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1、细细 胞胞 的的 物物 流流 机机 制制囊囊 泡泡 转转 运运一一. . George Emil Palade George Emil Palade 1960s 1960s 发现了囊泡转运发现了囊泡转运囊泡（vesicle）：由细胞器膜外凸或质膜内凹芽生而成，囊泡形成是一个主动的自我装配过程。囊泡卸货: 囊泡移动，定向地与另一种细胞器膜或质膜融合， 释放货物, 实现物质运输。细胞内物质定向运输囊泡转运（囊泡转运（ vesicular transport）细胞内外物质交换和信号传递George PaladeGeorge Palades s Noble Prize Lecture 1974 cv,
2、 condensing vacuolesgc, Golgi cisternaegv, Golgi vesicles; te, transitional elementsrer, rough endoplasmic reticulum电 镜 观 察 发 现 囊 泡 系 统Pulse-chase 分析发现了分泌蛋白的转运途径放射自显影技术进一步确定转运定位囊泡卸货需要细胞膜融合囊泡卸货需要细胞膜融合George Palades Noble Prize Lecture 1974 细细 胞胞 内内 运运 输输 模模 型型囊泡释放囊泡释放囊囊 泡泡 转转 运运内质网高尔基体分泌泡溶酶体囊囊 泡泡囊囊 泡
3、泡这些发现产生了一系列科学问题！二、囊二、囊 泡泡 衣衣 被被 的的 发发 现现Coat protein complex 囊泡的三种类型囊泡的三种类型 (A) CCV 电镜观察发现不同类型的囊泡囊泡囊泡的主要类型囊泡的主要类型承担细胞内物质定向运输的囊泡类型至少有10种以上。目前了解较多的主要有以下三种类型：3. 网格蛋白有被囊泡（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）
4、 产生于内质网特征：非网格蛋白有被囊泡功能： 介导从内质网到高尔基复合体的物质转运1. COPII有被囊泡特征：亦属于非网格蛋白有被囊泡类型功能：负责内质网逃逸蛋白的捕捉、回收转运及高尔基复合体膜内蛋白的逆向运输，行使从高尔基复合体到内质网的转移2. COP有被囊泡产生于高尔基复合体的COP有被囊泡产生于高尔基复合体、细胞膜的网格蛋白有被囊泡特征：直径在50100nm之间，外被以由网格蛋白纤维构成的网架结构在网格蛋白结构外框与囊膜之间填充、覆盖有大量的衔接蛋白功能：介导从高尔基复合体向溶酶体、胞内体或质膜外的物质转运将外来物质转送到细胞质或溶酶体3. 网格蛋白有被囊泡 Clathrin-coa
5、ted vesicles (CCV)网格蛋白有被小泡形态结构网格蛋白有被小泡形态结构 网格蛋白三、囊 泡 转 运 的 分 子机 制Randy Shekman1975s年代开始对年代开始对囊囊 泡泡 转转 运的机制运的机制感兴趣感兴趣选择酵母细胞作为模式生物开展了被许多科学家认为是“愚蠢的实验”24 C37 C for 1 hrHMSF 1 Secretion-defective mutant strain (sec 1-1)Sec1 to Sec23发现23个与细胞分泌相关基因CCVCOPIICOPICOPII的形成的形成 COPII的成分的成分Sec13/31Sec23/24Sar1-GTP
6、膜蛋白选择受体COPIICOPII的分子基础的分子基础The architecture of the COPII cage facilitates transport of diverse Electron micrographs of negatively stained Sec13/31 complex.Schematic view of the 15 nm Sec23/24Sar1 complex adhered to the surface of a 60 nm vesiclethe morphology of a COPII vesicle budded from microsome
7、s using purified coat proteins COPII 有被小泡的组装激活有被小泡的组装激活COPII 组装组装 COPII介导的内质网介导的内质网-高尔基复合体物质运输高尔基复合体物质运输 COPIICoatomer is a stable protein complex of seven subunits.COPI= CoatomerCOPICOPI的分子基础的分子基础 COPI COPI有被囊泡的主要功能有被囊泡的主要功能 COPIBidirectional transport between the ER and the GolgiAs the process con
8、tinues, the vesicles rounds up and pinches off.网格蛋白有被囊泡（网格蛋白有被囊泡（CCVCCV）Initially a clathrin coated pit forms. The cargo receptors extend through the membrane and interact with cargo molecules in the cytoplasm. The cargo receptors are interacting with particles containing protein and lipids that wil
9、l be incorporated. 网格蛋白有被囊泡（网格蛋白有被囊泡（CCVCCV）的分子基础的分子基础Clathrin网格蛋白CCVCCV有被囊泡有被囊泡Clathrin网格蛋白3 clathrin “heavy chains”(180-190 kDa)plus3 clathrin light chains(40 kDa)form“Triskelions”Clathrin “heavy chain”“Light chain”dynaminGTPGDPGTPase that regulates pinching offDynamin（发动蛋白） is a GTPase Explains w
