细胞生物学2010课件-chapter

Chapter7

Endomembranesystem,ProteinSortingandmembranetraffickingLearningobjective1.CompartmentalizationinEukaryoticCells;ThestructuralandfunctionalrelationshipbetweentheER,Golgicomplexes,lysosomesandplasmamembranesofeukaryoticcells;Thepathwaysofproteinstargetingandsorting,anditsmechanisms;Thewaysofproteinmodificationsandintracellularsitesaftertheyaresynthesized;Typesofvesicletransportandtheirfunctions.Endocytosisandexocytosis1.TheCompartmentalizationinEukaryoticCellsMembranesdividethecytoplasmofeukaryoticcellsintodistinctcompartments.

Treecategoriesineukaryoticcells:(1)theendomembranesystem:ER,Golgicomplex,Lys.,secretoryvesicles.(2)thecytosol.(3)mitochondria,chloroplasts,peroxisomes,andthenucleus.Membrane-boundstructures(organelles)arefoundinalleukaryoticcells.Cytosol:Theregionoffluidcontentofthecytoplasmoutsideofthemembranousorganelles.Aqueoussolutionoflargeandsmallmoleculesincludingfilamentsofcytoskeletonwhichactasorganizerforsomeorder.TheCytosolisthesiteofproteinsynthesisanddegradationormodification.Italsoperformsmostofthecell’sintermediarymetabolism.A.CytosolanditsfunctionsB.EndomembraneSystemEndomembraneSystem:ThestructuralandfunctionalrelationshiporganellesincludingER,Golgicomplex,lysosome,endosomes,secretoryvesicles.Membrane-boundstructures(organelles)arefoundinalleukaryoticcells.Intracellularcompartment%oftotalcellvolumeCytosol54Mittchondria22RoughERcisternae9SmoothERcisternaeplusGolgicisternae6Nucleus6Peroxisome1Lysosomes1Endosomes1RelativevolumesoccupiedbythemajorintracellularcompartmentsinLiverCellC.TheDynamicNatureoftheEndomembraneSystemMostorganellesarepartofadynamicsysteminwhichvesiclesmovebetweencompartments.Biosyntheticparthwaysmoveproteins,carbohydratesandlipidswithinthecell.Secretorypathwaysdischargeproteinsfromcells.Endocyticparthwaysmovematerialsintocells.Sortingsignalsarerecognizedbyreceptorsandtargetproteinstospecificsites.D.AfewapproachestothestudyofcytomembranesInsightsgainedfromautoradiography;Insightsgainedfromthebiochemicalanalysisofsubcellularfractions;Insightsgainedfromthestudyofgeneticmutants;Thedynamicactivitiesofendomembranesystemsarehighlyconserveddespitethestructuraldiversityofdifferentcelltypes.2023/1/129突变体使用2.ThestructureandfunctionsofEndoplasmicReticulum(ER)A.FunctionsoftherERProteinssynthesizedonribosomesofrERinclude:

secretoryproteins,integralmembraneproteins,solubleproteinsoforganelles.

