Protein a wɔhyehyɛ wɔ kokoam kwan no mu no ho hia na ama nkwammoaa no mu akyekyɛ ne homeostasis akɔ so. Wɔ akorade a wɔde yiyi nneɛma mu akyi no, dwuma a srade di wɔ kinesin a wɔhyehyɛ mu wɔ kokoam akwantu mu no yɛ asɛmmisa titiriw a akyɛ a wonnya mmuae. Ɛha yi, yɛyɛ 3D bere koro mu multicolor high-resolution real-resolution mfonini de kyerɛ wɔ vivo sɛ foforo synthesized glycosylphosphatidylinositol-immobilized proteins a ɛware yiye ceramide lipid moieties yɛ clustered na kyekyɛ mu soronko endoplasms Net exit site, a ɛsono nea transmembrane proteins de di dwuma. Afei nso, yɛkyerɛ sɛ nkɔnsɔnkɔnsɔn tenten a ɛwɔ ceramide mu wɔ endoplasmic reticulum membrane no mu no ho hia ma saa nhyehyɛe a wɔpaw yi. Yɛn nhwehwɛmu no de adanse a edi kan tẽẽ wɔ vivo mu de kyekyɛ protein nneɛma a egyina lipid nkɔnsɔnkɔnsɔn tenten so mu kɔ mmeae a wɔpaw a wɔde kɔ amannɔne wɔ kokoam kwan no mu ma.
Wɔ eukaryotic nkwammoaa mu no, afei wɔhyehyɛ protein ahorow a wɔayɛ wɔ endoplasmic reticulum (ER) no mu bere a wɔde fa kokoam kwan no so de kɔ baabi a ɛfata a nkwammoaa no kɔ (1). Wɔ coat-mediated sorting akyi no, na wɔasusuw bere tenten sɛ lipids bi nso betumi ayɛ adwuma sɛ mmeae a wɔpaw sɛ wobefi mu denam wɔn a wɔbɛboaboa ano ayɛ no membrane domains pɔtee bi a protein pɔtee bi (2-5) no so. Nanso, adanse a ɛkɔ so tẽẽ wɔ nipadua mu a ɛkyerɛ sɛ ebia saa adwinnade a egyina srade so yi da so ara nni hɔ. Sɛnea ɛbɛyɛ a yebedi ɔhaw titiriw yi ho dwuma no, yesuaa sɛnea wɔde glycosylphosphatidylinositol (GPI) anchored proteins (GPI-APs) fi ER kɔ amannɔne wɔ ɔkwan soronko so no ho ade wɔ mmɔkaw mu. GPI-APs yɛ nkwammoaa ani protein ahorow a ɛne lipid wɔ abusuabɔ ( 6, 7 ). GPI-AP yɛ protein a wɔde sie a ɛnam glycolipid fã (GPI anchor) no so bata plasma membrane no akyi nhaban ho. Wogye GPI anchors tom sɛ conservative post-translational nsakrae wɔ ER lumen (8). Sɛ ɛbata ho wie a, GPI-AP fa Golgi mfiri (5, 9) no mu fi ER no mu kɔ plasma membrane no mu. GPI anchors a ɛwɔ hɔ no ma wɔde GPI-AP fa protein ahorow a wɔde sie wɔ nipadua no mu (a protein afoforo a ɛwɔ nipadua no mu ka ho) ho fa kokoam kwan no so ( 5 , 9 , 10 ). Wɔ mmɔkaw nkwammoaa mu no, wɔtetew GPI-AP ahorow fi protein afoforo a wɔde sie wɔ endoplasmic reticulum no mu, na afei wɔkyekyere no gu ntini soronko a wɔde coat protein complex II (COPII) abɔ ho mu (6, 7). Nneɛma a ɛkyerɛ saa nkyekyɛmu nhyehyɛe yi wɔ ER export nhyehyɛe no mu no mu nna hɔ, nanso wɔsusuw sɛ ebia saa adwinnade yi behia lipids, titiriw structure remodeling a lipid fã a ɛwɔ GPI anchor ( 5 , 8 ). Wɔ mmɔkaw mu no, GPI srade a wɔsan yɛ no foforo fi ase bere a GPI no abata ho akyi pɛɛ, na wɔ nsɛm pii mu no, ɛma ceramide kyekyere 26-carbon long-chain saturated fatty acid (C26:0) no ho (11, 12). C26 ceramide ne ceramide titiriw a mmɔkaw nkwammoaa ayɛ de besi nnɛ. Wɔyɛ no wɔ ER mu na wɔde ne fã kɛse no ara kɔ Golgi mfiri no mu denam COPII vesicles so ( 13 ). ER export a GPI-AP pɔtee hwehwɛ sɛ kɔ so yɛ ceramide synthesis ( 14 , 15 ), na nea ɛbɛba no, nsakrae a ceramide dan inositol phosphate ceramide (IPC) wɔ Golgi mfiri no gyina GPI anchor synthesis ( 16 ). Biophysical nhwehwɛmu a wɔde artificial membranes ayɛ no ada no adi sɛ acyl nkɔnsɔnkɔnsɔn atenten paa ceramides tumi bom yɛ nhyehyɛe a wɔahyehyɛ a ɛwɔ honam fam su soronko ( 17 , 18 ). Saa nsɛm yi de adwene a ɛne sɛ C26 ceramide ne GPI-AP a C26 ceramide wom no de wɔn honam fam su di dwuma de bom yɛ mmeae anaa mmeae a ɛyɛ nhyehyɛe wɔ ER membrane lipid tebea a ɛyɛ basabasa kakra no mu. Ɛyɛ glycerolipids ntiantiaa ne nea enni ahoɔden (C16:1 ne C18:1) (19, 20) na ɛwom titiriw. Wɔbɛpaw saa mmeae yi wɔ mmeae pɔtee bi a wofi ER fi adi (ERES), baabi a wobetumi de ceramide ne ceramide a egyina GPI-AP so akɔ Golgi wɔ COPII vesicle koro no ara a wɔahyira so no mu (5).
Wɔ saa nhwehwɛmu yi mu no, yɛasɔ saa afiri a egyina srade so yi ahwɛ tẽẽ denam super-resolution confocal real-time imaging microscopy (SCLIM) a yɛde dii dwuma so, a ɛyɛ microscopy kwan a ɛyɛ nwonwa a ebetumi ahwɛ protein ahorow a wɔde fluorescent ahyɛ so bere koro mu Mfonini ahorow a ɛwɔ kɔla abiɛsa ne afã abiɛsa (3D) no wɔ nsusuwii ne ahoɔhare a ɛkorɔn yiye wɔ nkwammoaa a ɛte ase mu (21, 22).
Yɛdii kan de SCLIM mfiridwuma dii dwuma de kyerɛkyerɛɛ sɛnea wɔhwehwɛɛ GPI-AP a ɛteɛ a ɛwɔ C26 ceramide kuw no mu fii protein ahorow a wɔde sie wɔ transmembrane mu bere a wofii ER no mu wɔ S. cerevisiae mu no mu bio. Sɛnea ɛbɛyɛ a yɛbɛhwɛ ER nkyekyɛmu no, yɛde awosu nhyehyɛe a ebetumi ayɛ nneɛma foforo a wɔayɛ no ho mfonini tẽẽ a ɛhyɛn ERES mu wɔ vivo mu dii dwuma ( 7 , 23 ). Sɛ́ adesoa no, yɛpaw C26 ceramide-gyina GPI-AP Gas1 a wɔde green fluorescent protein (GFP) ahyɛ no agyirae ne transmembrane secreted protein Mid2 a wɔde near-infrared fluorescent protein (iRFP) ahyɛ no agyirae, a abien no nyinaa de wɔn ani si plasma membrane so (24-26). Wɔ sec31-1 temperature-sensitive mutant no mu no, wɔda saa nneɛma abien yi adi wɔ galactose-inducible promoter ne constitutive ERES marker ase. Wɔ ɔhyew a ɛboro so (37°C), esiane sɛ sec31-1 mutation no nya COPII atade fã Sec31 dwumadi so nkɛntɛnso ma esiw COPII fifi ne ER a wɔde kɔ amannɔne no ano nti, nneɛma a wɔayɛ no foforo boaboa ano wɔ ER (23). Bere a ɛyɛɛ nwini koduu ɔhyew a ɛba fam (24°C) akyi no, sec31-1 mutant nkwammoaa no san fii baabi a wɔde kokoam nneɛma gu no, na wofii ase de nneɛma foforo a wɔayɛ a wɔaboaboa ano no fii ER no kɔɔ amannɔne. CLIM mfonini kyerɛe sɛ Gas1-GFP ne Mid2-iRFP a wɔayɛ no foforo no mu dodow no ara da so ara boaboa ano wɔ sec31-1 mutant nkwammoaa no ER mu bere a wɔde ahyɛ 37°C mu akyi na afei wɔayi no adi wɔ 24°C simma 5 (Mfonini 1). Esiane sɛ Mid2-iRFP kyekyɛ wɔ ER membrane no nyinaa so, na Gas1-GFP no ayɛ kɛse na wɔaboaboa ano wɔ discontinuous ER membrane area nti, wɔn kyekyɛ yɛ soronko koraa (Mfonini 1, A kosi C ne Movie S1). Bio nso, sɛdeɛ wɔakyerɛ wɔ Mfonini 1D mu no, Gas1-GFP akuakuo no nni Mid2-iRFP. Saa nsunsuansoɔ yi kyerɛ sɛ wɔpaapaee GPI-AP ne transmembrane proteins mu kɔɔ ER membrane mpɔtam ahodoɔ mu ntɛm. Gas1-GFP akuakuo no bɛn ERES pɔtee bi a wɔde mCherry COPII coat protein Sec13 (Mfonini 1, E ne F, ne sini S1) (23) ahyɛ no agyirae.
