Protein mligbalamɔ yɛ secretory gbɛ lɛ nɔ lɛ he hiaa waa kɛha wala yibii lɛ amlijaa kɛ homeostasis. Kɛfata ŋmeii ni akɛtoɔ he lɛ ahe lɛ, gbɛfaŋnɔ ni lipids tsuɔ yɛ kinesin mli yɛ ŋmeii ni akɛyaa he kroko lɛ mli lɛ ji shishijee sanebimɔ ko ni ehi shi be kakadaŋŋ ni ahako hetoo lolo. Yɛ biɛ lɛ, wɔfeɔ 3D mfonirifeemɔ ni yɔɔ sui srɔtoi ni yɔɔ ŋwɛi ŋwɛi yɛ be kome mli koni wɔkɛma nɔ mi yɛ wala mli akɛ glycosylphosphatidylinositol-ni afee ehee ni bɛ hewalɛ ni yɔɔ ceramide lipid fãi ni kɛlɛ waa lɛ abua naa ni aja mli awo endoplasms krɛdɛɛi amli kɛtsɔ protein ni akɛtsuɔ nii kɛjɛɔ he ni akɛtsuɔ nii yɛ mli lɛ nɔ, ni ji. Kɛfata he lɛ, wɔtsɔɔ akɛ ceramide ni yɔɔ endoplasmic reticulum membrane lɛ mli lɛ sɛɛ tsɛɔ waa kɛha nɛkɛ srɔtofeemɔ ni akɛhalaa mɛi nɛɛ. Wɔ nikasemɔ lɛ haa klɛŋklɛŋ odaseyeli ni yɔɔ tɛ̃ɛ ni akɛjaraa protein jatsui ni damɔ lipid chain length nɔ lɛ mli kɛyaa hei ni ahala ni akɛyaa maŋsɛɛ yɛ secretory gbɛ lɛ nɔ.
Yɛ eukaryotic wala yibii lɛ amli lɛ, no sɛɛ lɛ, ajieɔ protein ni afee yɛ endoplasmic reticulum (ER) lɛ mli lɛ mli yɛ be mli ni akɛyaa he kroko kɛtsɔɔ tsɔne ni haa wala yibii lɛ shwieɔ shi lɛ nɔ koni akɛya he ni sa ni amɛyɔɔ lɛ (1). Kɛfata coat-mediated sorting lɛ he lɛ, asusu be kakadaŋŋ akɛ lipids komɛi hu baanyɛ atsu nii akɛ gbɛ ni atsɔɔ nɔ ajieɔ kɛjɛɔ mli kɛtsɔ amɛ ni abua naa awoɔ membrane domains pɔtɛɛ ni protein pɔtɛɛ komɛi yɔɔ mli lɛ nɔ (2-5). Shi kɛlɛ, odaseyeli ni yɔɔ tɛ̃ɛ ni tsɔɔ bɔ ni nɛkɛ gbɛ ni damɔ lipid nɔ lɛ baanyɛ aba lɛ bɛ lolo. Bɔni afee ni wɔtsu shishijee naagba nɛɛ he nii lɛ, wɔkase yɛ ŋmɔtɔ mli bɔ ni glycosylphosphatidylinositol (GPI) ni akɛwoɔ ŋmɔtɔ mli lɛ (GPI-APs) lɛ jeɔ kpo kɛjɛɔ ER lɛ mli yɛ gbɛi srɔtoi anɔ. GPI-APs ji lipid-ni kɛ amɛhe kpaa gbee ni yɔɔ wala yibii lɛ anɔ lɛ srɔtoi ( 6, 7 ). GPI-AP ji protein ko ni ajieɔ kɛjɛɔ mli ni akɛkpɛtɛɔ plasma fã ni yɔɔ kponɔgbɛ lɛ he kɛtsɔɔ glycolipid fã (GPI anchor) lɛ nɔ. Amɛkpɛlɛɔ GPI ŋwɛi ŋtayaai anɔ akɛ shishitsɔɔmɔ sɛɛ tsakemɔi ni yɔɔ ER lumen (8) lɛ mli. Kɛji akɛ GPI-AP lɛ fĩ sɛɛ lɛ, etsɔɔ Golgi tsɔne lɛ mli (5, 9) kɛjɛɔ ER lɛ mli kɛyaa plasma lɛ mli. GPI ankɔs ni yɔɔ lɛ haa GPI-AP lɛ yaa he kroko kɛjɛɔ transmembrane mli protein ni ajieɔ lɛ kpo lɛ he (ni plasma membrane mli protein krokomɛi fata he) yɛ sɛɛgbɛtsɔɔmɔ gbɛ lɛ nɔ (5, 9, 10). Yɛ yibii amli lɛ, ajieɔ GPI-APs lɛ kɛjɛɔ protein krokomɛi ni ajieɔ lɛ kpo yɛ endoplasmic reticulum lɛ mli lɛ amli, ni no sɛɛ lɛ akɛwoɔ tsɔnei ni yɔɔ srɔto ni coat protein complex II (COPII) (6, 7) lɛ ebɔle he lɛ amli. Nibii ni tsɔɔ nɛkɛ mligbalamɔ gbɛjianɔtoo nɛɛ mli yɛ ER nibii ni akɛyaa maŋsɛɛ lɛ mli lɛ mli kãaa shi faŋŋ, shi asusuɔ akɛ nɛkɛ gbɛ nɛɛ baanyɛ abi lipids, titri lɛ, lipids fã ni yɔɔ GPI ŋwɛi ŋta lɛ mli lɛ he gbɛjianɔtoo tsakemɔ (5, 8). Yɛ yeast mli lɛ, GPI lipid lɛ tsakemɔ jeɔ shishi amrɔ nɔŋŋ beni GPI lɛ ekpɛtɛ ehe lɛ sɛɛ, ni yɛ bei pii amli lɛ, ehaa ceramide lɛ kɛ 26-carbon ni ji fatty acid ni mli eyi obɔ lɛ (C26:0) lɛ kpaa gbee (11, 12). C26 ceramide ji ceramide titri ni ŋmɔtɔ mli wala yibii lɛ feɔ kɛbashi amrɔ nɛɛ. Afee lɛ yɛ ER lɛ mli ni akɛ efã babaoo yaa Golgi tsɔne lɛ mli kɛtsɔɔ COPII vesicles (13) lɛ anɔ. 14, 15), ni yɛ no najiaŋ lɛ, ceramide tsakemɔ kɛmiiya inositol phosphate ceramide (IPC) yɛ Golgi tsɔne lɛ mli lɛ damɔ GPI ŋwɛi ŋtayaa lɛ nɔ (16). Biophysical nikasemɔi ni akɛ nibii ni akɛfee lɛ tsuɔ nii yɛ mli lɛ etsɔɔ akɛ acyl chain ceramides ni kɛlɛ waa lɛ baanyɛ afee ekome ni efee domains ni ato he gbɛjianɔ ni yɔɔ gbɔmɔtsoŋ sui srɔtoi (17, 18). Nɛkɛ saji nɛɛ kɛ susumɔ ni ji akɛ C26 ceramide kɛ GPI-AP kɛ C26 ceramide kɛ amɛgbɔmɔtsoŋ nibii tsuɔ nii koni amɛkɛfee ekome kɛya hei ni ato he gbɛjianɔ loo hei ni yɔɔ ER membrane lipid shihilɛ ni mli ewo basabasa lɛ mli. Nɔ titri ni yɔɔ mli ji glycérolipids kukuji ni bɛ ŋmɔlɔ (C16:1 kɛ C18:1) (19, 20). Nɛkɛ kpokpai nɛɛ baahala ER kpojiemɔ hei pɔtɛɛ (ERES), he ni ceramide kɛ ceramide-based GPI-AP baanyɛ afee ekome kɛya Golgi yɛ COPII vesicle kome too lɛ nɔŋŋ mli (5).
Yɛ nikasemɔ nɛɛ mli lɛ, wɔka gbɛ ni damɔ lipid nɔ nɛɛ wɔkwɛ tɛ̃ɛ kɛtsɔ super-resolution confocal real-time imaging microscopy (SCLIM) ni akɛtsuɔ nii lɛ nɔ, ni ji tsɔne ko ni akɛkwɛɔ nibii bibii ni yɔɔ ŋmɛnɛŋmɛnɛ beaŋ ni baanyɛ akwɛ wala yibii ni yɔɔ hulutsoo mli lɛ yɛ be kome mli. (21 kɛ 22).
Wɔkɛ SCLIM ŋaalee tsu nii klɛŋklɛŋ koni wɔkɛtsɔɔ bɔ ni akwɛɔ GPI-AP ni yɔɔ C26 ceramide kuu lɛ mli kɛjɛɔ protein ni ajieɔ kɛjɛɔ mli lɛ mli beni ashi ER yɛ S. cerevisiae mli lɛ sɛɛ. Bɔni afee ni wɔkwɛ bɔ ni ER lɛ mlijaa yɔɔ ha lɛ, wɔkɛ tsɛŋemɔ hela he gbɛjianɔtoo ko ni baanyɛ afee nibii heei ni afee lɛ ni boteɔ ERES mli yɛ vivo mli lɛ tsu nii tɛ̃ɛ (7, 23). Akɛ jatsu lɛ, wɔhala C26 ceramide-ni damɔ GPI-AP Gas1 ni akɛ ŋmɔtɔ ni haa hulu kpɛɔ (GFP) kɛ ŋmɔtɔ ni haa hulu kpɛɔ ni bɛŋkɛɔ hulutsoo (iRFP) wo nɔ lɛ nɔ, ni amɛ fɛɛ amɛkwɛɔ plasma ŋmɔtɔ lɛ nɔ (24–26). Yɛ sec31-1 ni haa mɔ he jɔɔ lɛ mli lɛ, ajieɔ nɛkɛ jatsui enyɔ nɛɛ akpo yɛ galactose-inducible promoter kɛ ERES okadi ni feɔ nɔ ko lɛ shishi. Yɛ hulutsoo be ni mli wa waa mli (37°C), akɛni sec31-1 tsakemɔ lɛ saa COPII atade lɛ mli nibii ni ji Sec31 lɛ nitsumɔ he koni ekɛtsi COPII shweremɔ kɛ ER ni akɛyaa maŋsɛɛ lɛ naa hewɔ lɛ, jatsui heei ni afee lɛ buaa ehe naa yɛ ER lɛ mli (23). Beni edɔ kɛyashɛ he ko ni hulutsoo be lɛ baa shi (24°C) lɛ, sec31-1 mutant cells lɛ he wa kɛjɛ secretory he lɛ, ni synthetic cargo hee ni abua naa lɛ je shishi kɛjɛ ER lɛ mli. CLIM nibii ni anaa lɛ tsɔɔ akɛ Gas1-GFP kɛ Mid2-iRFP ni afee ehee lɛ ateŋ babaoo ebua amɛhe naa lolo yɛ ER ni ji sec31-1 mutant cells lɛ mli yɛ be mli ni ajie amɛ yɛ 37°C mli lɛ sɛɛ, ni no sɛɛ lɛ ajie amɛ kpo yɛ 24°C mli minitii 5 (Mfoniri 1). Akɛni Mid2-iRFP lɛ mlijaa yɛ ER membrane lɛ fɛɛ nɔ hewɔ lɛ, ni Gas1-GFP lɛ ebɔle ehe ni ebua ehe naa yɛ ER membrane lɛ kpokpaa ni bɛ nɔ lɛ nɔ hewɔ lɛ, amɛmlijaa lɛ yɛ srɔto kwraa (Mfoniri 1, A kɛyashi C kɛ Sini S1). Kɛfata he lɛ, taakɛ atsɔɔ yɛ mfoniri 1D lɛ mli lɛ, Gas1-GFP kuu lɛ bɛ Mid2-iRFP. Nibii nɛɛ tsɔɔ akɛ, ajie GPI-AP kɛ transmembrane proteins lɛ amli awo ER membrane kpokpai srɔtoi anɔ mra. Gas1-GFP kuu lɛ bɛŋkɛ ERES pɔtɛɛ ko ni akɛ mCherry COPII kootu protein Sec13 (Mfoniri 1, E kɛ F, kɛ sini S1) (23) ekadi lɛ.
