-label keng green fluorescent protein (GFP) ampo ing transmembrane secreted protein Mid2 a maka-label keng near-infrared fluorescent protein (iRFP), parehu lang maka-target keng plasma membrane (24-26). Ing sec31-1 temperature-sensitive mutant, deng adwang cargo a reni mipalabas la lalam ning galactose-inducible promoter ampo ing constitutive ERES marker. keng sobrang temperatura (37°C), uling ing sec31-1 mutation makaapektu ya keng function ning COPII coat component Sec31 para pigilan ing COPII germination ampo ing ER export, ing bayung synthesized a cargo mitipun ya keng ER (23). Kaybat nang melamig keng mababang temperatura (24°C), ing sec31-1 mutant cells mibawi ya keng secretory area, at ing mitipun a bayung synthetic cargo megumpisa yang mipalabas ibat keng ER. Ing CLIM visualization pepakit na ing keraklan kareng bayung me-synthesize a Gas1-GFP ampo Mid2-iRFP mitipun la pa keng ER da reng sec31-1 mutant cells kaybat dang me-incubate keng 37°C at kaybat mipalual la keng 24°C kilub ning 5 minutu (Figure 1). Uling ing Mid2-iRFP makalage ya keng mabilug a ER membrane, at ing Gas1-GFP makakonsentra ya at makatipun ya keng discontinuous ER membrane area, ing karelang pamikalat ganap yang miyayaliwa (Figure 1, A anggang C ampo Movie S1). Dagdag pa, antimo ing akakit keng Figure 1D, ing Gas1-GFP cluster ala yang Mid2-iRFP. Deng resulta papakit da na ing GPI-AP ampo reng transmembrane proteins mikakawani la kareng miyayaliwang ER membrane regions kanitang minuna. Ing Gas1-GFP cluster makalapit ya keng metung a ERES a makalabel keng mCherry's COPII coat protein Sec13 (Figure 1, E ampo F, ampo movie S1) (23).
sec31-1 cells magpalwal lang galactose-induced secretions, metung a makaba a acyl chain (C26) ceramide GPI-AP Gas1-GFP (GPI-AP, berdi) ampo ing transmembrane protein Mid2-iRFP (TMP, asul) ampo ining Constructive ERES labeling Sec13-mCherry (E) me-incubate ya para keng 30°C minutu, mipalitan ya keng 24°C, at me-image ya keng SCLIM 5 minutu kaybat. (A anggang C) papakit ne ing metung a representative merged o single 2D image ning metung a eroplanu (A), metung a 2D projection image ning 10 z-sections (B) o metung a 3D cell hemisphere image ning cargo ampo ERES markers (C). Scale bar 1μm (A ampo B). Ing scale unit 0.551μm (C). Ing Gas1-GFP meyakit ya keng discrete ER regions o clusters, kabang ing Mid2-iRFP meyakit ya at mipamye ya keng mabilug a ER membrane (C). (D) Ing graph papakit ne ing relatibung fluorescence intensity ning Gas1-GFP ampo Mid2-iRFP keng Gas1-GFP cluster keng white arrow line (kaili). AU, arbitrary unit. (E ampo F) magrepresenta la keng 3D a larawan na magsamut kareng pibandyan ampo ing marka ning ERES. Gas1-GFP clusters meyakit la malapit keng specific ERES. Ing scale unit 0.551μm ya. (F) Ing maputing solidung pana tatanda ne ing Gas1-GFP cluster a makasuglung keng ERES. Ing libutad ampo wanan a panel papakit na ing metung a meragul a 3D a larawan ampo ing metung a rotated a view ning mepiling Gas1-GFP cluster.
Ing malapit a relasyun ning Gas1-GFP cluster ampo ing metung a specific ERES papakit na ing Gas1-GFP malyari yang lungub keng selective ERES, na miyayaliwa keng selectivity a gagamitan ning Mid2-iRFP para mako ya keng ER. Para a-address ing posibilidad ayni, me-quantify mi ya ing ERES ratio para keng metung o adwang bage (Figure 2, A to C). ikit mi na keraklan kareng ERES (70%) atin lang metung a uri ning karga. Ing larawan king lalam ning Figure 2C papakit nala deng adwang tipikal a alimbawa ning ERES a maki Gas1-GFP (Figure 1) o Mid2-iRFP (Figure 2). Ing pamiyaliwa, manga 20% ning ERES atin yang adwang kargamento na mipapalibut keng metung a lugal. ikit da na deng mapilan a ERES (10%) atin lang adwang uri ning karga, pero makabukud la keng miyayaliwang lugal. Uli na niti, ining statistical analysis papakit na kaybat ning pamag-export ning ER, ing GPI-AP Gas1-GFP ampo ing transmembrane cargo Mid2-iRFP mepitna la kareng miyayaliwang ERES (Figure 2D). Ing pamag-sort efficiency ayni metung yang consistent keng minunang biochemical analysis (6) ampo ing morphological determination (7). Malyari tamu namang akit ing ugali da reng maka-quarantine a karga na lulub keng ERES (Figure 2E ampo Movie S2). Ing Figure 2E papakit na ing ditak mung dake ning Gas1-GFP (panel 3) o Mid2-iRFP (panel 4) lulub ya keng ERES ibat keng metung a gilid at makasukul ya keng metung a discrete a lugal. Ing Panel 5 ning Figure 2E papakit na ing Gas1-GFP ampo ing Mid2-iRFP misan mayayakit la keng parehung ERES, pero lulub la ibat kareng miyayaliwang panig ampo makakonsentra la kareng miyayaliwang lugal na malyaring magrepresenta kareng miyayaliwang COPII vesicles. Kinumpirma mi mu rin na ing meyakit a pamikawani ampo ing pamagklasipika ning C26 ceramide-based GPI-AP Gas1 bilang selective ERES specific ya uling ing metung pang transmembrane secretion cargo, ing GFP-tagged plasma membrane protein Axl2 (27 ), papakit na ing kalupa nang ugali ning Mid2-iRFP. (Larawan S1 ampo Pelikula S3). Ing bayung synthesized Axl2-GFP mipamye ya keng ER membrane kalupa ning Mid2-iRFP (Figure S1, A ampo B), ampo co-localized ya keng Mid2-iRFP keng keraklan a ERES (Figure S1, B anggang D). Deng panel 1 ampo 2 ning Figure 1. S1C papakit nala deng adwang tipikal a alimbawa ning ERES nung nukarin deng adwang transmembrane cargoes mipapalibut la. Kareng kasu a reni, parehu lang lungub deng bage keng ERES (Figure S1E, Panel 3 ampo Movie S3).