Modificationandprocessingofnewlysynthesizedproteins:glycosylationintherER;N-linked:linkedtotheamidenitrogenofasparagine(ER)O-linked:linkedtothehydroxylgroupserineorthreonineviaGalNac(inGolgi)Theprecursorof14residuesisthesameinplants,animals,andsingle-celledeukaryotesthenremove3glucosesand1mannoseintheER2023/1/1215N-linkedglycosylation2023/1/1216Additionofglycosyl-phosphatidyl-inositol(GPI,糖基磷脂酰肌醇)anchorsQualitycontrolofofnewlysynthesizedproteins---TheroleofN-linkedglycosylationinERproteinfoldingB.FunctionsofthesERSynthesisofmembranelipids.SynthesisofsteroidsinendocrinecellsDetoxificationoforganiccompoundsinlivercells.Systemofoxygenases---cytochromep450familyReleaseofglucose6-phosphateinlivercells.SequestrationofCa2+.Ca2+-ATPase2023/1/1222磷脂的合成Synthesis0fmembranelipidsMostmembranelipidsaresynthesizedenterelywithintheER.Therearetwoexceptions:(1)sphingomyelinandglycolipids,(beginsinER;completedinGolgi);(2)someoftheuniquelipidsoftheMitandChlmembranes(themself).Themembranesofdifferent0rganelleshavemarkdlydifferentlipidscomposition.Transportbybudding：ER→GC、Ly、PMTransportbyphospholipidexchangeproteins(PEP)：ER→otherorganelles（includingMitandChl）Theroleofphospholipidtranslocatorsinlipidbilayersynthesisphospholipidtranslocators=Scramblase(ABCtransporterFamily)2023/1/1225解毒作用◆光面内质网含有丰富的氧化酶系统(如细胞色素P450、NADH细胞色素C还原酶等)能使许多有害物质解毒,转化为易于排出的物质。◆混合功能的氧化酶(mixed-functionoxidase)●每分子的底物被氧化需要消耗一分子氧,并且将NADPH转变成NADP+●该氧化过程消耗的氧分子中有一个氧原子出现在水中，另一个氧原子出现在产物中●故此将催化该反应的酶称为混合功能的氧化酶。2023/1/1226光面内质网的氧化作用2023/1/1227糖原分解与游离葡萄糖释放

3.ThestructureandfunctionsofGolgicomplexA.ThepolarityofGolgicomplex2023/1/1229高尔基体的极性◆高尔基内侧网络(cis-Golginetwork,CGN)

顺面、形成面◆中间潴泡(medialcisternae)◆高尔基外侧网络

(transGoljinetwork,TGN)

外侧面、成熟面a)CiscisternaeofGolgicomplex:reducedosmiumtetroxide;b)ReactionforenzymemannosidaseII,localizedinthemedial;c)Reactionforenzymenucleosidediphosphatase,localizedinthetranscisternae.RegionaldifferencesinmembranecompositionacrosstheGolgistack2023/1/1234B.ThefunctionsofGolgiapparatusSecretion：ProteinsynthesizedinRER—ERlumen—COPII—CGNofGolgi—modificationinmedial—transportationvesiclesformedinTGN—transportation，fusionwithplasmamembraneO-linkedglycosylationN-linkedglycosylation进行膜的转化功能：内质网上合成的新膜脂转移至高尔基体后，经过修饰和加工，形成运输泡与质膜结合将蛋白水解为活性物质：将没有活性的蛋白原N端或两端的序列切除形成成熟的多肽（如胰岛素）；将含有多个相同氨基酸序列的前体水解为有活性的多肽，如神经肽参与形成溶酶体参与植物细胞壁的形成GlycosylationintheGolgicomplex

Golgicomplexplaysakeyroleintheassemblyofthecarbohydratecomponentofglycoproteinsandglycolipids.N-linkedoligosaccharides:Beginwithadditionofanoligosaccharidewith14sugars(dilichol)intheERSubsequentlycertainsugarresidduesareremovedandothersareaddedOnlyAsninAsn-x-ThrorAsn-x-SerwasmodifiedModificationstoN-linkedoligosaccharidesarecompletedintheGolgiapparatus◆O-linkedoligosaccharides:O-连接的糖基化

将糖链转移到多肽链的丝氨酸、苏氨酸或羟赖氨酸的羟基上，称为O-连接的糖基化。FormedbythesequentialtransferofsugarsfromnucleotideprecursorsAminoacids:-OHofSer、Thr、HylSugars:galactoseorN-acetylglucosamineShortsugarsresiduesGolgiapparatusStructureoftypicalO-andN-linkedoligosaccharidesCoreRegionWhatisthepurposeofglycosylation?N-linkedglycosylationisprevalentinalleucaryotes,butisabsentfromprocaryotes.Itdon’trequireatemplate.ThereisanimportantdifferencebetweentheconstructionofanoligosaccharideandthesynthesisofDNA,RNA,andprotein.Importantfunctions:

(1)Onemightsuspectthattheyfunctiontoaidfoldingandthetransportprocess;forexample,carbohydrateasamarkerduringproteinfoldinginERandtheuseofcarbohydrate-bindinglectinsinguidingER-to-Golgitransport.(2)Limittheapproachofothermacromoleculestotheproteinsurface,moreresistanttodigestionbyproteases.(3)Regulatoryrolesinsignalingthroughthecell-surfacereceptorNotch,toallowsthesecellstorespondselectivelytoactivatingstimuli.TheGolginetworksareprocessingandsortingstationswhereproteinsaremodified,segregatedandthenshippedindifferentdirections.中考题之一2023/1/1244高尔基体膜囊中进行的蛋白质糖基化和转运Golgicomplexandcell’ssecretionContinual,unregulateddischargeofmaterialfromthecellsThedischargeofproductsstoredincytoplasmicgranules,inresponsetoappropriatestimuli.VesivulartransportwithintheGolgiapparatus:

Twoviews:cisternalmaturationmodelandvesiculartransportmodelTwopossiblemodelsexplainingtheorganizationoftheGolgicomplexandthetransportfromonecisternatothenext.4.ThestructureandfunctionsofLysosomesA.CharacteristicsofLysosomes①Lysosomeisaheterogenousorganelle:endosomeslysosomesheterophagicautophagicResidualbodyvesicle.LysosomeDeDuve,A.ClaudeandG.Palade,1974NobelPlrize②Lysosomescontainplentyacidhydrolasesthatcandigesteverykindofbiologicalmolecule.---theprincipalsitesofintracellulardigestion.Markerenzyme:acidphosphatase③Lysosomemembrane:H+-pumps:internalprotonconcentrationiskepthighbyH+-ATPaseGlycosylatedproteins:mayprotectthelysosomefromself-digestion.

Transportproteins:transportingdigestedmaterials.溶酶体膜稳定性◆溶酶体的膜上嵌有质子泵◆溶酶体的膜上具有多种载体蛋白用于水解产物向外转运◆溶酶体的膜蛋白高度糖基化◆溶酶体的膜含有能促进膜稳定性的胆固醇PLANTCELLVACUOLESB.TheFunctionsofLysosomesLysosomesareinvolvedinthreemajorcellfunctions:①phagocytosis;②autophagy;③endocytosis.吞噬作用(Phagocytosis)Phagosome-phagolysosome自噬作用(Autophagy)autophagosome(自噬体)

-autophagolysosome溶酶体的其它功能

自溶作用(Autolysis)其它的生理功能◆胚胎发育和形态建成◆受精作用(fertilization)

C.BiogenesisofLysosomesThetransportofnewlysynthesizedlysosomalhydrolasestolysosomes.溶酶体酶的结构特征◆溶酶体的酶上都有一个特殊的标记∶6-磷酸甘露糖(mannose6-phosphate,M6P)。◆高尔基体外侧网络通过对M6P的识别将溶酶体的酶分选出来。◆这一标记是溶酶体在粗面内质网合成后通过糖基化和磷酸化添加上去的。◆甘露糖-6-磷酸途径◆非甘露糖-6-磷酸途径甘露糖-6-磷酸途径溶酶体酶的加工◆粗面内质网中进行N-连接糖基化，然后转运到高尔基体中进一步加工◆甘露糖-6-磷酸化●N-乙酰葡萄糖胺磷酸转移酶

(N-acetyglucosaminephosphotransferase)

