Nhazi protein n'ụzọ nzuzo dị oke mkpa maka ịnọgide na-enwe nhazi nke sel na homeostasis. Na mgbakwunye na nhazi nke shei na-eme, ọrụ nke lipids na nhazi kinesin na usoro njem nzuzo bụ ajụjụ dị ogologo oge nke a na-azabeghị. N'ebe a, anyị na-eme onyonyo oge dị elu nke nwere ọtụtụ agba 3D n'otu oge iji gosi na a na-ejikọta protein glycosylphosphatidylinositol nke emepụtara ọhụrụ nwere ogologo mkpụrụ ndụ lipid ceramide ma kewaa ya n'ime endoplasms pụrụ iche Ebe ọpụpụ Net, nke dị iche na nke protein transmembrane na-eji. Na mgbakwunye, anyị na-egosi na ogologo agbụ nke ceramide na akpụkpọ ahụ endoplasmic reticulum dị oke mkpa maka nhọrọ nhazi a. Ọmụmụ ihe anyị na-enye ihe akaebe mbụ kpọmkwem na vivo iji kewaa ibu protein dabere na ogologo agbụ lipid gaa na ebe mbupụ ahọpụtara na ụzọ nzuzo.
N'ime mkpụrụ ndụ eukaryotic, a na-ahazi protein ndị a na-emepụta na endoplasmic reticulum (ER) n'oge njem site na ụzọ nzuzo maka nnyefe na ebe ha na-aga (1). Na mgbakwunye na nhazi nke mkpuchi, a na-eche ogologo oge na ụfọdụ lipids nwekwara ike ịbụ ebe ọpụpụ nhọrọ site na ịchịkọta ha n'ime mpaghara membrane kpọmkwem nke protein ndị a kapịrị ọnụ (2-5). Agbanyeghị, enwere enweghị ihe akaebe kpọmkwem na vivo iji gosi usoro a nwere ike ịdabere na lipid. Iji dozie nsogbu a bụ isi, anyị mụrụ na yist otu esi ewepụta protein anchored glycosylphosphatidylinositol (GPI) (GPI-APs) dị iche iche site na ER. GPI-APs bụ ụdị protein dị iche iche nke lipid jikọtara na sel (6, 7). GPI-AP bụ protein e zoro ezo nke ejikọtara na mpempe akwụkwọ mpụta nke plasma membrane site na glycolipid moiety (GPI anchor). Ha na-anabata anchor GPI dị ka mgbanwe nchekwa mgbe ntụgharị okwu gasịrị na ER lumen (8). Mgbe ejikọtachara ya, GPI-AP na-agafe ngwa Golgi (5, 9) site na ER ruo na akpụkpọ ahụ plasma. Ọnụnọ nke ihe mgbochi GPI na-eme ka a na-ebufe GPI-AP iche site na protein ndị transmembrane wepụtara (gụnyere protein ndị ọzọ dị na plasma membrane) n'ụzọ nzuzo (5, 9, 10). Na mkpụrụ ndụ yist, a na-ekewapụ GPI-APs site na protein ndị ọzọ e zoro ezo na endoplasmic reticulum, wee kechie ha n'ime vesicles pụrụ iche nke ejiri kọt protein complex II (COPI) kpuchie (6, 7). Ihe ndị na-ekpebi usoro nhazi a na usoro mbupụ ER edoghị anya, mana a na-eche na usoro a nwere ike ịchọ lipids, karịsịa nhazi nhazi nke akụkụ lipid nke anchor GPI (5, 8). Na yist, mgbanwe lipid GPI na-amalite ozugbo GPI jikọtara, na n'ọtụtụ ọnọdụ, ọ na-eme ka ceramide jikọọ na fatty acid 26-carbon long-chain saturated (C26:0) (11, 12). C26 ceramide bụ isi seramidi nke mkpụrụ ndụ yist mepụtara ruo ugbu a. A na-emepụta ya na ER, a na-ebupụkwa ọtụtụ n'ime ya na ngwaọrụ Golgi site na vesicles COPII (13). Mbupụ ER nke GPI-AP chọrọ kpọmkwem njikọ ceramide na-aga n'ihu (14, 15), n'aka nke ya, mgbanwe nke ceramide ka ọ bụrụ inositol phosphate ceramide (IPC) na ngwaọrụ Golgi dabere na njikọ arịlịka GPI (16). Ọmụmụ ihe gbasara biophysical na membranes artificial egosila na ceramides acyl yinye dị ogologo nwere ike ijikọ iji mepụta ngalaba ndị a haziri ahazi nwere ihe pụrụ iche nke anụ ahụ (17, 18). Data ndị a na-eduga n'echiche na ceramide C26 na GPI-AP na ceramide C26 na-eji ihe onwunwe anụ ahụ ha ejikọta na mpaghara ma ọ bụ mpaghara ndị dị n'usoro na gburugburu ebe obibi lipid ER membrane dị nro. Ọ bụ isi ihe mejupụtara ya bụ glycerolipids dị mkpụmkpụ na nke na-enweghị afọ ojuju (C16:1 na C18:1) (19, 20). A ga-elekwasị anya na mpaghara ndị a kpọmkwem na ebe ọpụpụ ER (ERES), ebe a ga-ebuga ceramide na GPI-AP nke dabeere na ceramide gaa Golgi n'otu vesicle COPII raara onwe ya nye (5).
N'ọmụmụ ihe a, anyị anwalela usoro a dabere na lipid site na iji ihe ngosi onyonyo oge confocal dị elu (SCLIM), nke bụ usoro ihe ngosi dị elu nke nwere ike ịhụ protein ndị nwere akara fluorescent n'otu oge. Foto ndị nwere agba atọ na akụkụ atọ (3D) nwere mkpebi na ọsọ dị elu na mkpụrụ ndụ dị ndụ (21, 22).
Anyị buru ụzọ jiri teknụzụ SCLIM mee ihe iji kọwaa etu esi enyocha GPI-AP nkịtị na otu ceramide C26 site na protein transmembrane wepụtara mgbe ha hapụrụ ER na S. cerevisiae. Iji lelee nhazi nke ER, anyị jiri usoro mkpụrụ ndụ ihe nketa nke nwere ike ịhụ ozugbo ibu ọhụrụ emepụtara na-abanye na ERES na vivo (7, 23). Dịka ibu, anyị họọrọ C26 ceramide-based GPI-AP Gas1 nke ejiri protein fluorescent green (GFP) na protein transmembrane emepụtara Mid2 nke ejiri protein fluorescent near-infrared (iRFP) mee ihe, ha abụọ lekwasịrị anya na membrane plasma (24-26). Na sec31-1 nke na-emetụta okpomọkụ, a na-egosipụta ibu abụọ a n'okpuru onye na-akwalite galactose-inducible na akara ERES constitutive. Na oke okpomọkụ (37°C), n'ihi na mgbanwe sec31-1 na-emetụta ọrụ nke ihe mkpuchi COPII Sec31 iji gbochie COPII germination na mbupụ ER, ibu ọhụrụ emepụtara na-agbakọta na ER (23). Mgbe ha jụrụ oyi ruo obere okpomọkụ (24°C), a gbakechara mkpụrụ ndụ mutant sec31-1 site na mpaghara secretory, a malitekwara ibu ọhụrụ e ji aka chịkọtara site na ER. Ihe ngosi CLIM gosiri na ọtụtụ n'ime Gas1-GFP na Mid2-iRFP e mepụtara ọhụrụ ka na-agbakọ na ER nke mkpụrụ ndụ mutant sec31-1 mgbe ha kpọchara na 37°C wee hapụ ha na 24°C maka nkeji ise (Foto 1). Ebe ọ bụ na a na-ekesa Mid2-iRFP na akpụkpọ ahụ ER dum, a na-agbakọkwa Gas1-GFP ma na-achịkọta ya na mpaghara akpụkpọ ahụ ER na-adịghị akwụsị akwụsị, nkesa ha dị iche kpamkpam (Foto 1, A ruo C na Movie S1). Na mgbakwunye, dịka egosiri na Foto 1D, otu Gas1-GFP enweghị Mid2-iRFP. Nsonaazụ ndị a na-egosi na e kewara GPI-AP na protein transmembrane n'ime mpaghara akpụkpọ ahụ ER dị iche iche n'oge. Otu Gas1-GFP dị n'akụkụ otu ERES a kapịrị ọnụ nke e ji protein mkpuchi COPII nke mCherry's Sec13 mee (Foto 1, E na F, na ihe nkiri S1) (23).