Deng sec31-1 cells a magpalwal galactose inducible secretions, Gas1-GFP (GPI-AP, berdi) ampo Mid2-iRFP (TMP, asul) ampo ing constitutive ERES labeling Sec13-mCherry (ERES, magenta) mibili la keng 37 Kaybat dang mi-incubate kilub ning 30 minutu keng °C, 24 C, i-block ing imahe keng SCLIM kaybat ning 20 minutu. (A anggang C) Representative 2D projection images (A; scale bar, 1μm) o 3D cell hemisphere images (B ampo C; scale unit, 0.456μm) ning karga ampo 10 z-sections a makamarka keng ERES. Ing lalam a panel keng (B) ampo ing panel keng (C) papakit lang meprosesu a larawan para ipakit la mu reng bage a atyu keng ERES (magenta) [Gas1-GFP (gray) ampo Mid2-iRFP (light blue)]. (C) Open arrow: Ing ERES magdala yang metung mung karga (1 anggang 4). Gray arrow: ERES atin yang segregated cargo (5). Puting solidung pana: ERES a maki co-located a karga. king lalam: Ing mepiling ERES atin yang Gas1-GFP (1) o Mid2-iRFP (2). Scale bar, 100 nm. (D) Quantification ning photomicrograph a makalarawan keng (C). Ing average a porsyentu ning ERES na atin mung metung a karga (Gas1-GFP o Mid2-iRFP), segregated cargo ampo overlapping cargo. keng atlung independenting experiment, n=432 keng 54 cells. Error bar = SD. Two-tailed unpaired t test. *** P = 0.0002. (E) 3D a larawan da reng mepiling ERES da reng maka-quarantine a karga a makamarka king (C). Ing Gas1-GFP (berdi) (3) o Mid2-iRFP (asul) (4) lulub ya keng ERES (magenta) ibat keng metung a gilid at makabawal ya keng malating lugal kilub ning ERES. Minsan, parehu lang lulub deng adwang uri ning kargamentu keng parehung ERES (5) ibat keng parehung gilid ampo makakulong la keng metung a makabukud a lugal kilub ning ERES. Scale bar, 100 nm.
Kaybat, sinubukan mi ing hypothesis na ing makabang acyl chain ceramide (C26) a atyu keng ER membrane ing magdrive keng specific clustering ampo pamag-sort ning Gas1 keng selective ERES. Para kaniti, ginamit mi ing metung a modified yeast strain GhLag1, nung nukarin ding adwang endogenous ceramide synthases Lag1 ampo Lac1 mipalitan la king GhLag1 (ing Lag1 homolog ning kapas), a magresulta king metung a yeast strain a maki cell membrane Ceramide strain a mas makuyad kesa king wild type (Figure 3A) (28). Ing mass spectrometry (MS) analysis pepakit na keng wild-type strains, 95% ning kabilugan a ceramide makaba ya (C26) chain ceramide, kabang keng GhLag1, 85% ning ceramide makaba ya (C18 ampo C16). ), 2% mu ning ceramide ing makaba (C26) chain ceramide. Agyang deng C18 ampo C16 ceramides ila reng manimunang ceramides a akit keng GhLag1 membrane angga ngeni, ing MS analysis mekumpirma ya na ing GPI anchor ning Gas1-GFP a mipalabas keng GhLag1 strain atin yang C26 ceramide, na kalupa da reng wild-type lipids. Ing kalidad parehu ya (Fig. 3A) (26). Inya, buri ng sabyan niti na ing ceramide remodeling enzyme Cwh43 matas yang selective para keng C26 ceramide, antimo ing akakit keng Figure 26, mas buri na ing GPI anchor ibat keng ditak a C26 ceramide keng GhLag1 strain. S2 (29). Makanyan man, ing cell membrane ning GhLag1 atin yang C18-C16 ceramide, kabang ing Gas1-GFP atin ya pang C26 ceramide. Ing katutwan ayni gagawan neng masanting a kasangkapan ing strain ayni para a-solve ing problema ning acyl chain length ning membrane ceramide keng ER. Ing hypothetical a papil ning klasing at pamag-sort. Kaybat, mumuna ming inaral ing abilidad ning C26 Gas1-GFP para mitipun kareng clusters keng GhLag1 a maki temperature-sensitive mutant allele ning sec31-1 kapamilatan ning conventional fluorescence microscopy, nung nukarin ing makaba (C18-C16) chain mu ing atyu keng ER membrane Ceramide (Fig. 3). ikit mi na keng sec31-1, keraklan ning Gas1-GFP makakonsentra ya keng clusters, kabang ing Gas1-GFP keng sec31-1 GhLag1 a maki makaba (C18-C16) makaba a ceramide ER membrane keraklan e ya makacluster ampo mipamye ya keng mabilug a ER membrane. Para maging eksaktu, uling ing C26 ceramide-based clustering malapit yang makaugne keng specific ERES (Figure 1), tutuki ming siniasatan nung ing prosesung ayni malyari ya namang makiabe keng function ning ER export protein mechanism. Ing GPI-AP gagamit yang espesyal a COPII system para keng ER export, na aktibung mi-regulate keng Ted1′s structural remodeling ning glycan portion ning GPI anchor (30, 31). Ing recombinant GPI-glycan kanita akilala ne ning transmembrane cargo receptor p24 complex, na nung nukarin selectively recruit ne ing Lst1, na metung yang specific isoform ning major COPII cargo binding subunit Sec24, gagawang GPI-AP-rich COPII Vesicles are necessary (31-33). Uli na niti, ginawa kaming double mutant a misasanmetung king pamagbura kareng single proteins (ing p24 complex component Emp24, GPI-glycan remodeling enzyme Ted1 ampo ing specific COPII subunit Lst1) king sec31-1 mutant strain, at inaral mi la. ikit mi na keng sec31-1emp24Δ ampo sec31-1ted1Δ, ing Gas1-GFP keraklan e ya maka-cluster ampo mipamye ya keng mabilug a ER membrane, antimo ing minunang ikit keng sec31-1 GhLag1, kabang keng sec31-1lst1Δ, ing Gas1-GFP kalupa ne ning sec31-11-1. Deng resulta a reni papakit da na dagdag pa keng presensya ning C26 ceramide keng ER membrane, ing pamag-cluster ning Gas1-GFP kailangan ya namang mag-bind keng p24 complex, at e na kailangan ing specific Lst1 recruitment. Kaybat, linawe mi ing posibilidad na ing chain length ning ceramide keng ER membrane malyari yang magregulate keng pamagtali ning Gas1-GFP keng p24. Makanyan man, ikit mi na ing presensya ning C18-C16 ceramide keng membrane ali ya makaapektu kareng GPI-glycans a me-reconstruct ning p24 complex (Figures S3 ampo S4, A ampo B) o makatali keng GPI-AP ampo mag-export GPI-AP. abilidad. Recruit COPII subtype Lst1 (Figure S4C). Uli na niti, ing C26 ceramide-dependent clustering e na kailangan ing pamiugnayan da reng protina kareng miyayaliwang mekanismo ning protina ning pamaglual ning ER, dapot susuportan ne ing alternatibong mekanismo ning pamag-sort a tutulak ning kaba ning lipid. Kaybat, sinuri mi nung ing ceramide acyl chain length keng ER membrane importanti ya para keng epektibung klasipikasyun ning Gas1-GFP bilang selective ERES. Uling ing Gas1 keng GhLag1 strain a maki short-chain ceramide lulual ya keng ER at lulub ya keng plasma membrane (Figure S5), maniwala kami na nung ing pamag-sort mipatupad ya keng kaba ning ceramide acyl chain, ing Gas1 keng GhLag1 strain malyari yang mi-redirect at mi-cross. ERES goods na parehas lang membrane.
