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1、BiogenesisoftheGolgiapparatusinlivingparasites.ah,TransgenicparasitesstablyexpressingIMC1CFP(blue)weretransfectedwithplasmidDNAencodingGRASPYFP(green).After20hofinfectioninHFFs,fourparasiteswereimagedbytime-lapsevideofluorescencemicroscopy.Imagesweretakenevery10minfor7hat37C.Representativeimagesatth
2、eindicatedtimesareshown.NotethatT.gondii.Replicatessynchronouslyinagivenvacuole,whichpermitssimultaneousimagingofseveralcellsatthesamecell-cyclestage.i,j,TransgenicparasitesexpressingNAGTIYFP(green)wereimagedovertimeandsampleimageslateincelldivisionareshown.ForbothGolgimarkersnotetheinheritanceoftwo
3、structuresbyeachnascentdaughter(f,i,j)andtheireventualcoalescence(arrowingandh).k,Threedimensionalreconstructionoftwoparasitesduringmitosis.TheGolgiwasselectivelyoutlinedinredandotherelectron-densestructureswerecolouredingreenordarkbluetodifferentiatethetwoformingdaughtercells.Golgiareinheritedbybot
4、hcells,andinthecompletereconstructionofonedaughter(right)twoGolgistructuresarevisible(arrows).NotethattheotherdaughterwasonlyreconstructedpartiallyandcontainsasingleGolgistructure.4. The structure and functions of LysosomesA. Characteristics of Lysosomes Lysosome is a heterogenous organelle: Primary
5、 lysosomesSecond lysosomes heterophagic autophagicResidual bodyPrimary Lys.Second LysFigure6-19Histochemical visualization of lysosomes.Electronmicro-graphsoftwosectionsofacellstainedtorevealthelocationofacidphosphatase,amarkerenzymeforlysosomes.Thelargermembrane-boundedorganelles,containingdensepre
6、cipitatesofleadphosphate,arelysosomes,whosediversemorphologyreflectsvariationsintheamountandnatureofthematerialtheyaredigesting.Theprecipitatesareproducedwhentissuefixedwithglutaraldehydeisincubatedwithaphosphatasesubstrateinthepresenceofleadions.Twosmallvesiclesthoughttobecarryingacidhydrolasesfrom
7、theGolgiapparatusareindicatedbyred arrows inthetoppanel.(CourtesyofDanielS.Friend.) Lysosomes contain plenty acid hydrolases that can digest every kind of biological molecule. -the principal sites of intracellular digestion.Marker enzyme: acid phosphataseLysosome membrane: H+-pumps: internal proton
8、concentration is kept high by H+-ATPase Glycosylated proteins: may protect the lysosome from self-digestion. Transport proteins: transporting digested materials.Figure13-18The low pH in lysosomes and endosomes.ProteinslabeledwithapH-sensitivefluorescentprobe(fluorescein)andthenendocytosedbycellscanb
9、eusedtomeasurethepHinendosomesandlysosomes.ThedifferentcolorsreflectthepHthatthefluorescentprobeencountersintheseorganelles.ThepHinlysosomes(red)isabout5,whilethepHinvarioustypesofendosomes(blue andgreen)rangesfrom5.5to6.5.(CourtesyofFredMaxfieldandKennethDunn.)Figure13-20The plant cell vacuole.This
10、electronmicrographofcellsinayoungtobaccoleafshowsthatthecytosolisconfinedbytheenormousvacuoletoathinlayer,containingchloroplasts,pressedagainstthecellwall.Themembraneofthevacuoleiscalledthetonoplast.(CourtesyofJ.Burgess.)B. The Functions of LysosomesvLysosomes are involved in three major cell functi
11、ons: phagocytosis; autophagy; endocytosis.Primary lys fuse with either phagocytic or autophagic vesicles, forming residual bodies that either undergo exocytosis or are retained in the cell as lipofuscin granules.C. Lysosomes and DiseasesDisorders resulting from defects in lysosomal function:Autolysi
12、s: A break or leak in the membrane of lys releases digestive enzymes into the cell which damages the surrounding tissues (Silicosis). Lysosomal storage diseases are due to the absence of one or more lysosomal enzymes, and resulting in accumulation of material in lysosomes as large inclusions. One se
13、vere type of the disease is I-cell disease (inclusion cell disease, GlcNAc-Phosphotransferase gene mutant). Tay-Sachs disease results from a deficiency of the enzyme (-N-hexosaminidase A) whose function is to degrade gangliosides, a major component of brain cell membranes.表表1. 神经鞘脂神经鞘脂贮积病积病疾病缺失酶类主要贮