sec31-1 nkwammoaa da galactose-a ɛde secretions ba, acyl nkɔnsɔnkɔnsɔn tenten (C26) ceramide GPI-AP Gas1-GFP (GPI-AP, ahabammono) ne transmembrane protein Mid2-iRFP (TMP, bruu) na wɔde saa Constructive ERES labeling Sec13-mCherry (ERES, magenta) yi too 37°C simma 30, de kɔɔ 24°C, na SCLIM yɛɛ mfonini wɔ simma 5 akyi. (A kosi C) kyerɛ ananmusifoɔ a wɔaka abom anaa 2D mfonini baako a ɛkyerɛ wimhyɛn (A), 2D projection mfonini a ɛwɔ z-afã 10 (B) anaa 3D cell hemisphere mfonini a ɛkyerɛ nneɛma ne ERES agyiraeɛhyɛdeɛ (C). Nsεmfua 1μm (A ne B). Nsusuwii no yɛ 0.551μm (C). Wɔhunuu Gas1-GFP wɔ ER mpɔtam anaa akuakuo a ɛsono emu biara mu, berɛ a wɔhunuu Mid2-iRFP na wɔkyekyɛɛ mu wɔ ER membrane (C) no nyinaa mu. (D) Graf no kyerɛ Gas1-GFP ne Mid2-iRFP a ɛwɔ Gas1-GFP akuakuo no mu wɔ agyan fitaa no so (benkum so). AU, unit a wɔde di dwuma wɔ ɔkwan a wɔpɛ so. (E ne F) gyina hɔ ma 3D mfonini a ɛka nneɛma ne ERES agyiraehyɛde bom. Wɔhunuu Gas1-GFP akuakuo wɔ baabi a ɛbɛn ERES pɔtee no. Nsusuwii no yɛ 0.551μm. (F) Agyan fitaa a ɛyɛ den no hyɛ Gas1-GFP akuakuo a ɛbata ERES ho no agyiraeɛ. Mfinimfini ne nifa so ntama no kyerɛ 3D mfonini a wɔaka abom a wɔatrɛw mu ne Gas1-GFP akuw a wɔapaw no a wɔadan no.
Abusuabɔ a ɛbɛn a ɛda Gas1-GFP akuakuo ne ERES pɔtee bi ntam no kyerɛ sɛ Gas1-GFP tumi hyɛn ERES a wɔpaw mu, a ɛyɛ soronko wɔ paw a Mid2-iRFP de di dwuma de firi ER no mu. Sɛ yɛbɛdi saa asɛm yi ho dwuma a, yɛbuu ERES nsusuiɛ dodoɔ maa nneɛma baako anaa mmienu pɛ (Mfonini 2, A kɔsi C). Yehui sɛ ERES dodow no ara (70%) kura nneɛma biako pɛ. Mfonini 2C ase mfonini no kyerɛ nhwɛso abien a wɔtaa yɛ a ɛfa ERES a Gas1-GFP nkutoo na ɛwɔ mu (Mfonini 1) anaa Mid2-iRFP nkutoo (Mfonini 2). Nea ɛne eyi bɔ abira no, bɛyɛ 20% wɔ ERES mu no, nneɛma abien a ɛka bom wɔ beae koro. Wohui sɛ ERES bi (10%) kura nneɛma ahorow abien, nanso na wɔatew wɔn ho wɔ mmeae ahorow a ɛda adi pefee. Enti, saa akontabuo nhwehwɛmu yi kyerɛ sɛ, sɛ wɔde ER no kɔ amannɔne wie a, wɔkyekyɛ GPI-AP Gas1-GFP ne transmembrane cargo Mid2-iRFP mu yɛ no ERES ahodoɔ (Mfonini 2D). Saa nhyehyɛe a wɔde yiyi nneɛma mu yiye yi ne biochemical nhwehwɛmu a atwam (6) ne morphological determination (7) no hyia yiye. Yebetumi nso ahwɛ sɛnea nneɛma a wɔde ato nnipa mu a ɛrehyɛn ERES mu no yɛ wɔn ade (Mfonini 2E ne Sini S2). Mfonini 2E kyerɛ sɛ Gas1-GFP (panel 3) anaa Mid2-iRFP (panel 4) no fã ketewaa bi pɛ na efi ɔfã biako hyɛn ERES no mu na wɔatoto mu wɔ beae a ɛda nsow. Panel 5 a ɛwɔ Mfonini 2E mu no kyerɛ sɛ ɛtɔ da bi a wohu Gas1-GFP ne Mid2-iRFP wɔ ERES koro no ara mu, nanso efi afã horow na ɛhyɛn mu na ɛyɛ kɛse wɔ mmeae ahorow a ebia egyina hɔ ma COPII vesicles ahorow. Yɛsan nso sii so dua sɛ mpaapaemu a wɔahu ne nkyekyɛmu a ɛfa C26 ceramide-gyina GPI-AP Gas1 ho sɛ ERES a wɔpaw no yɛ pɔtee efisɛ transmembrane secretion adesoa foforo, GFP-tagged plasma membrane protein Axl2 (27 ), a ɛkyerɛ suban a ɛte sɛ Mid2-iRFP. (Mfonini S1 ne Sini S3). Axl2-GFP a wɔayɛ no foforo no nam ER membrane no so kyekyɛ te sɛ Mid2-iRFP (Mfonini S1, A ne B), na ɛne Mid2-iRFP bom wɔ ERES dodow no ara mu (Mfonini S1, B kosi D). Panel 1 ne 2 a ɛwɔ Mfonini 1. S1C kyerɛ ERES nhwɛsoɔ mmienu a ɛtaa ba a transmembrane cargoes mmienu ka bom. Wɔ saa nsɛm yi mu no, nneɛma abien no nyinaa bom hyɛn ERES mu (Mfonini S1E, Panel 3 ne Movie S3).
Wɔde sec31-1 nkwammoaa a ɛda galactose inducible secretions adi, Gas1-GFP (GPI-AP, ahabammono) ne Mid2-iRFP (TMP, bruu) ne constitutive ERES labeling Sec13-mCherry (ERES, magenta) no guu 37 Bere a wɔde ahyɛ mu simma 30 wɔ °C akyi, tu kɔ 24 °C na ama wɔagyae secretion block no, na wɔde SCLIM mfonini wɔ akyi Simma 20 na ɛyɛ. (A kosi C) Ananmusifo 2D projection mfonini (A; scale bar, 1μm) anaa 3D cell hemisphere mfonini (B ne C; scale unit, 0.456μm) a ɛfa adesoa no ho ne z-afã horow 10 a wɔde ERES ahyɛ no agyirae. Panel a ɛwɔ aseɛ wɔ (B) ne panel a ɛwɔ (C) no kyerɛ mfonini a wɔayɛ ho adwuma de akyerɛ nneɛma a ɛwɔ ERES (magenta) [Gas1-GFP (gray) ne Mid2-iRFP (light blue)] nkutoo. (C) Agyan a wɔabue: ERES soa nneɛma biako pɛ (1 kosi 4). Agyan a ɛyɛ fitaa: ERES kura nneɛma a wɔatew mu (5). Agyan fitaa a ɛyɛ den: ERES a nneɛma a ɛwɔ baabi koro wom. Ase hɔ: ERES biako a wɔapaw no kura Gas1-GFP (1) anaa Mid2-iRFP (2) nkutoo. Scale bar, 100 nm. (D) Photomicrograph a wɔaka ho asɛm wɔ (C) no dodow a wɔbɛkyerɛ. Sɛ wɔkyekyɛ mu a, ERES ɔha biara mu nkyekyɛmu a nneɛma baako pɛ na ɛwɔ mu (Gas1-GFP anaa Mid2-iRFP), nneɛma a wɔatew mu ne nneɛma a ɛka bom. Wɔ nhwehwɛmu abiɛsa a wɔde wɔn ho mu mu no, n=432 wɔ nkwammoaa 54 mu. Mfomso bar = SD. T sɔhwɛ a ɛwɔ dua abien a enni abien. *** P = 0.0002 na ɛwɔ hɔ. (E) 3D mfonini a ɛkyerɛ ERES a wɔapaw a ɛfa nneɛma a wɔde ahyɛ mu a wɔde (C) ahyɛ no agyirae. Gas1-GFP (ahabammono) (3) anaa Mid2-iRFP (blue) (4) fi ɔfã biako hyɛn ERES (magenta) mu na wɔabara wɔ beae ketewaa bi wɔ ERES mu. Ɛtɔ da bi a, nneɛma ahorow abien no nyinaa fi ɔfã koro na ɛkɔ ERES koro no ara mu (5) na wɔde to beae bi a atew ne ho wɔ ERES no mu. Scale bar, 100 nm.