(A) Ing cell membrane ning GhLag1 atin yang mas makuyad a C18-C16 ceramides, kabang ing GPI anchor ning Gas1-GFP atin yang pareung C26 IPC kareng wild-type cells. babo: acyl chain length analysis ning ceramide keng cell membrane ning wild-type (Wt) ampo GhLag1p strains kapamilatan ning mass spectrometry (MS). Ing data magrepresenta yang porsyentu ning kabilugan a ceramide. Ing average da reng atlung independenting experiment. Error bar = SD. Two-tailed unpaired t test. **** P ＜0.0001. Bottom panel: MS analysis ning acyl chain length ning IPC a atyu keng Gas1-GFP (GPI-IPC) GPI anchor a mipalabas keng wild-type ampo GhLag1p strains. Ing data magrepresenta yang porsyentu ning kabilugan a IPC signal. Average da reng limang independenting experiment. Error bar = SD. Two-tailed unpaired t test. ns, ali importanti. P = 0.9134. (B) Fluorescence micrographs ning sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ ampo sec31-1lst1Δ cells a mag-express galactose-induced Gas1-GFP me-incubate la keng 37°C kilub ning 30 minutu ampo me-perform lang fluoresce microscopy kaybat ning 24°C. Puting pana: ER Gas1-GFP cluster. Open arrow: Ing unclustered Gas1-GFP mipamye ya keng mabilug a ER membrane, papakit na ing ER characteristic nuclear ring staining. Scale bar, 5μm. (C) Quantification ning photomicrograph a makalarawan keng (B). Ing average a porsyentu da reng cell a maki punctate Gas1-GFP a istruktura. keng atlung independenting experiment, n≥300 cells. Error bar = SD. Two-tailed unpaired t test. **** P ＜0.0001.
Para aluswan ing problemang ayni, ginawa mi ing SCLIM visualization ning Gas1-GFP ampo Mid2-iRFP keng GhLag1 gamit ing sec31-1 temperature-sensitive mutant allele (Figure 4 ampo Movie S4). Kaybat nang menatili ing ER king 37°C at kaybat mipalual ya king 24°C, keraklan kareng bayung me-synthesize a Gas1-GFP e la me-cluster at mipamye la king mabilug a ER membrane, antimo ing ikit da reng conventional microscopes (Figure 4, A and B ). Dagdag pa, ing maragul a porsyentu ning ERES (67%) kayabe no reng adwang uri ning kargamento a makatuknang kaniti (Figure 4D). Deng panel 1 ampo 2 ning Figure 4C papakit da la reng adwang tipikal a alimbawa ning ERES a maki mipapalibut a Gas1-GFP ampo Mid2-GFP. Dagdag pa, parehu lang me-recruit deng goods keng parehung ERES (Figure 4E, panel 3 ampo movie S4). Uli na niti, ing kekaming resulta papakit na ing kaba ning ceramide acyl chain keng ER membrane metung yang importanting determinant ning ER protein aggregation ampo classification.
Sec31-1 GhLag1 cells a magpalwal galactose-induced secretions, Gas1-GFP (GPI-AP, berdi) ampo Mid2-iRFP (TMP, asul) ampo constitutive ERES-labeled Sec13-mCherry (ERES, magenta) I-incubate keng 37°C. Isundu me kilub ning 30 minutu, ibaba me keng 24°C para mipalabas ing secretions, at i-image me gamit ing SCLIM kaybat ning 20 minutu. (A anggang C) Representative 2D projection images (A; scale bar, 1μm) o 3D cell hemisphere images (B ampo C; scale unit, 0.45μm) da reng 10 z-sections a makamarka keng cargo ampo ERES. Ing lalam a panel keng (B) ampo ing panel keng (C) papakit lang meprosesu a larawan para ipakit la mu reng bage a atyu keng ERES (magenta) [Gas1-GFP (gray) ampo Mid2-iRFP (light blue)]. (C) White filled arrow: ERES, goods overlap. Open arrow: Ing ERES atin yang metung a bage. Lower panel: Ing mepiling ERES atin yang overlapping goods (1 ampo 2) a makamarka keng (C). Scale bar, 100 nm. (D) Quantification ning photomicrograph a makalarawan keng (C). keng sec31-1 ampo sec31-1 GhLag1 units, metung mung karga (Gas1-GFP o Mid2-iRFP) ing kayabe, ampo ing average a porsyentu ning ERES para keng makabukud a karga ampo mipapatung a karga. kareng atlung independenting experiment, n = 432 keng 54 cells (sec31-1) ampo n = 430 keng 47 cells (sec31-1 GhLag1). Error bar = SD. Two-tailed unpaired t test. *** P = 0.0002 (sec31-1) ampo ** P = 0.0031 (sec31-1 GhLag1). (E) 3D a larawan ning mepiling ERES a maki mipapatung a karga (3) a makamarka king (C). Gas1-GFP (berdi) ampo Mid2-iRFP (asul) lalapit la keng ERES (magenta) ibat keng parehung gilid ampo manatili la keng parehung ERES restricted area. Scale bar, 100 nm.