1-1-GFP (GPI-AP, eŋɔli) kɛ transmembrane protein Mid2-iRFP (TMP, bluu) kɛ nɛkɛ ERES ni tsɔɔ nɔ ni haa mɔ tsui nyɔɔ emli lɛ kpo, ni nɛkɛ ERES ni akɛkadiɔ Sec13-mCherry, minitii, ni ajie kɛtee 24°C, ni SCLIM fee mfoniri yɛ minitii 5 sɛɛ. (A kɛyashi C) tsɔɔ najiaŋdamɔlɔ ni akɛfee ekome loo 2D mfoniri kome ni kɔɔ kɔɔyɔŋ lɛlɛ he (A), 2D mfoniri ni tsɔɔ z-fãi 10 (B) loo 3D cell hemisphere mfoniri ni kɔɔ jatsui kɛ ERES okadii ahe (C). Skel bar 1μm (A kɛ B). Sɛkɛl lɛ ji 0.551μm (C). Ana Gas1-GFP yɛ ER kpokpai loo kui sɔrɔtoi amli, yɛ be mli ni ana Mid2-iRFP ni aja yɛ ER membrane (C) lɛ mli fɛɛ. (D) Graf lɛ tsɔɔ bɔ ni Gas1-GFP kɛ Mid2-iRFP lɛ mli la ni tsoɔ waa lɛ mli wa ha yɛ Gas1-GFP kuu lɛ mli yɛ gãimlibi yɛŋ lɛ he (abɛkugbɛ). AU, nɔ ni yɔɔ srɔto. (E kɛ F) damɔ shi kɛha 3D mfoniri ni kɛ nibii lɛ kɛ ERES okadi lɛ feɔ ekome lɛ. Ana Gas1-GFP kui ni bɛŋkɛ ERES pɔtɛɛ lɛ. Sɛkɛl lɛ ji 0.551μm. (F) Akpɔlɔi ni yɔɔ ŋmɔlɔŋmɔlɔ lɛ kadiɔ Gas1-GFP kuu ni kɛ ERES yɔɔ tsakpaa lɛ. Nibii ni yɔɔ teŋgbɛ kɛ ninejurɔgbɛ lɛ tsɔɔ 3D mfoniri ni atsake lɛ ni akɛfee ekome lɛ kɛ Gas1-GFP kuu ni ahala lɛ he mfoniri ni atsake lɛ.
Wekukpaa ni bɛŋkɛ kpaakpa ni ka Gas1-GFP kuu lɛ kɛ ERES pɔtɛɛ ko teŋ lɛ tsɔɔ akɛ Gas1-GFP baanyɛ abote ERES ni halaa lɛ mli, ni esoro no kwraa yɛ halamɔ ni Mid2-iRFP kɛtsuɔ nii koni ekɛshi ER lɛ he. Bɔni afee ni wɔtsu nɛkɛ gbɛ ni baanyɛ aba nɛɛ he nii lɛ, wɔsusu ERES ratio lɛ he kɛha nibii kome loo enyɔ pɛ (Mfoniri 2, A kɛyashi C). Wɔna akɛ ERES lɛ mli babaoo (70%) lɛ, jatsu henɔ kome pɛ yɔɔ mli. Mfoniri ni yɔɔ shishigbɛ ni yɔɔ mfoniri 2C lɛ mli lɛ tsɔɔ ERES he nɔkwɛmɔnii enyɔ ni afɔɔ namɔ ni Gas1-GFP pɛ yɔɔ mli (mfoniri 1) loo Mid2-iRFP pɛ (mfoniri 2). Nɔ ni tamɔɔɔ enɛ lɛ, aaafee ERES lɛ mli 20% yɛ jatsui enyɔ ni tsɔɔ amɛhe yɛ he kome. Ana akɛ ERES komɛi (10%) yɛ jatsui srɔtoi enyɔ, shi amɛtsi amɛhe kɛjɛ hei srɔtoi ni yɔɔ faŋŋ. No hewɔ lɛ, nɛkɛ akɔntaabuu mlipɛimɔ nɛɛ tsɔɔ akɛ, kɛ́ ajie ER lɛ kɛtee maŋsɛɛ lɛ, ajaa GPI-AP Gas1-GFP kɛ transmembrane cargo Mid2-iRFP lɛ mli awoɔ ERES srɔtoi amli (Mfoniri 2D). Nɛkɛ srɔtofeemɔ nitsumɔ nɛɛ kɛ tsutsu biochemical mlipɛimɔ (6) kɛ morphological yiŋkpɛɛ (7) lɛ kpãa gbee waa. Wɔbaanyɛ wɔkwɛ bɔ ni jatsui ni akɛwo ERES mli lɛ feɔ amɛnii amɛhaa lɛ hu (Mfoniri 2E kɛ Sini S2). 2E tsɔɔ akɛ Gas1-GFP (panel 3) loo Mid2-iRFP (panel 4) lɛ fã bibioo ko pɛ boteɔ ERES lɛ mli kɛjɛɔ he kome ni ekɛ ehe woɔ he ko ni yɔɔ srɔto. Panel 5 ni yɔɔ mfoniri 2E lɛ mli lɛ tsɔɔ akɛ bei komɛi lɛ, anaa Gas1-GFP kɛ Mid2-iRFP yɛ ERES kome mli, shi amɛboteɔ mli kɛjɛɔ hei srɔtoi ni amɛyɔɔ kpokpai srɔtoi ni baanyɛ adamɔ shi aha COPII vesicles srɔtoi lɛ amli. Wɔma nɔ mi hu akɛ, mligbalamɔ ni ana kɛ C26 ceramide-ni damɔ GPI-AP Gas1 nɔ lɛ mligbalamɔ akɛ ERES ni halaa lɛ ji nɔ pɔtɛɛ ejaakɛ transmembrane sɛɛkuu jatsu kroko, ni ji GFP-tagged plasma membrane protein Axl2 (27 ), ni tsɔɔ subaŋ ni tamɔ Mid2-iRFP lɛ nɔŋŋ. (Mfoniri S1 kɛ Sini S3). Axl2-GFP ni afee ehee lɛ mlijaa kɛtsɔɔ ER membrane lɛ nɔ tamɔ Mid2-iRFP (Mfoniri S1, A kɛ B), ni ekɛ Mid2-iRFP lɛ feɔ ekome yɛ ERES babaoo mli (Mfoniri S1, B kɛyashi D). S1C tsɔɔ ERES he nɔkwɛmɔnii enyɔ ni afɔɔ namɔ yɛ he ni transmembrane jatsui enyɔ tsɔɔ nɔ. Yɛ shihilɛi nɛɛ amli lɛ, nibii enyɔ lɛ fɛɛ boteɔ ERES mli ekome (Mfoniri S1E, Panel 3 kɛ Sini S3).
1-GFP (GPI-AP, eŋɔli) kɛ Mid2-iRFP (TMP, bluu) kɛ ERES ni feɔ ERES ni ji Sec13-mCherry (ERES, magenta) lɛ ji nɔ ni akɛwo 37 mli. block, kɛ mfoniri kɛ SCLIM yɛ minitii 20 sɛɛ. (A kɛyashi C) Najiaŋdamɔlɔ 2D mfoniri ni ajieɔ lɛ kpo (A; skel bar, 1μm) loo 3D cell hemisphere mfoniri (B kɛ C; skel unit, 0.456μm) ni kɔɔ jatsu lɛ kɛ z-fãi 10 ni ERES kadi lɛ he. Nɔ ni yɔɔ shishigbɛ yɛ (B) kɛ nɔ ni yɔɔ (C) lɛ jieɔ mfonirii ni atsu he nii koni akɛjie nibii ni yɔɔ ERES (magenta) [Gas1-GFP (ŋmɔŋ) kɛ Mid2-iRFP (bluu ni mli tse)] pɛ kpo. (C) Akpɔlɔi ni agbele: ERES woɔ jatsu kome pɛ (1 kɛyashi 4). Akpɔlɔ ni tsuɔ tamɔ ŋmɔtɔ: ERES lɛ mli jatsui ni mli egbala (5) yɔɔ mli. Akpɔlɔ yɛŋ ni wa: ERES ni jatsui ni yɔɔ he kome yɔɔ mli. Yɛ shishigbɛ: ERES kome ni ahala lɛ mli lɛ, Gas1-GFP (1) loo Mid2-iRFP (2) pɛ yɔɔ mli. Skel tso, 100 nm. (D) Fotomikrograf ni atsɔɔ mli yɛ (C) lɛ falɛ. ERES ni jatsu kome pɛ yɔɔ mli lɛ mlijaa oha mlijaa (Gas1-GFP loo Mid2-iRFP), jatsui ni mli egbala kɛ jatsui ni tsɔɔ amɛhe lɛ. Yɛ shikpamɔi etɛ ni jɛ amɛ diɛŋtsɛ amɛhe lɛ amli lɛ, n=432 yɛ 54 mli. Tɔmɔ bar = SD. Enyɔ-tails ni bɛ t kaa. *** P = 0.0002. (E) 3D mfoniri ni kɔɔ ERES ni ahala ni ji jatsui ni akɛto he ko ni akɛ (C) ekadi lɛ he. Gas1-GFP (eŋɔli) (3) loo Mid2-iRFP (bluu) (4) boteɔ ERES (eŋɔli) mli kɛjɛɔ he kome ni ekɛ ehe woɔ he bibioo ko ni yɔɔ ERES lɛ mli lɛ mli pɛ. Bei komɛi lɛ, jatsui srɔtoi enyɔ lɛ fɛɛ boteɔ ERES (5) kome mli kɛjɛɔ he kome ni amɛyaa he ko ni yɔɔ shɔŋŋ yɛ ERES lɛ mli. Skel tso, 100 nm.