Mkpụrụ ndụ sec31-1 na-egosipụta ihe ndị galactose na-emepụta, ogologo acyl yinye (C26) ceramide GPI-AP Gas1-GFP (GPI-AP, akwụkwọ ndụ akwụkwọ ndụ) na protein transmembrane Mid2-iRFP (TMP, acha anụnụ anụnụ) na akara ERES a na-ewu ewu Sec13-mCherry (ERES, magenta) ka etinyere na 37°C ruo nkeji iri atọ, bufee ya na 24°C, ma see ya na SCLIM nkeji ise ka e mesịrị. (A ruo C) na-egosi onyonyo 2D ejikọtara ma ọ bụ otu nke ụgbọelu (A), onyonyo 2D nke ngalaba z iri (B) ma ọ bụ onyonyo mkpụrụ ndụ 3D nke ibu na akara ERES (C). Ogwe nha 1μm (A na B). Ngalaba nha bụ 0.551μm (C). Achọpụtara Gas1-GFP na mpaghara ma ọ bụ ụyọkọ ER dị iche iche, ebe achọpụtara ma kesaa Mid2-iRFP n'ime membrane ER (C). (D) Grafụ ahụ na-egosi ike ọkụ Gas1-GFP na Mid2-iRFP dị n'ime otu Gas1-GFP n'akụkụ ahịrị akụ ọcha (aka ekpe). AU, otu na-enweghị usoro. (E na F) na-anọchite anya onyonyo 3D nke na-ejikọta ngwongwo na akara ERES. Achọpụtara otu Gas1-GFP n'akụkụ ERES kpọmkwem. Otu nha ahụ bụ 0.551μm. (F) Akụ siri ike ọcha na-egosi otu Gas1-GFP nke jikọtara ya na ERES. Ogwe etiti na aka nri na-egosi onyonyo 3D a kpụkọrọ akpụkọ na echiche agbagharị nke otu Gas1-GFP ahọpụtara.
Mmekọrịta dị n'etiti Gas1-GFP cluster na otu ERES a kapịrị ọnụ na-egosi na Gas1-GFP nwere ike itinye ERES nhọrọ, nke dị iche na nhọrọ nke Mid2-iRFP ji pụọ na ER. Iji dozie ohere a, anyị tụlere oke ERES maka naanị otu ma ọ bụ abụọ ngwaahịa (Foto 2, A ruo C). Anyị chọpụtara na ọtụtụ ERES (70%) nwere naanị otu ụdị ibu. Foto ala nke Foto 2C na-egosi ihe atụ abụọ nke ERES nwere naanị Gas1-GFP (Foto 1) ma ọ bụ naanị Mid2-iRFP (Foto 2). N'ụzọ dị iche, ihe dị ka 20% nke ERES nwere ibu abụọ nke na-adakọ n'otu mpaghara ahụ. A chọpụtara na ụfọdụ ERES (10%) nwere ụdị ibu abụọ, mana e kewapụrụ ha n'ebe dị iche iche doro anya. Ya mere, nyocha ọnụọgụgụ a na-egosi na mgbe ebupụchara ER, a na-ekewa GPI-AP Gas1-GFP na ibu transmembrane Mid2-iRFP n'ime ERES dị iche iche (Foto 2D). Arụmọrụ nhazi a kwekọrọ nnọọ na nyocha biochemical gara aga (6) na mkpebi morphological (7). Anyị nwekwara ike ịhụ omume nke ibu ndị a kpọchiri akpọchi na-abanye na ERES (Foto 2E na Movie S2). Foto 2E na-egosi na naanị obere akụkụ nke Gas1-GFP (panel 3) ma ọ bụ Mid2-iRFP (panel 4) na-abanye na ERES site n'otu akụkụ ma dị n'ime mpaghara dị iche iche. Panel 5 nke Foto 2E na-egosi na a na-ahụ Gas1-GFP na Mid2-iRFP mgbe ụfọdụ n'otu ERES, mana ha na-abanye site n'akụkụ dị iche iche ma na-etinye uche na mpaghara dị iche iche nke nwere ike ịnọchite anya vesicles COPII dị iche iche. Anyị kwadokwara na nkewa na nhazi nke C26 ceramide dabere na GPI-AP Gas1 dị ka ERES nhọrọ bụ kpọmkwem n'ihi na ibu ọzọ transmembrane secretion, protein plasma membrane nke GFP-tagged Axl2 (27), na-egosi omume yiri nke Mid2-iRFP. (Foto S1 na Movie S3). A na-ekesa Axl2-GFP ọhụrụ a na-emepụta site na akpụkpọ ahụ ER dị ka Mid2-iRFP (Foto S1, A na B), a na-ejikọta ya na Mid2-iRFP n'ọtụtụ ERES (Foto S1, B ruo D). Panel 1 na 2 nke Foto 1. S1C na-egosi ihe atụ abụọ nke ERES ebe ibu transmembrane abụọ na-ejikọta ọnụ. N'ọnọdụ ndị a, ngwongwo abụọ ahụ na-abanye ERES ọnụ (Foto S1E, Panel 3 na Movie S3).
E tinyere mkpụrụ ndụ sec31-1 nke na-egosipụta ihe ndị a na-akpọ galactose inducible sections, Gas1-GFP (GPI-AP, akwụkwọ ndụ akwụkwọ ndụ) na Mid2-iRFP (TMP, acha anụnụ anụnụ) na akara ERES Sec13-mCherry (ERES, magenta) na 37 Mgbe ha gbasasịrị ruo nkeji 30 na CelsiusC, gaa na 24 °C iji wepụta ihe ndị a na-akpọ "section blood", ma tinye onyonyo na SCLIM mgbe nkeji 20 gachara. (A ruo C) Foto ndị a na-anọchite anya 2D projection (A; scale bar, 1μm) ma ọ bụ onyonyo hemisphere sel 3D (B na C; scale unit, 0.456μm) nke ibu na ngalaba z 10 nke ERES mara. Panel dị ala na (B) na panel dị na (C) na-egosipụta onyonyo ndị a haziri iji gosipụta naanị ngwaahịa dị na ERES (magenta) [Gas1-GFP (isi awọ) na Mid2-iRFP (acha anụnụ anụnụ ọkụ)]. (C) Akụ mepere emepe: ERES na-ebu naanị otu ibu (1 ruo 4). Akụ isi awọ: ERES nwere ibu kewapụrụ iche (5). Akụ siri ike ọcha: ERES nwere ibu dị n'otu ebe. N'okpuru: Otu ERES ahọpụtara nwere naanị Gas1-GFP (1) ma ọ bụ Mid2-iRFP (2). Ogwe nha, 100 nm. (D) Ngụkọta nke fotomicrograph akọwara na (C). Pasentị nkezi nke ERES nke nwere naanị otu ibu (Gas1-GFP ma ọ bụ Mid2-iRFP), ibu kewapụrụ iche na ibu na-ekpuchi. Na nnwale atọ nọọrọ onwe ha, n=432 na sel 54. Ogwe njehie = SD. Nnwale t nke nwere ọdụ abụọ na-ejikọghị ọnụ. *** P = 0.0002. (E) Foto 3D nke ERES ahọpụtara nke ibu kewapụrụ iche nke ejiri (C) akara. Gas1-GFP (akwụkwọ ndụ akwụkwọ ndụ) (3) ma ọ bụ Mid2-iRFP (acha anụnụ anụnụ) (4) na-abanye ERES (magenta) site n'otu akụkụ ma bụrụ nke a kpaara ókè na obere mpaghara dị n'ime ERES. Mgbe ụfọdụ, ụdị ibu abụọ a na-abanye n'otu ERES (5) site n'otu akụkụ ma a na-etinye ha n'ebe dịpụrụ adịpụ n'ime ERES. Ogwe nha, 100 nm.