Ing pamagaral ayni mamye yang direktang in vivo a ebidensya na deng lipid-based protein cargoes makaclassify la kareng selective export sites keng secretory pathway, ampo papakit na ing importansya ning acyl chain length para keng classification selectivity. Gamit ing metung a masikan at makabayung paralan ning microscopy a ausan dang SCLIM, pepakit mi ing bayung me-synthesize a Gas1-GFP (metung a maragul a plasma membrane GPI-AP a maki makabang acyl chain (C26) ceramide lipid portion) king yeast ) Deng rehiyon a maka-cluster kareng discrete ERs makasuglung la kareng specific E, kabang deng transmembrane a protina mipalaganap la kapamilatan ning membrane ERRES (Figure 1). Dagdag pa, deng adwang uri da reng pibandyan a reni lulub la kareng miyayaliwang ERES a mapili (Figure 2). Ing acyl chain length ning cellular ceramide keng membrane mabawas ya manibat C26 angga C18-C16, ing Gas1-GFP cluster mesira ya keng discrete ER region, ampo ing Gas1-GFP mi-reroute ya para mako ya keng ER ampo ing transmembrane protein kapamilatan ning pareung ERES (Figure 3 ampo Figure 3). 4).
Agyang ing GPI-AP gagamit yang specialized protein mechanism para lumual keng ER, ikit mi na ing C26 ceramide-dependent separation ali ya magdependi keng differential protein interactions na malyaring magdalang ERES specialization (Figures S4 ampo S5). Bagkus, ing kekaming akit suportan ne ing alternatibong mekanismo ning pamagklasipika a gagawan ning lipid-based protein clustering ampo ing kayang pamaglakwan kareng aliwang karga. Ing kekaming obserbasyun papakit na ing Gas1-GFP region o cluster a makasuglung keng metung a specific ERES ala yang transmembrane secreted protein Mid2-iRFP, na papakit na ing C26 ceramide-dependent GPI-AP cluster pasikanan na ing karelang pamaglub keng kaugnayan a ERES, at keng parehung oras, ilako ne ing transmembrane Ding secretions lulub la kening partikular a ERES (Figures 1 and RES). Ing pamiyaliwa, ing presensya da reng C18-C16 ceramides keng ER membrane e ya magdalang GPI-AP para gawang rehiyon o clusters, inya e la mag-exclude o magpalitan kareng transmembrane secreted proteins keng pareung ERES (Figures 3 ampo 4). . Uli na niti, mumusbung mi na ing C26 ceramide ing magdrive king pamikawani at pamagklasipika kapamilatan ning pamagpasikan king pamisanmetung da reng protina a makasuglung kareng partikular a ERES.
makananu yang akwa ining C26 ceramide-dependent clustering keng metung a specific ER area? Ing tendency ning membrane ceramide na mikawani ya keng gilid malyari yang maging sangkan ning GPI-AP ampo ing C26 ceramide para gawang malati ampo agad a maka-order a lipid keng mas irregular a lipid a kapaligiran ning ER membrane a maki mas makuyad ampo e saturated a glycerolipids. Quality clusters (17, 18). Deting malating pansamantalang clusters malyari lang mas misasanmetung kareng mas maragul, mas matatag a clusters kaybat dang mitali keng p24 complex (34). Agpang kaniti, pepakit mi na ing C26 Gas1-GFP kailangan yang makipag-ugnayan king p24 complex ban gawang mas maragul a akakit a clusters (Figure 3). Ing p24 complex metung yang heterozygous oligomer a bibilugan da reng apat a miyayaliwang p24 transmembrane proteins keng yeast (35), na mamye multivalent binding, na malyaring magdalang cross-linking kareng malating GPI-AP clusters, inya sa't makagawa yang mas maragul a Stable cluster (34). Ing pamiugnayan da reng protein ectodomains da reng GPI-APs malyari ya namang makaambag king karelang pamisanmetung, antimo ing pepakit da king karelang Golgi transport kareng mammalian polarized epithelial cells (36). Makanyan man, nung ing C18-C16 ceramide atyu ya keng ER membrane, nung ing p24 complex makatali ya keng Gas1-GFP, e la mabuo deng mangaragul a miyayaliwang clusters. Ing makabasi a mekanismo malyari yang magdependi keng partikular a pisikal ampo chemical a katangian ning makaba a acyl chain ceramide. Deng biophysical a pamagaral kareng artipisyal a membrane papakit da na agyang pareung makaba (C24) ampo makuyad (C18-C16) a acyl chain ceramides malyari lang maging sangkan ning phase separation, deng makaba mung acyl chain ceramides (C24) ing makapromote matas a Curvature ampo film bending para i-reshape ing film. Kapamilatan ning mutual reference (17, 37, 38). Mepakit na ing transmembrane helix ning TMED2, ing human homologue ning Emp24, selectively makipag-ugnayan ya keng C18 ceramide-based sphingomyelin keng cytoplasmic lobules (39). Gamit ing molecular dynamics (MD) simulations, ikit mi na pareung C18 ampo C26 ceramides mitipun la keng paligid da reng cytoplasmic lobules ning Emp24 transmembrane helix, ampo atin lang parehung kagustuwan (Figure S6). Dapat papansinan na iti papakit na ing transmembrane helix ning Emp24 malyari yang magdalang asymmetric distribution da reng lipids keng membrane. Iti metung yang bayung resulta base kareng cell da reng mammal. Deng kalupa nang MD simulations papakit da ing presensya da reng ether lipids (40) . Uli na niti, aisip mi na ing C26 ceramide kareng adwang lobules ning ER26 lokal yang mipayaman. Nung ing GPI-AP keng luminal lobules diretsu yang makagapus keng multivalent p24 ampo ing pamitipun ning C26 ceramide keng paligid ning p24 keng cytoplasmic lobules, malyari yang i-promote ing kayabe nang Protein aggregation ampo ing membrane curvature a gagawan da kapamilatan da reng taliri (41), a sangkan nung bakit ing GPI-AP mikakawani ya kareng discrete a lugal a makalapit keng E, a pabor mu naman kareng matas a curvature a rehiyon ing ER membrane (42). Deng minunang ulat suportan de ing mipanukalang mekanismo (43, 44). Ing multivalent binding da reng oligolectins, pathogens o antibodies kareng ceramide-based glycosphingolipids (GSL) keng plasma membrane mag trigger yang maragul a GSL aggregation, magpasanting keng phase separation ampo magdalang membrane deformation ampo internalization (44). Iwabuchi etc. (43) Ikit da na keng presensya da reng makaba (C24) pero ali makuyad (C16) acyl chains, ing multivalent ligand a makagapus keng GSL lactosylceramide ing meging sangkan ning pamaglalang da reng mangaragul a clusters ampo ing membrane invagination, ampo ing cytoplasm Lyn-mediated signal transduction kareng leaflets mipapagsalita ya kareng neutrophils couplels.