Afei, yɛsɔɔ adwene bi a ɛne sɛ acyl nkɔnsɔnkɔnsɔn tenten ceramide (C26) a ɛwɔ ER membrane no mu no ma Gas1 akuwakuw pɔtee ne nea wɔhyehyɛ no kɔ ERES a wɔpaw mu no hwɛe. Nea ɛbɛyɛ na yɛatumi ayɛ eyi no, yɛde mmɔkaw a wɔayɛ no foforo GhLag1 dii dwuma, a wɔde GhLag1 (Lag1 homolog a ɛwɔ kotoku mu) sii endogenous ceramide synthases abien Lag1 ne Lac1 ananmu, na ɛde mmɔkaw a ɛwɔ nkwammoaa mu ntini Ceramide ɔkwan a ɛyɛ tiaa sen wuram su (Mfonini 3A) (28) bae. Mass spectrometry (MS) nhwehwɛmu kyerɛɛ sɛ wɔ wuram-su ahorow mu no, ceramide nyinaa mu 95% yɛ nkɔnsɔnkɔnsɔn ceramide tenten paa (C26), bere a wɔ GhLag1 mu no, ceramide no 85% yɛ tenten yiye (C18 ne C16). ), ceramide 2% pɛ na ɛyɛ nkɔnsɔnkɔnsɔn ceramide a ɛware yiye (C26). Ɛwom sɛ C18 ne C16 ceramides ne ceramides titiriw a wɔahu wɔ GhLag1 membrane no mu de besi nnɛ de, nanso MS nhwehwɛmu nso sii so dua sɛ GPI anchor a ɛwɔ Gas1-GFP a wɔda no adi wɔ GhLag1 ɔkwan no mu no kura C26 ceramide, a wɔde toto wuram-su lipids ho. Su no yɛ pɛ (Mfonini 3A) (26). Enti, eyi kyerɛ sɛ ceramide remodeling enzyme Cwh43 no paw C26 ceramide kɛse, sɛnea wɔakyerɛ wɔ Mfonini 26 mu no, ɛpɛ sɛ ɛde GPI anchor a efi C26 ceramide kakraa bi mu wɔ GhLag1 ɔkwan no mu no ka ho. S2 (29) (29) no. Ne nyinaa mu no, C18-C16 ceramide nkutoo na ɛwɔ GhLag1 nkwammoaa mu ntini no mu titiriw, bere a Gas1-GFP da so ara wɔ C26 ceramide. Saa nokwasɛm yi ma saa nhyɛso yi yɛ adwinnade a eye a wɔde bedi ɔhaw a ɛfa acyl nkɔnsɔnkɔnsɔn tenten a ɛwɔ membrane ceramide mu wɔ ER no ho dwuma pɔtee. Dwuma a adesuakuw ne nhyehyɛe di wɔ nsusuwii hunu mu. Afei, yedii kan suaa tumi a C26 Gas1-GFP tumi boaboa ano wɔ akuwakuw mu wɔ GhLag1 mu denam ɔhyew-te nka mutant allele a ɛyɛ sec31-1 denam amanne kwan so fluorescence microscopy so, baabi a nkɔnsɔnkɔnsɔn tenten (C18-C16) nkutoo na ɛwɔ ER membrane Ceramide (Mfonini 3). Yɛhunuu sɛ wɔ sec31-1 mu no, na Gas1-GFP dodoɔ no ara ayɛ akuwakuw, berɛ a Gas1-GFP a ɛwɔ sec31-1 GhLag1 a ɛwɔ ceramide ER membrane tenten (C18-C16) tenten mu no, titire no na ɛnyɛ akuakuo na wɔakyekyɛ wɔ Wɔ ER membrane no nyinaa mu. Sɛ yɛbɛka no pɛpɛɛpɛ a, esiane sɛ C26 ceramide-based clustering ne ERES pɔtee bi wɔ abusuabɔ kɛse (Mfonini 1) nti, afei yɛhwehwɛɛ mu sɛ ebia saa adeyɛ yi nso betumi afa ER export protein mechanism no dwumadi ho anaa. GPI-AP de COPII nhyehyɛe titiriw bi di dwuma ma ER export, a ɛyɛ nnam regulated denam Ted1′s structural remodeling a glycan fã a GPI anchor ( 30 , 31 ). Afei wohu GPI-glycan a wɔasan ayɛ no foforo no denam transmembrane cargo receptor p24 complex no so, a ɛno nso paw Lst1, a ɛyɛ isoform pɔtee bi a ɛyɛ COPII adesoa binding subunit titiriw Sec24, a ɛyɛ GPI-AP-rich COPII Vesicles ho hia (31-33). Enti, yɛyɛɛ mutant abien a ɛkaa protein biako yi a wɔpopa (p24 complex component Emp24, GPI-glycan remodeling enzyme Ted1 ne COPII subunit pɔtee Lst1) ne sec31-1 mutant strain no boom, na yesuaa wɔn ho ade So ɛyɛ yiye sɛ wɔbɛhyehyɛ Gas1-cluster GFP (Mfonini 3). Yɛhunuu sɛ wɔ sec31-1emp24Δ ne sec31-1ted1Δ mu no, Gas1-GFP titire no yɛ unclustered na ɛkyekyɛ wɔ ER membrane no nyinaa mu, sɛdeɛ yɛadi kan ahunu wɔ sec31-1 GhLag1 mu, berɛ a wɔ sec31-1lst1Δ mu no, Gas1-GFP Te sɛ sec31-1. Saa nsunsuansoɔ yi kyerɛ sɛ, wɔ C26 ceramide a ɛwɔ ER membrane no mu akyi no, ɛhia sɛ Gas1-GFP a ɛboaboa ano no nso kyekyere p24 complex no, na ɛnhia sɛ wɔfa Lst1 pɔtee. Afei, yɛhwehwɛɛ sɛnea ebetumi aba sɛ nkɔnsɔnkɔnsɔn tenten a ɛwɔ ceramide mu wɔ ER membrane no mu no betumi ahwɛ Gas1-GFP a ɛkyekyere p24 no so. Nanso, yehui sɛ C18-C16 ceramide a ɛwɔ membrane no mu no nnya GPI-glycans a p24 complex no san ayɛ no foforo (Mfonini S3 ne S4, A ne B) anaasɛ ɛkyekyere GPI-AP na ɛde GPI-AP kɔ amannɔne no so nkɛntɛnso. tumi. Fa COPII subtype Lst1 (Mfonini S4C) no. Enti, C26 ceramide-gyina clustering nhwehwɛ sɛ protein nkitahodi ne ER export protein akwan horow, na mmom ɛfoa nhyehyɛe foforo a wɔde hyehyɛ nneɛma a lipid tenten na ɛkanyan. Afei, yɛyɛɛ nhwehwɛmu sɛ ebia ceramide acyl nkɔnsɔnkɔnsɔn tenten a ɛwɔ ER membrane no mu no ho hia ma Gas1-GFP nkyekyɛmu a etu mpɔn sɛ ERES a wɔpaw. Esiane sɛ Gas1 a ɛwɔ GhLag1 ɔkwan a ɛwɔ nkɔnsɔnkɔnsɔn tiawa ceramide mu no fi ER no mu na ɛkɔ plasma membrane no mu (Mfonini S5) nti, yegye di sɛ sɛ wɔde ceramide acyl nkɔnsɔnkɔnsɔn no tenten na ɛma wɔhyehyɛ no a, wobetumi asan de Gas1 a ɛwɔ GhLag1 ɔkwan no mu no akɔ baabi foforo na wɔatwa. ERES nneɛma a ɛwɔ membrane koro no ara.
