北京大學(xué)細(xì)胞生物學(xué)課件-6jizhiyuneimo

1、Chapter 6 Cytoplasmic matrix, Endomembrane system, Protein Sorting and membrane traffickingLearning objective1. Compartmentalization in Eukaryotic Cells; The structural and functional relationship among the ER, Golgi complexes, lysosomes and plasma membranes of eukaryotic cells;The pathways of prote

2、ins targeting and sorting, and its mechanisms; The ways of protein modifications and intracellular sites after they are synthesized;Types of vesicle transport and their functions.1. The Compartmentalization in Eukaryotic CellsMembranes divide the cytoplasm of eukaryotic cells into distinct compartme

3、nts. Three categories in eukaryotic cells: (1) the endomembrane system: ER, Golgi complex, Lys., secretory vesicles. (2) the cytosol. (3) mitochondria, chloroplasts, peroxisomes, and the nucleus. Membrane-bound structures (organelles) are found in all eukaryotic cells.Cytoplasmic matrix and its func

4、tionsCytoplasmic Matrix: The region of fluid content of the cytoplasm outside of the membranous organelles. Aqueous solution of large and small molecules including filaments of cytoskeleton which act as organizer for some order.The Cytosol is the site of protein synthesis and degradation or modifica

5、tion. It also performs most of the cells intermediary metabolism.Cytoplasmic matrix (Cytosol) and Endomembrane SystemFunctions of cytoplasmic matrix:The protein synthesis, degradation and modification.Cells carefully monitor the amount of misfolded proteins. An accumulation of misfolded proteins in

6、the cytosol triggers a heat-shock response, which stimulates the transcription of genes encoding cytosolic chaperones that help to refold the proteins.B. Endomembrane SystemEndomembrane System : The structural and functional relationship organelles including ER,Golgi complex, lysosome, endosomes, se

7、cretory vesicles.Membrane-bound structures (organelles) are found in all eukaryotic cells.Intracellular compartment % of total cell volumeCytosol 54Mittchondria 22Rough ER cisternae 9Smooth ER cisternae plus Golgi cisternae 6Nucleus 6Peroxisome 1Lysosomes 1Endosomes 1Relative volumes occupied by the

8、 major intracellular compartments in Liver CellC. The Dynamic Nature of the Endomembrane SystemMost organelles are part of a dynamic system in which vesicles move between compartments.Biosynthetic parthways move proteins, carbohydrates and lipids within the cell.Secretory pathways discharge proteins

9、 from cells.Endocytic parthways move materials into cells.Sorting signals are recognized by receptors and target proteins to specific sites.D. A few approaches to the study of cytomembranes Insights gained from autoradiography; Insights gained from the biochemical analysis of subcellular fractions;I

10、nsights gained from the study of genetic mutants;The dynamic activities of endomembrane systems are highly conserved despite the structural diversity of different cell types.De Duve, A.Claude and G.Palade,1974 Nobel Plrize2. The structure and functions of Endoplasmic Reticulum(ER)Rough endoplasmic r

11、eticulum and Smooth endoplasmic reticulum RER has ribosomes on the cytosolic side of continuous, flattened sacs(cisternae); SER is an interconnecting network of tubular membrane elements.Microsome(100-200nm)rER of pancreatic cellsMicrosomes are heterogeneous mixtures of similar-sized vesicles, forme

12、d from membranes of the ER and Golgi complex. Microsomes retain activity during purification, allowing studies of function and composition.A. Functions of the rERProteins synthesized on ribosomes of rER include: secretory proteins, integral membrane proteins, soluble proteins of organelles. Modifica

13、tion and processing of newly synthesized proteins: glycosylation in the rER;N-linked: linked to the amide nitrogen of asparagine (ER)O-linked: linked to the hydroxyl group serine or threonine via GalNac (in Golgi)The precursor of 14 residues is the same in plants, animals, and single-celled eukaryot