kareng mammalian polarized epithelial cells, ing konsentrasyun ning anti-Golgi network (TGN) angga keng antas ning apical plasma membrane ing magkontrol keng pamikawani ampo pamag-sort ning GPI-AP (10, 45). Ing aggregation ayni metung yang GPI-AP oligomerization (36), pero malyari ya namang magdependi keng ceramide chain length a akit tamu keng yeast. Agyang ing mammalian GPI-AP atin yang ether lipid-based anchor, at ing kayang chemical structure miyayaliwa ya keng sobrang makabang acyl chain ceramide, ing bayung pamagaral menakit yang pareung lipid atin lang evolutionary similar physical ampo chemical properties ampo function (40). Uli na niti, ing ether lipid part kareng mammalian cells malyari yang kalupa ning C26 ceramide keng yeast, at ing kayang papil yapin ing makiabe ya keng long-chain ceramide keng membrane para isulong ing GPI-AP aggregation ampo ing pamag-sort. Agyang kailangan ya pa muring subukan diretsu ing posibilidad a ini, deng minunang akit susuportan da na ing pamagdala ning long acyl chain ceramide papunta keng Golgi body ali ya gagawan da reng cytoplasmic transfer proteins, pero makadependi ya keng synthesis da reng GPI anchors kalupa ning yeast. Uli na niti, ing evolutionary conservative mechanism balamu agyu nang i-selective co-transport ing makabang acyl chain ceramide ampo ing GPI-AP (13, 16, 20, 46, 47) king pareung transport vesicle.
keng yeast ampo mammalian polarized epithelial cell systems, ing GPI-AP aggregation ampo ing pamikawani da reng aliwang plasma membrane proteins malilyari la ngan bayu la miras keng cell surface. Paladino at aliwa pa. (48) ikit da na keng TGN da reng mammalian polarized epithelial cells, ing GPI-AP clustering ali ya mu kailangan para keng selective classification da reng GPI-APs keng apical plasma membrane, nune magregulate ya naman keng clustering organization da reng GPI-APs ampo ing kayang biological activity. Cell surface. keng yeast, ining pamagaral pepakit na ing C26 ceramide-dependent GPI-AP cluster keng ER malyari yang magregulate keng cluster organization ampo ing functional activity ning GPI-AP keng plasma membrane (24, 49). Agpang keng model ayni, deng GhLag1 cells allergic la kareng GPI inhibitors o panulu na makaapektu keng cell wall integrity (28), ampo ing pamangailangan para kareng functional Gas1-GFP clusters (49) ning tip ceramide a mipalabas keng pamakipag-asawa da reng yeast cells papakit na ing G Possible physiological consequences da reng hLag1 cells. GPI-AP error. Makanyan man, ing karagdagang pamagsuri nung ing functional organization ning cell surface me-program ya ibat keng ER kapamilatan ning metung a paralan ning pamag-sort base keng lipid length ing maging subject ning kekaming daratang a pamanyaliksik.
Deng Saccharomyces cerevisiae strains a ginamit kening obra makalista la keng Table S1. Ing MMY1583 ampo MMY1635 strains ning SCLIM para keng live cell imaging megawa la keng background ning W303. Deting strain a mag-express Sec13-mCherry a maki fluorescent protein tag megawa la gamit ing polymerase chain reaction (PCR)-based a paralan a maki pFA6a plasmid bilang template (23). Ing strain a magpalwal Mid2-iRFP a makalabel keng fluorescent protein lalam ning kontrol ning GAL1 promoter megawa ya anti kanini. PCR amplification ning iRFP-KanMx sequence ibat keng pKTiRFP-KAN vector (regalu nang E. O'Shea, Addgene plasmid number 64687; http://n2t.net/addgene: 64687; research resource identifier (RRID): Addgene_64687) At milub ya keng endogenous Ctermind ning 22. Kaybat ning Mid2-iRFP genome sequence me-amplify ya at me-clone ya keng GAL1 promoter, me-integrate ya keng Not I-Sac I site ning integration plasmid pRS306. Ing resulta ning plasmid pRGS7 me linearize ya keng Pst I para maka integrate ya keng URA3 locus.
Ing Gas1-GFP fusion gene mipalabas ya lalam ning kontrol ning GAL1 promoter keng centromere (CEN) plasmid, na megawa anti kanini. Ing Gas1-GFP sequence me-amplify ya kapamilatan ning PCR ibat king pRS416-GAS1-GFP plasmid (24) (regalu nang L. Popolo) at me-clone ya king Xma I–Xho I site ning CEN plasmid pBEVY-GL LEU2 (regalu nang C) . Miller; Addgene plasmid number 51225; http://n2t.net/addgene: 51225; RRID: Addgene_51225). Ing plasmid a meyaus pRGS6. Ing Axl2-GFP fusion gene mipalabas ya naman lalam ning kontrol ning GAL1 promoter ning pBEVY-GL LEU2 vector, at ing kayang pangagawa anti kanini. Ing Axl2-GFP sequence me-amplify ya ibat keng pRS304-p2HSE-Axl2-GFP plasmid (23) kapamilatan ning PCR, at me-clone ya keng Bam HI-Pst I site ning pBEVY-GL LEU2 vector. Ing plasmid a meyaus pRGS12. Ing sequence da reng oligonucleotides a ginamit kening pamagaral makalista ya keng Table S2.