(A) C18-C16 ceramides ntiantiaa na ɛwɔ GhLag1 nkwammoaa mu ntini no mu titiriw, bere a Gas1-GFP GPI anchor no da so ara wɔ C26 IPC koro no ara a ɛwɔ wuram-su nkwammoaa no. Atifi hɔ: acyl nkɔnsɔnkɔnsɔn tenten nhwehwɛmu a ɛfa ceramide a ɛwɔ nkwammoaa mu ntini a ɛwɔ wuram-su (Wt) ne GhLag1p ahorow mu denam mass spectrometry (MS) so. Data no gyina hɔ ma ceramide nyinaa ɔha biara mu nkyem 100. Sɛ wɔkyekyem pɛpɛɛpɛ a, sɔhwɛ abiɛsa a wɔde wɔn ho ayɛ. Mfomso bar = SD. T sɔhwɛ a ɛwɔ dua abien a enni abien. **** P <0.0001 na ɛwɔ hɔ. Bottom panel: MS nhwehwɛmu a ɛfa acyl nkɔnsɔnkɔnsɔn tenten a ɛwɔ IPC a ɛwɔ Gas1-GFP (GPI-IPC) GPI anchor a wɔada no adi wɔ wuram-su ne GhLag1p ahorow no mu. Data no gyina hɔ ma ɔha biara mu nkyem 100 a ɛwɔ IPC sɛnkyerɛnne nyinaa mu. Sɛ wɔkyekyem pɛpɛɛpɛ a, sɔhwɛ ahorow anum a wɔde wɔn ho ayɛ. Mfomso bar = SD. T sɔhwɛ a ɛwɔ dua abien a enni abien. ns, ɛnyɛ nea ɛho hia. P = 0.9134 na ɛwɔ hɔ. (B) Fluorescence micrographs of sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ and sec31-1lst1Δ cells expressing galactose-induced Gas1-GFP were incubated at 37°C for 30 minutes and passed down to Perform routine fluorescence microscopy after 24°C na ɛyɛ hyew. Agyan fitaa: ER Gas1-GFP akuw. Agyan a wɔabue: Wɔakyekyɛ Gas1-GFP a ɛnyɛ akuwakuw wɔ ER ntini no nyinaa so, na ɛkyerɛ ER su nuklea ring staining. Scale bar, 5μm. (C) Photomicrograph a wɔaka ho asɛm wɔ (B) no dodow a wɔbɛkyerɛ. Sɛ wɔkyekyem pɛpɛɛpɛ a, nkwammoaa a wɔwɔ punctate Gas1-GFP nhyehyɛe no ɔha biara mu nkyem 100. Wɔ nhwehwɛmu abiɛsa a wɔde wɔn ho mu no, n≥300 nkwammoaa. Mfomso bar = SD. T sɔhwɛ a ɛwɔ dua abien a enni abien. **** P <0.0001 na ɛwɔ hɔ.
Sɛnea ɛbɛyɛ a yebedi ɔhaw yi ho dwuma tẽẽ no, yɛyɛɛ SCLIM mfonini a ɛkyerɛ Gas1-GFP ne Mid2-iRFP wɔ GhLag1 mu a sec31-1 ɔhyew-te nka mutant allele (Mfonini 4 ne Sini S4). Bere a wɔkoraa ER no so wɔ 37°C na akyiri yi woyii no adi wɔ 24°C akyi no, wɔanboaboa Gas1-GFP a wɔayɛ no foforo no mu dodow no ara ano na wɔakyekyɛ wɔ ER ntini no nyinaa mu, sɛnea wɔde afiri a wɔde hwɛ nneɛma nketenkete a wɔtaa de di dwuma hui no (Mfonini 4, A ne B ). Bio nso, ERES ɔha mu nkyekyɛmu kɛseɛ (67%) ka nneɛma ahodoɔ mmienu a ɛwɔ mu a ɛka bom (Mfonini 4D). Panel 1 ne 2 a ɛwɔ Mfonini 4C mu no kyerɛ nhwɛsoɔ mmienu a ɛyɛ soronko a ɛfa ERES a ɛwɔ Gas1-GFP ne Mid2-GFP a ɛka bom ho. Bio nso, wɔfaa nneɛma mmienu no nyinaa kɔɔ ERES koro no ara mu (Mfonini 4E, panel 3 ne sini S4). Enti, nea yenya fii mu no kyerɛ sɛ ceramide acyl nkɔnsɔnkɔnsɔn a ɛwɔ ER membrane no mu tenten yɛ ade titiriw a ɛkyerɛ ER protein a wɔboaboa ano ne nkyekyɛmu.
Sec31-1 GhLag1 nkwammoaa a ɛda galactose-a ɛma nsuo a ɛba, Gas1-GFP (GPI-AP, ahabammono) ne Mid2-iRFP (TMP, bruu) ne constitutive ERES-labeled Sec13-mCherry (ERES, magenta) adi no Toto 37°C. Toa so simma 30, kɔ fam kodu 24°C na ama nsu a efi mu aba no afi hɔ, na fa SCLIM yɛ mfonini wɔ simma 20 akyi. (A kosi C) Ananmusifo 2D projection mfonini (A; scale bar, 1μm) anaa 3D cell hemisphere mfonini (B ne C; scale unit, 0.45μm) a ɛwɔ z-afã horow 10 a wɔde nneɛma ne ERES ahyɛ no agyirae no mu. Panel a ɛwɔ aseɛ wɔ (B) ne panel a ɛwɔ (C) no kyerɛ mfonini a wɔayɛ ho adwuma de akyerɛ nneɛma a ɛwɔ ERES (magenta) [Gas1-GFP (gray) ne Mid2-iRFP (light blue)] nkutoo. (C) Agyan a wɔahyɛ no ma fitaa: ERES, nneɛma a ɛka bom. Open arrow: ERES no kura ade biako pɛ. Lower panel: ERES a wɔapaw no wɔ nneɛma a ɛkata so (1 ne 2) a wɔahyɛ no agyirae wɔ (C) mu. Scale bar, 100 nm. (D) Photomicrograph a wɔaka ho asɛm wɔ (C) no dodow a wɔbɛkyerɛ. Wɔ sec31-1 ne sec31-1 GhLag1 akuo no mu no, nneɛma baako pɛ (Gas1-GFP anaa Mid2-iRFP) na ɛka ho, na sɛ wɔkyekyɛ mu a, ERES ɔha biara mu nkyekyɛmu a ɛfa nneɛma a atew ne ho ne nneɛma a ɛka bom ho. Wɔ nhwehwɛmu abiɛsa a wɔde wɔn ho mu no, n = 432 wɔ nkwammoaa 54 mu (sec31-1) ne n = 430 wɔ nkwammoaa 47 mu (sec31-1 GhLag1). Mfomso bar = SD. T sɔhwɛ a ɛwɔ dua abien a enni abien. *** P = 0.0002 (sec31-1) ne ** P = 0.0031 (sec31-1 GhLag1) na ɛwɔ hɔ. (E) 3D mfonini a ɛkyerɛ ERES a wɔapaw a nneɛma a ɛkata so (3) a wɔahyɛ no agyirae wɔ (C) mu. Gas1-GFP (ahabammono) ne Mid2-iRFP (blue) bɛn ERES (magenta) fi ɔfã koro na wɔtra beae koro no ara a wɔabara ERES. Scale bar, 100 nm.
Saa nhwehwɛmu yi de adanseɛ tẽẽ ma wɔ vivo mu sɛ wɔkyekyɛ protein nneɛma a egyina lipid so mu kɔ mmeaeɛ a wɔpaw sɛ wɔde kɔ amannɔne wɔ kokoam kwan no mu, na ɛda hia a acyl nkɔnsɔnkɔnsɔn tenten ho hia ma nkyekyɛmu a wɔpaw adi. Yɛde afiri a wɔde hwɛ nneɛma nketenkete a tumi wom na ɛyɛ nwonwa a wɔfrɛ no SCLIM dii dwuma de kyerɛɛ Gas1-GFP a wɔayɛ no foforo (plasma membrane GPI-AP titiriw a ɛwɔ acyl nkɔnsɔnkɔnsɔn tenten paa (C26) ceramide lipid fã) wɔ mmɔkaw mu ) Mpɔtam a wɔaboaboa ano wɔ ER ahorow a ɛsono emu biara mu no ne ERES pɔtee bi wɔ abusuabɔ, bere a protein ahorow a wɔde sie wɔ nipadua no mu no kyekyɛ wɔ ER ntini no nyinaa mu (Mfonini 1). Bio nso, saa nneɛma ahodoɔ mmienu yi hyɛn ERES ahodoɔ mu a wɔpaw (Mfonini 2). Acyl nkɔnsɔnkɔnsɔn tenten a ɛwɔ nkwammoaa mu ceramide a ɛwɔ membrane no mu no so tew fi C26 kɔ C18-C16, wɔsɛe Gas1-GFP akuw no kɔ ER mpɔtam a ɛda nsow no mu, na wɔsan de Gas1-GFP kwan ma efi ER no mu a transmembrane protein no nam ERES koro no ara so (Mfonini 3 ne Mfonini 3). 4).
Ɛwom sɛ GPI-AP de protein kwan titiriw bi di dwuma de fi ER mu de, nanso yehui sɛ mpaapaemu a egyina C26 ceramide so no nnyina protein nkitahodi soronko a ebetumi ama ERES ayɛ soronko so (Mfonini S4 ne S5). Mmom no, nea yɛahu no foa ɔkwan foforo a wɔfa so kyekyɛ nneɛma mu a protein a egyina srade so a wɔboaboa ano ne nea akyiri yi wɔyi nneɛma afoforo fi mu na ɛkanyan no so. Yɛn nhwɛsoɔ kyerɛ sɛ Gas1-GFP mantam anaa akuakuo a ɛbata ERES pɔtee bi ho no nni transmembrane secreted protein Mid2-iRFP, a ɛkyerɛ sɛ C26 ceramide-dependent GPI-AP cluster no bɛma ayɛ mmerɛw sɛ wɔbɛkɔ ERES a ɛfa ho no mu, na bere koro no ara mu no, wɔayi transmembrane The secretions enter this particular ERES (Mfonini 1 ne 2). Nea ɛne eyi bɔ abira no, C18-C16 ceramides a ɛwɔ ER membrane no mu no mma GPI-AP nnyɛ mpɔtam anaa akuwakuw, enti ɛnnyi protein a transmembrane fi mu no mfi mu anaasɛ ɛnsi ananmu nkɔ ERES koro no ara mu (Mfonini 3 ne 4). . Enti, yɛhyɛ nyansa sɛ C26 ceramide ma mpaapaemu ne nkyekyɛmu ba denam protein ahorow a ɛbata ERES pɔtee bi ho a wɔboaboa ano a ɛma ɛyɛ mmerɛw no so.
