細(xì)胞生物學(xué)第五章-細(xì)胞內(nèi)膜系統(tǒng)與囊泡轉(zhuǎn)運(yùn)課件

1、細(xì) 胞 的 物 流 機(jī) 制囊 泡 轉(zhuǎn) 運(yùn)一. George Emil Palade 1960s 發(fā)現(xiàn)了囊泡轉(zhuǎn)運(yùn)囊泡（vesicle）：由細(xì)胞器膜外凸或質(zhì)膜內(nèi)凹芽生而成，囊泡形成是一個主動的自我裝配過程。囊泡卸貨: 囊泡移動，定向地與另一種細(xì)胞器膜或質(zhì)膜融合， 釋放貨物, 實現(xiàn)物質(zhì)運(yùn)輸。細(xì)胞內(nèi)物質(zhì)定向運(yùn)輸囊泡轉(zhuǎn)運(yùn)（ vesicular transport）細(xì)胞內(nèi)外物質(zhì)交換和信號傳遞George Palades Noble Prize Lecture 1974 cv, condensing vacuolesgc, Golgi cisternaegv, Golgi vesicles; te, tra

2、nsitional elementsrer, rough endoplasmic reticulum電 鏡 觀 察 發(fā) 現(xiàn) 囊 泡 系 統(tǒng)Pulse-chase 分析發(fā)現(xiàn)了分泌蛋白的轉(zhuǎn)運(yùn)途徑放射自顯影技術(shù)進(jìn)一步確定轉(zhuǎn)運(yùn)定位囊泡卸貨需要細(xì)胞膜融合George Palades Noble Prize Lecture 1974 細(xì) 胞 內(nèi) 運(yùn) 輸 模 型囊泡釋放囊 泡 轉(zhuǎn) 運(yùn)內(nèi)質(zhì)網(wǎng)高爾基體分泌泡溶酶體囊 泡囊 泡這些發(fā)現(xiàn)產(chǎn)生了一系列科學(xué)問題！二、囊 泡 衣 被 的 發(fā) 現(xiàn)Coat protein complex 囊泡的三種類型 (A) CCV 電鏡觀察發(fā)現(xiàn)不同類型的囊泡囊泡的主要類型承擔(dān)細(xì)胞內(nèi)物質(zhì)定

3、向運(yùn)輸?shù)哪遗蓊愋椭辽儆?0種以上。目前了解較多的主要有以下三種類型：3. 網(wǎng)格蛋白有被囊泡（clathrin-coated vesicle， CCV）2. COPI 有被囊泡（ coat protein complex I vesicle，COPI ）1. COPII有被囊泡 （coat protein complex II vesicle， COPII） 產(chǎn)生于內(nèi)質(zhì)網(wǎng)特征：非網(wǎng)格蛋白有被囊泡功能： 介導(dǎo)從內(nèi)質(zhì)網(wǎng)到高爾基復(fù)合體的物質(zhì)轉(zhuǎn)運(yùn)1. COPII有被囊泡特征：亦屬于非網(wǎng)格蛋白有被囊泡類型功能：負(fù)責(zé)內(nèi)質(zhì)網(wǎng)逃逸蛋白的捕捉、回收轉(zhuǎn)運(yùn)及高爾基復(fù)合體膜內(nèi)蛋白的逆向運(yùn)輸，行使從高爾基復(fù)合體到內(nèi)質(zhì)網(wǎng)的

4、轉(zhuǎn)移2. COP有被囊泡產(chǎn)生于高爾基復(fù)合體的COP有被囊泡產(chǎn)生于高爾基復(fù)合體、細(xì)胞膜的網(wǎng)格蛋白有被囊泡特征：直徑在50100nm之間，外被以由網(wǎng)格蛋白纖維構(gòu)成的網(wǎng)架結(jié)構(gòu)在網(wǎng)格蛋白結(jié)構(gòu)外框與囊膜之間填充、覆蓋有大量的銜接蛋白功能：介導(dǎo)從高爾基復(fù)合體向溶酶體、胞內(nèi)體或質(zhì)膜外的物質(zhì)轉(zhuǎn)運(yùn)將外來物質(zhì)轉(zhuǎn)送到細(xì)胞質(zhì)或溶酶體3. 網(wǎng)格蛋白有被囊泡 Clathrin-coated vesicles (CCV)網(wǎng)格蛋白有被小泡形態(tài)結(jié)構(gòu) 網(wǎng)格蛋白三、囊 泡 轉(zhuǎn) 運(yùn) 的 分 子機(jī) 制Randy Shekman1975s年代開始對囊 泡 轉(zhuǎn) 運(yùn)的機(jī)制感興趣選擇酵母細(xì)胞作為模式生物開展了被許多科學(xué)家認(rèn)為是“愚蠢的實驗”2