●N-乙酰葡萄糖苷酶。Mannose6-phosphateresiduestargetproteinstolysosomesTargetingofsolublelysosomalenzymestoendosomesandlysosomesbyM-6-PtagPhosphorylationofmannoseresiduesonlysosomalenzymescatalyzedbytwoenzymesRecognitionsitebindstoSignalpatchGlcNAcphosphotransferasephosphodiesteraseThemannose6-phosphatepathway:M-6-Preceptorbindsitsspecificoligo.atpH6.5-6.7inTGNandreleasesitatpH6.0inlateendosomes.pH6.0pH6.5-6.7pH5.02023/1/1268溶酶体形成的非M6P途径◆发现:I细胞病(GlcNAC磷酸转移酶）黏脂病(mucolipidosis)◆种类:●溶酶体膜中的糖蛋白:β-葡糖脑苷脂酶(β-glucocerebrosidedase)●作为溶酶体膜结合蛋白前体被合成的溶酶体酶:

酸性磷酸酶

◆信号:酪氨酸(胞质结构域）

D.LysosomesandDiseasesDisordersresultingfromdefectsinlysosomalfunction:①Autolysis:Abreakorleakinthemembraneoflysreleasesdigestiveenzymesintothecellwhichdamagesthesurroundingtissues(Silicosis,矽肺病).类风湿性关节炎②

Lysosomalstoragediseasesareduetotheabsenceofoneormorelysosomalenzymes,andresultinginaccumulationofmaterialinlysosomesaslargeinclusios.OneseveretypeofthediseaseisI-celldisease(inclusion–celldisease,GlcNAc-Phosphotransferasegenemutant).

Tay-Sachsdisease台-萨氏综合征，又叫黑蒙性家族痴呆症，

resultsfromadeficiencyoftheenzyme(-N-hexosaminidaseA)whosefunctionistodegradegangliosides,amajorcomponentofbraincellmembranes.表1.神经鞘脂贮积病疾病缺失酶类主要贮积底物后果GM1神经节苷脂贮积症GM1-半乳糖苷酶神经节苷脂GM1智力迟钝，肝脏肥大，骨骼受累，2岁前死亡泰－萨二氏病己糖胺酶A神经节苷脂GM2智力迟钝，失明，3岁前死亡法布莱氏病-半乳糖苷酶A三己糖神经酰胺皮疹，肾功能丧失，下肢疼痛山霍夫氏病己糖胺酶A和B神经节苷脂GM2和红细胞糖苷酯与泰－萨氏疾病症状相似，但发展更快高歇氏病葡糖脑苷酯酶葡糖脑苷脂肝脏和脾脏肿大，长骨腐蚀，只在婴儿期发生智力迟钝尼-皮二氏病鞘磷脂水解酶鞘磷脂肝脏和脾脏肿大，智力迟钝Farber’s脂肪肉芽肿病神经酰胺水解酶神经酰胺疼痛性与退行性的关节变形，皮肤瘤，几年内死亡Krabbe’s病半乳糖脑苷酯酶半乳糖脑苷脂髓磷脂缺失，智力迟钝，2岁前死亡脑硫脂沉积芳基硫酸酯酶脑硫脂智力迟钝，前十年死亡E.过氧化物酶体(peroxisome)又称微体(microbody)，是由单层膜围绕的内含一种或几种氧化酶类的异质性细胞器Containenzymesthatusemolecularoxygentooxidizevarioussubstrates,forminghydrogenperoxide(H2O2)Catalase,aperoxisome-localizedenzyme,efficientlyposesH2O2intoH2O.过氧化物酶体与溶酶体的区别过氧化物酶体和初级溶酶体的形态与大小类似，但过氧化物酶体中的尿酸氧化酶等常形成晶格状结构，可作为电镜下识别的主要特征。通过离心可分离过氧化物酶体和溶酶体微体与初级溶酶体的特征比较

过氧化物酶体的功能动物细胞（肝细胞或肾细胞）中过氧化物酶体可氧化分解血液中的有毒成分，起到解毒作用。 过氧化物酶体中常含有两种酶： 依赖于黄素（FAD）的氧化酶，其作用是将底物氧化形成H2O2； 过氧化氢酶，作用是将H2O2分解，形成水和氧气。过氧化物酶体分解脂肪酸等高能分子向细胞直接提供热能。在植物细胞中过氧化物酶体的功能：