14、esthen remove 3 glucoses and 1 mannose in the ER Quality control of of newly synthesized proteins-The role of N-linked glycosylation in ER protein foldingQuality control: ensuring that misfolded proteins do not leave ERThe lumen of rER contains:Bip and calnexin (chaperones) : that recognize and bind

15、 to unfolded or misfolded proteins and give them correct conformation;Protein disulfide isomerase ( PDI ) ;GT(glucosyl-transferase, monitoring enenzyme ) recognize unfolded or misfolded proteins and adds a glucose to the end of oligo. Synthesis of membrane lipidsMost membrane lipids are synthesized

16、enterly within the ER. There are two exceptions:sphingomyelin and glycolipids, (begins in ER; completed in Golgi); (2) some of the unique lipids of the Mit and Chl membranes (themself).The membranes of different 0rganelles have markedly different lipids composition.Transport by budding：ERGC、Ly、PMTra

17、nsport by phospholipid exchange proteins(PEP)：ERother organelles（including Mit and Chl）The role of phospholipid translocators in lipid bilayer synthesisphospholipid translocators =Scramblase(ABC transporter Family)B. Functions of the sERSynthesis of steroids in endocrine cells.Detoxification of orga

18、nic compounds in liver cells. System of oxygenases-cytochrome p450 familyRelease of glucose 6-phosphate in liver cells.Sequestration of Ca2+. Ca2+-ATPase3. The structure and functions of Golgi complexA.The polarity of Golgi complexa) Cis cisternae of Golgi complex: reduced osmium tetroxide(OsO4);b)

19、Reaction for enzyme mannosidase II , localized in the medial;c) Reaction for enzyme nucleoside diphosphatase , localized in the trans cisternae.Regional differences in membrane composition across the Golgi stackB. The Functions of Golgi complexGlycosylation in the Golgi complexGolgi complex plays a

20、key role in the assembly of the carbohydrate component of glycoproteins and glycolipids. The core carbohydrate of N-linked oligosaccharides is assembled in the rER. Modifications to N-linked oligosaccharides are completed in the Golgi complex. O-linked oligosaccharides takes place in Golgi complex.S

21、tructure of typical O- and N-linked oligosaccharidesCore RegionAfter R. Kornfeld and S. Kornfeld, 1985, Annu. Rev. Biochem. 45:631What is the purpose of glycosylation?N-linked glycosylation is prevalent in all eucaryotes, but is absent from procaryotes.It dont require a template. There is an importa

22、nt difference between the construction of an oligosaccharide and the synthesis of DNA,RNA,and protein.Important functions: (1) One might suspect that they function to aid folding and the transport process; for example, carbohydrate as a marker during protein folding in ER and the use of carbohydrate

23、-binding lectins in guiding ER-to-Golgi transport. (2) Limit the approach of other macromolecules to the protein surface, more resistant to digestion by proteases. (3) Regulatory roles in signaling through the cell-surface receptor Notch, to allows these cells to respond selectively to activating st

24、imuli.The Golgi networks are processing and sorting stations where proteins are modified, segregated and then shipped in different directions.Golgi complex and cells secretionContinual,unregulated discharge of material from the cellsThe discharge of products stored in cytoplasmic granules, in respon

25、se to appropriate stimuli. Vesivular transport within the Golgi apparatus: Two views: cisternal maturation model and vesicular transport modelTwo possible models explaining the organization of the Golgi complex and the transport from one cisterna to the next.十 十 十 C. Golgi BiogenesisStages of Golgi

26、growth and division. Shown are thin section electron micrographs of T. gondii RH tachyzoites replicating by endodyogeny in HFF cells. Cells were placed in one of four categories according to the number and size of the Golgi: a, single Golgi; b, single, elongated Golgi; c, two Golgi; d, Golgi, often

27、more vesiculated, ineach nascent daughter cell, delineated by the growing inner membrane complex (IMC). a, apicoplast; dg, dense granules; er, ER; es, ER exit sites on the outer flattened part of the nuclear envelope; G, Golgi; m, micronemes; mit, mitochondria; r, rhoptries. Scale bar, 0.5mm.Stable