5、4 C37 C for 1 hrHMSF 1 Secretion-defective mutant strain (sec 1-1)Sec1 to Sec23發(fā)現(xiàn)23個與細(xì)胞分泌相關(guān)基因CCVCOPIICOPICOPII的形成 COPII的成分Sec13/31Sec23/24Sar1-GTP膜蛋白選擇受體COPIICOPII的分子基礎(chǔ)The architecture of the COPII cage facilitates transport of diverse Electron micrographs of negatively stained Sec13/31 complex.Sche

6、matic view of the 15 nm Sec23/24Sar1 complex adhered to the surface of a 60 nm vesiclethe morphology of a COPII vesicle budded from microsomes using purified coat proteins COPII 有被小泡的組裝激活COPII 組裝 COPII介導(dǎo)的內(nèi)質(zhì)網(wǎng)-高爾基復(fù)合體物質(zhì)運(yùn)輸 COPIICoatomer is a stable protein complex of seven subunits.COPI= CoatomerCOPI的分子

7、基礎(chǔ) COPI有被囊泡的主要功能 COPIBidirectional transport between the ER and the GolgiAs the process continues, the vesicles rounds up and pinches off.網(wǎng)格蛋白有被囊泡（CCV）Initially a clathrin coated pit forms. The cargo receptors extend through the membrane and interact with cargo molecules in the cytoplasm. The cargo

8、receptors are interacting with particles containing protein and lipids that will be incorporated. 網(wǎng)格蛋白有被囊泡（CCV）的分子基礎(chǔ)Clathrin網(wǎng)格蛋白CCV有被囊泡Clathrin網(wǎng)格蛋白3 clathrin “heavy chains”(180-190 kDa)plus3 clathrin light chains(40 kDa)form“Triskelions”Clathrin “heavy chain”“Light chain”dynaminGTPGDPGTPase that reg

9、ulates pinching offDynamin（發(fā)動蛋白） is a GTPase Explains why non-hydrolyzable GTP analogues block endocytosis經(jīng)由胞吞作用的網(wǎng)格蛋白有被小泡之形成過程 貨運(yùn)分子衣被組裝及 貨運(yùn)選擇 有被小窩 的形成 有被小泡 的分離 有被小泡 的形成 脫衣被 釋放小泡網(wǎng)格蛋白有被小泡之來源與形成 CCV網(wǎng)格蛋白四、囊 泡 轉(zhuǎn) 運(yùn) 的 特 異 性囊泡轉(zhuǎn)運(yùn)是細(xì)胞內(nèi)物質(zhì)定向運(yùn)輸?shù)闹匾緩胶突拘问?囊泡轉(zhuǎn)運(yùn)特異性的分子基礎(chǔ)1.轉(zhuǎn)運(yùn)物質(zhì)的分選：使囊泡轉(zhuǎn)運(yùn)成為一個高度有序、 受到嚴(yán)格選擇和精密控制的物質(zhì)運(yùn)輸過程2.特異

10、性識別融合： 使囊泡物質(zhì)能夠定向轉(zhuǎn)運(yùn)和 準(zhǔn)確卸載Quality control: ensuring that mis-folded proteins do not proceed forward內(nèi) 質(zhì) 網(wǎng) 的 分 選Mannose-6-P targets proteins from Golgi to lysosomeCis GolgiNetwork (CGN)Trans GolgiNetwork (TGN)RERM6P receptor recycling back to GolgiTransport via clathrin-coated vesicles toLysosomeM6P rec

11、eptor in TGN directs transport of enzymes to lysosome via clathrin-coated vesiclesAddition of M6P to lysosomal enzymes in cis-GolgiLysosomal hydrolase (precursor)Addition of M6P Removal of phosphate &proteolytic processingMaturehydrolaseM6P receptorClathrin coatUncoupling(pH 5)高爾基復(fù)合體的分選Lipid micelle

12、:800 phospholipids500 molecules of cholesterolExample: Low-density lipoprotein (LDL), structure in which cholesterol is transported through our bodies“Receptor-mediated endocytosis” How do cells take up specific macromolecules?Total mass: 3 x 106 Da1500 molecules of cholesterol ester1 copy of apopro

13、tein BOverview of receptor-mediated uptake of LDLLow pH of endosome (6) causes LDL to dissociate from receptorReceptor is recycled back to surface (cycles about every 10 min!)LDL is transferred to lysosome (fusion of vesicles from TGN)Hydrolytic enzymes cleave LDL, releasing cholesterol to cytoplasm