在绿色植物叶肉细胞中，它催化CO2固定反应副产物的氧化， 即所谓光呼吸反应；

乙醛酸循环的反应，在种子萌发过程中，过氧化物酶体 降解储存的脂肪酸乙酰辅酶A琥珀酸葡萄糖。过氧化物酶体的发生

氧化物酶体经分裂后形成子代的细胞器，子代的过氧化物酶体 还需要进一步装配形成成熟的细胞器。

组成过氧化物酶体的蛋白均由核基因编码，主要在细胞质基质中合成，然后转运到过氧化物酶体中。

过氧化物酶体蛋白分选的信号序列（Peroxisomal-targetingsignal，PTS）：

PTS1为SKL(Ser-lys-leu)，多存在于基质蛋白的C端。

PTS2为Arg/Lys-Leu/lle-5X-His/Gln-leu，存在于某些基质蛋白N-端。

过氧化物酶体膜上存在几种可与信号序列相识别的可能的受体蛋白。

过氧化物酶体的膜脂可能在内质网上合成后转运而来。

内质网也参与氧化物酶体的发生SynthesisandtargetingofperoxisomalproteinsProteinswithSKLsignalattheveryC-terminalbindtoasolublereceptorPTS1ROtherperoxisomalmatrixproteinswithanN-terminaluptake-targetingsequencebindtoPTS2RBothPTS1RandPTS2Rthenbindtoareceptorprotein,Pex14p,inthemembrane,andproteinsaretransferredinward.N-terminalwillberemovedandSKLwillnotberemoved5.ProteinSorting

Overviewofsortingofnuclear-encodedproteinsineukaryoticcellsProteinsareimportedintoorganellesbythreemechanisms:GatedTransport:TransportthroughnuclearporesTransmembranetransport:ER,Mit,Chl,PerVesiculartransport:ER-Golgi-PM-Lys,Endosome

Roadmapofproteinsorting

Proteinsorting:Proteinmoleculesmovefromthecytosoltotheirtargetorganellesorcellsurfacedirectedbythesortingsignalsintheproteins.SignalpeptidesandSignalpatchesGatedtransport:

Throughgatedpores—Nuclearpores;Nuclearlocalizationsignal(NLS);Foldedandassemblyformtotransport.TransmembranetransportERsignalsequence,Mit,Chl,Per:Leadersequence;Throughtranslocononthemembrane;SignalandUnfoldform;HelpedbymolecularchaperonsVesiculartransportBudding,transporting,dockingandatlastfusionwithtargetmembrane;Assemblycoatedproteinsonthevesicles(Clathrin,COPIIandCOPI);OnlyProperlyfoldedandassembledproteins;Theorientationoftransportedproteinsandlipidsisnotchangedduringtransporting.B.SignalHypothesis

--G.Blobel&D.Sabatini,1971.AmodelfortheSignalMechanismofCotranslationalImportMilsteinetal:StudyingthesynthesisoflightchainofIgG(incell-freesystems,20AalongeratN-terminalendthantheauthenticlightchain);AddingERmembranestothissystemleadstotheproductionofanIgGlightchainofthecorrectsize.2023/1/1287信号识别颗粒与停靠蛋白◆signalrecognitionpartical,SRP●SRPisacytosolicribonucleoproteinparticle●Sixdiscretepolypeptides●A300-nucleotide(7S)RNA●三个功能域:▲翻译暂停结构域(P9/P14)▲信号肽识别结合位点(P54)▲SRP受体蛋白结合位点(P68/P72)。ERsignalsequence:Typically15-30aminoacids:Consistofthreedomains:apositivelychargedN-terminalregion,acentralhydrophobicregion,andapolarregionadjoiningthesitewherecleavagefromthematureproteinwilltakeplace.AsignalsequenceonnascentseretoryproteinstargetsthemtotheERandisthencleavedoffSRPhavethreemainactivesites:OnethatrecognizesandbindstoERsignalsequence;Onethatinteractswiththeribosometoblockfurthertranslation;OnethatbindstotheERmembrane(dockingprotein))2023/1/1289◆SRPreceptor(GTPbindingprotein),Dockingprotein,DP