No sɛɛ lɛ, wɔka susumɔ ko wɔkwɛ akɛ, acyl chain ceramide (C26) ni yɔɔ ER membrane lɛ mli lɛ tsirɛɔ Gas1 lɛ kuu pɔtɛɛ kɛ ejaramɔ kɛyaa ERES ni ahalaa lɛ mli. Kɛha enɛ feemɔ lɛ, wɔkɛ ŋmɔtɔ ni atsake lɛ mli tsofa ko ni atsɛɔ lɛ GhLag1 lɛ tsu nii, ni akɛ GhLag1 (ni ji Lag1 homolog ni yɔɔ ŋmɔtɔ mli lɛ) tsake endogenous ceramide synthases enyɔ ni ji Lag1 kɛ Lac1 lɛ, ni no ha ŋmɔtɔ ŋmɔtɔ ni yɔɔ cell membrane Ceramide ŋmɔtɔ ni yɔɔ kuku fe koo mli ŋmɔtɔ lɛ (Mfoniri 3A) (2) ba. Mass spectrometry (MS) mlipɛimɔ tsɔɔ akɛ yɛ koo mli ŋmeii lɛ amli lɛ, 95% yɛ ŋmeii lɛ fɛɛ mli lɛ ji ŋmeii ni kɛlɛ waa (C26) ŋmeii ni yɔɔ ŋmeii lɛ amli lɛ, yɛ be mli ni yɛ GhLag1 mli lɛ, ŋmeii lɛ amli 85% lɛ kɛlɛ waa (C18 kɛ C16). ), 2% pɛ ni ji ceramide lɛ ji ceramide ni kɛlɛ waa (C26) lɛ. Eyɛ mli akɛ C18 kɛ C16 ceramides ji ceramides titri ni ana yɛ GhLag1 membrane lɛ mli kɛbashi amrɔ nɛɛ moŋ, shi MS mlipɛimɔ hu ma nɔ mi akɛ GPI anchor ni ji Gas1-GFP ni ajieɔ lɛ kpo yɛ GhLag1 fã lɛ mli lɛ yɛ C26 ceramide, ni akɛtoɔ koo mli lipids he. Su lɛ ji nɔ kome (Mfoniri 3A) (26). No hewɔ lɛ, enɛ tsɔɔ akɛ, ceramide tsakemɔ enzyme Cwh43 lɛ halaa C26 ceramide lɛ waa, taakɛ atsɔɔ yɛ mfoniri 26 lɛ mli lɛ, esumɔɔ ni ekɛ GPI ŋwɛi ŋta ni jɛ C26 ceramide fioo ko ni yɔɔ GhLag1 mli lɛ mli lɛ awo mli. S2 (29). Shi kɛlɛ, nɔ titri ni yɔɔ GhLag1 mli wala yibii lɛ amli lɛ ji C18-C16 ceramide pɛ, yɛ be mli ni Gas1-GFP lɛ yɛ C26 ceramide lolo. Nɛkɛ anɔkwale nɛɛ haa nɛkɛ tsofa nɛɛ feɔ dɛŋdade ni hi jogbaŋŋ ni akɛbaatsu naagba ni kɔɔ acyl chain length ni ji membrane ceramide ni yɔɔ ER lɛ he lɛ he nii tɛ̃ɛ. Klas kɛ mligbalamɔ nitsumɔ ni asusuɔ akɛ eji lɛ. No sɛɛ lɛ, wɔkase nyɛmɔ ni C26 Gas1-GFP yɔɔ ni ekɛbuaa ehe naa yɛ kui amli yɛ GhLag1 mli kɛ hulutsoo-henumɔ mutant allele ni ji sec31-1 kɛtsɔ ŋmɛnɛŋmɛnɛ beaŋ hulutsoo nibii bibii ni akwɛɔ nɔ lɛ nɔ, he ni (C18-C16) ŋwɛi ŋta ni kɛlɛ pɛ yɔɔ ER membrane Ceramide (Mfoniri 3) lɛ mli lɛ. Wɔna akɛ yɛ sec31-1 lɛ mli lɛ, Gas1-GFP lɛ mli babaoo lɛ ebɔle kui ahe, yɛ be mli ni Gas1-GFP ni yɔɔ sec31-1 GhLag1 ni yɔɔ ceramide ER membrane kakadaŋŋ (C18-C16) lɛ mli lɛ ji nɔ titri ni abuaaa naa ni ajaa yɛ ER membrane lɛ mli fɛɛ. Kɛji akɛ wɔbaawie lɛ pɛpɛɛpɛ lɛ, akɛni C26 ceramide-based clustering lɛ kɛ ERES pɔtɛɛ ko yɔɔ tsakpaa waa hewɔ lɛ (Mfoniri 1), wɔtao mli yɛ sɛɛ mli akɛ nɛkɛ gbɛ nɛɛ baanyɛ akɔ ER export protein gbɛjianɔtoo lɛ nitsumɔ hu he. GPI-AP kɛ COPII gbɛjianɔtoo krɛdɛɛ ko tsuɔ nii kɛhaa ER ni akɛyaa maŋsɛɛ, ni Ted1′s structure remodeling ni ji glycan fã ni yɔɔ GPI ŋwɛi ŋta lɛ mli lɛ kudɔɔ lɛ jogbaŋŋ (30, 31). No sɛɛ lɛ, transmembrane jatsu hemɔ p24 lɛ yɔseɔ GPI-glycan ni akɛtoɔ he lɛ, ni no hu halaa Lst1, ni ji isoform pɔtɛɛ ko ni yɔɔ COPII jatsu wulu ni fiɔ Sec24 lɛ mli lɛ, ni efeɔ GPI-AP-nibii COPII Vesicles ni he hiaa (33). No hewɔ lɛ, wɔfee mutant ni ji enyɔ ni kɛ nɛkɛ protein kome nɛɛ ni ajieɔ lɛ (p24 ni yɔɔ srɔto ni ji Emp24, GPI-glycan ni tsakeɔ enzyme Ted1 kɛ COPII subunit pɔtɛɛ Lst1) kɛ sec31-1 mutant strain lɛ fata he, ni wɔkase amɛhe Ani abaanyɛ afee GFP1-clu3). Wɔna akɛ yɛ sec31-1emp24Δ kɛ sec31-1ted1Δ mli lɛ, Gas1-GFP lɛ ji nɔ titri ni bɛ kuu mli ni aja mli yɛ ER fã lɛ mli fɛɛ, taakɛ ana tsutsu yɛ sec31-1 GhLag1 lɛ mli lɛ, yɛ be mli ni yɛ sec31-1lst1Δ lɛ mli lɛ, Gas1-GFP Tamɔ sec3. Nibii nɛɛ tsɔɔ akɛ, kɛfata C26 ceramide ni yɔɔ ER membrane lɛ mli lɛ he lɛ, ehe miihia ni Gas1-GFP lɛ kuu lɛ hu akpɛtɛ p24 complex lɛ he, ni ehe ehiaaa ni Lst1 nitsumɔ pɔtɛɛ ko aba. No sɛɛ lɛ, wɔtao bɔ ni ebaanyɛ eba akɛ ceramide ni yɔɔ ER membrane lɛ mli lɛ baanyɛ akudɔ Gas1-GFP kɛ p24 lɛ. Shi, wɔna akɛ C18-C16 ceramide ni yɔɔ tsɔne lɛ mli lɛ enaaa GPI-glycans ni p24 complex lɛ etswa ekoŋŋ lɛ nɔ hewalɛ (Mfoniri S3 kɛ S4, A kɛ B) loo ekɛ GPI-AP lɛ kpãa gbee ni ekɛ GPI-AP lɛ yaa maŋsɛɛ. nɔni akɛbaafe. Tsɔɔmɔ COPII henɔ bibioo Lst1 (Mfoniri S4C). No hewɔ lɛ, C26 ceramide-ni damɔ kuu nɔ lɛ biɔ ni protein kɛ ER protein ni akɛyaa maŋsɛɛ lɛ atsu nii, shi moŋ eyeɔ ebuaa gbɛ kroko ni akɛtoɔ nibii ahe ni lipid lɛlɛ tsirɛɔ lɛ. No sɛɛ lɛ, wɔpɛi mli kɛji akɛ ceramide acyl chain lɛ sɛɛ tsɛɔ yɛ ER membrane lɛ mli lɛ he hiaa kɛha Gas1-GFP lɛ mligbalamɔ ni mɔɔ shi jogbaŋŋ akɛ ERES ni halaa. Akɛni Gas1 ni yɔɔ GhLag1 shwiemɔ ni yɔɔ ceramide ni yɔɔ kuku lɛ mli lɛ shĩɔ ER lɛ mli ni eboteɔ plasma membrane lɛ mli hewɔ lɛ (Mfoniri S5) hewɔ lɛ, wɔheɔ wɔyeɔ akɛ kɛji akɛ ceramide acyl shwiemɔ lɛ sɛɛ tsɛɔ lɛ ji nɔ ni tsirɛɔ srɔtofeemɔ lɛ, Gas1 ni yɔɔ GhLag1 shwiemɔ mli lɛ baanyɛ atsɔ gbɛ kroko nɔ ni efo mli. ERES nibii ni yɔɔ membrane kome too lɛ nɔŋŋ.