10、hy non-hydrolyzable GTP analogues block endocytosis经由胞吞作用的网格蛋白有被小泡之形成过程经由胞吞作用的网格蛋白有被小泡之形成过程 货运分子衣被组装及衣被组装及 货运选择货运选择 有被小窝有被小窝 的形成的形成 有被小泡有被小泡 的分离的分离 有被小泡有被小泡 的形成的形成 脱衣被脱衣被 释放小泡释放小泡网格蛋白有被小泡之来源与形成网格蛋白有被小泡之来源与形成 CCVCCV网格蛋白网格蛋白四、四、囊囊 泡泡 转转 运运 的的 特特 异异 性性囊泡转运是细胞内物质定向运输的重要途径和基本形式 囊泡转运囊泡转运特异性的分子基础的分子基础1.转运物
11、质的分选：使囊泡转运成为一个高度有序、 受到严格选择和精密控制的物质运输过程2.特异性识别融合： 使囊泡物质能够定向转运和 准确卸载Quality control: ensuring that mis-folded proteins do not proceed forward内内 质质 网网 的的 分分 选选Mannose-6-P targets proteins from Golgi to lysosomeCis GolgiNetwork (CGN)Trans GolgiNetwork (TGN)RERM6P receptor recycling back to GolgiTranspor
12、t via clathrin-coated vesicles toLysosomeM6P receptor 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 receptorCla
13、thrin coatUncoupling(pH 5)高尔基复合体的分选高尔基复合体的分选Lipid micelle: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
14、106 Da1500 molecules of cholesterol ester1 copy of apoprotein 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)Hydrolyt
15、ic enzymes cleave LDL, releasing cholesterol to cytoplasm 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 term
16、inus binds adaptinNH2Recruitment of LDL-R to coated pits 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 adapt
17、in complexes; bind different endocytosis signals on receptorsAdaptins 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 coa
18、ted pitSame coated pits used for pinocytosis!LDL-RLow density 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
19、 makes hundreds of trips (10 min/cycle)Free cholesterolpH 7.2pH 6LDL-RpH 7-.7.2Low density lipoprotein (LDL)Proton pump in endosome acidifies endosome lumen causing LDL to dissociate from receptordynamin囊泡的停靠对接（docking）的机理James RothmanBalch WE, Glick BS, Rothman JE: Sequential intermediates in the p
20、athway of intercompartmental transport in a cell-free system. 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
21、modification（ H-N-acetylglucosamine (GlcNAc)）When it reaches 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
22、approach, he purified essential components of the vesicle 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, Bloc
23、k MR, Ullrich A, Rothman JE: A fusion protein required for 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 92:759-7
24、72, 1998 sec18, corresponded to NSF sec17，equivalence to 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
25、, VAMP/Synaptobrevin, SNAP-25 and syntaxin, were found in 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 se
26、t of sequential steps of synaptic docking, activation and 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 i
27、ntegral-membrane proteins that pull membranes together.Neuronal 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-memb
28、rane proteinSnares (20) and Rabs Snares also promote membrane fusion囊 泡 的 停 靠 对 接 的 特 异 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 GTPase