●是SRP在内质网膜上的受体蛋白●两个亚基:亲水的α亚基,疏水的β亚基◆易位子(translocon)●MultiplecopiesoftheSec61complexassembletoformacylindertransloconchannel●5～6nmhighand8.5nmindiameter,withacentralpore,roughly2nmindiameter●哺乳动物细胞中有三种类型的Sec61,即α、β和γ●Sec61α有10个跨膜α螺旋2023/1/1291体外实验证明SRP颗粒、SRP受体的作用2023/1/1294单次跨膜蛋白的定向I?2023/1/1295单次跨膜蛋白的定向IITheOrientationofNascentPolypeptide

TheNascentpolypeptideisorientedwithintransloconsothatthepositivelychargedflankingsequencefacesthecytosolCorrectfoldingofnewlymadeproteinsisfacilitatedbyseveralERproteinsProteinswithoutanysignalsequencearecytosolresidualproteins6.MembranetraffickingA.TypesofVesicleTransportandTheirFunctionsA.Thethreedifferenttypesofcoatedvesicles.Differentcoatproteinsselectdifferentcargoandshapethetransportvesiclesthatmediatethevariousstepsinthebiosynthetic-secretoryandendocyticpathways.2023/1/12110运输小泡的类型被膜小泡(coatedvesicles)类型◆披网格蛋白小泡(clathrin-coatedvesicle)◆COPII被膜小泡(COPIIcoatedvesicles)◆COPI被膜小泡(COPIcoatedvesicles)2023/1/121134.6.2Clathrin-coatedvesicles

披网格蛋白小泡Transportation：plasmamembrane—endosome；Golgiapparatus—lysosome；Golgi—plantvacuoleClathrin：threeheavychainsandthreelighttrainsAdapterprotein:adaptin(AP1,AP2,AP3)SmallGTP-bindingprotein:ARFGTPhydrolysisbydynaminisrequiredforpinchingoffofclathrin-coatedvesicles2023/1/12114网格蛋白及被膜亚基(coatsubunits)◆二聚体(Dimer):◆三联体骨架(triskelion):◆被膜亚基(coatsubunits):五（六）边形网络结构◆披网格蛋白小泡Modelfortheformationofaclathrin-coatedpitandtheselectiveincorporationofintegralmembraneproteinsintoclathrin-coatedvesicles2023/1/12119披网格蛋白小泡的形成过程◆披网格蛋白小窝(Clathrin-CoatedPit)◆披网格蛋白小泡(Clathrin-CoatedVesicles)

有被小泡(CoatVesicles)◆无被小泡(UncoatVesicles)◆分子伴侣Hsp70蛋白催化外被去聚合，并且需要ATP。另外Ca2+也参与了包被的形成和去被的过程。2023/1/12121衔接蛋白(adaptin)与发动蛋白◆衔接蛋白的作用●Ontheirouter(cytosolic)surface,theadaptorsbindtoclathrinmolecules,holdingtheclathrinscaffoldingontothesurfaceofthevesicle.●Ontheirinnersurface,theadaptorsbindtosortingsignalsinthecytosolictailsofintegralmembraneproteins.