(A) GhLag1 mli wala yibii lɛ amli tsɔne lɛ titri ji C18-C16 ceramides kukuji, yɛ be mli ni GPI anchor ni ji Gas1-GFP lɛ yɛ C26 IPC lɛ nɔŋŋ lolo tamɔ wala yibii ni yɔɔ koo mli lɛ. Yɛ ŋwɛi: acyl ŋwɛi ŋta lɛ mlipɛimɔ ni kɔɔ ceramide ni yɔɔ ŋtayaai amli lɛ mli lɛ mli lɛ, kɛ GhLag1p ŋtayaai lɛ amli kɛtsɔ ŋwɛi ŋtayaai (MS) lɛ anɔ. Data lɛ damɔ shi kɛha ceramide fɛɛ mlijaa oha mlijaa. Shikpamɔi etɛ ni yɔɔ amɛ diɛŋtsɛ amɛhe lɛ mlijaa. Tɔmɔ bar = SD. Enyɔ-tails ni bɛ t kaa. **** P <0.0001. MS mlipɛimɔ ni kɔɔ acyl ŋwɛi ŋta ni yɔɔ IPC ni yɔɔ Gas1-GFP (GPI-IPC) GPI ŋwɛi ŋta ni ajieɔ lɛ kpo yɛ koo mli-henɔ kɛ GhLag1p ŋta lɛ mli lɛ he. Data lɛ damɔ shi kɛha IPC okadi lɛ fɛɛ mlijaa oha mlijaa. Shikpamɔi enumɔ ni yɔɔ amɛ diɛŋtsɛ amɛhe lɛ mlijaa. Tɔmɔ bar = SD. Enyɔ-tails ni bɛ t kaa. ns, ehe ehiaaa. P = 0.9134. (B) Akɛ 31-1, 31-1 GhLag1, 31-1emp24Δ, 31-1ted1Δ kɛ 31-1lst1Δ wala yibii ni jieɔ galactose-ni tsirɛɔ Gas1-GFP lɛ kpo lɛ wo mli yɛ 37°C kɛyaa minitii 30 mli. yɛ 24°C sɛɛ. Akpɔlɔi yɛŋ: ER Gas1-GFP kuu. Gbelemɔ gãimlibi: Ajara Gas1-GFP ni abuuu naa lɛ mli yɛ ER membrane lɛ fɛɛ nɔ, ni etsɔɔ ER su ni ji nuklea ring staining lɛ. Skel bar, 5μm. (C) Fotomikrograf ni atsɔɔ mli yɛ (B) lɛ falɛ. Nɔ ni fe fɛɛ lɛ, wala yibii ni yɔɔ Gas1-GFP gbɛjianɔtoo lɛ mlijaa oha mlijaa. Yɛ shikpamɔi etɛ ni jɛ amɛ diɛŋtsɛ amɛhe lɛ amli lɛ, n≥300 wala yibii. Tɔmɔ bar = SD. Enyɔ-tails ni bɛ t kaa. **** P <0.0001.
Kɛha naagba nɛɛ naa tsabaa tɛ̃ɛ lɛ, wɔfee SCLIM mfonirifeemɔ ni kɔɔ Gas1-GFP kɛ Mid2-iRFP he yɛ GhLag1 mli kɛ sec31-1 hulutsoo-henumɔ mutant allele (Mfoniri 4 kɛ Sini S4). Beni ER lɛ hi 37°C ni no sɛɛ lɛ ajie lɛ kpo yɛ 24°C sɛɛ lɛ, Gas1-GFP ni afee ehee lɛ mli babaoo lɛ ebɔko he ni ejaraaa ER membrane lɛ mli fɛɛ, taakɛ tsɔnei ni akɛkwɛɔ nibii bibii ni afɔɔ nitsumɔ lɛ naa lɛ (Mfoniri 4, A kɛ B ). Kɛfata he lɛ, ERES lɛ mlijaa oha mlijaa 67 (67%) lɛ mli jatsui srɔtoi enyɔ ni yɔɔ mli lɛ fata he (Mfoniri 4D). kɛ 2 ni yɔɔ mfoniri 4C lɛ mli lɛ tsɔɔ ERES he nɔkwɛmɔnii enyɔ ni Gas1-GFP kɛ Mid2-GFP ni feɔ ekome lɛ yɔɔ mli. Kɛfata he lɛ, aŋɔ nibii enyɔ lɛ fɛɛ awo ERES kome lɛ mli (Mfoniri 4E, panel 3 kɛ sini S4). No hewɔ lɛ, nɔ ni wɔna lɛ tsɔɔ akɛ, ceramide acyl chain ni yɔɔ ER membrane lɛ mli lɛ sɛɛ tsɛɔ lɛ ji nɔ ko ni he hiaa ni tsɔɔ ER protein lɛ ni abua naa kɛ mligbalamɔ.
1-GFP (GPI-AP, eŋɔli) kɛ Mid2-iRFP (TMP, bluu) kɛ ERES-mCherry (ERES, magenta) ni yɔɔ 37°C lɛ mli lɛ. Tsa nɔ minitii 30, ni okɛba shi kɛyashɛ 24°C koni ejie ŋmɔtɔ, ni okɛ SCLIM afee mfoniri yɛ minitii 20 sɛɛ. (A kɛyashi C) Najiaŋdamɔlɔ 2D mfoniri ni ajieɔ lɛ kpo (A; skel bar, 1μm) loo 3D cell hemisphere mfoniri (B kɛ C; skel unit, 0.45μm) ni ji z-fãi 10 ni cargo kɛ ERES kadi lɛ. Nɔ ni yɔɔ shishigbɛ yɛ (B) kɛ nɔ ni yɔɔ (C) lɛ jieɔ mfonirii ni atsu he nii koni akɛjie nibii ni yɔɔ ERES (magenta) [Gas1-GFP (ŋmɔŋ) kɛ Mid2-iRFP (bluu ni mli tse)] pɛ kpo. (C) Akpɔlɔi ni eyi obɔ kɛ futafuta: ERES, nibii ni tsɔɔ nɔ. Gbelemɔ gãimlibi: ERES lɛ mli nibii kome pɛ yɔɔ. Panɛl ni yɔɔ shishigbɛ: ERES ni ahala lɛ yɛ nibii ni feɔ ekome (1 kɛ 2) ni akadi yɛ (C) mli. Skel tso, 100 nm. (D) Fotomikrograf ni atsɔɔ mli yɛ (C) lɛ falɛ. Yɛ sec31-1 kɛ sec31-1 GhLag1 kui lɛ amli lɛ, jatsu kome pɛ (Gas1-GFP loo Mid2-iRFP) fata he, kɛ ERES mlijaa oha mlijaa kɛha jatsui ni yɔɔ srɔtoi kɛ jatsui ni tsɔɔ amɛhe lɛ. Yɛ shikpamɔi etɛ ni jɛ amɛ diɛŋtsɛ amɛhe lɛ amli lɛ, n = 432 yɛ 54 mli (sec31-1) kɛ n = 430 yɛ 47 mli (sec31-1 GhLag1). Tɔmɔ bar = SD. Enyɔ-tails ni bɛ t kaa. *** P = 0,0002 (sɛɛk 31-1) kɛ ** P = 0,0031 (sɛɛk 31-1 GhLag1). (E) 3D mfoniri ni kɔɔ ERES ni ahala lɛ he kɛ jatsui ni feɔ ekome (3) ni akadi yɛ (C) lɛ mli. Gas1-GFP (eŋɔli) kɛ Mid2-iRFP (bluu) tsiɔ amɛhe kɛbɛŋkɛɔ ERES (eŋɔli) kɛjɛɔ he kome ni amɛhiɔ he kome ni ERES tsiɔ naa lɛ. Skel tso, 100 nm.
Nikasemɔ nɛɛ haa odaseyeli ni yɔɔ tɛ̃ɛ yɛ wala mli akɛ, ajaa protein cargos ni damɔ lipid nɔ lɛ mli awoɔ hei ni ahala ni akɛyaa maŋsɛɛ yɛ secretory gbɛ lɛ nɔ, ni ejieɔ bɔ ni acyl chain length he hiaa ha kɛha srɔtofeemɔ halamɔ lɛ kpo. Kɛtsɔ hewalɛ ni yɔɔ ŋwɛi nibii bibii ni atsɛɔ lɛ SCLIM lɛ nɔ lɛ, wɔjie Gas1-GFP (plasma membrane GPI-AP wulu ko ni yɔɔ acyl chain (C26) ceramide lipid fã kakadaŋŋ waa) ni afee lɛ ehee lɛ kpo yɛ yeast mli ) Kpokpai ni abua naa yɛ teemɔŋ protein ni yɔɔ srɔtoi lɛ amli lɛ kɛ ERs pɔtɛɛ ni aja mli lɛ yɔɔ tsakpaa, ER tsɔne lɛ mli (Mfoniri 1). Kɛfata he lɛ, nɛkɛ nibii enyɔ nɛɛ boteɔ ERES srɔtoi amli yɛ gbɛ ni amɛhalaa nɔ (Mfoniri 2). Akɛ acyl chain lɛ sɛɛ tsɛɔ ni ji cellular ceramide ni yɔɔ membrane lɛ mli lɛ baa shi kɛjɛɔ C26 kɛyaa C18-C16, ni Gas1-GFP kuu lɛ mli gbalaa kɛyaa ER kpokpaa ni yɔɔ srɔto lɛ mli, ni Gas1-GFP lɛ tsakeɔ gbɛ koni eshi ER lɛ kɛ transmembrane protein lɛ kɛtsɔ ERES lɛ nɔŋŋ nɔ (Mfoniri 3 kɛ Mfoniri 3). 4).
Eyɛ mli akɛ GPI-AP kɛ protein gbɛjianɔtoo krɛdɛɛ ko tsuɔ nii ni ekɛjeɔ ER mli moŋ, shi wɔna akɛ C26 ceramide-ni damɔ mligbalamɔ nɔ lɛ damɔɔɔ protein nifeemɔi srɔtoi ni baanyɛ aha ERES krɛdɛɛ aba lɛ nɔ (Mfoniri S4 kɛ S5). Shi moŋ, nɔ ni wɔna lɛ fiɔ gbɛ kroko ni atsɔɔ nɔ ajaraa nibii amli ni lipid-based protein clustering kɛ jatsui krokomɛi ni ajieɔ kɛjɛɔ mli lɛ tsirɛɔ lɛ sɛɛ. Nibii ni wɔkwɛɔ lɛ tsɔɔ akɛ Gas1-GFP kpokpaa lɛ loo kuu ni kɛ ERES pɔtɛɛ ko yɔɔ tsakpaa lɛ bɛ protein ni ajieɔ kɛjɛɔ mli lɛ mli Mid2-iRFP, ni tsɔɔ akɛ C26 ceramide-ni damɔ GPI-AP kuu lɛ nɔ lɛ baaha amɛbote ERES ni sa lɛ mli, ni yɛ nakai beaŋ nɔŋŋ lɛ, ejie secretions pɔtɛɛ nɛɛ kɛ transmembrane TheFig1 2). Nɔ ni tamɔɔɔ enɛ lɛ, C18-C16 ceramides ni yɔɔ ER membrane lɛ mli lɛ haaa GPI-AP lɛ afee kpokpai loo kui, no hewɔ lɛ amɛjieee loo amɛtsakeee transmembrane secreted proteins lɛ kɛyaa ERES kome lɛ nɔŋŋ mli (Mfoniri 3 kɛ 4). . No hewɔ lɛ, wɔkɛɔ akɛ C26 ceramide lɛ kudɔɔ mligbalamɔ kɛ mligbalamɔ kɛtsɔ protein ni kɛ ERES pɔtɛɛ ko yɔɔ tsakpaa lɛ akuu ni ehaa efeɔ mlɛo lɛ nɔ.