14、 for continued membrane biosynthesis in smooth ERECB 15-32Domains in LDL receptorAdaptin complex (four polypeptides)Plasma membraneValTyrProAsnLDL-RLDLBased on MBoC (3) figure 13-53HOOCN terminus of LDL receptor binds apoprotein B in LDLC terminus binds adaptinNH2Recruitment of LDL-R to coated pits

15、requires an “endocytosis signal” in cytoplasmic domain Adaptin complex (four polypeptides)Plasma membraneAdaptin complex binds endocytosis signal in cytoplasmic domain of receptor:-NPXY- (Asn-Pro-Val-Tyr) in LDL-RAt least three different adaptin complexes; bind different endocytosis signals on recep

16、torsAdaptins recruit clathrin and initiate coated pit/vesicle formationValTyrProAsnLDL-REndocytosis signalLDLBased on MBoC (3) figure 13-53HOOCA single coated pit has many different receptors and cargos1,000s of receptors of many types per coated pitSame coated pits used for pinocytosis!LDL-RLow den

17、sity lipoprotein (LDL)Summary of “receptor-mediated” endocytosis of LDLATPADP+PiH+LysosomeEarly endosomeATPADP+PiUncoating(HSP70 family)GTPGDP+PiCoatedvesicle Fusion(Snares)Cholesterol ester cleavedCholesterol released for useA single receptor makes hundreds of trips (10 min/cycle)Free cholesterolpH

18、 7.2pH 6LDL-RpH 7-.7.2Low density lipoprotein (LDL)Proton pump in endosome acidifies endosome lumen causing LDL to dissociate from receptordynamin囊泡的?？繉樱╠ocking）的機(jī)理James RothmanBalch WE, Glick BS, Rothman JE: Sequential intermediates in the pathway of intercompartmental transport in a cell-free sys

19、tem. Cell 39:525-536, 1984 Large amounts of a particular viral protein, the VSV-G protein, are produced in infected cells. Reconstitution assay system based on vesicular stomatitis virus (VSV) The VSV-G protein is marked by a particular sugar modification（ H-N-acetylglucosamine (GlcNAc)）When it reac

20、hes the Golgi compartment, which makes it possible to identify when it reaches its destination. 3 Cell-free reconstitution assay an in vitro reconstitution assay to dissect events involved in intracellular vesicle transport proteinsUsing this approach, he purified essential components of the vesicle

21、 fusion process. Clary DO, Griff IC, Rothman JE: SNAPs, a family of NSF attachment proteins involved in intracellular membrane fusion in animals and yeast. Cell 61:709-721, 1990 Wilson DW, Wilcox CA, Flynn GC, Chen E, Kuang WJ, Henzel WJ, Block MR, Ullrich A, Rothman JE: A fusion protein required fo

22、r vesicle-mediated transport in both mammalian cells and yeast. Nature 339:355-359, 1989 Weber T, Zemelman BV, McNew JA, Westermann B, Gmachl M, Parlati F, Sollner TH, Rothman JE: SNAREpins: minimal machinery for membrane fusion. Cell 92:759-772, 1998 sec18, corresponded to NSF sec17，equivalence to

23、SNAP Other sec genes were shown to correspond to genes encoding fusion proteins were identified by other methods. Using the NSF and SNAP proteins as bait，Rothman discovered SNAREs (soluble NSF-attachment protein receptors) Three SNARE proteins, VAMP/Synaptobrevin, SNAP-25 and syntaxin, were found in

24、 stoichiometric amounts VAMP/Synaptobrevin resided on the vesicle, whereas SNAP-25 and syntaxin were found at the plasma membrane. SNARE hypothesis Target and vesicle SNAREs (t-SNAREs and v-SNAREs) were critical for vesicle fusion through a set of sequential steps of synaptic docking, activation and

25、 fusion A particular t-SNARE only interacted with one or a few of the large number of potential v-SNAREs Snares guide vesicular transportVesicle-surface markers that direct vesicles to the correct placeV=vesicle and t=target SNARESSnares are integral-membrane proteins that pull membranes together.Ne

26、uronal snares are the targets of neural toxin proteases (botulism)Coiled coilThe coiled-coil is a tightly intertwined set of 4 a-helix domainsThree are contributed by t-snares, and 1 by the v-snareAt least 1 of the t-snares is an integral-membrane proteinSnares (20) and Rabs Snares also promote memb

