2023/4/161Chapter4:Intracellularcompartmentandtransport4.1Intruction4.2TheEndoplasmicReticulum4.3Golgicomplex4.4Lysosomes4.5SecretoryandEndocytosis4.6TheMechanismofVesicularTransport2023/4/1624.1Intruction

Organellesandtheirfunctions2023/4/163Themainfunctionsofthemembrane-enclosedcompartmentsofaeukaryoticcell

Compartment MainfunctionCytosol Containsmanymetabolicpathways,proteinsynthesisNucleus Containsmaingenome;DNAandRNAsynthesisER Synthesisofmostlipids,synthesisofproteinsfordistributiontomanyorganellesandtotheplasmamembraneGolgiapparatus Modification,sorting,andpackagingofproteinsandlipidsforeithersecretionordeliverytoanotherorganelleLysosomes IntracellulardegradationEndosomes SortingofendocytosedmaterialMitochondria ATPsynthesisbyoxidativephosphorylationChloroplasts ATPsynthesisandcarbonfixationbyphotosynthesisPeroxisomes Oxidationoftoxicmolecules2023/4/164EndomembraneSystem

2023/4/165內(nèi)膜結(jié)構(gòu)特點與動態(tài)性質(zhì)

◆獨立性

●內(nèi)膜封閉的區(qū)室●執(zhí)行獨立的功能◆協(xié)作性:動態(tài)性質(zhì)●

Biosyntheticpathway●Secretorypathway

●Endocyticpathway:materialsmovefromtheoutersurfaceofthecelltocompartments,suchasendosomesandlysosomes,locatedwithinthecytoplasm2023/4/1662023/4/167內(nèi)膜系統(tǒng)的生物學(xué)意義◆形成了一些特定的功能區(qū)域和微環(huán)境(細(xì)胞內(nèi)功能分化)●合理使用資源●集團化管理●提高工作效率◆動態(tài)性質(zhì)●保證了膜結(jié)構(gòu)的一致性●使細(xì)胞“青春常在”

?2023/4/168內(nèi)膜系統(tǒng)的研究方法◆放射自顯影術(shù)(autoradiography)

●胰泡細(xì)胞●脈沖示蹤實驗(pulse-chaseexperiment)◆亞細(xì)胞組分的分離●微粒體(microsomes)◆突變體使用2023/4/169Isolationofmicrosome(微粒體)?2023/4/1610突變體使用2023/4/1611ProteinsortinginsidecellTransportthroughnuclearpores:SolublenuclearproteinsenterthroughnuclearporesTransportacrossmembranes:Proteinscanenteranorganellesuchasmitochondrion,chloroplast,Peroxisomeandnuclearmembranebypassingthroughthemembrane.Transportbyvesicles:Proteinscanenteranorganellebytransportandfusionofvesicles.Golgiapparatus,lysosome,endosome,bytransportandfusionofvesicles.2023/4/1612Threemechanismsthatproteinsareimportedintoorganelles2023/4/1613信號序列的引導(dǎo)作用◆與生俱來的三種信號序列

●壽命信號●加工信號●定位信號

▲核定位信號▲引導(dǎo)肽▲信號肽。2023/4/1614

信號序列

在蛋白質(zhì)定位中的作用?2023/4/1615TableSometypicalsignalsequencesTargetorganelle Signallocation Signalremoval NatureofsignalERN-terminal(+) ‘Core’of6-12mostlyhydrophobicaminoacids,oftenprecededbyoneormorebasicaminoacidsERretentionC-terminal(-) KDELMitochondrionN-terminal(+) 3-5nonconsecutiveArgorLysresidues,oftenwithSerandThr;noGluorAspresiduesChloroplastN-terminal(+) Nocommonsequencemotifs;generallyrichinSer,Thr,andsmallhydrophobicaminoacidresiduesandpoorinGluandAspresiduesPeroxisomeC-terminal(-) UsuallySer-Lys-LeuatextremeC-terminusNucleusInternal(-) Oneclusterof5basicaminoacids,ortwosmallerclustersofbasicresiduesseparatedby10aminoacids2023/4/1616Sometypicalproteinsortingsignals2023/4/1617ProteinSortingin

EndomembraneSystem

Becauseofthecomplexinternalorganizationofeukaryoticcells,thesortingandtargetingofproteinstotheirappropriatedestinationsareconsiderabletasks.