Te abaafee tɛŋŋ aná nɛkɛ C26 ceramide-ni damɔ shi kɛha kuu nɛɛ kɛya ER kpokpaa pɔtɛɛ ko nɔ? Nibii ni haa mɔ tsui nyɔɔ emli lɛ baanyɛ aha GPI-AP kɛ C26 ceramide lɛ afee lipids bibii ni ato he gbɛjianɔ amrɔ nɔŋŋ yɛ lipids shihilɛ ni bɛ gbɛjianɔtoo mli yɛ ER membrane ni glycerolipids kukuji ni bɛ mli lɛ yɔɔ mli lɛ mli. Kuu ni hi jogbaŋŋ (17, 18). Nɛkɛ be kukuoo mli kui bibii nɛɛ baanyɛ afee ekome ekoŋŋ ni amɛfee kui wuji ni yɔɔ shiŋŋ fe tsutsu lɛ yɛ be mli ni amɛkɛ p24 complex lɛ ebɔ he lɛ sɛɛ (34). Kɛ enɛ kpãa gbee lɛ, wɔtsɔɔ akɛ ehe miihia ni C26 Gas1-GFP lɛ kɛ p24 complex lɛ atsu nii koni efee kui wuji ni anaa (Mfoniri 3). p24 complex lɛ ji heterozygous oligomer ni p24 transmembrane proteins sɔrɔtoi ejwɛ ni yɔɔ yeast mli lɛ yɔɔ mli (35), ni haa multivalent tsakpaa, ni baanyɛ aha GPI-AP kui bibii lɛ afee ekome, ni no haa Stable kuu wulu baa (34). Nibii ni tee nɔ yɛ protein ectodomains ni ji GPI-APs lɛ ateŋ lɛ hu baanyɛ aye abua ni amɛbua amɛhe naa, taakɛ atsɔɔ yɛ amɛ Golgi gbɛfaa lɛ mli yɛ kooloi ni yɔɔ fɔmɔ kotoku mli lɛ amli lɛ mli (36). Shi, kɛ́ C18-C16 ceramide lɛ yɛ ER membrane lɛ mli, kɛ́ p24 complex lɛ kɛ Gas1-GFP lɛ kpã gbee lɛ, kui wuji ni yɔɔ srɔtoi lɛ efeŋ. Gbɛ ni yɔɔ shishi lɛ baanyɛ adamɔ gbɔmɔtsoŋ kɛ tsofafeemɔ mli nibii pɔtɛɛ ni yɔɔ acyl chain ceramide kakadaŋŋ lɛ mli lɛ nɔ. Nibii ni kɔɔ gbɔmɔtso lɛ he lɛ tsɔɔ akɛ, eyɛ mli akɛ acyl chain ceramides ni kɛlɛ (C24) kɛ kuku (C18-C16) lɛ fɛɛ baanyɛ aha fã mligbalamɔ aba moŋ, shi acyl chain ceramides kakadaŋŋ (C24) pɛ baanyɛ aha Curvature ni nɔ kwɔ kɛ sini ni shwieɔ shi lɛ aya hiɛ koni ekɛtsake sini lɛ. Kɛtsɔ amɛhe ni amɛtsɔɔ mli lɛ nɔ (17, 37, 38). Atsɔɔ akɛ, TMED2 ni ji gbɔmɔtso lɛ mli tsɔne ni haa mɔ he jɔɔ lɛ lɛ, kɛ C18 ceramide-ni damɔ sphingomyelin nɔ lɛ tsuɔ nii yɛ tsɔne ni haa mɔ he jɔɔ lɛ mli lɛ mli (39). Kɛtsɔ molecular dynamics (MD) he mfonirifeemɔ nɔ lɛ, wɔna akɛ C18 kɛ C26 ceramides lɛ fɛɛ buaa amɛhe naa yɛ Emp24 transmembrane helix lɛ mli tsɔnei ni haa mɔ tsui nyɔɔ emli lɛ ahe, ni amɛyɛ nibii ni amɛsumɔɔ ni tamɔ amɛhe (Mfoniri S6). E sa nɛ wa kai kaa enɛ ɔ tsɔɔ kaa transmembrane helix nɛ ngɛ Emp24 ɔ mi ɔ ma nyɛ ma ha nɛ lipids nɛ ngɛ membrane ɔ mi ɔ nɛ a gba a he blɔ nya tomi ɔ. Enɛ ji nɔ ni jɛ mli ba nyɛsɛɛ nɛɛ ni damɔ kooloi ni yɔɔ fɔmɔ kotoku mli lɛ amli wala yibii anɔ. MD he mfonirifeemɔi ni tamɔ nakai nɔŋŋ hu tsɔɔ akɛ ether lipids (40) yɛ. No hewɔ lɛ, wɔsusuɔ akɛ C26 ceramide ni yɔɔ ER26 lɛ lobules enyɔ lɛ mli lɛ ji nɔ ni yɔɔ he ni eyɔɔ lɛ. Kɛji akɛ GPI-AP ni yɔɔ luminal lobules lɛ mli lɛ kɛ multivalent p24 lɛ kpãa gbee tɛ̃ɛ kɛ C26 ceramide ni abua naa yɛ p24 he lɛ yɛ cytoplasmic lobules lɛ mli lɛ, ebaanyɛ ewo Protein ni fata he lɛ he hewalɛ ni ekɛ membrane curvature lɛ baa kɛtsɔ niji anɔ (41), ni no haa GPI-AP lɛ hu mli gbalaa kɛyaa kpokpaa ni nɔ kwɔ nɔ. kpokpai ni yɔɔ ER lɛ mli lɛ (42). Amaniɛbɔi ni eho lɛ fi gbɛ ni atsɔɔ nɔ atsuɔ nii lɛ sɛɛ (43, 44). Nibii ni haa mɔ he jɔ̃ɔ ehe ni ji oligolectins, helai loo nibii ni gbeɔ helai ni yɔɔ ceramide-based glycosphingolipids (GSL) lɛ anɔ lɛ haa GSL lɛ feɔ ekome babaoo, ehaa fãi amli gbalamɔ yaa hiɛ ni ehaa fã lɛ mligbalamɔ kɛ emlibotemɔ baa ( 44 ). Iwabuchi kɛ ekrokomɛi (43) Ana akɛ yɛ acyl ŋwɛi ŋtayaai ni kɛlɛ (C24) shi jeee kukuji (C16) lɛ ahiɛ lɛ, ŋwɛi ŋtayaai ni yɔɔ srɔtoi ni akɛ GSL lactosylceramide lɛ kpãa gbee lɛ haa ŋtayaai wuji kɛ ŋtayaai amlibotemɔ baa, ni cytoplasm Lyn-mediated signal transduction ni yɔɔ chagittropyl lɛ nɔ lɛ ji interface.
Yɛ kooloi ni yɔɔ fɔmɔ kotoku mli lɛ amli lɛ, bɔ ni anti-Golgi network (TGN) lɛ fa ha kɛyashɛɔ apical plasma membrane lɛ he lɛ kudɔɔ GPI-AP lɛ mligbalamɔ kɛ egbɛjianɔtoo (10, 45). GPI-AP oligomerization (36) lɛ ji nɔ ni tsirɛɔ nɛkɛ nibii ni abua naa nɛɛ, shi ebaanyɛ edamɔ ceramide ŋwɛi ŋta ni wɔnaa yɛ ŋmɔtɔ mli lɛ hu nɔ. Eyɛ mli akɛ mammalian GPI-AP lɛ yɛ ether lipid-based anchor, ni etsofafeemɔ gbɛjianɔtoo lɛ yɛ srɔto kwraa yɛ acyl chain ceramide ni kɛlɛ waa lɛ he moŋ, shi nyɛsɛɛ nɛɛ nikasemɔ ko na akɛ lipids enyɔ lɛ fɛɛ yɛ gbɔmɔtsoŋ kɛ tsofafeemɔ mli nibii ni tamɔ amɛhe yɛ sutsakemɔ mli Kɛ nitsumɔ (40). No hewɔ lɛ, ether lipid fã ni yɔɔ kooloi ni yɔɔ fɔmɔ kotoku mli lɛ amli lɛ baanyɛ afee tamɔ C26 ceramide ni yɔɔ yeast mli lɛ, ni egbɛfaŋnɔ ji ni ekɛ long-chain ceramide ni yɔɔ membrane lɛ mli lɛ abɔ koni ekɛwo GPI-AP lɛ buamɔ kɛ srɔtofeemɔ he hewalɛ. Eyɛ mli akɛ ehe miihia ni aka bɔ ni ebaanyɛ eba lɛ akwɛ tɛ̃ɛ moŋ, shi nibii ni ana tsutsu lɛ fiɔ sɛɛ akɛ, jeee cytoplasmic transfer proteins ji nɔ ni kɛ acyl chain ceramide kakadaŋŋ lɛ yaa Golgi gbɔmɔtso lɛ mli, shi moŋ edamɔ GPI ŋwɛi ŋtayaai tamɔ yeast lɛ afeemɔ nɔ. No hewɔ lɛ, etamɔ nɔ ni sutsakemɔ gbɛjianɔtoo lɛ baanyɛ ahala nɔ ni ekɛ acyl chain ceramide ni kɛlɛ waa kɛ GPI-AP (13, 16, 20, 46, 47) lɛ aya tsɔne kome mli.
Yɛ yeast kɛ kooloi ni yɔɔ fɔmɔ kotoku mli lɛ amli lɛ, GPI-AP ni abua naa kɛ mligbalamɔ kɛjɛɔ plasma membrane protein krokomɛi amli lɛ fɛɛ baa mli dani eshɛɔ wala yibii lɛ anɔ. Paladino kɛ ekrokomɛi. ( 48 ) Na akɛ yɛ TGN ni ji mammalian polarized epithelial cells lɛ nɔ lɛ, GPI-AP kuu lɛ he ehiaaa kɛha GPI-APs lɛ mligbalamɔ ni ahalaa kɛyaa apical plasma membrane lɛ pɛ, shi moŋ ekwɛɔ GPI-APs lɛ kuu gbɛjianɔtoo lɛ kɛ E-biological nitsumɔ lɛ hu nɔ. Cell hiɛ. Yɛ yeast mli lɛ, nikasemɔ nɛɛ tsɔɔ akɛ C26 ceramide-ni damɔ GPI-AP kuu ni yɔɔ ER lɛ nɔ lɛ baanyɛ akudɔ kuu lɛ gbɛjianɔtoo kɛ nitsumɔ nitsumɔ ni GPI-AP yɔɔ plasma membrane lɛ nɔ lɛ (24, 49). Yɛ nɛkɛ nɔkwɛmɔnɔ nɛɛ naa lɛ, GhLag1 wala yibii lɛ bɛ GPI inhibitors loo tsofai ni saa wala yibii lɛ gbogbo lɛ he lɛ ahe (28), ni hiamɔ ni yɔɔ Gas1-GFP kui ni tsuɔ nii (49) ni ji tip ceramide ni atsɔɔ mli yɛ yeast wala yibii lɛ ateŋ lɛ tsɔɔ G Possible physiological consequences of hLa cells. GPI-AP tɔmɔ. Shi kɛlɛ, kaa kroko ni abaakwɛ kɛji akɛ ato gbɛjianɔtoo ni tsuɔ nii ni yɔɔ wala yibii lɛ anɔ lɛ he gbɛjianɔ kɛjɛ ER lɛ mli kɛtsɔ gbɛ ni atsɔɔ nɔ ahalaa nibii ni yɔɔ mli lɛ amli lɛ nɔ lɛ baafee nɔ ni wɔsɛɛ be mli niiamlitaomɔ lɛ baasusu he.