Mgbe nke ahụ gasịrị, anyị nwalere echiche na ceramide ogologo acyl chain (C26) dị na membrane ER na-akpali nchịkọta na nhazi nke Gas1 ka ọ bụrụ nhọrọ ERES. Iji mezuo nke a, anyị jiri ụdị yist GhLag1 gbanwere agbanwe, nke ejiri GhLag1 (Lag1 homolog nke owu) dochie Lag1 na Lac1 abụọ nke endogenous ceramide synthases, nke butere ụdị yist nwere akpụkpọ ahụ sel Ceramide dị mkpụmkpụ karịa ụdị anụ ọhịa (Foto 3A) (28). Nnyocha Mass spectrometry (MS) gosiri na n'ụdị anụ ọhịa, 95% nke ceramide zuru oke dị ogologo (C26) yinye ceramide, ebe na GhLag1, 85% nke ceramide dị ogologo (C18 na C16). ), naanị 2% nke ceramide dị ogologo (C26) yinye ceramide. Ọ bụ ezie na ceramides C18 na C16 bụ ceramides bụ isi achọpụtara na akpụkpọ ahụ GhLag1 ruo ugbu a, nyocha MS gosikwara na ihe nkwado GPI nke Gas1-GFP nke a na-egosipụta na ụdị GhLag1 nwere ceramide C26, nke yiri lipids ụdị ọhịa. Ọdịdị ya bụ otu (Fig. 3A) (26). Ya mere, nke a pụtara na enzyme mgbanwe ceramide Cwh43 dị oke mma maka ceramide C26, dịka egosiri na Foto 26, ọ na-etinye ihe nkwado GPI site na obere ceramide C26 na ụdị GhLag1. S2 (29). Agbanyeghị, akpụkpọ ahụ sel nke GhLag1 nwere naanị ceramide C18-C16, ebe Gas1-GFP ka nwere ceramide C26. Eziokwu a na-eme ka ụdị a bụrụ ngwaọrụ zuru oke iji dozie nsogbu nke ogologo acyl nke membrane ceramide na ER. Ọrụ echiche nke ọkwa na nhazi. Mgbe ahụ, anyị buru ụzọ mụọ ikike C26 Gas1-GFP iji chịkọta na mkpokọta na GhLag1 site na allele mutant nke sec31-1 site na microscopy fluorescence nkịtị, ebe naanị ogologo (C18-C16) yinye dị na membrane ER Ceramide (Foto 3). Anyị hụrụ na na sec31-1, ọtụtụ n'ime Gas1-GFP dị na mkpokọta, ebe Gas1-GFP dị na sec31-1 GhLag1 nwere ogologo (C18-C16) akpụkpọ ahụ ceramide ER ogologo anaghị ejikọta ma kesaa na membrane ER dum. Iji bụrụ nke ziri ezi, n'ihi na mkpokọta ceramide dabere na C26 nwere njikọ chiri anya na ERES kpọmkwem (Foto 1), anyị nyochachara ma usoro a nwekwara ike ịgụnye ọrụ nke usoro protein mbupụ ER. GPI-AP na-eji sistemụ COPII pụrụ iche maka mbupụ ER, nke na-achịkwa nke ọma site na nhazi nhazi nke Ted1 nke akụkụ glycan nke arịlịka GPI (30, 31). Mgbe ahụ, a na-amata GPI-glycan recombinant site na transmembrane cargo receptor p24 complex, nke na-ahọpụta Lst1, nke bụ isoform kpọmkwem nke nnukwu COPII cargo binding subunit Sec24, na-emepụta COPII bara ọgaranya nke GPI-AP dị mkpa (31-33). Ya mere, anyị wuru mutant abụọ nke jikọtara nhichapụ nke otu protein ndị a (ihe mejupụtara p24 complex Emp24, GPI-glycan remodeling enzyme Ted1 na kpọmkwem COPII subunit Lst1) na ụdị mutant sec31-1, ma mụọ ha Ò kwere omume ịmepụta Gas1-cluster GFP (Foto 3). Anyị hụrụ na na sec31-1emp24Δ na sec31-1ted1Δ, Gas1-GFP bụ ihe a na-anaghị ejikọta ọnụ ma kesaa n'ime akpụkpọ ahụ ER niile, dịka a hụrụ na sec31-1 GhLag1, ebe na sec31-1lst1Δ, Gas1-GFP Dịka sec31-1. Nsonaazụ ndị a na-egosi na na mgbakwunye na ọnụnọ nke ceramide C26 na akpụkpọ ahụ ER, nchịkọta nke Gas1-GFP kwesịkwara ijikọ na mgbagwoju anya p24, ọ chọghịkwa ka a kpọbata Lst1 kpọmkwem. Mgbe ahụ, anyị nyochachara ohere na ogologo yinye nke ceramide na akpụkpọ ahụ ER nwere ike ịchịkwa njikọ nke Gas1-GFP na p24. Agbanyeghị, anyị chọpụtara na ọnụnọ nke ceramide C18-C16 na akpụkpọ ahụ anaghị emetụta GPI-glycans nke mgbagwoju anya p24 rụgharịrị (Foto S3 na S4, A na B) ma ọ bụ njikọ na GPI-AP na mbupụ ikike GPI-AP. Nweta ụdị COPII Lst1 (Foto S4C). Ya mere, nchịkọta nke dabere na ceramide C26 anaghị achọ mmekọrịta protein na usoro protein mbupụ ER dị iche iche, kama ọ na-akwado usoro nhazi ọzọ nke ogologo lipid na-akwalite. Mgbe ahụ, anyị nyochachara ma ogologo yinye ceramide acyl dị na membrane ER dị mkpa maka nhazi dị irè nke Gas1-GFP dị ka ERES nhọrọ. Ebe ọ bụ na Gas1 dị na ụdị GhLag1 nwere ceramide dị mkpụmkpụ na-ahapụ ER ma banye na akpụkpọ ahụ plasma (Foto S5), anyị kwenyere na ọ bụrụ na nhazi ahụ na-akpali site na ogologo nke yinye ceramide acyl, enwere ike ibugharị Gas1 dị na ụdị GhLag1 ma gafere ya. Ngwaahịa ERES nwere otu akpụkpọ ahụ.