Ing strain midagdag ya keng 0.2% adenine ampo 2% glucose [YP-dextrose (YPD)], 2% raffinose [YP-raffinose] rich yeast extract protein p (YP) medium (1 % Yeast extract ampo 2% protein ept). (YPR)] o 2% galactose [YP-galactose (YPG)] bilang carbon source, o keng synthetic minimal medium (0.15% yeast nitrogen base ampo 0.5% ammonium sulfate) para dagdagan la reng amino acid ampo base a kailangan para keng nutrisyon, ampo atin yang 2% glucose (2% glucose glucose) o minimal medium galactose (synthetic galactose minimal medium) bilang carbon source.
Para keng real-time imaging, deng temperature-sensitive sec31-1 mutant cells a magpalwal keng construct lalam ning GAL1 promoter meragul la keng YPR medium keng 24°C magdamag anggang mid-log phase. Kaybat ning induction keng YPG keng 24°C kilub ning 1 oras, ding cells mi-incubate la keng SG keng 37°C kilub ning 30 minutus, at kaybat mipalitan la keng 24°C para mipalabas la keng secretion block. Ing Concanavalin A ginamit ya para ayusan la reng cell keng glass slide ampo me-image ya keng SCLIM. Ing SCLIM metung yang kombinasyun ning Olympus IX-71 inverted fluorescence microscope ampo ing UPlanSApo 100×1.4 numerical aperture oil lens (Olympus), high-speed ampo high-signal-to-noise ratio rotating disc confocal scanner (Yokogawa Electric), custom spectrometer, intensive systems ampo ing custom cotofier systems (Hamamatsu Photonics) malyari yang mamye magnifying lens system a maki tauling magnification a ×266.7 ampo ing charge-coupled device camera a magparakal kareng electron (Hamamatsu Photonics) (21). Ing pamagkua kareng larawan gagawan de kapamilatan ning custom software (Yokogawa Electric). Para kareng 3D a larawan, ginamit kaming custom-made piezoelectric actuator para i-vibrate ing objective lens vertically, at tinipun mi la reng optical parts 100 nm ing pamiyaliwa da keng metung a stack. Ing Z-stack image mibayu ya keng 3D voxel data, at ing theoretical point spread function a gagamitan para keng rotating disc confocal microscope gagamitan de para keng deconvolution processing ning Volocity software (PerkinElmer). Gamit ing Volocity software para automatic yang threshold para keng co-location analysis, ing ERES kayabe na ing cargo mesukad ya. Ing line scan analysis megawa ya gamit ing MetaMorph software (Molecular Devices).
Gamitan me ing GraphPad Prism software para abalu ing statistical significance. Para keng two-tailed Student's t-test ampo ing ordinaryung one-way analysis of variance (ANOVA) test, ing pamiyaliwa da reng grupu metung yang makabaldugan a epektu keng P <0.05 (*).
Para keng fluorescence microscopy ning Gas1-GFP, deng log phase cells meragul la keng bengi keng YPD ampo mekolekta la kapamilatan ning centrifugation, me hugas lang adwang besis keng phosphate buffered saline, ampo me incubate la keng yelo kilub ning e kumulang 15 minutus, at kaybat misundu la lalam ning microscope antimo ing minunang misulat Check (24). Ing Leica DMi8 microscope (HCX PL APO 1003/1.40 oil PH3 CS) a ating objective lens, L5 (GFP) filter, Hamamatsu camera ampo Application Suite X (LAS X) software ing ginamit para keng pamag-acquire. .
Deng sample me-denatured la keng SDS sample buffer keng 65°C kilub ning 10 minutu, at kaybat me-separate la kapamilatan ning SDS-polyacrylamide gel electrophoresis (PAGE). Para keng immunoblotting analysis, 10 μl ning sample ing mikarga balang lane. Primary antibody: Gamitan me ing rabbit polyclonal anti-Gas1 keng dilution a 1:3000, rabbit polyclonal anti-Emp24 keng dilution a 1:500, ampo rabbit polyclonal anti-GFP (metung a regalu ibat kang H. Riezman) keng dilution a 1:3000. Ing mouse monoclonal anti-Pgk1 antibody ginamit ya keng dilution a 1:5000 (metung a regalu ibat kang J. de la Cruz). Secondary antibody: Horseradish peroxidase (HRP) conjugated goat anti-rabbit immunoglobulin G (IgG) ginamit keng dilution a 1:3000 (Pierce). Ing HRP-conjugated goat anti-mouse IgG ginamit ya keng dilution a 1:3000 (Pierce). Ing immune response zone me-obserban ya kapamilatan ning chemiluminescence a paralan ning SuperSignal West Pico reagent (Thermo Fisher Scientific).
Antimo ing makasulat keng (31), metung a natural a immunoprecipitation experiment ing megawa keng enriched ER fraction. king makuyad a salita, hugasan la reng yeast cells gamit ing TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride ampo protease inhibitor mixture) keng 600 nm ( OD600) keng 100 optical density adwang besis. Mesira ya kareng glass beads, at kaybat ing cell debris ampo glass beads mewala la kapamilatan ning centrifugation. Ing supernatant me centrifuge ya keng 17,000 g kilub ning 15 minutus keng 4°C. Ing pellet mibalik ya keng TNE ampo ing digitalis saponin midagdag ya keng tauling konsentrasyun a 1%. Ing suspension mi-incubate ya kilub ning 1 oras a maki rotation keng 4°C, at kaybat ding e matunaw a sangkap mewala la kapamilatan ning centrifugation keng 13,000 g keng 4°C kilub ning 60 minutu. Para keng Gas1-GFP immunoprecipitation, mumuna i-pre-incubate ing sample kareng alang laman a agarose beads (ChromoTek) keng 4°C kilub ning 1 oras, at kaybat i-incubate keng GFP-Trap_A (ChromoTek) keng 4°C kilub ning 3 oras. Deng immunoprecipitated beads mehugas lang limang besis keng TNE a maki 0.2% digoxigenin, me-elute la keng SDS sample buffer, me-separate la keng SDS-PAGE, ampo me-analisa la kapamilatan ning immunoblotting.
Antimo ing makasulat keng (31), ing cross-linking determination megawa ya keng enriched ER fraction. king makuyad a salita, ing me-enriched ER fraction me-incubate ya keng 0.5 mM dithiobis(succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, USA; 20°C, 20 min). Ing crosslinking reaction me-quench ya kapamilatan ning pamagdagdag glycine (50 mM final concentration, 5 minutes, 20°C).