◆衔接蛋白的种类●AP1:识别M6P受体●AP2:识别细胞质膜受体和M6P受体（αβ亚基）2023/1/12122衔接蛋白的作用2023/1/12123AP2在网格蛋白小泡装配中的作用2023/1/12124◆发动蛋白(dynamin)

胞质溶胶G蛋白,又叫：assemblyreactionfactor,ARF)发动蛋白在网格蛋白小泡形成过程中同出芽的颈部结合，一旦小泡装配完成，发动蛋白立即水解其本身结合的GTP从而将小泡与质膜切离。2023/1/12125发动蛋白的作用2023/1/12126COP-被膜小泡形成的机理非选择性小泡运输，ER-CGN-TGN,Golgi-ERCOPI:胞质溶胶蛋白，7个亚基，类似clathrinKDELreceptor:Golgi膜囊中结合KDEL；在胞质面有lys-lys-X-X序列作为COPI识别信号COPII：多亚基，sec23/24,sec13/31,等

COPII装配识别信号：di-acidicsortingsignal:eg.Asp-X-GluCOPII小泡融合成大泡，以微管微运输轨道2023/1/12127SmallGTP-bindingproteins◆单体G蛋白(monomericGTP-bindingprotein)◆活性调节GTP-binding:活性状态，GDP-binding:非活性状态●鸟嘌呤核苷释放蛋白(guanine-nucleotide-releasingprotein,GNRP),orguanine-nucleotide-exchangefactor,GEF:

供体膜上●GTP酶激活蛋白(GTPase-activatingprotein,GAP)2023/1/12128◆Types●ARF:Assemblyreactionfactor,

参与COPI被膜小泡的装配●Sar1

参与COPII被膜小泡的装配◆功能

Whichappeartoregulatethebindingofcoatproteinstothemembrane.

2023/1/12129ARF在COP被膜小泡形成中作用外被体COPII-coatedvesiclesmovematerialsfromtheERtotheGolgi.TheassemblyofaCOPII-coatedvesicles.Sar—GTPbindingprotein:Sar-GTPbindstotheER;Sar-GDPdissociatesfromtheERAntibodiesisabletoblockthebuddingofvesiclefromERbuthavenoeffectonvesicletransportfromoneGolgicompartmenttoanotherinmammaliancell.COPI-coatedvasiclestransportingEscapedERresidentProteinsBacktotheER.TheassemblyofaCOPI-coatismediatedbyADP-ribosylationfactor(ARF),GTPbindingprotein,whichisrequiredforvesicletransferbetweencisternae.COPIcoatedvesiclesmayselectspecificcargo.

ERisanopenprison.SolubleERproteinbearRetrievingsignal—KDEL(Lys-Asp-Glu-Leu)inmammalandHDELinyeast,whereasERmembraneproteinsbearthesignalKKXX.TheKDELreceptorpresentinvesiculartubularclustersandtheGolgiapparatus..(3)COPI-coatedvesiclewerefirstidentifiedbytreatmentofGTPanalogues---COPI-coatedvesicleaccumulatedwithinthecellandcouldbeisolatedbycentrifugation.Clathrin-coatedvesicle:TransportingCargofromtheTGNtoendosomes,Lysosomes,andplantvacuolesandalsoMovematerialsfromthePMtocytoplasmiccompartmentsalongtheendocyticpathway.TheTGNofGolgiistheSourseofClathrin-coatedvesicle.(2)Clathrin-coatscontain:

proteinclathrin-----whichformsastructuralscaffold,adaptors----multisubunit,whichformsaninnershell.

2023/1/12135三种类型被膜小泡运输途径?2023/1/12136被膜小泡运输的信号及相关性质D.caveolae-mediatedendocytosis2023/1/12139B.TheMechanismofVesicular

Transport

小泡运输的三个关键问题∶

◆小泡是如何形成的?为什么有些膜整合蛋白能够被选择性地包入小泡?◆不同类型的运输小泡具有什么样的信号标记，这些标记如何帮助它们同特定类型细胞器的膜结合?◆运输小泡的膜同靶膜相互融合的机理是什么?2023/1/12140小泡的定向运输、

停靠和融合机理SNAREhypothesis◆NSF(N-ethylmaleimide-sensitivefusionprotein,NSF)，N-乙基马来酰亚胺敏感融合蛋白,可溶性蛋白◆SNAP(可溶性NSF附着蛋白,solubleNSFattachmentprotein,SNAP)