Ɔkwan bɛn so na wobetumi anya saa C26 ceramide-dependent clustering yi akɔ ER beae pɔtee bi? Su a membrane ceramide wɔ sɛ ɛbɛtetew wɔ nkyɛnkyɛn no betumi ama GPI-AP ne C26 ceramide ayɛ lipids nketewa a wɔahyehyɛ no ntɛm ara wɔ lipid tebea a ɛnyɛ pɛpɛɛpɛ kɛse wɔ ER membrane a glycerolipids ntiantiaa ne nea enni mu wom no mu. Nneɛma a ɛyɛ papa akuwakuw (17, 18). Wobetumi de saa bere tiaa mu akuwakuw nketewa yi afrafra bio ayɛ no akuwakuw akɛse a ɛyɛ den bere a wɔakyekyere p24 a ɛyɛ den no awie no ( 34 ). Nea ɛne eyi hyia no, yɛkyerɛe sɛ ɛsɛ sɛ C26 Gas1-GFP ne p24 complex no di nkitaho na ama ayɛ akuwakuw akɛse a wotumi hu (Mfonini 3). p24 complex no yɛ heterozygous oligomer a ɛyɛ p24 transmembrane protein ahorow anan a ɛwɔ mmɔkaw mu ( 35 ), a ɛma multivalent binding, a ebetumi ama cross-linking GPI-AP clusters nketewa, na ɛnam so ama Stable cluster akɛse aba ( 34 ). Nkitahodi a ɛda protein ectodomains a ɛwɔ GPI-APs ntam nso betumi aboa ma wɔaboaboa ano, sɛnea wɔada no adi wɔ wɔn Golgi akwantu mu wɔ nufuboa polarized epithelial nkwammoaa mu ( 36 ). Nanso, sɛ C18-C16 ceramide wɔ ER membrane no mu, bere a p24 complex no kyekyere Gas1-GFP no, akuw akɛse a wɔatew wɔn ho renhyehyɛ. Ebia adwinnade a ɛhyɛ ase no begyina nipadua ne nnuru pɔtee a ɛwɔ acyl nkɔnsɔnkɔnsɔn tenten ceramide no mu so. Biophysical nhwehwɛmu a wɔayɛ wɔ artificial membranes ho no kyerɛ sɛ ɛwom sɛ acyl chain ceramides atenten (C24) ne tiawa (C18-C16) nyinaa betumi ama phase separation aba de, nanso acyl chain ceramides atenten (C24) nkutoo na ebetumi ama Curvature a ɛkorɔn ne film bending anya nkɔso de asan ayɛ film no. Ɛnam wɔn ho wɔn ho a wɔde wɔn ho bɛhyɛ mu (17, 37, 38). Wɔada no adi sɛ transmembrane helix a TMED2, onipa homologue a Emp24, paw sɛ ɛne C18 ceramide-gyina sphingomyelin di nkitaho wɔ cytoplasmic lobules ( 39 ). Yɛde molecular dynamics (MD) simulations dii dwuma no, yehui sɛ C18 ne C26 ceramides nyinaa boaboa ano twa cytoplasmic lobules a ɛwɔ Emp24 transmembrane helix no ho hyia, na wɔwɔ nea wɔpɛ a ɛte saa ara (Mfonini S6). Ɛfata sɛ yɛhyɛ no nsow sɛ eyi kyerɛ sɛ Emp24 transmembrane helix no betumi ama lipids a ɛwɔ membrane no mu no akyekyɛ asymmetric. Eyi yɛ nea afi mu aba nnansa yi a egyina nufuboa nkwammoaa so. MD simulations a ɛte saa ara nso kyerɛ sɛ ether lipids wɔ hɔ (40) . Enti, yɛsusu sɛ C26 ceramide a ɛwɔ ER26 lobules mmienu no mu no yɛ nea ɛwɔ mpɔtam hɔ. Sɛ GPI-AP a ɛwɔ luminal lobules no mu no kyekyere multivalent p24 tẽẽ na C26 ceramide a ɛboaboa ano twa p24 ho hyia wɔ cytoplasmic lobules no mu a, ebetumi ama Protein a ɛka ho no ayɛ kɛse na membrane curvature no nam nsateaa no so na ɛba ( 41 ), na ɛma GPI-AP tetew kɔ mmeae a ɛsono emu biara a ɛbɛn ERES, a ɛno nso boa ma ɛyɛ curved kɛse no mmeae a ɛwɔ ER ntini no mu ( 42 ). Amanneɛbɔ ahorow a atwam no foaa ɔkwan a wɔahyɛ ho nyansa no so ( 43 , 44 ). Multivalent a ɛkyekyere oligolectins, pathogens anaa antibodies to ceramide-based glycosphingolipids (GSL) wɔ plasma membrane no kanyan GSL aggregation kɛse, ɛma phase mpaapaemu yɛ kɛse na ɛma membrane deformation ne internalization ( 44 ). Iwabuchi ne nea ɛkeka ho (43) Wohui sɛ wɔ acyl nkɔnsɔnkɔnsɔn atenten (C24) nanso ɛnyɛ tiawa (C16) a ɛwɔ hɔ no, multivalent ligand a ɛkyekyere GSL lactosylceramide no maa akuwakuw akɛse ne membrane invagination hyehyɛe, na cytoplasm Lyn-ntamgyinafo sɛnkyerɛnne transduction wɔ nkrataa no so no yɛ interdigitated denam acyl nkɔnsɔnkɔnsɔn a wɔaka abom so neutrophils a ɛma nipadua no yɛ adwuma.
Wɔ nufuboa polarized epithelial nkwammoaa mu no, anti-Golgi network (TGN) no dodow a ɛkɔ soro kodu apical plasma membrane no gyinabea no na ɛhwɛ GPI-AP a wɔpaapae ne nea wɔhyehyɛ no so ( 10 , 45 ). Saa nkabom yi nam GPI-AP oligomerization ( 36 ) so na ɛba, nanso ebetumi agyina ceramide nkɔnsɔnkɔnsɔn tenten a yehu wɔ mmɔkaw mu nso so. Ɛwom sɛ nufuboa GPI-AP wɔ ether lipid-based anchor, na ne nnuru nhyehyɛe yɛ soronko koraa wɔ acyl nkɔnsɔnkɔnsɔn a ɛware yiye ceramide no ho de, nanso nhwehwɛmu bi a wɔyɛe nnansa yi kyerɛ sɛ lipids abien no nyinaa wɔ adannandi mu a ɛte sɛ honam fam ne nnuru su Ne dwumadi ( 40 ). Enti, ebia ether lipid fã a ɛwɔ nufuboa nkwammoaa mu no bɛyɛ te sɛ C26 ceramide a ɛwɔ mmɔkaw mu no, na ne dwumadi ne sɛ ɛne ceramide a ɛwɔ nkɔnsɔnkɔnsɔn tenten a ɛwɔ ntini no mu no bɛbɔ de ahyɛ GPI-AP a wɔboaboa ano ne nea wɔhyehyɛ no ho nkuran. Ɛwom sɛ ɛda so ara hia sɛ wɔsɔ saa asɛm yi hwɛ tẽẽ de, nanso nea wɔadi kan ahu no foa so sɛ ɛnyɛ cytoplasmic transfer proteins na ɛde acyl nkɔnsɔnkɔnsɔn tenten no kɔ Golgi nipadua no mu, na mmom egyina GPI ankora te sɛ mmɔkaw a wɔyɛ so. Enti, ɛte sɛ nea adannandi afiri a ɛhwɛ nneɛma so no tumi paw sɛ ɛde acyl nkɔnsɔnkɔnsɔn atenten yiye ceramide ne GPI-AP (13, 16, 20, 46, 47) kɔ baabiara wɔ akwantu vesicle koro no ara mu.