Atsɔɔ Saccharomyces cerevisiae henɔi ni akɛtsu nii yɛ nitsumɔ nɛɛ mli lɛ yɛ Okadi S1. Afee MMY1583 kɛ MMY1635 henɔi ni ji SCLIM kɛha wala yibii amli mfonirifeemɔ yɛ W303 sɛɛ. Akɛ nɛkɛ tsɛŋemɔ helai ni jieɔ Sec13-mCherry kpo ni yɔɔ hulutsoo protein tag lɛ tsu nii kɛtsɔ gbɛ ni damɔ polymerase chain reaction (PCR) nɔ ni akɛ pFA6a plasmid tsuɔ nii akɛ nɔkwɛmɔnɔ (23) lɛ nɔ. Afee tsofa ni jieɔ Mid2-iRFP ni akɛ fluorescent protein ewo nɔ yɛ GAL1 nɔkwɛlɔ lɛ kudɔmɔ shishi lɛ tamɔ nɛkɛ. PCR mlilɛɛmɔ ni kɔɔ iRFP-KanMx sɛɛkuu kɛjɛ pKTiRFP-KAN tsɔne lɛ mli (E. O’Shea nikeenii, Addgene plasmid yibɔ 64687; Beni atswa Mid2-iRFP genome lɛ he adafi ni afee lɛ tamɔ nɔ ni akɛwo GAL1 hiɛnyiɛlɔ lɛ mli lɛ, akɛwo Not I-Sac I he ni akɛfataa plasmid pRS306 he lɛ mli. Akɛ plasmid pRGS7 ni jɛ mli ba lɛ to Pst I he koni ekɛfata URA3 he lɛ he.
Ajieɔ Gas1-GFP fusion tsɛŋemɔ hela lɛ kpo yɛ GAL1 sɛɛnyiɛlɔ lɛ kudɔmɔ shishi yɛ centromere (CEN) plasmid lɛ mli, ni afee lɛ tamɔ nɛkɛ. Akɛ PCR ha Gas1-GFP sɛɛkuu lɛ tee hiɛ kɛjɛ pRS416-GAS1-GFP plasmid (24) (L. Popolo nikeenii) ni afee lɛ tamɔ nɔ ni yɔɔ Xma I–Xho I he ni yɔɔ CEN plasmid pBEVY-GL LEU2 (C nikeenii) lɛ mli. Mila; Plasmid ni akɛwoɔ mli lɛ yibɔ ni ji 51225; 51225; RRID: Nɔ ni fata he_51225). Atsɛɔ plasmid ni jɛ mli ba lɛ akɛ pRGS6. Axl2-GFP fusion gene lɛ hu jieɔ ehe kpo yɛ GAL1 pBEVY-GL LEU2 vector lɛ nɔkwɛlɔ lɛ kudɔmɔ shishi, ni etswaa lɛ ji nɔ ni nyiɛ sɛɛ nɛɛ. Akɛ PCR tsu nii ni akɛwo Bam HI-Pst I he ni pBEVY-GL LEU2 tsɔne lɛ yɔɔ lɛ mli. Atsɛɔ plasmid ni jɛ mli ba lɛ akɛ pRGS12. Atsɔɔ oligonucleotides ni akɛtsu nii yɛ nikasemɔ nɛɛ mli lɛ mli yɛ Table S2.
Akɛ 0.2% adenine kɛ 2% glucose [YP-dextrose (YPD)]. (YPR)] loo 2% galactose [YP-galactose (YPG)] akɛ carbon jɛɛhe, loo yɛ nɔ ko ni afee lɛ bibioo ko mli (0.15% yeast nitrogen shishitoo kɛ 0.5% ammonium sulfate) koni akɛfata amino acid kɛ shishitoo ni sa ni he hiaa kɛha niyenii kpakpa he , Ni nɔ ni yɔɔ mli lɛ ji 2% glucose bibioo ko loo glucose bibioo ko (galactose ni afee lɛ diɛŋtsɛ) akɛ carbon jɛɛhe.
Kɛha be-mli mfonirifeemɔ, hulutsoo-henumɔ sec31-1 mutant wala yibii ni jieɔ tsu lɛ kpo yɛ GAL1 hiɛnyiɛlɔ lɛ shishi lɛ dara yɛ YPR mli yɛ 24°C gbɛkɛ kɛyashi mid-log phase. Beni akɛwo YPG mli yɛ 24°C mli ŋmɛlɛtswaa kome sɛɛ lɛ, akɛ wala yibii lɛ wo SG mli yɛ 37°C mli minitii 30, ni no sɛɛ lɛ ajie amɛ kɛtee 24°C koni ajie amɛ kɛjɛ secretion block lɛ mli. Akɛ Concanavalin A tsu nii ni akɛsaa wala yibii lɛ yɛ glase slide nɔ ni SCLIM fee mfoniri. SCLIM ji Olympus IX-71 ni akɛkwɛɔ nibii bibii ni haa hulu kpɛɔ kɛ UPlanSApo 100×1.4 yibɔi ni akɛkwɛɔ nibii bibii amli (Olympus), oyaiyeli kɛ okadi-kɛ-gbee ni yaa hiɛ ni tsɔɔ diski ni haa hulu tsoɔ waa (Yokogawa Electric kɛ kusum naa mfonirifeemɔ gbɛjianɔtoo) (Hamamatsu Photonics) baanyɛ aha ŋmɛlɛtswaa ni akɛkwɛɔ nibii amli lɛ afee nɔ ni naagbee ŋmɛlɛtswaa ni ji ×266.7 kɛ tsɔne ni akɛwoɔ tsɔne mli ni haa ŋmɛlɛtswai ni haa hulutsoo lɛ yaa hiɛ lɛ (Hamamatsu Photonics) (21) aha. Akɛ tsɔne ni akɛtsuɔ nii (Yokogawa Electric) tsuɔ nii kɛheɔ mfoniri. Kɛha 3D mfonirii lɛ, wɔkɛ piezoelectric actuator ko ni afee lɛ diɛŋtsɛ tsu nii ni ekɛwo yiŋtoo lɛ mli ŋmɛlɛtswaa lɛ mli, ni wɔbua ŋmɛlɛtswaa ni akɛkwɛɔ nii lɛ fãi ni yɔɔ 100 nm lɛ anaa yɛ ŋmɛlɛtswaa kome mli. Atsakeɔ Z-stack mfoniri lɛ awoɔ 3D voxel data mli, ni akɛ tsɔɔmɔ ni gbɛɔ eshwaa nitsumɔ ni akɛtsuɔ nii kɛhaa tsɔne ni akɛkwɛɔ nibii bibii ni tsɔɔ ŋwɛigbɛ lɛ tsuɔ nii kɛhaa Volocity tsɔne ni akɛtsuɔ nii (PerkinElmer) lɛ he nitsumɔ. Kɛtsɔ Volocity tsɔne ni akɛtsuɔ nii lɛ nɔ lɛ, asusu ERES ni jatsu fata he lɛ he. Akɛ MetaMorph tsɔne (Molecular Devices) tsu nii kɛpɛi line mli.
Kɛ GraphPad Prism sɔftwɛɛ lɛ atsu nii ni okɛna akɔntaabuu shishinumɔ. Kɛha nikaselɔ t-test ni yɔɔ sɛɛ enyɔ lɛ kɛ gbɛ kome ni atsɔɔ nɔ akwɛɔ srɔtofeemɔ mli (ANOVA) kaa lɛ, abuɔ srɔtofeemɔi ni yɔɔ kui ateŋ lɛ akɛ eyɛ hewalɛ ni sa kadimɔ waa yɛ P <0.05 (*) nɔ.
Kɛha fluorescence microscopy ni ji Gas1-GFP lɛ, akwɛ log phase cells lɛ anɔ nyɔɔŋ kome yɛ YPD mli ni abua naa kɛtsɔ centrifugation nɔ, afɔ he shii enyɔ kɛ phosphate buffered saline, ni akɛwo ais nɔ kɛ hoo lɛ minitii 15, ni no sɛɛ lɛ aya nɔ yɛ microscope lɛ shishi taakɛ atsɔɔ mli tsutsu lɛ (Check24). 1003/1.40 oil PH3 CS) ni akɛ nɔ ko ni akɛkwɛɔ nii amli, L5 (GFP) filta, Hamamatsu kamera kɛ Application Suite X (LAS X) tsɔne lɛ tsu nii kɛhe nii. .
Akɛ SDS nɔkwɛmɔnɔ buffer ni yɔɔ 65°C lɛ tsake nɔkwɛmɔnii lɛ minitii 10, ni no sɛɛ lɛ akɛ SDS-polyacrylamide gel electrophoresis (PAGE) gbala mli. Kɛha immunoblotting mlipɛimɔ lɛ, akɛ nɔkwɛmɔnɔ 10 μl wo gbɛ fɛɛ gbɛ nɔ. Nɔ ni gbeɔ hela: Kɛ ŋmɔtɔ ni gbeɔ hela ni ji Gas1 lɛ atsu nii yɛ 1:3000 mli, ŋmɔtɔ mli tsofa ni gbeɔ Emp24 yɛ 1:500 mli, kɛ ŋmɔtɔ mli tsofa ni gbeɔ GFP (nikeenii ni jɛ H. Riezman ŋɔɔ) yɛ 1:3000 mli. Akɛ tsofa ni gbeɔ Pgk1 ni yɔɔ ŋmɔtɔ mli lɛ tsu nii yɛ ŋmɔtɔ ni ji 1:5000 mli (nikeenii ni jɛ J. de la Cruz ŋɔɔ). Nɔ ni ji enyɔ ni gbeɔ hela: Horseradish peroxidase (HRP) ni akɛ too ni gbeɔ ŋmɔtɔ G (IgG) ni akɛtsuɔ nii yɛ 1:3000 (Pierce) mli lɛ mli. Akɛ HRP-ni akɛfee too ni gbeɔ ŋmɔtɔ IgG lɛ tsu nii yɛ 1:3000 (Pierce) mli. Akɛ tsofa ni ji SuperSignal West Pico reagent (Thermo Fisher Scientific) lɛ tsuɔ nii kɛkwɛɔ bɔ ni gbɔmɔtso lɛ mli hewalɛ ni tsiɔ hela naa lɛ tsuɔ nii ehaa lɛ lɛ nɔ.