(A) Akpụkpọ ahụ mkpụrụ ndụ nke GhLag1 nwere ceramides C18-C16 dị mkpụmkpụ, ebe arịlịka GPI nke Gas1-GFP ka nwere otu C26 IPC dị ka mkpụrụ ndụ ụdị ọhịa. N'elu: nyocha ogologo acyl nke ceramide na akpụkpọ ahụ mkpụrụ ndụ nke ụdị ọhịa (Wt) na ụdị GhLag1p site na mass spectrometry (MS). Data ahụ na-anọchite anya pasentị nke ceramide zuru oke. Nkezi nke nnwale atọ nọọrọ onwe ha. Ogwe njehie = SD. Nnwale t nke ọdụ abụọ na-ejikọghị ọnụ. **** P ＜0.0001. Ala panel: Nyocha MS nke ogologo acyl yinye nke IPC dị na arịlịka GPI Gas1-GFP (GPI-IPC) nke egosiri na ụdị ọhịa na GhLag1p. Data ahụ na-anọchite anya pasentị nke ngụkọta akara IPC. Nkezi nke nnwale ise nọọrọ onwe ha. Ogwe njehie = SD. Nnwale t nke ọdụ abụọ na-ejikọghị ọnụ. ns, ọ dịghị mkpa. P = 0.9134. (B) E tinyere obere ihe ndị na-egosi ìhè ọkụ nke sec31-1, sec31-1 GhLag1, sec31-1emp24Δ, sec31-1ted1Δ na sec31-1lst1Δ nke na-egosipụta Gas1-GFP nke galactose kpatara na okpomọkụ na 37°C ruo nkeji iri atọ ma nyefee ya na Mee obere ihe ndị na-egosi ìhè ọkụ mgbe okpomọkụ 24°C gasịrị. Akụ ọcha: ER Gas1-GFP cluster. Akụ mepere emepe: A na-ekesa Gas1-GFP nke a na-anaghị ejikọta ọnụ na akpụkpọ ahụ ER dum, na-egosi agba mgbanaka nuklia nke ER. Ogwe nha, 5μm. (C) Ọnụọgụ nke fotomicrograph akọwara na (B). Pasent nkezi nke mkpụrụ ndụ nwere nhazi Gas1-GFP punctate. Na nnwale atọ nọọrọ onwe ha, mkpụrụ ndụ n≥300. Ogwe njehie = SD. Nnwale t nke nwere ọdụ abụọ na-enweghị njikọ. **** P ＜0.0001.
Iji dozie nsogbu a ozugbo, anyị mere ihe ngosi SCLIM nke Gas1-GFP na Mid2-iRFP na GhLag1 na allele mutant nke sec31-1 nwere mmetụta okpomọkụ (Foto 4 na Movie S4). Mgbe ejidere ER na 37°C ma mesịa wepụta ya na 24°C, ọtụtụ n'ime Gas1-GFP emepụtara ọhụrụ ejikọtaghị ma kesaa ya n'ime membrane ER, dịka a hụrụ site na microscopes ọdịnala (Foto 4, A na B). Na mgbakwunye, nnukwu pasentị nke ERES (67%) gụnyere ụdị ibu abụọ dị na ya (Foto 4D). Panels 1 na 2 nke Foto 4C na-egosi ihe atụ abụọ nke ERES nwere Gas1-GFP na Mid2-GFP na-ekpuchi. Na mgbakwunye, a kpọbatara ngwongwo abụọ ahụ n'otu ERES (Foto 4E, panel 3 na fim S4). Ya mere, nsonaazụ anyị na-egosi na ogologo nke ceramide acyl yinye na membrane ER bụ ihe dị mkpa na-ekpebi nchịkọta na nhazi protein ER.
Mkpụrụ ndụ GhLag1 nke Sec31-1 na-egosipụta ihe ndị galactose kpatara, Gas1-GFP (GPI-AP, akwụkwọ ndụ akwụkwọ ndụ) na Mid2-iRFP (TMP, acha anụnụ anụnụ) na Sec13-mCherry (ERES, magenta) nke nwere akara ERES na 37°C. Gaa n'ihu ruo nkeji iri atọ, daa ruo 24°C iji wepụta ihe ndị na-apụta, wee see foto na SCLIM mgbe nkeji iri abụọ gachara. (A ruo C) Foto ndị na-egosi 2D (A; ogwe nha, 1μm) ma ọ bụ onyonyo mkpụrụ ndụ 3D (B na C; nkeji nha, 0.45μm) nke ngalaba z iri nke ibu na ERES mara. Ogwe ala dị na (B) na panel dị na (C) na-egosipụta onyonyo ndị a haziri iji gosipụta naanị ngwaahịa dị na ERES (magenta) [Gas1-GFP (isi awọ) na Mid2-iRFP (acha anụnụ anụnụ ọkụ)]. (C) Akụ ọcha jupụtara: ERES, ngwongwo na-ejikọta ọnụ. Akụ mepere emepe: ERES nwere naanị otu ihe. Ogwe ala: ERES ahọpụtara nwere ngwongwo ndị na-ekpuchi (1 na 2) nke akara na (C). Ogwe nha, 100 nm. (D) Ngụkọta nke fotomicrograph akọwara na (C). Na nkeji sec31-1 na sec31-1 GhLag1, naanị otu ibu (Gas1-GFP ma ọ bụ Mid2-iRFP) ka etinyere, yana pasentị nkezi nke ERES maka ibu dịpụrụ adịpụ na ibu jikọtara ọnụ. Na nnwale atọ nọọrọ onwe ha, n = 432 na sel 54 (sec31-1) na n = 430 na sel 47 (sec31-1 GhLag1). Ogwe njehie = SD. Nnwale t nke nwere ọdụ abụọ na-ejikọghị ọnụ. *** P = 0.0002 (sec31-1) na ** P = 0.0031 (sec31-1 GhLag1). (E) Foto 3D nke ERES ahọpụtara nwere ibu jikọtara ọnụ (3) nke akara na (C). Gas1-GFP (akwụkwọ ndụ akwụkwọ ndụ) na Mid2-iRFP (acha anụnụ anụnụ) na-abịaru ERES (magenta) site n'otu akụkụ ma nọgide n'otu mpaghara ERES a kpaara ókè. Ogwe nha, 100 nm.
Ọmụmụ ihe a na-enye ihe akaebe kpọmkwem na vivo na-egosi na a na-ekewa ibu protein dabere na lipid n'ime ebe a na-ebupụ ihe n'ụzọ nzuzo, ma na-ekpughe mkpa ogologo acyl yinye maka nhọrọ nhazi. Site na iji usoro microscopy dị ike ma dị elu nke a na-akpọ SCLIM, anyị gosipụtara Gas1-GFP emepụtara ọhụrụ (GPI-AP nke nwere nnukwu plasma membrane nwere oke acyl yinye (C26) ceramide lipid part) na yist. Mpaghara ndị a chịkọtara na discrete ERs jikọtara ya na ERES kpọmkwem, ebe a na-ekesa protein transmembrane na membrane ER niile (Foto 1). Na mgbakwunye, ụdị ngwaahịa abụọ a na-abanye na ERES dị iche iche nhọrọ (Foto 2). Ogologo acyl yinye nke ceramide cell dị na membrane ahụ na-ebelata site na C26 gaa na C18-C16, a na-emebi ụyọkọ Gas1-GFP n'ime mpaghara ER dị iche, a na-emegharị Gas1-GFP ka ọ hapụ ER na protein transmembrane site na otu ERES ahụ (Foto 3 na Foto 3). 4).
Ọ bụ ezie na GPI-AP na-eji usoro protein pụrụ iche iji pụọ na ER, anyị chọpụtara na nkewa nke dabere na ceramide C26 anaghị adabere na mmekọrịta protein dị iche iche nke nwere ike ibute ọkachamara ERES (Foto S4 na S5). Kama nke ahụ, nchọpụta anyị na-akwado usoro nhazi ọzọ nke nchịkọta protein dabere na lipid na mwepụ nke ibu ndị ọzọ na-esote. Ihe anyị chọpụtara na-egosi na mpaghara Gas1-GFP ma ọ bụ otu ERES jikọtara ya na ERES kpọmkwem enweghị protein transmembrane nke Mid2-iRFP wepụtara, nke na-egosi na otu C26 ceramide-dependent GPI-AP ga-eme ka ha banye na ERES dị mkpa, ma n'otu oge ahụ, wepụ transmembrane Ihe nzuzo ndị ahụ na-abanye na ERES a kpọmkwem (Foto 1 na 2). N'ụzọ dị iche, ọnụnọ nke ceramides C18-C16 na membrane ER anaghị eme ka GPI-AP mepụta mpaghara ma ọ bụ ụyọkọ, yabụ ha anaghị ewepụ ma ọ bụ dochie protein transmembrane zoro ezo n'ime otu ERES (Foto 3 na 4). Ya mere, anyị na-atụ aro na C26 ceramide na-akwalite nkewa na nhazi site n'inyere aka ịhazi protein ndị jikọtara na ERES kpọmkwem.