27、rane fusion囊 泡 的 停 靠 對 接 的 特 異 性Rab GTPases ensure the specificity of vesicular docking30 members-each bind a particular vesicle-On the cytoplasmic face Rabs interact with SnaresVariation in Rab C-terminal tailsVariation in effectorsRab GTPases How are Snares separated?NSF is an ATPase that dissocia

28、tes Snare pairs由囊泡轉(zhuǎn)運(yùn)介導(dǎo)的細(xì)胞內(nèi)膜流示意圖 總結(jié)囊泡轉(zhuǎn)運(yùn)與神經(jīng)沖動的傳遞Rothman and Schekman had provided fundamental machinery for vesicle fusion, but how vesicle fusion was temporally controlled remained enigmatic. Sdhof was intrigued by the rapid exocytosis of synaptic vesicles, which is under tight temporal control and r

29、egulated by the changes in the cytoplasmic free calcium concentration. Complexin and synaptotagmin are two critical proteins in calcium-mediated vesicle fusion Munc18-1=sec1 Synaptotagmin-1 as a calcium sensor for rapid synaptic fusion 內(nèi)膜系統(tǒng)與醫(yī)學(xué)的關(guān)系一、內(nèi)質(zhì)網(wǎng)的病理改變.腫脹、肥大或囊池塌陷是最為常見的內(nèi)質(zhì)網(wǎng)形態(tài)結(jié)構(gòu)改變鈉離子和水的滲入、內(nèi)流；低氧、輻射、

30、阻塞是引起腫脹的常見原因膜的過氧化損傷所致的合成障礙，則往往造成內(nèi)質(zhì)網(wǎng)囊池的塌陷 2. 內(nèi)質(zhì)網(wǎng)囊腔中包涵物的形成和出現(xiàn)是某些疾病或病理過程的表現(xiàn)特征 在藥物中毒、腫瘤及某些遺傳性疾病所致的代謝障礙情況下，內(nèi)質(zhì)網(wǎng)中經(jīng)常會形成、出現(xiàn)不同形式的包涵物 3. 在不同腫瘤細(xì)胞中，內(nèi)質(zhì)網(wǎng)可發(fā)生多種形式的異常改變二、病理狀態(tài)下高爾基復(fù)合體的異常改變 功能亢進(jìn)導(dǎo)致高爾基復(fù)合體的代償性肥大2. 毒性物質(zhì)作用下常導(dǎo)致高爾基復(fù)合體的萎縮與損壞3. 在腫瘤細(xì)胞中,高爾基復(fù)合體的數(shù)量分布、形態(tài)結(jié)構(gòu)極其發(fā)達(dá)程度, 會因腫瘤細(xì)胞的分化狀態(tài)不同而呈現(xiàn)顯著的差異Patients with I-cell disease lack

31、 phosphotransferase needed for addition of M-6-P to lysosomal proteins in fibroblasts secretedI-細(xì)胞疾?。∕ucolipidosis II）是一種遺傳病，其會導(dǎo)致phosphotransferase的缺失，引起細(xì)胞結(jié)構(gòu)不正常。三、溶酶體與人類疾病溶酶體缺乏或缺陷疾病多為先天性疾病 已經(jīng)發(fā)現(xiàn)有40多種先天性溶酶體病是由溶酶體中某些酶的缺乏或缺陷所引起2.溶酶體酶的異常釋放或外泄會造成的細(xì)胞或組織損傷性疾病四、過氧化物酶體異常與疾病原發(fā)性過氧化物酶體缺陷相關(guān)的大多是一些遺傳性疾病2. 過氧化物酶體病理性

32、改變相關(guān)疾病 過氧化物酶體的病理性改變表現(xiàn)為數(shù)量、體積、 形態(tài)等多種異常HIV enters through membrane fusionInfluenza enters through receptor-mediated endocytosisSnare-likeIM-likeProteinsHydrophobicTails exposedVesicle transport and fusion is essential for physiological processes ranging from control of nerve cell communication in the b

33、rain to immunological responses and hormone section. Deregulation of the transport system is associated with disease in these areas. 思考題 1.何謂內(nèi)膜系統(tǒng)？ 2.真核生物內(nèi)膜系統(tǒng)的出現(xiàn)、形成具有哪些重要的生物學(xué)意義？ 3.是通過內(nèi)膜系統(tǒng)各結(jié)構(gòu)組分在結(jié)構(gòu)、功能上的相互轉(zhuǎn)化易行關(guān)系來闡明細(xì)胞的整體性。 Comparing normal (left) with genetically mutated yeast cells (right) in which vesicle traffic was disturbed, he identified genes that control transport to different compartments and to the cell surface.Protein complex (pictured in orange) enables vesicles to fuse with their target membranes. James E. Rothman discovered