28、expression of mammalian Golgi proteins. a, b, Overlaid immunofluorescence and phase images of GRASPYFP (a) and NAGTIYFP (b) in stable, transgenic cell lines of Toxoplasma gondii. ch, Immunofluorescence images of a transgenic cell line expressing both GRASPCFP (green) and NAGTIYFP (red) before (ce) o

29、r after (fh) treatment with 5mg /ml BFA for 10 min at 37C. Merged images are shown on the right. Asterisks indicate a secreted form of NAGTIYFP that accumulates in the parasitophorous vacuole. Scale bars, 5mm.Immunoelectron microscopy of transgenic parasites. ac, Cryosections of GRASPYFP (a, c) or N

30、AGTIYFP (b) transgenic parasites, pretreated for 2 h with 50mg/ml cycloheximide, before being fixed and immunolabelled for YFP using polyclonal antibodies against GFP followed by protein A coupled to 5-nm gold particles. Note the high density of labelling restricted to Golgi membranes. In c, GRASPYF

31、P transgenic parasites were treated with BFA (5mg/ml) for 30 min before immunolabelling. Note the tubulo-vesicular appearance of the Golgi caused by loss of Golgi enzymes to the ER. d, Quantification of images in a and b. Results are presented as mean s.d. gold particles /um2.Biogenesis of the Golgi

32、 apparatus in living parasites. ah, Transgenic parasites stably expressing IMC1CFP (blue) were transfected with plasmid DNA encoding GRASPYFP (green). After 20 h of infection in HFFs, four parasites were imaged by time-lapse video fluorescence microscopy. Images were taken every 10 min for 7 h at 37

33、 C. Representative images at the indicated times are shown. Note that T. gondii. Replicates synchronously in a given vacuole, which permits simultaneous imaging of several cells at the same cell-cycle stage. i, j, Transgenic parasites expressing NAGTIYFP (green) were imaged over time and sample imag

34、es late in cell division are shown. For both Golgi markers note the inheritance of two structures by each nascent daughter (f, i, j) and their eventual coalescence (arrow in g and h). k, Threedimensional reconstruction of two parasites during mitosis. The Golgi was selectively outlined in red and ot

35、her electron-dense structures were coloured in green or dark blue to differentiate the two forming daughter cells. Golgi are inherited by both cells, and in the complete reconstruction of one daughter (right) two Golgi structures are visible (arrows). Note that the other daughter was only reconstruc

36、ted partially and contains a single Golgi structure.4. The structure and functions of LysosomesA. Characteristics of Lysosomes Lysosome is a heterogenous organelle: Primary lysosomesSecond lysosomes heterophagic autophagicResidual bodyPrimary Lys.Second LysFigure6-19Histochemical visualization of ly

37、sosomes.Electron micro-graphs of two sections of a cell stained to reveal the location of acid phosphatase, a marker enzyme for lysosomes. The larger membrane-bounded organelles, containing dense precipitates of lead phosphate, are lysosomes, whose diverse morphology reflects variations in the amoun

38、t and nature of the material they are digesting. The precipitates are produced when tissue fixed with glutaraldehyde is incubated with a phosphatase substrate in the presence of lead ions. Two small vesicles thought to be carrying acid hydrolases from the Golgi apparatus are indicated by red arrows

39、in the top panel. (Courtesy of Daniel S. 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 concentration is kept high by H+-A

40、TPase Glycosylated proteins: may protect the lysosome from self-digestion. Transport proteins: transporting digested materials.Figure13-18The low pH in lysosomes and endosomes.Proteins labeled with a pH-sensitive fluorescent probe (fluorescein) and then endocytosed by cells can be used to measure th

41、e pH in endosomes and lysosomes. The different colors reflect the pH that the fluorescent probe encounters in these organelles. The pH in lysosomes (red) is about 5, while the pH in various types of endosomes (blue and green) ranges from 5.5 to 6.5. (Courtesy of Fred Maxfield and Kenneth Dunn.) Figu