Kedu otu esi enweta nchịkọta nke dabere na ceramide C26 a n'ime mpaghara ER kpọmkwem? Ọdịmma nke membrane ceramide kewapụrụ n'akụkụ nwere ike ime ka GPI-AP na ceramide C26 mepụta obere lipids na-ahazi ozugbo n'ebe dị jụụ nke akpụkpọ ahụ ER nwere glycerolipids dị mkpụmkpụ na nke na-enweghị afọ ojuju. Ụyọkọ dị mma (17, 18). Enwere ike ijikọ obere obere obere ndị a n'ime nnukwu, nke kwụsiri ike mgbe ejikọtara ya na mgbagwoju anya p24 (34). N'ikwekọ na nke a, anyị gosiri na C26 Gas1-GFP kwesịrị ịkpakọrịta na mgbagwoju anya p24 iji mepụta nnukwu ụyọkọ a na-ahụ anya (Foto 3). Mgbakwụnye p24 bụ oligomer heterozygous nke mejupụtara protein transmembrane p24 anọ dị iche iche na yist (35), nke na-enye njikọta multivalent, nke nwere ike ibute njikọ nke obere ụyọkọ GPI-AP, si otú a na-emepụta nnukwu ụyọkọ Stable (34). Mmekọrịta dị n'etiti ectodomains protein nke GPI-APs nwekwara ike inye aka na nchịkọta ha, dịka egosiri n'oge njem Golgi ha na mkpụrụ ndụ epithelial polarized mammalian (36). Agbanyeghị, mgbe ceramide C18-C16 dị na membrane ER, mgbe mgbagwoju anya p24 na-ejikọ na Gas1-GFP, a gaghị emepụta nnukwu ụyọkọ dị iche iche. Usoro dị n'okpuru nwere ike ịdabere na njirimara anụ ahụ na kemịkalụ kpọmkwem nke ceramide ogologo acyl yinye. Ọmụmụ ihe gbasara akpụkpọ ahụ arụrụ arụ na-egosi na ọ bụ ezie na ceramides ogologo (C24) na obere (C18-C16) acyl yinye ceramides nwere ike ibute nkewa nkewa, naanị ceramides ogologo acyl yinye (C24) nwere ike ịkwalite mgbagọ dị elu na mgbagọ ihe nkiri iji gbanwee ọdịdị ihe nkiri ahụ. Site na ntụnyere aka (17, 37, 38). E gosila na helix transmembrane nke TMED2, nke bụ njikọ mmadụ nke Emp24, na-emekọrịta ihe nke ọma na sphingomyelin nke dabeere na C18 ceramide na lobules cytoplasmic (39). Site na iji ihe atụ molekul dynamics (MD), anyị chọpụtara na ma ceramides C18 na C26 na-agbakọta gburugburu lobules cytoplasmic nke helix transmembrane Emp24, ha nwekwara mmasị yiri nke ahụ (Foto S6). Ọ dị mkpa ịmara na nke a na-egosi na helix transmembrane nke Emp24 nwere ike ibute nkesa lipids na membrane ahụ. Nke a bụ nsonaazụ ọhụrụ dabere na mkpụrụ ndụ anụmanụ. Ihe atụ MD ndị yiri ya na-egosikwa ọnụnọ nke lipids ether (40). Ya mere, anyị na-eche na C26 ceramide na lobules abụọ nke ER26 bara ọgaranya n'ógbè. Mgbe GPI-AP dị na lobules luminal na-ejikọ kpọmkwem na multivalent p24 na mkpokọta nke C26 ceramide gburugburu p24 na cytoplasmic lobules, ọ nwere ike ịkwalite nchịkọta protein na-eso ya na mkpịsị aka na-emepụta mkpụkọ akpụkpọ ahụ (41), na-eme ka GPI-AP kewaa n'ime mpaghara dị iche iche dị n'akụkụ ERES, nke na-akwadokwa mpaghara gbagọrọ agbagọ nke membrane ER (42). Akụkọ ndị gara aga kwadoro usoro a tụrụ aro (43, 44). Njikọ multivalent nke oligolectins, nje ma ọ bụ ọgwụ mgbochi na glycosphingolipids (GSL) nke dabeere na ceramide na membrane plasma na-akpali nnukwu mkpokọta GSL, na-eme ka nkewa nkebi dịkwuo mma ma na-akpata mmebi akpụkpọ ahụ na ime ya (44). Iwabuchi wdg. (43) A chọpụtara na n'ihu ogologo (C24) mana ọ bụghị mkpụmkpụ (C16) acyl chains, multivalent ligand ejikọtara na GSL lactosylceramide kpaliri mmepụta nke nnukwu ụyọkọ na mgbochi akpụkpọ ahụ, na cytoplasm Lyn-mediated signal transduction na akwụkwọ nta na-ejikọta site na acyl chains na njikọ neutrophils.
N'ime mkpụrụ ndụ epithelial nke a na-akpọ mammalian polarized, ntinye nke netwọk anti-Golgi (TGN) ruo n'ọkwa nke akpụkpọ ahụ plasma apical na-achịkwa nkewa na nhazi nke GPI-AP (10, 45). Nchịkọta a na-akwalite site na GPI-AP oligomerization (36), mana ọ nwekwara ike ịdabere na ogologo yinye ceramide anyị na-ahụ na yist. Ọ bụ ezie na mammalian GPI-AP nwere arịlịka dabere na ether lipid, nhazi kemịkalụ ya dịkwa iche na ceramide acyl chain dị ogologo, nnyocha e mere n'oge na-adịbeghị anya chọpụtara na lipids abụọ ahụ nwere ihe onwunwe anụ ahụ na kemịkalụ yiri nke evolushọn (40). Ya mere, akụkụ ether lipid na mkpụrụ ndụ mammalian nwere ike ịdị ka ceramide C26 na yist, ọrụ ya bụkwa ijikọ ya na ceramide ogologo agbụ dị na akpụkpọ ahụ iji kwalite nchịkọta na nhazi GPI-AP. Ọ bụ ezie na a ka kwesịrị ịnwale ohere a ozugbo, ihe ndị a chọpụtara n'oge gara aga na-akwado na ibuga ceramide ogologo acyl yinye gaa n'ahụ Golgi abụghị site na protein nnyefe cytoplasmic, kama ọ dabere na njikọta nke arịlịka GPI dị ka yist. Ya mere, usoro nchekwa evolushọn yiri ka ọ nwere ike ijikọ aka na ibuga ceramide ogologo acyl yinye na GPI-AP (13, 16, 20, 46, 47) n'otu vesicle njem.
N'ime sistemụ sel epithelial nke yist na nke mammalian polarized, nchịkọta GPI-AP na nkewa site na protein membrane plasma ndị ọzọ niile na-eme tupu ha erute elu sel ahụ. Paladino et al. (48) chọpụtara na na TGN nke sel epithelial polarized mammalian, nchịkọta GPI-AP abụghị naanị ihe dị mkpa maka nhazi nhọrọ nke GPI-APs na membrane plasma apical, kamakwa ọ na-achịkwa nhazi nke GPI-APs na ọrụ bayọlọji Ya. Elu sel. Na yist, ọmụmụ ihe a gosiri na C26 ceramide-dependent GPI-AP cluster na ER nwere ike ịchịkwa nhazi cluster na ọrụ ọrụ nke GPI-AP na membrane plasma (24, 49). Dịka ụdị a si dị, mkpụrụ ndụ GhLag1 na-enwe nfụkasị ahụ na ndị na-egbochi GPI ma ọ bụ ọgwụ ndị na-emetụta iguzosi ike n'ezi ihe nke mgbidi sel (28), na mkpa maka ụyọkọ Gas1-GFP na-arụ ọrụ (49) nke ceramide tip nke a tụrụ anya na njikọta nke mkpụrụ ndụ yist na-egosi G​​​ Nsonaazụ physiological nwere ike ịpụta na mkpụrụ ndụ hLag1. Njehie GPI-AP. Agbanyeghị, nnwale ọzọ ma ọ bụrụ na ER enyela nhazi ọrụ nke elu sel site na usoro nhazi dabere na ogologo lipid ga-abụ isiokwu nke nnyocha anyị n'ọdịnihu.