Wɔ mmɔkaw ne nufuboa polarized epithelial cell systems mu no, GPI-AP a ɛboaboa ano ne nea ɛtetew fi plasma membrane protein afoforo mu nyinaa ba ansa na adu nkwammoaa no ani. Paladino ne afoforo. (48) hui sɛ wɔ TGN a nufuboa polarized epithelial nkwammoaa, GPI-AP clustering nyɛ sɛ ɛho hia ma selective classification of GPI-APs to apical plasma membrane, na mmom ɛhwɛ clustering nhyehyɛe a GPI-APs ne Ne biological dwumadi nso. Nkwammoaa no ani. Wɔ mmɔkaw mu no, nhwehwɛmu yi kyerɛe sɛ C26 ceramide-dependent GPI-AP cluster wɔ ER no betumi ahwɛ cluster nhyehyɛe ne dwumadi dwumadi a GPI-AP wɔ wɔ plasma membrane so ( 24 , 49 ). Nea ɛne saa nhwɛso yi hyia no, GhLag1 nkwammoaa no ho yɛ wɔn hyew wɔ GPI inhibitors anaa nnuru a ɛka nkwammoaa fasu mudi mu kura ho ( 28 ), na hia a ehia sɛ wɔyɛ Gas1-GFP akuwakuw a ɛyɛ adwuma ( 49 ) a ɛwɔ tip ceramide a wɔahyɛ ho nkɔm wɔ mmɔkaw nkwammoaa aware mu no kyerɛ G Possible physiological consequences of hLag1 cells. GPI-AP mfomso. Nanso, sɔhwɛ foforo a wɔbɛyɛ sɛ ebia wɔayɛ nkwammoaa no ani dwumadi nhyehyɛe ho nhyehyɛe afi ER no mu denam ɔkwan a wɔfa so hyehyɛ nneɛma a egyina srade tenten so so no bɛyɛ yɛn daakye nhwehwɛmu asɛmti.
Wɔabobɔ Saccharomyces cerevisiae ahorow a wɔde dii dwuma wɔ adwuma yi mu no din wɔ Table S1 mu. Wɔyɛɛ SCLIM ahorow a wɔde yɛ nkwammoaa a nkwa wom mfonini wɔ MMY1583 ne MMY1635 ahorow no wɔ W303 akyi. Wɔde polymerase chain reaction (PCR)-gyina kwan a pFA6a plasmid yɛ template ( 23 ) na ɛyɛɛ saa nkwaboaa yi a ɛda Sec13-mCherry adi a ɛwɔ fluorescent protein tag ( 23 ). Wɔyɛɛ ɔkwan a ɛda Mid2-iRFP a wɔde fluorescent protein ahyɛ no agyirae wɔ GAL1 promoter no tumi ase adi sɛnea edidi so yi. PCR a wɔde ma iRFP-KanMx ntoatoasoɔ a ɛfiri pKTiRFP-KAN vector (akyɛdeɛ a ɛfiri E. O’Shea, Addgene plasmid nɔma 64687; http://n2t.net/addgene: 64687; nhwehwɛmu ahodeɛ a ɛkyerɛ (RRID): Addgene_64687) Na wɔde ahyɛ C-terminus a ɛwɔ endogenous Mid2 mu. Bere a wɔmaa Mid2-iRFP genome ntoatoaso no yɛɛ kɛse na wɔyɛɛ no ​​sɛnea ɛte wɔ GAL1 promoter no mu akyi no, wɔde kaa Not I-Sac I beae a ɛwɔ integration plasmid pRS306 no mu. Wɔde Pst I yɛɛ plasmid pRGS7 a efii mu bae no linearized sɛnea ɛbɛyɛ a ɛbɛka URA3 beae no ho.
Wɔda Gas1-GFP fusion gene no adi wɔ GAL1 promoter no tumi ase wɔ centromere (CEN) plasmid no mu, a wɔayɛ no sɛnea edidi so yi. Wɔnam PCR so maa Gas1-GFP ntoatoaso no yɛɛ kɛse fii pRS416-GAS1-GFP plasmid (24) (L. Popolo akyɛde) mu na wɔyɛɛ no ​​CEN plasmid pBEVY-GL LEU2 (C akyɛde) no Xma I–Xho I beae no. Miller; Fa gene plasmid nɔma 51225 ka ho; 10. Ɔkwan bɛn so na wobetumi ayɛ eyi. 51225; RRID: Addgene_51225) a ɛwɔ hɔ no. Wɔtoo plasmid a efii mu bae no din pRGS6. Axl2-GFP fusion gene no nso da adi wɔ GAL1 promoter a ɛwɔ pBEVY-GL LEU2 vector no tumi ase, na ne nhyehyɛe te sɛ nea edidi so yi. Wɔnam PCR so maa Axl2-GFP ntoatoaso no yɛɛ kɛse fii pRS304-p2HSE-Axl2-GFP plasmid (23) mu, na wɔde yɛɛ clone wɔ Bam HI-Pst I beae a pBEVY-GL LEU2 vector no wɔ no. Wɔtoo plasmid a efii mu bae no din pRGS12. Wɔabobɔ oligonucleotide ahorow a wɔde dii dwuma wɔ nhwehwɛmu yi mu no nnidiso nnidiso wɔ Table S2 mu.
Wɔde adenine 0.2% ne glucose 2% [YP-dextrose (YPD)], raffinose [YP-raffinose] 2% a ɛdɔɔso wɔ mmɔkaw mu protein p (YP) aduru (1 % Yeast extract ne 2% protein ept) kaa ɔkwan a wɔfa so yɛ no ho. (YPR)] anaa 2% galactose [YP-galactose (YPG)] sɛ carbon fibea, anaasɛ wɔ synthetic minimal medium (0.15% yeast nitrogen base ne 0.5% ammonium sulfate) de ka amino acids ne bases a ɛfata a ɛho hia ma aduannuru , Na ɛwɔ 2% glucose (synthetic glucose minimal medium) anaa 2% galactose (synthetic galactose minimal medium) sɛ carbon fibea.
Wɔ bere ankasa mu mfoniniyɛ mu no, wɔde sec31-1 mutant nkwammoaa a ɛte ɔhyew nka a ɛda construct no adi wɔ GAL1 promoter ase no nyin wɔ YPR medium mu wɔ 24°C anadwo kosii mid-log phase. Bere a wɔde nkwammoaa no ahyɛ YPG mu wɔ 24°C mu dɔnhwerew 1 akyi no, wɔde nkwammoaa no too SG mu wɔ 37°C mu simma 30, na afei wɔde kɔɔ 24°C mu ma wogyaee fii secretion block no mu. Wɔde Concanavalin A siesiee nkwammoaa no wɔ ahwehwɛ slide so na wɔde SCLIM yɛɛ mfonini. SCLIM yɛ Olympus IX-71 inverted fluorescence microscope ne UPlanSApo 100×1.4 akontabuo aperture ngo lens (Olympus), ahoɔhare kɛseɛ ne kɛseɛ-signal-to-noise ratio rotating disc confocal scanner (Yokogawa Electric), custom spectrometer, ne custom cooling a wɔaka abom The system’s image intensifier (Hamamatsu Photonics) betumi ama wɔanya ahwehwɛ a ɛma nneɛma kɛse yɛ kɛse a etwa to a ɛma ɛyɛ kɛse ×266.7 ne mfiri a wɔde charge-coupled device camera a ɛma ɛlɛtrɔnik dɔɔso (Hamamatsu Photonics) (21). Wɔde software a wɔahyɛ da ayɛ (Yokogawa Electric) na ɛyɛ mfonini a wonya. Wɔ 3D mfonini ahorow ho no, yɛde piezoelectric actuator a wɔayɛ no sɛnea wɔpɛ dii dwuma de wosow botae ahwehwɛ no vertically, na yɛboaboaa optical afã horow no ano 100 nm a ɛda ntam wɔ stack mu. Wɔdane Z-stack mfonini no kɔ 3D voxel data mu, na wɔde theoretical point spread function a wɔde di dwuma ma rotating disc confocal microscope no di dwuma ma deconvolution processing denam Volocity software (PerkinElmer) so. Ɛdenam Volocity software a wɔde dii dwuma de yɛɛ threshold ankasa ma co-location analysis so no, wɔsusuu ERES a nneɛma ka ho. Wɔde MetaMorph software (Molecular Devices) na ɛyɛɛ line scan nhwehwɛmu.
Fa GraphPad Prism software di dwuma de kyerɛ akontaabu mu ntease. Wɔ Student’s t-test a ɛwɔ dua abien ne ɔkwan biako so nhwehwɛmu a wɔyɛ no daa (ANOVA) sɔhwɛ no mu no, wobu nsonsonoe a ɛda akuw ahorow ntam sɛ ɛwɔ nkɛntɛnso kɛse wɔ P <0.05 (*) so.
Wɔ fluorescence microscopy a Gas1-GFP, log phase nkwammoaa no nyin anadwo biako wɔ YPD na boaboaa ano denam centrifugation, hohoroo mprenu ne phosphate buffered saline, na wɔde too nsukyenee so anyɛ yiye koraa no simma 15, na afei wɔkɔɔ so wɔ microscope ase sɛnea wɔadi kan aka ho asɛm Check (24). Wɔde Leica DMi8 microscope (HCX PL APO 1003/1.40 oil PH3 CS) a wɔde objective lens, L5 (GFP) filter, Hamamatsu camera ne Application Suite X (LAS X) software ahyɛ mu no dii dwuma de gyee. .