2023/4/16182023/4/1619●ERisanetworkofmembrane-enclosedtubulesandsacs(cisternae)thatextendsfromthenuclearmembranethroughoutthecytoplasm.●ER

isthelargestorganelleofmosteukaryoticcells.●Itsmembranemayaccountforabouthalfofallcellmembranes.●thespaceenclosedbytheERmayrepresentabout10%ofthetotalcellvolume.

4.2Endoplasmicreticulum(ER)2023/4/1620Endoplasmicreticulum2023/4/1621SmoothER(sER)androughER(rER)sER:synthesisoffattyacidsandphospholipids.rER:ribosomesboundtotherERsynthesizecertainmembraneandorganelleproteinsandallproteinstobesecretedfromthecell.2023/4/16222023/4/16232023/4/16242023/4/1625

內(nèi)質(zhì)網(wǎng)的化學(xué)組成

◆內(nèi)質(zhì)網(wǎng)的標(biāo)志酶是葡萄糖-6-磷酸酶。

◆細(xì)胞色素P450在內(nèi)質(zhì)網(wǎng)膜中最為豐富。2023/4/1626◆Roughendoplasmicreticulum(RER)◆Smoothendoplasmicreticulum(SER)

▲內(nèi)質(zhì)網(wǎng)的外表面稱為胞質(zhì)溶膠面

(sytosolicspace)▲內(nèi)表面稱為潴泡面(cisternalspace)◆特殊類型的內(nèi)質(zhì)網(wǎng)●微粒體(microsome)●肌質(zhì)網(wǎng)(sarcoplasmicreticulum)2023/4/1627FunctionofER光面內(nèi)質(zhì)網(wǎng)的酶類

◆糖代謝酶類

◆脂代謝酶類

◆藥物脫毒與相關(guān)的氧化酶

◆蛋白質(zhì)的加工酶類2023/4/1628糖原分解與游離葡萄糖釋放脂類的合成與轉(zhuǎn)運解毒作用Ca2+離子濃度的調(diào)節(jié)作用2023/4/1629糖原分解與游離葡萄糖釋放

2023/4/1630磷脂的合成2023/4/16312023/4/1632◆磷脂轉(zhuǎn)運的三種方式2023/4/1633磷脂交換蛋白的作用2023/4/1634解毒作用◆光面內(nèi)質(zhì)網(wǎng)含有豐富的氧化酶系統(tǒng)(如細(xì)胞色素P450、NADH細(xì)胞色素C還原酶等)能使許多有害物質(zhì)解毒,轉(zhuǎn)化為易于排出的物質(zhì)?！艋旌瞎δ艿难趸?mixed-functionoxidase)●每分子的底物被氧化需要消耗一分子氧,并且將NADPH轉(zhuǎn)變成NADP+●該氧化過程消耗的氧分子中有一個氧原子出現(xiàn)在水中，另一個氧原子出現(xiàn)在產(chǎn)物中●故此將催化該反應(yīng)的酶稱為混合功能的氧化酶。2023/4/1635光面內(nèi)質(zhì)網(wǎng)的氧化作用2023/4/1636

粗面內(nèi)質(zhì)網(wǎng)的功能

--信號肽與蛋白質(zhì)運輸

2023/4/1637核糖體循環(huán)?2023/4/1638信號序列(signalsequence)◆研究過程●1960s,GeorgePalade示蹤技術(shù)研究培養(yǎng)細(xì)胞中蛋白質(zhì)的分泌●Redman和Sabatini用分離的RER小泡研究膜結(jié)合核糖體合成的蛋白質(zhì)去向●1971年美國洛克菲勒大學(xué)的Blobel等提出了信號序列(signalsequence)的概念。2023/4/1639同位素示蹤:分泌途徑研究2023/4/1640蛋白質(zhì)進入核糖體實驗2023/4/1641◆信號序列的間接證據(jù)1972年,Milstein和他的同事發(fā)現(xiàn):●從細(xì)胞質(zhì)分離的IgG輕鏈:

多20個氨基酸殘基●釋放到細(xì)胞外的IgG輕鏈:

少20個氨基酸殘基2023/4/1642◆信號肽的證實G.Blobel等用離體實驗證實了信號肽的存在:●RER對產(chǎn)物的影響●蛋白水解酶實驗●多聚核糖體的離體翻譯2023/4/1643體外合成實驗2023/4/1644信號肽的特性◆序列特征

15-35個氨基酸殘基，其中含有4-12個疏水殘基◆特異性:◆位置:●N-端突出的一段肽●內(nèi)含信號肽。2023/4/1645早期信號假說(signalhypothesis)1975年，Blobel等正式提出了信號假說,要點是:◆合成始于游離核糖體;◆N端信號序列靠自由碰撞與內(nèi)質(zhì)網(wǎng)膜接觸,然后插入內(nèi)質(zhì)網(wǎng)的膜;◆蛋白質(zhì)以袢環(huán)形式穿過內(nèi)質(zhì)網(wǎng)的膜;◆蛋白的存在方式:●若信號序列被信號肽酶切除:完全進入腔●若是停止轉(zhuǎn)移信號:膜蛋白

2023/4/1646信號識別顆粒與?？康鞍住魋ignalrecognitionpartical,SRP●SRPisacytosolicribonucleoproteinparticle●Sixdiscretepolypeptides●A300-nucleotideRNA●三個功能域:

▲翻譯暫停結(jié)構(gòu)域(P9/P14)▲信號肽識別結(jié)合位點(P54)▲SRP受體蛋白結(jié)合位點(P68/P72)。2023/4/1647Signalrecognitionpartical2023/4/1648◆Dockingprotein,DP

●是SRP在內(nèi)質(zhì)網(wǎng)膜上的受體蛋白●兩個亞基:親水的α亞基,疏水的β亞基◆易位子(translocon)●MultiplecopiesoftheSec61complexassembletoformacylindertransloconchannel●5～6nmhighand8.5nmindiameter,withacentralpore,roughly2nmindiameter●哺乳動物細(xì)胞中有三種類型的Sec61,即α、β和γ●Sec61α有10個跨膜α螺旋2023/4/1649Signalhypothesis2023/4/1650Signalhypothesis2023/4/1651體外實驗證明SRP顆粒、SRP受體的作用2023/4/1652SRP-DP識別的GDP-GTP交換循環(huán)2023/4/1653Orientationofmembraneproteins?2023/4/1654跨膜信號與膜蛋白的定向◆單次跨膜蛋白?●起始轉(zhuǎn)移信號(starttransfersignal)●終止轉(zhuǎn)移序列(stoptransfersequence)

●可切割的信號序列(Cleavablesignalsequence)(N-端)●內(nèi)部信號序列(internalsignalsequence)(internalnoncleavablesignalsequences)2023/4/1655信號肽與起始跨膜序列?2023/4/1656Stoptransfersequence?2023/4/1657◆單次跨膜蛋白的定向●Cleavablesignalsequenceaminoterminusexposedtothecytosol●Internalsignalsequence

▲Aminoterminusexposedtothecytosol▲Carboxyterminusexposedtothecytosol

2023/4/1658單次跨膜蛋白的定向I?2023/4/1659單次跨膜蛋白的定向II2023/4/1660◆停止轉(zhuǎn)運信號與多次跨膜蛋白的定向●產(chǎn)生:Proteinsthatspanthemembranemultipletimesarethoughttobeinsertedasaresultofanalternatingseriesofinternalsignalsequencesandtransmembranestop-transfersequences.

●定向:?2023/4/16612023/4/1662MajortopologicalclassesofintegralmembraneproteinssynthesizedontheroughER.2023/4/1663信號序列與膜蛋白定向?2023/4/1664ProteinFoldingand

ProcessingintheER

◆ProteinfoldingintheER

●Bip蛋白∶

Bip是一類分子伴侶(Molecularchaperone),是重鏈結(jié)合蛋白的簡稱(heavy-chainbindingprotein)●BiPisthoughttobindtotheunfoldedpolypeptidechainasitcrossesthemembraneandthenmediatesproteinfoldingandtheassemblyofmultisubunitproteinswithintheER2023/4/1665BiP蛋白在ER中的作用2023/4/1666◆蛋白質(zhì)在內(nèi)質(zhì)網(wǎng)中的加工與修飾●Theformationofdisulfidebondsbetweenthesidechainsofcysteineresidues:proteindisulfideisomerase