E depụtara ụdị Saccharomyces cerevisiae ejiri mee ihe n'ọrụ a na Tebụl S1. E wuru ụdị MMY1583 na MMY1635 nke SCLIM maka onyonyo mkpụrụ ndụ n'azụ W303. E ji usoro polymerase chain reaction (PCR) nke dabere na polymerase chain reaction (PCR) rụọ ụdị ndị a na-egosipụta site na iji pFA6a plasmid dị ka ihe nlereanya (23). E wuru ụdị Mid2-iRFP nke ejiri protein fluorescent mee akara n'okpuru njikwa nke onye na-akwalite GAL1 dị ka ndị a. Mmelite PCR nke usoro iRFP-KanMx sitere na vektọ pKTiRFP-KAN (onyinye nke E. O'Shea, nọmba plasmid Addgene 64687; http://n2t.net/addgene: 64687; ihe nchọpụta akụrụngwa (RRID): Addgene_64687) Ma tinye ya n'ime C-terminus nke endogenous Mid2 Mid2. Mgbe e mepechara usoro mkpụrụ ndụ ihe nketa Mid2-iRFP ma mepụta ya n'ime onye na-akwalite GAL1, e tinyere ya n'ime ebe Not I-Sac I nke njikọta plasmid pRS306. E jiri Pst I hazie plasmid pRGS7 nke si na ya pụta ka ọ banye n'ime ebe URA3.
A na-egosipụta mkpụrụ ndụ ihe nketa Gas1-GFP n'okpuru njikwa nke onye na-akwalite GAL1 na plasmid centromere (CEN), nke e wuru dịka ndị a. E jiri PCR mee ka usoro Gas1-GFP dịkwuo elu site na PCR site na plasmid pRS416-GAS1-GFP (24) (onyinye nke L. Popolo) ma kụọ ya n'ime ebe Xma I–Xho I nke CEN plasmid pBEVY-GL LEU2 (onyinye nke C). Miller; Nọmba plasmid Addgene 51225; http://n2t.net/addgene: 51225; RRID: Addgene_51225). A kpọrọ plasmid ahụ pRGS6. A na-egosipụtakwa mkpụrụ ndụ ihe nketa Axl2-GFP n'okpuru njikwa nke onye na-akwalite GAL1 nke vektọ pBEVY-GL LEU2, owuwu ya dịkwa ka ndị a. E si na pRS304-p2HSE-Axl2-GFP plasmid (23) mee ka usoro Axl2-GFP dịkwuo elu, wee kụọ ya n'ime ebe Bam HI-Pst I nke pBEVY-GL LEU2 vector. Aha plasmid ahụ a nwetara bụ pRGS12. E depụtara usoro nke oligonucleotides ejiri mee ihe n'ọmụmụ ihe a na Tebụl S2.
E tinyere 0.2% adenine na 2% glucose [YP-dextrose (YPD)], 2% raffinose [YP-raffinose] rich yist extract protein p (YP) ọkara (1% Yist extract na 2% protein ept). (YPR)] ma ọ bụ 2% galactose [YP-galactose (YPG)] dị ka isi iyi carbon, ma ọ bụ na obere ihe e ji emepụta ihe (0.15% yist nitrogen base na 0.5% ammonium sulfate) iji gbakwunye amino acid na ntọala kwesịrị ekwesị dị mkpa maka nri, Nakwa nwere 2% glucose (synthetic glucose minimally medium) ma ọ bụ 2% galactose (synthetic galactose minimally medium) dị ka isi iyi carbon.
Maka onyonyo oge a na-ahụ anya, a zụlitere mkpụrụ ndụ mutant sec31-1 nke na-egosipụta okpomọkụ nke na-egosipụta ihe owuwu ahụ n'okpuru onye na-akwalite GAL1 na YPR na 24°C n'abalị ruo n'etiti usoro. Mgbe etinyere YPG na 24°C ruo otu awa, a kpọchiri mkpụrụ ndụ ndị ahụ na SG na 37°C ruo nkeji iri atọ, wee bufee ha na 24°C ka ha si na ngọngọ mmiri pụta. Ejiri Concanavalin A dozie mkpụrụ ndụ ndị ahụ na slide iko ma jiri SCLIM see foto ha. SCLIM bụ ngwakọta nke igwe onyonyo ọkụ ọkụ Olympus IX-71 nke a gbanwere agbanwe na oghere mmanụ ọnụọgụgụ UPlanSApo 100×1.4 (Olympus), ihe nyocha diski confocal nke na-agbagharị ọsọ ọsọ na nke dị elu (Yokogawa Electric), ihe nlele custom, na oyi kpọnwụrụ akpọnwụ. Ihe na-eme ka onyonyo dịkwuo elu na sistemụ ahụ (Hamamatsu Photonics) nwere ike inye sistemụ oghere ọkụ ọkụ nwere mmụba ikpeazụ nke ×266.7 na igwefoto ngwaọrụ ejiri chajị jikọta nke na-amụba elektrọn (Hamamatsu Photonics) (21). A na-eji ngwanrọ omenala (Yokogawa Electric) eme nnweta onyonyo. Maka onyonyo 3D, anyị jiri ihe na-eme piezoelectric emebere iji mee ka oghere anya ahụ maa jijiji n'akụkụ kwụ ọtọ, wee chịkọta akụkụ anya 100 nm iche iche na otu stack. A na-agbanwe onyonyo Z-stack ka ọ bụrụ data voxel 3D, a na-ejikwa ọrụ mgbasa nke echiche ejiri maka microscope confocal diski na-agbagharị maka nhazi deconvolution site na ngwanrọ Volocity (PerkinElmer). Site na iji ngwanrọ Volocity iji chọpụta oke na nyocha ọnọdụ otu, a tụrụ ERES tinyere ibu. Ejiri ngwanrọ MetaMorph (Ngwaọrụ Molecular) mee nyocha nyocha ahịrị.
Jiri ngwanrọ GraphPad Prism chọpụta mkpa ọnụọgụgụ dị. Maka ule t-ụmụ akwụkwọ nwere ọdụ abụọ na ule nyocha otu ụzọ nkịtị nke mgbanwe (ANOVA), a na-ewere ọdịiche dị n'etiti otu dị iche iche dị ka mmetụta dị ukwuu na P <0.05 (*).
Maka nyocha fluorescence nke Gas1-GFP, a kụrụ mkpụrụ ndụ log phase n'abalị na YPD ma chịkọta ha site na centrifugation, saa ha ugboro abụọ na saline phosphate buffered, ma tinye ha na ice ruo opekata mpe nkeji iri na ise, wee gaa n'ihu n'okpuru microscope dịka akọwara na mbụ Lelee (24). Ejiri igwe microscope Leica DMi8 (HCX PL APO 1003/1.40 mmanụ PH3 CS) nke nwere oghere anya, nzacha L5 (GFP), igwefoto Hamamatsu na ngwanrọ Application Suite X (LAS X) zụta ha.