29、s How are Snares separated?NSF is an ATPase that dissociates Snare pairs由囊泡转由囊泡转运介导的运介导的细胞内膜细胞内膜流示意图流示意图 总结囊泡转运转运与神经冲动的传递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 e
30、xocytosis of synaptic vesicles, which is under tight temporal control and regulated 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 synap
31、tic fusion 内膜系统与医学的关系内膜系统与医学的关系一、内质网的病理改变一、内质网的病理改变.肿胀、肥大或囊池塌陷是最为常见的内质网形态结构改变钠离子和水的渗入、内流；低氧、辐射、阻塞是引起肿胀的常见原因膜的过氧化损伤所致的合成障碍，则往往造成内质网囊池的塌陷 2. 内质网囊腔中包涵物的形成和出现是某些疾病或病理过程的表现特征 在药物中毒、肿瘤及某些遗传性疾病所致的代谢障碍情况下，内质网中经常会形成、出现不同形式的包涵物 3. 在不同肿瘤细胞中，内质网可发生多种形式的异常改变二、病理状态下高尔基复合体的异常改变二、病理状态下高尔基复合体的异常改变1. 功能亢进导致高尔基复合体的代偿性
32、肥大2. 毒性物质作用下常导致高尔基复合体的萎缩与损坏3. 在肿瘤细胞中,高尔基复合体的数量分布、形态结构极其发达程度, 会因肿瘤细胞的分化状态不同而呈现显著的差异Patients with I-cell disease lack phosphotransferase needed for addition of M-6-P to lysosomal proteins in fibroblasts secretedI-细胞疾病细胞疾病（Mucolipidosis II）是一种遗传病，其会导致phosphotransferase的缺失，引起细胞结构不正常。三、溶酶体与人类疾病三、溶酶体与人类疾病
33、1.溶酶体缺乏或缺陷疾病多为先天性疾病 已经发现有40多种先天性溶酶体病是由溶酶体中某些酶的缺乏或缺陷所引起2.溶酶体酶的异常释放或外泄会造成的细胞或组织损伤性疾病四、过氧化物酶体异常与疾病四、过氧化物酶体异常与疾病1.原发性过氧化物酶体缺陷相关的大多是一些遗传性疾病2. 过氧化物酶体病理性改变相关疾病 过氧化物酶体的病理性改变表现为数量、体积、 形态等多种异常HIV enters through membrane fusionInfluenza enters through receptor-mediated endocytosisSnare-likeIM-likeProteinsHydro
34、phobicTails exposedVesicle transport and fusion is essential for physiological processes ranging from control of nerve cell communication in the brain to immunological responses and hormone section. Deregulation of the transport system is associated with disease in these areas. 思考题思考题 1.何谓内膜系统？ 2.真核
35、生物内膜系统的出现、形成具有哪些重要的生物学意义？ 3.是通过内膜系统各结构组分在结构、功能上的相互转化易行关系来阐明细胞的整体性。 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 or
36、ange) enables vesicles to fuse with their target membranes. James E. Rothman discovered a fusion protein complex for vesicular transportHe identified molecular machinery (pictured in purple) that senses calcium ions (Ca2+) and triggers vesicle fusion, thereby explaining how temporal precision is ach
37、ieved and how signaling substances can be released from the vesicles on command. Ca2+The Nobel Prize in Physiology or Medicine 2013 was awarded jointly to James E. Rothman, Randy W. Schekman and Thomas C. Sdhof for their discoveries of machinery regulating vesicle traffic, a major transport system i
38、n our cells.Congratulation The Nobel Prize in Physiology or Medicine 2013James E. RothmanRandy W. Schekmanthe neuronscommunication at synapses, specialized junctions that allow neurons to exchange information on a millisecond timescale.Elucidating the underlying mechanisms of vesicular transport wit
39、hin cells and the secretion of proteins.Thomas C. SdhofMembrane assembly, vesicular transport, and membrane fusion among organelles of the secretory pathway.You are one, and they are many. If you do not work day and night, they will crush you like a roach.- Gnter Blobel -每个人都应该有梦想，没有梦想你就会很快枯萎死去。祝大家一生追求梦想好梦联翩！谢谢再见