Taakɛ atsɔɔ mli yɛ (31) lɛ, afee adebɔɔ mli immunoprecipitation kaa ko yɛ ER fã ni awo mli nii lɛ nɔ. Yɛ kukufoo mli lɛ, okɛ TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride kɛ protease inhibitor ni akɛfutuɔ nii) afɔ ŋmɔtɔ mli wala yibii lɛ ahe yɛ 600 nm ( OD600) yɛ de 100 optically shii enyɔ. Akɛ glase ŋmeii shwieɔ nɔ, ni no sɛɛ lɛ, ajie wala yibii lɛ amli ŋmeii kɛ glase ŋmeii lɛ kɛtsɔ centrifugation nɔ. No sɛɛ lɛ, akɛ nu ni hoɔ lɛ woɔ 17,000 g mli minitii 15 yɛ 4°C. Akɛ pellet lɛ wo TNE mli ekoŋŋ ni akɛ digitalis saponin fata he kɛyashɛ naagbee 1% mli. Akɛ tsɔne ni akɛwoɔ mli lɛ wo mli ŋmɛlɛtswaa kome kɛ sɛɛtsimɔ yɛ 4°C, ni no sɛɛ lɛ ajie nibii ni nyɛɛɛ atsɔ mli lɛ kɛtsɔ centrifugation nɔ yɛ 13,000 g yɛ 4°C mli minitii 60. Kɛha Gas1-GFP tsofa ni tsiɔ hela naa lɛ, klɛŋklɛŋ lɛ, okɛ agarose ŋmeii ni bɛ nɔ ko (ChromoTek) awo nɔkwɛmɔnɔ lɛ mli yɛ 4°C ŋmɛlɛtswaa kome, ni no sɛɛ lɛ okɛ GFP-Trap_A (ChromoTek) awo mli yɛ 4°C ŋmɛlɛtswai etɛ. Akɛ TNE ni digoxigenin yɔɔ mli lɛ tsuɔ nii shii enumɔ, ni akɛ SDS nɔkwɛmɔnɔ buffer shwieɔ nɔ, ni ajara mli yɛ SDS-PAGE nɔ, ni akɛ immunoblotting kwɛɔ mli.
Taakɛ atsɔɔ mli yɛ (31) lɛ, afee cross-linking yiŋkpɛɛ yɛ ER fã ni awo lɛ nii babaoo lɛ nɔ. Yɛ kukufoo mli lɛ, akɛ 0.5 mM dithiobis (succinimidyl propionate) wo mli. Akɛ glycine (50 mM naagbee shweremɔ, minitii 5, 20°C) ni akɛfata he lɛ tsu nii ni ekɛgbe crosslinking nifeemɔ lɛ naa.
Taakɛ atsɔɔ mli kɛtsɔ hiɛ lɛ (50), afee MS mlipɛimɔ ni kɔɔ ceramide ni yɔɔ koo mli-henɔ kɛ GhLag1 mli lɛ he. Yɛ kukufoo mli lɛ, akwɛɔ wala yibii lɛ anɔ kɛyashɛɔ ŋwɛi ŋta (3 kɛyashi 4 OD600 units/ml) yɛ YPD mli yɛ 30°C, ni akpa wala yibii 25×107. Akɛ trichloroacetic acid gbeɔ amɛ niyenii ni tsakeɔ lɛ. 15:15:5:1:0.018 v/v) kɛ 1.2 nmol ni ji mligbɛ shishitoo mla ni ji C17 ceramide (860517, Avanti polar lipid) ni hi jogbaŋŋ lɛ atsu nii. Kɛ monomethylamine reagent [methanol, nu, n-butanol kɛ methylamine solution (4:3:1:5 v/v)] atsu nii ni okɛfee nɔ ni ajieɔ lɛ mli nu ni haa mɔ he jɔɔ lɛ fioo, ni no sɛɛ lɛ okɛ n-butanol ni nu eyi mli obɔ lɛ atsu nii ni okɛjie ŋmei. Yɛ naagbee lɛ, ajie nɔ ni ajie lɛ kpo lɛ mli ekoŋŋ yɛ tsofa ko ni haa mɔ he jɔɔ lɛ mli [klorofɔm/methanol/nu (2:7:1) + 5 mM ammonium acetate] ni akɛwoɔ tsɔne ni akɛkwɛɔ nibii ni haa mɔ he jɔɔ lɛ mli. Afee nibii babaoo ni akwɛɔ nɔ (MRM) koni akɛyoo sphingolipid molecules lɛ ayibɔ. TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) lɛ kɛ robot nanoflow ion jɛɛhe Nanomate HD (Advion Biosciences, Ithaca, NY) ewo mli kɛha lipid mlipɛimɔ. Akɛ gbeekpamɔ hewalɛ lɛ tsuɔ nii jogbaŋŋ kɛhaa ceramide henɔi lɛ eko fɛɛ eko. MS data lɛ ji nɔ ni ana yɛ gbɛ ni hi nɔ. Kɛha nibii ni wala yɔɔ amɛmli lɛ eko fɛɛ eko lɛ, lipid okadi lɛ ji susumɔi etɛ ni yɔɔ amɛ diɛŋtsɛ amɛhe lɛ ateŋ teŋgbɛ.
Taakɛ atsɔɔ mli yɛ (31) lɛ, akɛ wala yibii (800×107) ni jieɔ Gas1-GFP kpo lɛ wo adebɔɔ mli nibii ni haa mɔ he waa lɛ amli. Akɛ SDS-PAGE gbala Gas1-GFP ni atsuu he lɛ mli ni akɛtee tsɔne ko ni akɛwoɔ tsɔne mli ni akɛwoɔ tsɔne mli lɛ mli. Akwɛ protein lɛ kɛtsɔ PVDF ni akɛ amide diŋ wo mli lɛ nɔ. Afo Gas1-GFP kuu lɛ mli kɛjɛ PVDF lɛ mli ni akɛ methanol tsu he shii enumɔ, ni akɛ nu ni ji nu ni akɛkwɛɔ nibii amli-MS (LC-MS) grade lɛ tsu he shikome. Kɛtsɔ 500μl 0.3 M NaOAc (pH 4.0), buffer kɛ 500μl ni akɛfutuɔ ehee 1 M sodium nitrite ni akɛfutuɔ nii yɛ 37°C nɔ ŋmɛlɛtswai etɛ lɛ nɔ lɛ, ajieɔ lipid fã lɛ kɛjɛɔ Gas1-GFP kɛ inophamine ni ajieɔ lɛ kpo lɛ mli. (51). No sɛɛ lɛ, akɛ nu ni ji LC-MS grade lɛ tsu membrane strip lɛ he shii ejwɛ, ni egbi yɛ tsu mli latsaa mli, ni akɛto nitrogen kɔɔyɔɔ ni mli edɔ yɛ -80°C mli kɛyashi be mli ni akwɛ mli. Akɛ nɔ ni akɛkwɛɔ nɔ lɛ, akɛ PVDF membrane nɔkwɛmɔnɔ ni bɛ nɔ ko tsu nii kɛha kaa fɛɛ kaa. No sɛɛ lɛ, MS lɛ kwɛ lipid ni ajie kɛjɛ Gas1-GFP lɛ mli lɛ mli taakɛ atsɔɔ mli lɛ (50). Yɛ kukufoo mli lɛ, akɛ PVDF strips ni GPI-lipid yɔɔ mli lɛ wo 75μl ni ji ŋmɔtɔ ni ehiii [chloroform/methanol (1:2) + 5 mM ammonium acetate] mli ekoŋŋ, ni atsɔ electrospray ionization (ESI)-MRM/MS Analysis of sphingolipid henɔi (TSQ Vantage) lɛ nɔ. Yɛ shihilɛ nɛɛ mli lɛ, ana MS saji lɛ yɛ ion ni ehiii mli.
Taakɛ atsĩ tã kɛtsɔ hiɛ lɛ, ajie GPI ŋwɛi ŋta lɛ mli lipid fã lɛ kɛjɛ [3H]-inositol-ni aŋmala GPI-AP (16) lɛ mli. Akɛ tsɔne ni haa mɔ he jɔɔ lɛ (55:45:10 klorofɔm-methanol-0.25% KCl) tsu nii kɛjara lipidii lɛ amli, ni akɛ FLA-7000 (Fujifilm) tsu nii kɛkwɛ mli.
Akɛ TNE buffer ni TNE buffer yɔɔ mli lɛ tsu wala yibii ni jieɔ Gas1-GFP (600×107) lɛ kpo lɛ ahe shii enyɔ, ni akɛ glase ŋmeii shwie mli, ni no sɛɛ lɛ akɛwo centrifuge mli koni ajie wala yibii lɛ amli ŋmeii kɛ glase ŋmeii lɛ. No sɛɛ lɛ, akɛ nu ni hoɔ lɛ woɔ 17,000 g mli ŋmɛlɛtswaa kome yɛ 4°C. Akɛ 1 U PI-PLC (Invitrogen) ni yɔɔ TNE ni digitalis saponin 0.2% yɔɔ mli lɛ wo mli ŋmɛlɛtswaa kome yɛ 37°C. Yɛ enzyme tsofafeemɔ lɛ sɛɛ lɛ, ajie tsɔne ni akɛwoɔ mli lɛ kɛtsɔ 17,000 g ni akɛwoɔ mli yɛ 4°C nɔ ŋmɛlɛtswaa kome nɔ. Kɛha Gas1-GFP ni tsiɔ hela naa lɛ, akɛ nɔ ni yɔɔ mli lɛ wo GFP-Trap_A (ChromoTek) mli yɛ 4°C mli nyɔɔŋ kome. Akɛ Coomassie bluu ni kpɛɔ lɛ wo Gas1-GFP ni atsuu he ni SDS-PAGE gbala mli lɛ he. Afo Gas1-GFP ni akɛwoɔ mli lɛ mli kɛjɛ ŋmɔtɔ ni bɔle nu gbɛ lɛ he lɛ mli, ni no sɛɛ lɛ, beni akɛ iodoacetamide fee alkylation kɛ dithiothreitol ni akɛba shi lɛ sɛɛ lɛ, akɛ trypsin fee in-gel mli ŋmɔtɔ. Ajieɔ tryptic peptides kɛ peptides ni akɛ GPI-glycans tsuɔ nii yɛ mli lɛ ni agbiɔ. Akɛ peptide ni egbi lɛ wo nu 20 μl mli. Okɛ fã ko (8μl) awo LC lɛ mli. Akɛ oktadecylsilane (ODS) ko (Develosil 300ODS-HG-5; mligbɛ ŋwɛi ŋta ni ji 150 mm×1.0 mm; Nomura Chemical, Aichi Kpokpaa lɛ nɔ, Japan) tsu nii ni akɛgbala peptides lɛ amli yɛ shihilɛi pɔtɛɛ komɛi ni tsɔɔ ŋwɛi ŋta lɛ shishi. Nibii ni yaa nɔ lɛ ji tsɔne ni haa nu mli tseɔ A (0.08% fɔmik asid) kɛ nɔ ni haa nu mli tsɔɔ B (0.15% fɔmik asid ni yɔɔ 80% asetonitrile mli). Akɛ Accela HPLC gbɛjianɔtoo (Thermo Fisher Scientific, Boston, Massachusetts) tsu nii ni akɛjie ŋwɛi ŋta ni akɛ tsofa ni haa mɔ he jɔɔ lɛ A lɛ tsu nii yɛ minitii 55 mli yɛ 50 μl min-1 ni hoɔ oya lɛ mli minitii 5, ni no sɛɛ lɛ, ajie tsofa ni haa mɔ he jɔɔ lɛ B lɛ mlibotemɔ kɛyashɛ 40%. , Amerika). Akɛ eluate lɛ woɔ ESI ion jɛɛhe lɛ mli be fɛɛ be, ni akɛ LTQ Orbitrap XL (ni ji tsɔne ni akɛkwɛɔ nibii ni yɔɔ ŋwɛi lɛ amli lɛ mli lɛ) kwɛɔ tryptic peptides kɛ peptides ni yɔɔ GPI-glycans lɛ mli. Yɛ MS gbɛjianɔtoo lɛ mli lɛ, ato voltage ni yɔɔ kapilɛi jɛɛhe lɛ mli lɛ he gbɛjianɔ yɛ 4.5 kV, ni akɛ kapilɛi ni akɛyaa he kroko lɛ mli latsaa lɛ to 300°C. Akɛ tsɔne ni akɛwoɔ tsɔne mli lɛ kɛ tsɔne ni akɛwoɔ tsɔne mli lɛ mli tsɔne lɛ to 15 V kɛ 50 V. MS saji lɛ ji nɔ ni ana yɛ ion gbɛ ni hi jogbaŋŋ nɔ (60,000 yiŋkpɛɛ; fãi 10 ni falɛ ja yɛ akpekpei amli) yɛ fãi 300/m/z fãi/shika mlijaa (m/z) 3000 mli. gbeekpamɔ ni kɛ gbeekpamɔ baa (CID)].