E jiri ihe nchekwa SDS sample buffer hichaa ihe nlele ndị a na okpomọkụ 65°C ruo nkeji iri, wee kewaa ha site na SDS-polyacrylamide gel electrophoresis (PAGE). Maka nyocha immunoblotting, e tinyere 10 μl nke ihe nlele kwa ụzọ. Ọgwụ mgbochi mbụ: Jiri ọgwụ mgbochi polyclonal rabbit na mgbaze nke 1:3000, ọgwụ mgbochi polyclonal rabbit na mgbaze nke 1:500, na ọgwụ mgbochi polyclonal rabbit (onyinye sitere na H. Riezman) na mgbaze nke 1:3000. E jiri ọgwụ mgbochi monoclonal anti-Pgk1 òké mee ihe na mgbaze nke 1:5000 (onyinye sitere na J. de la Cruz). Ọgwụ mgbochi nke abụọ: Horseradish peroxidase (HRP) nke e ji agwọ nkịta mgbochi rabbit immunoglobulin G (IgG) nke ejiri mee ihe na mgbaze nke 1:3000 (Pierce). E jiri IgG nke e ji HRP mee ihe na ngwakọta nke 1:3000 (Pierce). A hụrụ mpaghara mmeghachi omume ahụ ji alụso ọrịa ọgụ site na usoro chemiluminescence nke SuperSignal West Pico reagent (Thermo Fisher Scientific).
Dịka akọwara na (31), emere nnwale immunoprecipitation eke na akụkụ ER bara ụba. Na nkenke, saa mkpụrụ ndụ yist na TNE buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride na ngwakọta protease inhibitor) na 600 nm (OD600) na 100 optical njupụta ugboro abụọ. E jiri iko beads gbajie ya, wee wepụ ihe mkpofu sel na beads iko site na centrifugation. E mesịa centrifugation tinye centrifugation na 17,000 g maka nkeji 15 na 4°C. E tinyere pellet ahụ ọzọ na TNE ma tinye digitalis saponin na mkpokọta ikpeazụ nke 1%. E tinyere nkwusioru ahụ ruo otu awa na ntụgharị na 4°C, wee wepụ ihe ndị na-anaghị agbaze site na centrifugation na 13,000 g na 4°C maka nkeji 60. Maka Gas1-GFP immunoprecipitation, buru ụzọ tinye ihe nlele ahụ na mkpụrụ agarose efu (ChromoTek) na 4°C ruo otu awa, wee tinye GFP-Trap_A (ChromoTek) na 4°C ruo awa atọ. E jiri TNE nwere 0.2% digoxigenin sachaa mkpụrụ ndị ahụ nwere immunoprecipitated ugboro ise, jiri ihe nchekwa SDS sample buffer kpuchie ha, kewapụ ha na SDS-PAGE, wee nyochaa ha site na immunoblotting.
Dịka akọwara na (31), e mere mkpebi njikọta cross-link na ER nke emelitere. N'ụzọ dị mkpirikpi, e tinyere 0.5 mM dithiobis(succinimidyl propionate) n'ime 0.5 mM nke ER nke emelitere (Pierce, Thermo Fisher Scientific, Rockford, IL, USA; 20°C, nkeji 20). E tinyere glycine (ntinye ikpeazụ 50 mM, nkeji 5, 20°C) n'ime crosslink ahụ.
Dịka e kwuru na mbụ (50), e mere nyocha MS nke ceramide n'ụdị ọhịa na ụdị GhLag1. Na nkenke, e toro mkpụrụ ndụ ruo n'ọkwa exponential (3 ruo 4 OD600 units/ml) na YPD na 30°C, e wee gbute mkpụrụ ndụ 25×107. A na-eji trichloroacetic acid emechi metabolism ha. Jiri ihe mgbaze mwepụta [ethanol, mmiri, ether, pyridine na 4.2 N ammonium hydroxide (15:15:5:1:0.018 v/v)] na 1.2 nmol nke C17 ceramide dị n'ime (860517, Avanti polar lipid) àgwà). Jiri monomethylamine reagent [methanol, mmiri, n-butanol na methylamine solution (4:3:1:5 v/v)] mee obere alkaline hydrolysis nke ihe ewepụtara, wee jiri n-butanol nke mmiri jupụtara na ya mee ka ọ dị jụụ. N'ikpeazụ, e tinyere ihe e si na ya wepụta ọzọ n'ime ihe mgbaze mode dị mma [chloroform/methanol/mmiri (2:7:1) + 5 mM ammonium acetate] ma gbanye ya n'ime igwe spectrometer. Emere nlekota mmeghachi omume multi-reaction (MRM) iji chọpụta ma tụọ ọnụọgụ nke molekul sphingolipid. TSQ Vantage tertiary quadrupole mass spectrometer (Thermo Fisher Scientific) nwere isi iyi nanoflow ion robotic Nanomate HD (Advion Biosciences, Ithaca, NY) maka nyocha lipid. Ike nkwekọ ka mma maka ụdị ceramide ọ bụla. E nwetara data MS na ọnọdụ dị mma. Maka ihe ọ bụla e ji mee ihe na-eme na bayọlọji, akara lipid bụ etiti nke nha atọ nwere onwe ha.
Dịka akọwara na (31), a na-etinye mkpụrụ ndụ (800×107) na-egosipụta Gas1-GFP n'ọnọdụ immunoprecipitation eke. E kewara Gas1-GFP dị ọcha site na SDS-PAGE wee bufee ya na akpụkpọ ahụ polyvinylidene fluoride (PVDF). E jiri amide black mee ka protein ahụ dị ka ihe na-acha ọcha. E bepụrụ eriri Gas1-GFP site na PVDF ma saa ya ugboro ise na methanol, otu ugboro kwa, jiri mmiri mmiri chromatography-MS (LC-MS) sachaa ya. Site n'itinye eriri akpụkpọ ahụ na ngwakọta 500μl 0.3 M NaOAc (pH 4.0), buffer na 500μl nke 1 M sodium nitrite agbazere ọhụrụ na 37°C ruo awa atọ, a na-ewepụta akụkụ lipid site na Gas1-GFP ma kpụchaa ya. Nwepụta nke inosine phosphate ceramide n'etiti glucosamine na inositol (51). Mgbe nke ahụ gasịrị, a sachara eriri akpụkpọ ahụ ugboro anọ na mmiri ọkwa LC-MS, kpọọ nkụ na okpomọkụ ụlọ, ma chekwaa ya na ikuku nitrogen na -80°C ruo mgbe a ga-enyocha ya. Dịka njikwa, ejiri ihe nlele efu nke akpụkpọ ahụ PVDF mee ihe maka nnwale ọ bụla. MS wee nyochaa lipid ewepụtara na Gas1-GFP dịka akọwara ya (50). Na nkenke, a kpọchiri mpempe PVDF nwere GPI-lipid ọzọ na 75μl negative mold solvent [chloroform/methanol (1:2) + 5 mM ammonium acetate] ma gafere electrospray ionization (ESI)-MRM/MS Nyocha nke ụdị sphingolipid (TSQ Vantage). N'okwu a, enwetara data MS na ọnọdụ ion negative.
Dịka ekwuru na mbụ, e kewapụrụ akụkụ lipid nke anchor GPI na [3H]-inositol-labeled GPI-AP (16). E jiri usoro solvent (55:45:10 chloroform-methanol-0.25% KCl) kewaa lipids ndị ahụ ma jiri FLA-7000 (Fujifilm) mee ihe ngosi.