NSF/SNAP介导不同的小泡融合◆V-SNARE(vesicle-SNAPreceptor)◆T-SNARE(target-SNAPreceptor)2023/1/12141FusionoftransportvesiclestotargetmembraneSNARE:SolubleNSFattachmentproteinreceptorv-SNAREsonthesurfaceoftransportvesiclet-SNAREsonthesurfaceoftargetmembraneTheinteractionbetweenv-SNAREsandt-SNAREsholdsthevesiclesclosetoacceptormembraneforvesicletargetingandfusionNSFproteinscatalyzethedissociationoft-SNAREsandv-SNAREs2023/1/121422023/1/12143Rab-protein◆TheRabproteinsareafamilyofsmallGTP-bindingproteinsthatarerelatedtotheRasproteins.◆Morethan30differentRabproteinshavebeenidentifiedandshowntofunctioninspecificvesicletransportprocesses.◆Theymayfunctioninseveralstepsofvesicletrafficking,includinginteractingwithSNAREstoregulateandfacilitatetheformationofv-SNARE/t-SNAREcomplexes.2023/1/12144膜泡运输、融合及Rab蛋白的作用2023/1/12145MembraneFusion需要V-SNARE,T-SNARE,SNAP25存在2023/1/12146NSF在膜泡运输中的作用2023/1/12147同型膜融合2023/1/12148MembraneBiogenesis

膜合成的两种模型∶

◆自我装配模型

(Spontaneousself-assembly)。

◆膜扩展模型∶

(Membraneextensionmodel)

2023/1/12149

膜脂的来源及不对称分布◆大多数磷脂在ER上合成●通过出芽形成小泡，将磷脂转移到其它的膜结构上●通过磷脂转运蛋白运输到其它的膜结构。◆磷脂在膜上的不对称分布●磷脂交换蛋白对磷脂的运输和插入是选择性的；●热动力学驱使磷脂的不对称分布，因为膜两侧的环境是不同的。2023/1/12150膜合成中脂的来源2023/1/12151膜整合蛋白和外周蛋白的形成◆膜整合蛋白的方向◆脂锚定蛋白●糖脂锚定蛋白●脂肪酸锚定蛋白◆外周蛋白●内侧外周蛋白●外侧外周蛋白2023/1/12152糖蛋白与脂锚定蛋白7.Endocytosis:

LargemoleculesenterintocellsA.EndocytosisimportsextracellularmoleculesdissolvedorsuspendedinfluidbyformingvesiclesfromtheplasmamembraneBulk-phaseendocytosisdoesnotrequiresurfacemembranerecognition.Itisthenonspecificuptakeofextracellularfluids.Receptor-mediatedendocytosis(RME)followsthebindingofsubstancestomembranereceptors.2023/1/121547CellSecretionandEndocytosis2023/1/12155A.Secretorypathways细胞分泌概述◆概念◆细胞分泌活动的过程●内质网:●高尔基体:●细胞质膜:2023/1/12156CharacteristicsofvesicletransportationFormationofvesiclesrequireadapterproteinsGTPisthemainenergyFusionofavesicletotargetrequirescomplementaryproteinspairlocatedonvesicleandtargetmembrane.2023/1/12157SecretorypathwaysERGolgiLysosomePlasmamembraneCOPIIClathrinClathrin Formationofdisulfidebonds Properfolding Additionandprocessingofcarbohydrates Specificproteolyticcleavages Assemblyintomultimericproteins2023/1/12158ConstitutiveandregulatedsecretionpathwaysConstitutiveexocytosispathway:vesiclescontinuouslyformandcarryproteinsfromtheGolgitothecellsurface.SupplytheplasmamembranewithnewlysynthesizedlipidsandproteinsRegulatedexocytosispathway:hormones,mucusordigestiveenzymesarestoredinsecretoryvesiclesuntilanextracellularsignalstimulatestheirsecretion.Specializedsecretorycelled2023/1/121592023/1/12160Proteinsortinginsecretorypathway