14、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Farbers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Krabbes病半乳糖脑苷酯酶
15、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D. Biogenesis of LysosomesFigure6-23The transport of newly synthesized lysosomal hydrolases to lysosomes.Theprecursorsoflysosomalhydrolasesarecovalentlymodifiedbytheadditionofmannose6-phosphateintheCGN.Theythenbecomesegregatedfromallothertypesofproteinsi
16、ntheTGNbecauseaspecificclassoftransportvesiclesbuddingfromtheTGNconcentratesmannose6-phosphate-specificreceptors,whichbindthemodifiedlysosomalhydrolases.Thesevesiclessubsequentlyfusewithlateendosomes.AtthelowpHofthelateendosomethehydrolasesdissociatefromthereceptors,whicharerecycledtotheGolgiapparat
17、usforfurtherroundsoftransport.Inlateendosomesthephosphateisremovedfromthemannoseonthehydrolases,furtherensuringthatthehydrolasesdonotreturntotheGolgiapparatuswiththereceptor.Mannose 6-phosphate residues target proteins to lysosomesTargeting of soluble lysosomal enzymes to endosomes and lysosomes by
18、M-6-P tag Phosphorylation of mannose residues on lysosomal enzymes catalyzed by two enzymesRecognition site binds to Signal patchGlcNAc phosphotransferasephosphodiesteraseFigure 6-40. The mannose 6-phosphate (M6P) pathway, the major route for targeting lysosomal enzymes to lysosomes. Precursors of l
19、ysosomal enzymes migrate from the rER to the cis-Golgi where mannose residues are phosphorylated. In the TGN, the phosphorylated enzymes bind to M6P receptors, which direct the enzymes into vesicles coated with the clathrin. The clathrin lattice surrounding these vesicles is rapidly depolymerized to
20、 its subunits, and the uncoated transport vesicles fuse with late endosomes. Within this low-pH compartment, the phosphorylated enzymes dissociate from the M6P receptors and then are dephosphorylated. The receptors recycle back to the Golgi, and the enzymes are incorporated into a different transpor
21、t vesicle that buds from the late endosome and soon fuses with a lysosome. The sorting of lysosomal enzymes from secretory proteins thus occurs in the TGN, and these two classes of proteins are incorporated into different vesicles, which take different routes after they bud from the Golgi.G. Griffit
22、hs et al., Cell 52:329; S. Kornfeld, Annu. Rev. Biochem. 61:307; and G. Griffiths and J. Gruenberg, Trends Cell Biol. 1:55. Protein Sorting A.Overview of sorting of nuclear-encodedB. proteins in eukaryotic cellsvProteins are imported into organelles by three mechanisms:Gated Transport: Transport thr
23、ough nuclear poresTransmembrane transport: ER, Mit, Chl, PerVesicular transport: ER-Golgi-PM-Lys, Endosome RoadmapofproteinsortingRoadmapofproteinsorting vProtein sorting: Protein molecules move from the cytosol to their target organelles or cell surface directed by the sorting signals in the protei
24、ns.Signal peptides and Signal patchesFigure6-8Two ways that a sorting signal can be built into a protein.(A)Thesignalresidesinasinglediscretestretchofaminoacidsequence,calledasignal peptide, thatisexposedinthefoldedprotein.Signalpeptidesoftenoccurattheendofthepolypeptidechain,buttheycanalsobelocated
25、elsewhere.(B)Asignal patchcanbeformedbythejuxtapositionofaminoacidsfromregionsthatarephysicallyseparatedbeforetheproteinfolds;alternatively,separatepatchesonthesurfaceofthefoldedproteinthatarespacedafixeddistanceapartcouldformthesignal.Gated transport:Through gated poresNuclear pores;Nuclear localiz
26、ation signal (NLS);Folded and assembly form to transport.Transmembrane transportER signal sequence, Mit, Chl, Per: Leader sequence;Through translocon on the membrane;Single and Unfold form; Helped by molecular chaperonsVesicular transportBudding, transporting, docking and at last fusion with target membrane;Assembly coated proteins on the vesicles (Clathrin, COPII and COPI);Only Properly folded and assembled proteins;The orientation of transported proteins and lipids is not changed during transporting.