Antimo ing minunang misulat (50), ing MS analysis ning ceramide keng wild-type ampo GhLag1 strains megawa ya. king makuyad a salita, deng cells meragul la keng exponential phase (3 anggang 4 OD600 units/ml) keng YPD keng 30°C, ampo 25×107 a cells ing me-ani. Ing karelang metabolismu mipapate ya kapamilatan ning trichloroacetic acid. Gamitan ing extraction solvent [ethanol, danum, ether, pyridine ampo 4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] ampo 1.2 nmol ning internal standard C17 ceramide (860517, Avanti polar lipid) a kalidad). Gamitan me ing monomethylamine reagent [methanol, danum, n-butanol ampo methylamine solution (4:3:1:5 v/v)] para gawan ing mild alkaline hydrolysis ning extract, at kaybat gamitan me ing water-saturated n-butanol para mag-desalt. Katataulian, ing extract mibalik ya keng positibong mode a solvent [chloroform/methanol/danum (2:7:1) + 5 mM ammonium acetate] at mi-inject ya keng mass spectrometer. Ing multi-reaction monitoring (MRM) megawa ya para keng pamangilala ampo pamag-quantify kareng sphingolipid molecules. Ing TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) atin yang robotic nanoflow ion source Nanomate HD (Advion Biosciences, Ithaca, NY) para keng lipid analysis. Ing collision energy me-optimize ya para kareng balang ceramide category. Ing MS data mekwa ya keng positibong mode. Para kareng balang biological replicate, ing lipid signal ya ing median da reng atlung independenting sukad.
Antimo ing makasulat keng (31), deng cell (800×107) a magpalwal Gas1-GFP mipailalam la keng natural a immunoprecipitation. Ing melinis a Gas1-GFP mekawani ya kapamilatan ning SDS-PAGE at mipalitan ya king polyvinylidene fluoride (PVDF) a membrane. Ing protina me-visualize ya kapamilatan ning pamag-stain keng PVDF gamit ing amide black. Ing Gas1-GFP band meputut ya keng PVDF ampo mehugas yang 5 besis keng methanol ampo misan keng liquid chromatography-MS (LC-MS) grade water. King pamag-incubate king membrane strip a maki 500μl 0.3 M NaOAc (pH 4.0), buffer ampo 500μl a bayung me-dissolved a 1 M sodium nitrite mixture king 37°C kilub ning 3 oras, ing lipid fraction mipalual ya ibat king Gas1-GFP at mipalabas ya ing pamaglual ning ininphoosine phoosphamise king pilatan ning ceramiosphamioldeit ampo ing gluoseit (51). Kaybat na nita, ing membrane strip mehugas yang apat a besis keng LC-MS grade a danum, mepatuyu ya keng room temperature, ampo mitagan ya keng nitrogen atmosphere keng -80°C angga na keng pamagsuri. Bilang control, metung a blankung sample ning PVDF membrane ing ginamit para keng balang experiment. Ing lipid a mekwa keng Gas1-GFP me-analisa ya keng MS antimo ing makasulat (50). Ing makuyad a salita, deng PVDF strips a maki GPI-lipid mibalik la keng 75μl negative mold solvent [chloroform/methanol (1:2) + 5 mM ammonium acetate] ampo mipasa la keng electrospray ionization (ESI)-MRM/MS Analysis of sphingolipid species (TSQ Vantage). keng kasu ayni, ing MS data mekwa ya keng negative ion mode.
Antimo ing sinabi ku na, ing lipid portion ning GPI anchor mekawani ya keng [3H]-inositol-labeled GPI-AP (16). Deng lipids mekawani la kapamilatan ning thin-layer chromatography gamit ing solvent system (55:45:10 chloroform-methanol-0.25% KCl) at me-visualize la gamit ing FLA-7000 (Fujifilm).
Deng cell a magpalwal Gas1-GFP (600×107) me hugas lang adwang besis keng TNE buffer a maki TNE buffer, ampo mesira la kareng glass beads, at kaybat me centrifuge la para milako la reng cell debris ampo glass beads. Ing supernatant me centrifuge ya keng 17,000 g kilub ning 1 oras keng 4°C. Ing pellet me hugas ya keng TNE ampo me incubate ya keng 1 U PI-PLC (Invitrogen) keng TNE na ating 0.2% digitalis saponin kilub ning 1 oras keng 37°C. Kaybat ning enzyme treatment, ing membrane mewala ya kapamilatan ning centrifugation keng 17,000 g keng 4°C kilub ning 1 oras. Para immunoprecipitate ing Gas1-GFP, ing supernatant mi-incubate ya keng GFP-Trap_A (ChromoTek) keng 4°C magdamag. Ing purified Gas1-GFP a me-separate keng SDS-PAGE me-stain ya keng Coomassie brilliant blue. Ing Gas1-GFP staining band meputut ya keng gray a makapadurut keng aqueduct, at kaybat ning alkylation keng iodoacetamide ampo reduction keng dithiothreitol, ing in-gel digestion keng trypsin megawa ya. I-extract at i-dry ing tryptic peptides ampo peptides gamit ing GPI-glycans. Ing metuling a peptide metunaw ya keng 20 μl ning danum. I-inject me ing metung a portion (8μl) keng LC. Ing octadecylsilane (ODS) column (Develosil 300ODS-HG-5; inner diameter 150 mm×1.0 mm; Nomura Chemical, Aichi Prefecture, Japan) ing ginamit para mikawani la reng peptides lalam ning partikular a gradient a kundisyun. Ing mobile phase yapin ing solvent A (0.08% formic acid) ampo ing solvent B (0.15% formic acid keng 80% acetonitrile). Ing Accela HPLC system (Thermo Fisher Scientific, Boston, Massachusetts) ginamit ya para i-elute ing column keng solvent A kilub ning 55 minutu keng flow rate a 50 μl min-1 kilub ning 5 minutu, at kaybat ing konsentrasyun ning solvent B miragdagan ya angga 40%. , United States). Ing eluate patuloy yang mipalub keng ESI ion source, at ding tryptic peptides ampo peptides a maki GPI-glycans me-analisa la kapamilatan ning LTQ Orbitrap XL (hybrid linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific). keng MS setup, ing voltage ning capillary source mitakda ya keng 4.5 kV, ampo ing temperatura ning transfer capillary menatili ya keng 300°C. Ing capillary voltage ampo ing tube lens voltage maka set la keng 15 V ampo 50 V, agpang kareti. Ing MS data mekwa ya keng positibong ion mode (resolution a 60,000; mass accuracy a 10 parts per million) keng mass range a 300/m/z mass/charge ratio (m/z) 3000. Ing MS/MS data mekwa ya kapamilatan ning ion trap keng LTQ Orbit [XL XL) nung nukarin ing mumunang digit makadependi ya keng digit collision induced dissociation (CID)].