E ji TNE buffer sachaa mkpụrụ ndụ ndị na-egosipụta Gas1-GFP (600×107) ugboro abụọ, jirikwa TNE buffer, ma jiri iko beads gbajie ha, wee jiri centrifuge kpụọ ha iji wepụ ihe ndị dị n'ime sel na iko beads. E mesịa, e tinyere centrifuge na 17,000 g maka otu awa na 4°C. E jiri TNE sachaa pellet ahụ ma tinye 1 U PI-PLC (Invitrogen) na TNE nke nwere 0.2% saponin digitalis maka otu awa na 37°C. Mgbe e gwọchara enzyme ahụ, e wepụrụ akpụkpọ ahụ site na centrifugation na 17,000 g na 4°C maka otu awa. Iji gbochie Gas1-GFP ime ka ahụ gbakee, e tinyere GFP-Trap_A (ChromoTek) na 4°C n'abalị. E ji Coomassie brilliant blue tee Gas1-GFP e mere ka ọ dị ọcha nke SDS-PAGE kewara. E bepụrụ eriri agba Gas1-GFP site na isi awọ gbara okirikiri mmiri ahụ gburugburu, wee mechaa mgbe alkylation na iodoacetamide ma belata ya na dithiothreitol, e mere mgbaze in-gel na trypsin. Wepụta ma kpọọ nkụ peptides na peptides tryptic na GPI-glycans. E gbazere peptide kpọrọ nkụ na 20 μl mmiri. Tinye otu akụkụ (8μl) n'ime LC. E jiri kọlụm octadecylsilane (ODS) (Develosil 300ODS-HG-5; dayameta ime 150 mm × 1.0 mm; Nomura Chemical, Aichi Prefecture, Japan) kewaa peptides n'okpuru ọnọdụ gradient kpọmkwem. Usoro mkpanaka bụ solvent A (0.08% formic acid) na solvent B (0.15% formic acid na 80% acetonitrile). E jiri usoro Accela HPLC (Thermo Fisher Scientific, Boston, Massachusetts) wepụ kọlụm ahụ na ihe mgbaze A n'ime nkeji 55 na ọnụego mmiri nke 50 μl min-1 maka nkeji 5, wee mụbaa njupụta nke ihe mgbaze B ruo 40%. , United States). A na-etinye eluate ahụ n'ime isi iyi ion ESI mgbe niile, a na-enyochakwa peptides tryptic na peptides nwere GPI-glycans site na LTQ Orbitrap XL (hybrid linear ion trap-orbitrap mass spectrometer; Thermo Fisher Scientific). Na ntọala MS, a na-edozi voltaji nke isi iyi capillary ka ọ bụrụ 4.5 kV, a na-edebekwa okpomọkụ nke capillary mbufe na 300°C. A na-edozi voltaji capillary na voltaji lenzi tube ka ọ bụrụ 15 V na 50 V, n'otu n'otu. E nwetara data MS n'ụdị ion dị mma (mkpebi nke 60,000; izi ezi nke akụkụ 10 kwa nde) na oke oke nke 300/m/z oke oke/charge (m/z) 3000. A na-enweta data MS/MS site na ọnyà ion dị na LTQ Orbitrap XL [nọmba atọ mbụ nke data dabere na ya, nkewa nke nkwekọrịta kpatara (CID)].
E jiri ngwanrọ GROMACS (52) na MARTINI 2 force field (53-55) mee ihe ngosi MD. E jirizie CHARMM GUI Membrane Builder (56, 57) wuo bilayer nwere dioleoylphosphatidylcholine (DOPC) na Cer C18 ma ọ bụ DOPC na Cer C26. E si na DXCE wepụta topology na coordinates nke Cer C26 site na iwepụ beads ndị ọzọ na ọdụ sphingosine. Jiri usoro akọwara n'okpuru mee ka oyi akwa abụọ ahụ dị mma ma gbaa ya, wee jiri coordinates ikpeazụ nke sistemụ ahụ wuo sistemụ nwere Emp24. E wuru mpaghara transmembrane nke yist Emp24 (ihe fọdụrụ 173 ruo 193) dị ka α-helix site na iji usoro molekul ngwaọrụ visual MD (VMD) (58). Mgbe ahụ, mgbe ewepụsịrị lipids ndị na-ekpuchi, e tinyere protein ahụ n'ime bilayer site na iji CHARMM GUI. Sistemụ ikpeazụ nwere 1202 DOPC na 302 Cer C26 ma ọ bụ 1197 DOPC na 295 Cer C18 na Emp24. Mee ka sistemụ ahụ dị ion ruo njupụta nke 0.150M. Emere ihe anọ n'onwe ha maka nhazi abụọ dị ka agba abụọ.
A na-eji usoro CHARMM GUI eme ka lipid bilayer ahụ dị n'otu nha nha, nke gụnyere ibelata ma dozie usoro 405,000, ebe a na-ebelata ma wepụ ihe mgbochi ọnọdụ ahụ nwayọ nwayọ, a na-amụbakwa usoro oge site na 0.005 ps ruo 0.02 ps. Mgbe nhazi ya gasịrị, ọ na-emepụta 6 µs na usoro oge nke 0.02 ps. Mgbe itinyechara Emp24, jiri otu usoro CHARMM GUI ahụ belata ma dozie sistemụ ahụ, wee gbaa ọsọ maka sekọnd 8 na mmepụta.
Maka sistemụ niile, n'oge nhazi, Berendsen barostat (59) na-achịkwa nrụgide ahụ, n'oge mmepụta, Parrinello-Rahman barostat (60) na-achịkwa nrụgide ahụ. N'ọnọdụ niile, nrụgide nkezi bụ 1 bar ma jiri usoro njikọ nrụgide semi-isotropic. N'ime usoro nhazi na mmepụta, a na-eji thermostat (61) nwere nhazi ọsọ iji jikọta okpomọkụ nke protein, lipid na ihe mgbaze n'otu n'otu. N'oge ọrụ ahụ dum, okpomọkụ ebumnuche bụ 310K. A na-agbakọ mmekọrịta na-abụghị njikọ site na ịmepụta ndepụta njikọta site na iji atụmatụ Verlet nwere ndidi nchekwa 0.005. A na-agbakọ okwu Coulomb site na iji ubi mmeghachi omume na anya njedebe nke 1.1 nm. Okwu Vander Waals na-eji usoro njedebe nwere anya njedebe nke 1.1 nm, a na-ejikwa atụmatụ njedebe Verlet maka ntụgharị nwere ike (62).
Site na iji VMD, ogologo oge dị n'etiti DOPC phosphate beads ma ọ bụ ceramide AM1 beads na protein ahụ bụ 0.7 nm, a na-agbakọkwa ọnụọgụ lipids ndị na-emekọ ihe na protein ahụ. Dịka usoro na-esonụ si dị, gbakọọ ihe na-ebelata-ịba ụba (DE) dịka ọ dị na (63): DE factor = (ọnụọgụ lipids zuru oke na protein 0.7) na protein 0.7 (ọnụọgụ Cer na mkpokọta lipids)
A na-enweta uru akọwara dị ka nkezi, ogwe njehie ahụ bụkwa mbipụta anọ nke SE n'onwe ha. A na-agbakọ mkpa ọnụọgụgụ nke ihe DE site na ule t [(nkeziDE-factor-1)/SE]. Gbakọọ uru P site na nkesa otu-ọdụ.
E jiri ngwa GROMACS gbakọọ maapụ njupụta 2D nke sistemụ nwere Emp24 n'ime ns 250 ikpeazụ nke akara ahụ. Iji nweta maapụ mmụba/mmebi nke ceramide, a na-ekewa maapụ njupụta nke Cer site na mkpokọta maapụ Cer na DOPC, wee kewaa ya site na mkpokọta nke Cer n'ahụ. A na-eji otu nha maapụ agba ahụ.
Maka ihe ndị ọzọ gbasara akụkọ a, biko lee 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 Wakoga), Argio Waako (Sergio Wape) (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Foto onyonyo dị elu nke 3D n'oge dị adị na-ekpughe mkpa ogologo yinye ceramide dị maka nhazi protein na saịtị mmepụta ahọpụtara.
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 Wakoga), Argio Waako (Sergio Wape) (Misako Arman), Miyako Riman (Miyako Riman), Prow Akira, Stefano Fanny, Akihiko Nakano, Manuel Muniz
Foto onyonyo dị elu nke 3D n'oge dị adị na-ekpughe mkpa ogologo yinye ceramide dị maka nhazi protein na saịtị mmepụta ahọpụtara.
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