Wɔde SDS nhwɛsode buffer yɛɛ nhwɛsode ahorow no denatured wɔ 65°C simma 10, na afei wɔde SDS-polyacrylamide gel electrophoresis (PAGE) tetew mu. Wɔ immunoblotting nhwehwɛmu mu no, wɔde nhwɛsode 10 μl guu kwan biara so. Nnuru a ɛko tia nyarewa titiriw: Fa kraman polyclonal anti-Gas1 a ɛyɛ 1:3000, kraman polyclonal anti-Emp24 a ɛyɛ 1:500, ne kraman polyclonal anti-GFP (akyɛde a efi H. Riezman hɔ) a ɛyɛ 1:3000 di dwuma. Wɔde mouse monoclonal anti-Pgk1 antibody no dii dwuma wɔ dilution a ɛyɛ 1:5000 (akyɛde a efi J. de la Cruz hɔ). Nkwammoaa a ɛto so abien a ɛko tia nyarewa: Horseradish peroxidase (HRP) a wɔde afrafra abirekyi anti-rabbit immunoglobulin G (IgG) a wɔde di dwuma wɔ nsu a ɛyɛ 1:3000 (Pierce). Wɔde HRP-conjugated goat anti-mouse IgG dii dwuma wɔ dilution a ɛyɛ 1:3000 (Pierce). Wɔnam chemiluminescence kwan a wɔfa so de SuperSignal West Pico reagent (Thermo Fisher Scientific) dii dwuma so na ɛhwɛɛ beae a nipadua no tumi ko tia nyarewa no.
Sɛnea wɔaka ho asɛm wɔ (31) mu no, wɔyɛɛ abɔde mu immunoprecipitation sɔhwɛ wɔ ER fã a wɔahyɛ no den no so. Ne tiawa mu no, fa TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride ne protease inhibitor afrafra) hohoro mmɔkaw nkwammoaa wɔ 600 nm ( OD600) wɔ 100 optical density mprenu. Wɔde ahwehwɛ nhwiren bubuu mu, na afei wɔde centrifugation yii nkwammoaa mu nneɛma a asɛe ne ahwehwɛ nhwiren no. Afei wɔde nsu a ɛwɔ soro no guu centrifuged mu wɔ 17,000 g mu simma 15 wɔ 4°C. Wɔsan de pellet no guu TNE mu na wɔde digitalis saponin kaa ho ma ɛyɛɛ 1% a etwa to. Wɔde aduru a wɔde ahyɛ mu no too mu dɔnhwerew 1 a wɔdannan no wɔ 4°C, na afei wɔde centrifugation yii nneɛma a entumi nkɔ mu no wɔ 13,000 g wɔ 4°C mu simma 60. Sɛ wopɛ Gas1-GFP immunoprecipitation a, di kan fa nhwɛsode no to agarose beads (ChromoTek) a hwee nni mu wɔ 4°C mu dɔnhwerew 1, na afei fa GFP-Trap_A (ChromoTek) to 4°C mu nnɔnhwerew 3. Wɔde TNE a digoxigenin 0.2% wom hohoroo nhwiren a immunoprecipitated no mpɛn anum, de SDS sample buffer yii mu, yii mu wɔ SDS-PAGE so, na wɔde immunoblotting yɛɛ mu nhwehwɛmu.
Sɛnea wɔaka ho asɛm wɔ (31) mu no, wɔyɛɛ cross-linking gyinaesi wɔ ER fã a wɔahyɛ no den no so. Ne tiawa mu no, wɔde ER fã a wɔahyɛ no den no guu 0.5 mM dithiobis(succinimidyl propionate) (Pierce, Thermo Fisher Scientific, Rockford, IL, U.S.A.; 20°C, simma 20) mu. Wɔde glycine (50 mM a etwa to, simma 5, 20°C) a wɔde kaa ho no dum crosslinking reaction no.
Sɛnea yɛadi kan aka ho asɛm (50) no, wɔyɛɛ MS nhwehwɛmu a wɔyɛe wɔ ceramide a ɛwɔ wuram-su ne GhLag1 ahorow mu. Ne tiawa mu no, wɔmaa nkwammoaa no nyin koduu exponential phase (3 kosi 4 OD600 units/ml) wɔ YPD mu wɔ 30°C, na wotwaa nkwammoaa 25×107. Wɔde trichloroacetic acid dum wɔn nipadua mu nneɛma a ɛsakra no. Fa aduru a wɔde yiyi mu [ethanol, nsu, ether, pyridine ne 4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] ne 1.2 nmol a ɛyɛ emu gyinapɛn C17 ceramide (860517, Avanti polar lipid) su) di dwuma. Fa monomethylamine reagent [methanol, nsu, n-butanol ne methylamine solution (4:3:1:5 v/v)] yɛ alkaline hydrolysis a ɛnyɛ den wɔ extract no mu, na afei fa n-butanol a nsu ayɛ mu ma di nkyene fi mu. Awiei koraa no, wɔsan de nea wɔayiyi no guu positive mode solvent [chloroform/methanol/nsu (2:7:1) + 5 mM ammonium acetate] mu na wɔde guu mass spectrometer no mu. Wɔyɛɛ multi-reaction monitoring (MRM) de huu sphingolipid molecules na wɔkyerɛɛ dodow. TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) no wɔ robɔt nanoflow ion fibea Nanomate HD (Advion Biosciences, Ithaca, NY) a wɔde yɛ lipid nhwehwɛmu. Wɔayɛ ahoɔden a ɛbɔ ho ban no yiye ama ceramide kuw biara. Wɔnyaa MS data wɔ positive mode mu. Wɔ abɔde a nkwa wom mu nsɛso biara mu no, srade sɛnkyerɛnne no yɛ susuw abiɛsa a ɛde ne ho no mfinimfini.
Sɛnea wɔaka ho asɛm wɔ (31) mu no, wɔde nkwammoaa (800×107) a ɛda Gas1-GFP adi no yɛɛ abɔde mu immunoprecipitation. Wɔde SDS-PAGE yii Gas1-GFP a wɔatew ho no mu na wɔde guu polyvinylidene fluoride (PVDF) aduru mu. Wɔnam amide tuntum a wɔde PVDF so yɛɛ protein no ho mfonini wɔ wɔn adwene mu. Wɔtwaa Gas1-GFP band no fii PVDF no mu na wɔde methanol hohoroo ho mpɛn 5 na wɔde nsuo a ɛyɛ nsuo chromatography-MS (LC-MS) grade nsuo hohoroo ho pɛnkoro. Ɛdenam membrane strip no a wɔde 500μl 0.3 M NaOAc (pH 4.0), buffer ne 500μl 1 M sodium nitrite afrafra a wɔapete no foforo wɔ 37°C mu nnɔnhwerew 3 so no, srade fã no fi Gas1-GFP ne lysed Release of inosine phosphate ceramide ntam glucosamine ne inositol (51). Ɛno akyi no, wɔde nsu a ɛyɛ LC-MS hohoroo membrane strip no mpɛn anan, wee wɔ dan mu hyew mu, na wɔde siee nitrogen wim a ɛyɛ -80°C kosii sɛ wɔbɛyɛ nhwehwɛmu. Sɛ́ nea wɔde bedi dwuma no, wɔde PVDF membrane nhwɛsode a ɛnyɛ hwee dii dwuma wɔ sɔhwɛ biara mu. Afei wɔde MS yɛɛ lipid a woyii fii Gas1-GFP mu no mu nhwehwɛmu sɛnea wɔaka ho asɛm no ( 50 ). Ne tiawa mu no, wɔsan de PVDF nhama a GPI-lipid wom no guu 75μl negative mold solvent [chloroform/methanol (1:2) + 5 mM ammonium acetate] mu na wɔde electrospray ionization (ESI)-MRM/MS Analysis of sphingolipid species (TSQ Vantage) twaam. Wɔ eyi mu no, wonya MS data wɔ negative ion mode mu.
Sɛnea yɛadi kan aka no, wɔtew GPI ankora no fã a srade wom no fii GPI-AP a wɔde [3H]-inositol ahyɛ no agyirae no ho ( 16 ). Wɔnam thin-layer chromatography a wɔde solvent system (55:45:10 chloroform-methanol-0.25% KCl) dii dwuma so yii srade no mu na wɔde FLA-7000 (Fujifilm) yɛɛ ho mfonini.