Akɛ GROMACS (52) tsɔne kɛ MARTINI 2 hewalɛ ŋmɔ (53-55) tsu nii kɛfee MD he mfonirifeemɔi. No sɛɛ lɛ, akɛ CHARMM GUI Membrane Builder (56, 57) lɛ tsu nii ni akɛma ŋwɛi ŋta ni yɔɔ ŋwɛi ŋta ni ji dioleoylphosphatidylcholine (DOPC) kɛ Cer C18 loo DOPC kɛ Cer C26. Ajie Cer C26 lɛ he gbɛjianɔtoo kɛ egbɛjianɔtoo lɛ kɛjɛ DXCE kɛtsɔ ŋmeii krokomɛi ni ajieɔ kɛjɛɔ sphingosine tsitsi lɛ mli lɛ nɔ. Kɛ gbɛ ni atsɔɔ mli yɛ shishigbɛ nɛɛ atsu nii ni okɛto ŋwɛi ŋta ni ji enyɔ lɛ he ni okɛtsu nii, no sɛɛ lɛ okɛ gbɛjianɔtoo lɛ naagbee ŋta lɛ atsu nii ni okɛma gbɛjianɔtoo ni Emp24 yɔɔ mli. 173 kɛyashi 193) lɛ ji α-helix ni akɛ MD (VMD) dɛŋdade ni anaa lɛ tsuɔ nii (58). Kɛkɛ ni, beni ajie lipids ni ebɔle amɛhe lɛ sɛɛ lɛ, afee protein lɛ bibii bibii ni akɛwo bilayer lɛ mli kɛtsɔ CHARMM GUI nɔ. 1202 DOPC kɛ 302 Cer C26 loo 1197 DOPC kɛ 295 Cer C18 kɛ Emp24 yɛ naagbee gbɛjianɔtoo lɛ mli. Ionize gbɛjianɔtoo lɛ kɛyashɛ 0.150M mli. Afee nɔkwɛmɔnii ejwɛ ni yɔɔ amɛ diɛŋtsɛ amɛhe kɛha bilayer nibii enyɔ.
Akɛ CHARMM GUI gbɛjianɔtoo lɛ tsuɔ nii kɛtoɔ lipid bilayer lɛ he, ni no biɔ ni ajie 405,000 steps lɛ shi ni no sɛɛ lɛ afee lɛ pɛpɛɛpɛ, he ni ajieɔ gbɛhe ni tsiɔ mɔ naa lɛ shi fiofio ni ajieɔ kɛjɛɔ mli lɛ, ni be step lɛ yaa hiɛ kɛjɛɔ 0.005 ps kɛyaa 0.02 ps. Yɛ sɛɛ mli lɛ, ekɛ 6 μs kɛ be ni ji 0.02 ps baa. Kɛji akɛ okɛ Emp24 wo mli sɛɛ lɛ, okɛ CHARMM GUI gbɛjianɔtoo lɛ nɔŋŋ atsu nii ni okɛba gbɛjianɔtoo lɛ shi ni ekɛto he, ni no sɛɛ lɛ okɛ 8 s atsu nii yɛ nifeemɔ mli.
Yɛ gbɛjianɔtoi fɛɛ ahe lɛ, yɛ be mli ni akɛtoɔ nii ahe lɛ, Berendsen barostat (59) lɛ kudɔɔ nɔnyɛɛ lɛ, ni yɛ nifeemɔ be mli lɛ, Parrinello-Rahman barostat (60) lɛ kudɔɔ nɔnyɛɛ lɛ. Yɛ shihilɛi fɛɛ amli lɛ, ŋwɛi ŋta lɛ ji 1 bar ni akɛ ŋwɛi ŋta ni ji semi-isotropic lɛ he gbɛjianɔtoo tsuɔ nii. Yɛ ŋmɛlɛtswaa kɛ nifeemɔ gbɛjianɔtoo lɛ mli lɛ, akɛ thermostat (61) ni yɔɔ oyaiyeli ni akɛtsakeɔ nii lɛ tsuɔ nii koni akɛto protein, lipid kɛ solvent mli nibii bibii lɛ amli latsaa lɛ he. Yɛ nitsumɔ lɛ fɛɛ mli lɛ, hulutsoo ni ato he gbɛjianɔ lɛ ji 310K. Akɛ tsakpaa ni bɛ tsakpaa lɛ buɔ he akɔntaa kɛtsɔ tsakpaa ni yɔɔ Verlet gbɛjianɔtoo ni yɔɔ 0.005 buffer tsuishitoo lɛ nɔ. Akɛ reaction field kɛ 1.1 nm ni afo mli lɛ buɔ Coulomb wiemɔ lɛ he akɔntaa. Vander Waals wiemɔ lɛ kɛ tsɔne ni akɛfoɔ nii ni tsɔne ni akɛfoɔ nii lɛ teŋ jɛkɛmɔ ji 1.1 nm tsuɔ nii, ni akɛ Verlet tsɔne ni akɛfoɔ nii lɛ tsuɔ nii kɛhaa tsɔne ni baanyɛ atsɔ mli (62).
Kɛ akɛ VMD tsu nii lɛ, DOPC phosphate ŋmeii loo ceramide AM1 ŋmeii lɛ kɛ protein lɛ teŋ lɛ ji 0.7 nm, ni abuɔ lipids ni kɛ protein lɛ tsuɔ nii lɛ ayibɔ he akɔntaa. Taakɛ fɔmula ni nyiɛ sɛɛ nɛɛ tsɔɔ lɛ, buɔ nɔ ni haa mɔ he jɔ̃ɔ ehe (DE) lɛ he akɔntaa tamɔ nɔ ni yɔɔ (63) lɛ mli lɛ: DE nɔ = (lipids fɛɛ ni yɔɔ protein lɛ mli lɛ ayibɔ 0.7) yɛ protein lɛ mli 0.7 (Cer ni yɔɔ lipids lɛ fɛɛ mli lɛ ayibɔ) .
Anaa sɛɛnamɔ ni abɔ he amaniɛ lɛ akɛ mlijaa, ni tɔmɔi ni yɔɔ mli lɛ ji SE lɛ ekomɛi ejwɛ ni yɔɔ amɛ diɛŋtsɛ amɛhe. Akɛ t kaa [(averageDE-factor-1)/SE] buɔ DE nibii ahe akɔntaabuu lɛ he akɔntaa. Buu P sɛɛnamɔ lɛ he akɔntaa kɛjɛ sɛɛgbɛ kome mlijaa lɛ mli.
Akɛ GROMACS dɛŋdade lɛ tsu nii ni akɛbu 2D lateral density map ni kɔɔ gbɛjianɔtoo ni Emp24 yɔɔ mli lɛ he akɔntaa yɛ naagbee 250 ns ni nyiɛɔ sɛɛ lɛ mli. Bɔni afee ni anyɛ ana ceramide lɛ he mfoniri ni haa mɔ he jɔɔ lɛ/egbɔjɔ lɛ, ajaa Cer lɛ he mfoniri ni haa mɔ he jɔɔ lɛ mli kɛ Cer kɛ DOPC he mfoniri lɛ fɛɛ, ni no sɛɛ lɛ akɛ Cer ni yɔɔ gbɔmɔtso lɛ mli lɛ mlijaa. Akɛ shikpɔŋ he mfoniri ni yɔɔ su kome too lɛ nɔŋŋ tsuɔ nii.
Kɛji ootao nibii krokomɛi ni fata he kɛha niŋmaa nɛɛ, ofainɛ kwɛmɔ http://advances.sciencemag.org/cgi/content/full/6/50/eaba8237/DC1.
Enɛ ji niŋmaa ko ni anyɛɔ ashɛɔ he ni ajaa yɛ Creative Commons Attribution-Non-Commercial License lɛ mlai lɛ ashishi, ni ŋmɛɔ gbɛ akɛ akɛtsuɔ nii, ajaa ni afee ekoŋŋ yɛ gbɛ fɛɛ gbɛ nɔ, kɛji akɛ naagbee nitsumɔ lɛ jeee kɛha jarayeli sɛɛnamɔ ni nɔ ni yɔɔ mli ji akɛ shishijee nitsumɔ lɛ ja. Kaimɔ nii.
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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 Lorgihope -Linero), Waga), Misako Arman, Miyako Riman, Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
3D mfonirifeemɔ ni yɔɔ ŋwɛi ŋwɛi lɛ jieɔ bɔ ni ceramide ŋwɛi ŋta lɛ he hiaa ha kɛha protein mligbalamɔ yɛ hei ni ahalaa nibii ni ajieɔ lɛ kpo lɛ kpo.
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 Lorgihope -Linero), Waga), Misako Arman, Miyako Riman, Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
3D mfonirifeemɔ ni yɔɔ ŋwɛi ŋwɛi lɛ jieɔ bɔ ni ceramide ŋwɛi ŋta lɛ he hiaa ha kɛha protein mligbalamɔ yɛ hei ni ahalaa nibii ni ajieɔ lɛ kpo lɛ kpo.
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