●N-linkedglycosylation(Asn-X-Ser/Thr)

●Additionofglycosylphosphatidylinositol(GPI,糖基磷脂酰肌醇)anchors

●ExportofProteinsfromtheER2023/4/1667Theformationofdisulfidebonds2023/4/1668N-linkedglycosylation2023/4/1669AdditionofGPIanchors2023/4/1670ExportofProteinsfromtheER2023/4/16714.3Golgiapparatus◆高爾基器(Golgiapparatus)或高爾基復(fù)合體(Golgicomplex)◆意大利科學(xué)家CamilloGolgi在1898年發(fā)現(xiàn)的。2023/4/1672Golgiapparatus4-8flattenedmembranevesiclesorsacssurroundedbyanumberofsphericalmembranevesicles.Cisregion(closetorER);medialregionandtransregion.2023/4/1673高爾基體的結(jié)構(gòu)和極性形態(tài)結(jié)構(gòu)連續(xù)的整體膜結(jié)構(gòu)?！舯馄侥遗?sacuules)◆小囊泡(vesicle)◆大囊泡(vacuoles)2023/4/1674Golgiapparatus2023/4/1675高爾基體的極性◆高爾基內(nèi)側(cè)網(wǎng)絡(luò)(cis-Golginetwork,CGN)

順面、形成面◆中間潴泡(medialcisternae)◆高爾基外側(cè)網(wǎng)絡(luò)

(transGoljinetwork,TGN)

外側(cè)面、成熟面2023/4/1676

RegionsoftheGolgiapparatus

2023/4/1677

數(shù)量與化學(xué)組成◆數(shù)量分泌功能越旺盛的細(xì)胞,越多?！舻鞍踪|(zhì)和脂

高爾基復(fù)合體高于內(nèi)質(zhì)網(wǎng)和質(zhì)膜:

質(zhì)膜:蛋白質(zhì)40%,脂60%;

內(nèi)質(zhì)網(wǎng):蛋白質(zhì)20%,脂50-60%;

高爾基體:蛋白質(zhì)60%,脂40%;◆高爾基復(fù)合體的標(biāo)志酶是糖基轉(zhuǎn)移酶。2023/4/1678ThefunctionsofGolgiapparatusSecretion：ProteinsynthesizedinRER—ERlumen—COPII—CGNofGolgi—modificationinmedial—transportationvesiclesformedinTGN—transportation，fusionwithplasmamembraneO-linkedglycosylation。進行膜的轉(zhuǎn)化功能：內(nèi)質(zhì)網(wǎng)上合成的新膜脂轉(zhuǎn)移至高爾基體后，經(jīng)過修飾和加工，形成運輸泡與質(zhì)膜結(jié)合將蛋白水解為活性物質(zhì)：將沒有活性的蛋白原N端或兩端的序列切除形成成熟的多肽（如胰島素）；將含有多個相同氨基酸序列的前體水解為有活性的多肽，如神經(jīng)肽參與形成溶酶體參與植物細(xì)胞壁的形成2023/4/1679ProteinsarefurthermodifiedandsortedintheGolgiapparatus2023/4/1680高爾基體的蛋白運輸作用2023/4/1681◆ER駐守蛋白的駐守機制●內(nèi)質(zhì)網(wǎng)駐守信號(ERretentionsignal):

Lys-Asp-Glu-Leu-COO-，即KDEL信號序列●KDEL信號受體▲內(nèi)質(zhì)網(wǎng)膜

▲高爾基體膜2023/4/1682ER保留蛋白的運輸2023/4/1683ProteinGlycosylationwithintheGolgi

◆O-連接的糖基化

將糖鏈轉(zhuǎn)移到多肽鏈的絲氨酸、蘇氨酸或羥賴氨酸的羥基上，稱為O-連接的糖基化?！鬘-連接的寡聚糖進一步加工2023/4/1684O-linkedoligosaccharides:FormedbythesequentialtransferofsugarsfromnucleotideprecursorsAminoacids:-OHofSer、Thr、HypSugars:galactoseorN-acetylglucosamineShort,1-4sugarsresiduesGolgiapparatusN-linkedoligosaccharides:Beginwithadditionofanoligosaccharid