Wɔde TNE buffer a ɛwɔ TNE buffer hohoroo nkwammoaa a ɛda Gas1-GFP (600×107) adi no mprenu, na wɔde ahwehwɛ nhwiren bubuu mu, na afei wɔde centrifuged yii nkwammoaa mu nneɛma a asɛe ne ahwehwɛ nhwiren. Afei wɔde nsu a ɛwɔ soro no guu centrifuge mu wɔ 17,000 g mu dɔnhwerew 1 wɔ 4°C. Wɔhohoroo pellet no wɔ TNE mu na wɔde 1 U PI-PLC (Invitrogen) a ɛwɔ TNE a digitalis saponin 0.2% wom mu no too mu dɔnhwerew 1 wɔ 37°C. Bere a wɔde enzyme ayɛ no akyi no, wɔde centrifugation yii membrane no wɔ 17,000 g wɔ 4°C dɔnhwerew 1. Sɛnea ɛbɛyɛ na Gas1-GFP ayɛ immunoprecipitate no, wɔde nsu a ɛwɔ soro no too GFP-Trap_A (ChromoTek) mu wɔ 4°C anadwo. Wɔde Coomassie bruu a ɛhyerɛn yɛɛ Gas1-GFP a wɔatew ho a wɔde SDS-PAGE atew mu no ho fĩ. Wɔtwaa Gas1-GFP staining band no fii grey a atwa nsuo kwan no ho ahyia no ho, na afei wɔde iodoacetamide ayɛ alkylation na wɔde dithiothreitol atew so akyi no, wɔde trypsin yɛɛ in-gel digestion. Yi tryptic peptides ne peptides a wɔde GPI-glycans yi fi mu na ɛyow. Wɔde peptide a ayow no guu nsu 20 μl mu. Fa ɔfã bi (8μl) gu LC no mu. Wɔde octadecylsilane (ODS) adum (Develosil 300ODS-HG-5; emu ntwemu 150 mm×1.0 mm; Nomura Chemical, Aichi Mantam, Japan) dii dwuma de yii peptide ahorow mu wɔ tebea pɔtee bi a ɛkɔ soro mu. Nneɛma a ɛkɔ baabiara no yɛ aduru A (0.08% formic acid) ne aduru B (0.15% formic acid a ɛwɔ 80% acetonitrile mu). Wɔde Accela HPLC nhyehyɛe (Thermo Fisher Scientific, Boston, Massachusetts) dii dwuma de hohoroo adum no mu wɔ simma 55 mu wɔ nsu a ɛsen 50 μl min-1 mu simma 5, na afei wɔmaa aduru B dodow kɔɔ soro koduu 40%. , United States). Wɔde eluate no kɔɔ ESI ion fibea no mu bere nyinaa, na wɔde LTQ Orbitrap XL (hybrid linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific) yɛɛ tryptic peptides ne peptides a GPI-glycans wom no mu nhwehwɛmu. Wɔ MS nhyehyɛe no mu no, wɔde ntini a ɛwɔ ntini no mu no ahoɔden sii hɔ sɛ 4.5 kV, na wɔmaa ntini a wɔde fa nipadua no mu no hyew yɛɛ 300°C. Wɔde capillary voltage ne tube lens voltage sii hɔ sɛ 15 V ne 50 V. Wɔnyaa MS data wɔ positive ion mode (resolution of 60,000; mass accuracy of 10 parts per million) wɔ mass range a ɛyɛ 300/m/z mass/charge ratio (m/z) 3000. Wonya MS/MS data no denam ion trap a ɛwɔ LTQ Orbitrap XL [digit 3 a edi kan a data no gyina so, collision induced dissociation (CID)].
Wɔde GROMACS (52) software ne MARTINI 2 ahoɔden field (53-55) na ɛyɛɛ MD simulations. Afei wɔde CHARMM GUI Membrane Builder (56, 57) yɛɛ bilayer a dioleoylphosphatidylcholine (DOPC) ne Cer C18 anaa DOPC ne Cer C26 wom. Wɔnya topology ne coordinates a ɛwɔ Cer C26 mu no firi DXCE mu denam beads a ɛboro soɔ a wɔyi firi sphingosine dua no mu no so. Fa ɔkwan a wɔaka ho asɛm wɔ aseɛ ha no kari pɛ wɔ layer mmienu no mu na fa tu mmirika, afei fa nhyehyɛeɛ no coordinates a ɛtwa toɔ no yɛ nhyehyɛeɛ a Emp24 wom. Wɔyɛɛ transmembrane domain a mmɔkaw Emp24 (nkae 173 kosi 193) sɛ α-helix denam aniwa MD (VMD) adwinnade molecule nhyehyɛe ( 58 ). Afei, bere a woyii srade a ɛkata so no fii hɔ akyi no, wɔde protein no yɛɛ granulated a ɛyɛ mmerɛw na wɔde CHARMM GUI guu bilayer no mu. Nhyehyɛe a etwa to no kura 1202 DOPC ne 302 Cer C26 anaa 1197 DOPC ne 295 Cer C18 ne Emp24. Ionize nhyehyɛe no ma ɛyɛ 0.150M. Wɔyɛɛ replicates anan a ɛde ne ho maa bilayer compositions abien.
Wɔde CHARMM GUI nhyehyɛe no na ɛkari pɛ wɔ lipid bilayer no mu, a ɛhwehwɛ sɛ wɔtew anammɔn 405,000 so na afei wɔkari pɛ, baabi a wɔtew gyinabea anohyeto ahorow no so nkakrankakra na woyi fi hɔ, na wɔma bere anammɔn no kɔ soro fi 0.005 ps kosi 0.02 ps. Afei equilibration, ɛma 6 μs a bere anammɔn 0.02 ps. Sɛ wode Emp24 hyɛ mu wie a, fa CHARMM GUI nhyehyɛe koro no ara di dwuma de brɛ nhyehyɛe no ase na kari pɛ, na afei tu mmirika s 8 wɔ adwumayɛ mu.
Wɔ nhyehyɛe ahorow nyinaa mu no, bere a wɔreyɛ kari pɛ no, Berendsen barostat (59) na ɛhwɛ nhyɛso no so, na bere a wɔreyɛ no, Parrinello-Rahman barostat (60) na ɛhwɛ nhyɛso no so. Wɔ tebea horow no nyinaa mu no, nhyɛso a ɛwɔ hɔ no yɛ 1 bar na wɔde semi-isotropic pressure coupling scheme na edi dwuma. Wɔ kari pɛ ne adeyɛ nhyehyɛe mu no, wɔde thermostat (61) a ahoɔhare san yɛ no di dwuma de ka protein, lipid ne solvent asinasin no hyew bom. Wɔ oprehyɛn no nyinaa mu no, ɔhyew a wɔde asi wɔn ani so no yɛ 310K. Wɔnam pairing list a wɔde Verlet nhyehyɛe a ɛwɔ 0.005 buffer tolerance di dwuma so na ebu nkitahodi a ɛnyɛ bonding no ho akontaa. Wɔde reaction field ne cut-off distance a ɛyɛ 1.1 nm na ɛbu Coulomb term no. Vander Waals asɛmfua no de ntwitwiridii nhyehyɛe a ntwamu kwan a ɛyɛ 1.1 nm di dwuma, na Verlet ntwamu nhyehyɛe no de di dwuma ma tumi a ɛbɛtwetwe (62).
Sɛ wɔde VMD di dwuma a, asorɔkye tenten a wɔatwa wɔ DOPC phosphate beads anaa ceramide AM1 beads ne protein no ntam yɛ 0.7 nm, na wobu lipids dodow a ɛne protein no di nkitaho. Sɛnea nhyehyɛe a edidi so yi kyerɛ no, bu depletion-enrichment (DE) factor no ho akontaa sɛnea ɛwɔ (63): DE factor = (srade dodow a ɛwɔ protein no mu nyinaa 0.7) wɔ protein no mu 0.7 (Cer dodow a ɛwɔ srade nyinaa mu) .
Wɔnya boɔ a wɔabɔ ho amanneɛ no sɛ nkyɛmu, na mfomsoɔ nkyerɛwdeɛ no yɛ SE no mfonini anan a ɛde ne ho. Wɔde t sɔhwɛ [(averageDE-factor-1)/SE] na ɛbu DE factor no akontabuo mu nteaseɛ. Bu P bo no ho akontaa fi dua biako nkyekyɛmu no mu.
Wɔde GROMACS adwinnade no dii dwuma de buu 2D lateral density map a ɛwɔ nhyehyɛe a Emp24 wom no mu wɔ 250 ns a etwa to wɔ trace no mu. Sɛnea ɛbɛyɛ a wobenya ceramide no enrichment/depletion map no, wɔde Cer ne DOPC map no nyinaa kyekyɛ Cer density map no mu, na afei wɔde Cer dodow a ɛwɔ nipadua no mu no kyekyɛ mu. Wɔde kɔla map nsenia koro no ara na edi dwuma.
Sɛ wopɛ nneɛma foforo a wode bɛka saa asɛm yi ho a, yɛsrɛ wo hwɛ http://advances.sciencemag.org/cgi/content/full/6/50/eaba8237/DC1
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Sofia Rodriguez-Gallardo, Kazuo Kurokawa, Susana Sabido-Bozo, Alejandro Cortez · Gomez (Alejandro Cortes-Gomez), Atsuko Ikeda (Atsuko Ikeda), Valeria Zoni (Valeria Zoni), Auxiliadora Aguilera-Romero, Ana Maria Perez -Linero), Sergio Lopez (Sergio Lopez), Miho Waga (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
3D high-resolution real-time imaging da hia a ceramide nkɔnsɔnkɔnsɔn tenten ho hia ma protein a wɔhyehyɛ wɔ mmeae a wɔpaw nea efi mu ba no adi.
Sofia Rodriguez-Gallardo, Kazuo Kurokawa, Susana Sabido-Bozo, Alejandro Cortez · Gomez (Alejandro Cortes-Gomez), Atsuko Ikeda (Atsuko Ikeda), Valeria Zoni (Valeria Zoni), Auxiliadora Aguilera-Romero, Ana Maria Perez -Linero), Sergio Lopez (Sergio Lopez), Miho Waga (Miho Waga), Misako Arman (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
3D high-resolution real-time imaging da hia a ceramide nkɔnsɔnkɔnsɔn tenten ho hia ma protein a wɔhyehyɛ wɔ mmeae a wɔpaw nea efi mu ba no adi.
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