Deng MD simulations megawa la gamit ing GROMACS (52) software ampo ing MARTINI 2 force field (53-55). Ing CHARMM GUI Membrane Builder (56, 57) ginamit de para gawan ing bilayer a maki dioleoylphosphatidylcholine (DOPC) ampo Cer C18 o DOPC ampo Cer C26. Ing topology ampo reng coordinates ning Cer C26 menibatan la keng DXCE kapamilatan ning pamaglako kareng dagdag a beads keng sphingosine tail. Gamitan me ing prosesung makalarawan lalam para i-balance ing double layer at i-run me, kaybat gamitan me ing tauling coordinates ning sistema para gawang sistemang ating Emp24. Ing transmembrane domain ning yeast Emp24 (residues 173 anggang 193) megawa ya bilang α-helix gamit ing visual MD (VMD) tool molecular structure (58). Kaybat, kaybat dang leko deng mipapalibut a lipids, ing protina me-granulate yang magaspang at milub ya keng bilayer gamit ing CHARMM GUI. Ing tauling sistema atin yang 1202 DOPC ampo 302 Cer C26 o 1197 DOPC ampo 295 Cer C18 ampo Emp24. Ionize ing sistema keng konsentrasyun a 0.150M. Apat a independenting replicates ing megawa para kareng adwang bilayer a komposisyun.
Ing lipid bilayer makabalanse ya gamit ing CHARMM GUI process, na maglaman yang pamagbawas at kaybat pamagbalanse 405,000 steps, nung nukarin ing position constraints bagya bagya yang mabawas at milako, at ing time step daragul ya manibat 0.005 ps angga 0.02 ps. Kaybat ning equilibration, magproduce yang 6 μs a maki time step a 0.02 ps. Kaybat meng isulud ing Emp24, gamitan me ing pareung CHARMM GUI a prosesu para mabawas at mabalanse ing sistema, at kaybat magdalagan ka kilub ning 8 s keng produksyun.
Para kareng eganaganang sistema, keng prosesu ning pamagbalanse, ing pressure kontroladu ne ning Berendsen barostat (59), at keng prosesu ning pamaglalang, ing pressure kontroladu ne ning Parrinello-Rahman barostat (60). Kareng eganaganang kasu, ing average a pressure 1 bar ya at ing semi-isotropic pressure coupling scheme ing gagamitan. keng prosesu ning pamagtimbang ampo pamaglalang, ing thermostat (61) a maki speed recalibration ing gagamitan para a-couple ing temperatura da reng protina, lipid ampo solvent particles. keng mabilug a operasyun, ing target a temperatura 310K ya. Ing non-bonding interaction makalkula ya kapamilatan ning pamaglalang pairing list gamit ing Verlet scheme a maki 0.005 buffer tolerance. Ing Coulomb term makalkula ya gamit ing reaction field ampo ing cut-off distance a 1.1 nm. Ing Vander Waals term gagamit yang cut-off scheme a maki cut-off distance a 1.1 nm, at ing Verlet cut-off scheme gagamit ya para keng potential drift (62).
Gamit ing VMD, ing cutoff wavelength pilatan da reng DOPC phosphate beads o ceramide AM1 beads ampo ing protina 0.7 nm ya, at ing bilang da reng lipid a makipag-ugnayan king protina makalkula ya. Agpang keng makatuking formula, kalkula me ing depletion-enrichment (DE) factor antimo ing (63): DE factor = (ing dagul ning kabilugan a lipids keng protina 0.7) keng protina 0.7 (ing dagul ning Cer keng kabilugan a lipids)
Ing mibalita a ulaga mekwa ya bilang average, at ding error bar apat lang independenting kopya ning SE. Ing statistical significance ning DE factor mekwa ya kapamilatan ning t test [(averageDE-factor-1)/SE]. Karkulan me ing P value ibat keng one-tailed distribution.
Ing GROMACS tool ginamit ya para makalkula ing 2D lateral density map ning sistemang ating Emp24 kilub ning tauling 250 ns ning trace. Para akwa ing enrichment/depletion map ning ceramide, ing density map ning Cer mepitna ya keng sum ning map ning Cer ampo DOPC, at kaybat mepitna ya keng concentration ning Cer keng katawan. Ing pareung kule ning mapa ing gagamitan.
Para kareng karagdagang materyales para kening artikulu, lawan me ing http://advances.sciencemag.org/cgi/content/full/6/50/eaba8237/DC1
Iti metung yang makabuklat a articulu a mipamye lalam ning tuntunan ning Creative Commons Attribution-Non-Commercial License, a magpaintulut king pamangamit, pamikalat ampong pamagparakal king nanu mang paralan, basta ing tauling pamangamit e ya para king komersyal a pakinabang at ing premise ya pin ing ustu ya ing orihinal a obra. Reperensya.
Tandanan: Anyaran mi mu ing ibie me ing kekang email address bang kanita abalu na ning taung irekomenda mu king page a buri meng akit ing email at e ya spam. E kami mag capture nanu mang email address.
Ing kutang ayni gamitan ya para subukan nung bisita ka at aiwasan ing automatic a pamagsubmit ning spam.
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 Lope (Sergio Lopez), Logazhope (Serho), (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Ing 3D high-resolution real-time imaging papakit na ing importansya ning ceramide chain length para keng protein sorting kareng selective 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 -Linero Lope (Sergio Lopez), Logazhope (Serho), (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Ing 3D high-resolution real-time imaging papakit na ing importansya ning ceramide chain length para keng protein sorting kareng selective output sites.
©2020 American Association for the Advancement of Science. makareserba la ngan deng karapatan. AAAS metung yang partner ning HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef ampo COUNTER. ScienceAdvances ISSN 2375-2548.