42、re13-20The plant cell vacuole.This electron micrograph of cells in a young tobacco leaf shows that the cytosol is confined by the enormous vacuole to a thin layer, containing chloroplasts, pressed against the cell wall. The membrane of the vacuole is called the tonoplast. (Courtesy of J. Burgess.) B

43、. The Functions of LysosomesLysosomes are involved in three major cell functions: 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. Lysosome

44、s and DiseasesDisorders resulting from defects in lysosomal function:Autolysis: 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 r

45、esulting in accumulation of material in lysosomes as large inclusions. One severe 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 gangli

46、osides, a major component of brain cell membranes.表1. 神經(jīng)鞘脂貯積病疾病缺失酶類(lèi)主要貯積底物后果GM1神經(jīng)節(jié)苷脂貯積癥GM1-半乳糖苷酶神經(jīng)節(jié)苷脂GM1智力遲鈍，肝臟肥大，骨骼受累，2歲前死亡泰薩二氏病己糖胺酶A神經(jīng)節(jié)苷脂GM2智力遲鈍，失明，3歲前死亡法布萊氏病-半乳糖苷酶A三己糖神經(jīng)酰胺皮疹，腎功能喪失，下肢疼痛山霍夫氏病己糖胺酶A和B神經(jīng)節(jié)苷脂GM2和紅細(xì)胞糖苷酯與泰薩氏疾病癥狀相似，但發(fā)展更快高歇氏病葡糖腦苷酯酶葡糖腦苷脂肝臟和脾臟腫大，長(zhǎng)骨腐蝕，只在嬰兒期發(fā)生智力遲鈍尼-皮二氏病鞘磷脂水解酶鞘磷脂肝臟和脾臟腫大，智力遲鈍Farbe

47、rs 脂肪肉芽腫病神經(jīng)酰胺水解酶神經(jīng)酰胺疼痛性與退行性的關(guān)節(jié)變形，皮膚瘤，幾年內(nèi)死亡Krabbes 病半乳糖腦苷酯酶半乳糖腦苷脂髓磷脂缺失，智力遲鈍，2歲前死亡腦硫脂沉積芳基硫酸酯酶腦硫脂智力遲鈍，前十年死亡D. Biogenesis of LysosomesFigure6-23The transport of newly synthesized lysosomal hydrolases to lysosomes.The precursors of lysosomal hydrolases are covalently modified by the addition of mannose 6

48、-phosphate in the CGN. They then become segregated from all other types of proteins in the TGN because a specific class of transport vesicles budding from the TGN concentrates mannose 6-phosphate-specific receptors, which bind the modified lysosomal hydrolases. These vesicles subsequently fuse with

49、late endosomes. At the low pH of the late endosome the hydrolases dissociate from the receptors, which are recycled to the Golgi apparatus for further rounds of transport. In late endosomes the phosphate is removed from the mannose on the hydrolases, further ensuring that the hydrolases do not retur

50、n to the Golgi apparatus with the receptor. Mannose 6-phosphate residues target proteins to lysosomesTargeting of soluble lysosomal enzymes to endosomes and lysosomes by M-6-P tag Phosphorylation of mannose residues on lysosomal enzymes catalyzed by two enzymesRecognition site binds to Signal patchG

51、lcNAc phosphotransferasephosphodiesteraseFigure 6-40. The mannose 6-phosphate (M6P) pathway, the major route for targeting lysosomal enzymes to lysosomes. Precursors of lysosomal enzymes migrate from the rER to the cis-Golgi where mannose residues are phosphorylated. In the TGN, the phosphorylated e

52、nzymes bind to M6P receptors, which direct the enzymes into vesicles coated with the clathrin. The clathrin lattice surrounding these vesicles is rapidly depolymerized to its subunits, and the uncoated transport vesicles fuse with late endosomes. Within this low-pH compartment, the phosphorylated en

53、zymes dissociate from the M6P receptors and then are dephosphorylated. The receptors recycle back to the Golgi, and the enzymes are incorporated into a different transport vesicle that buds from the late endosome and soon fuses with a lysosome. The sorting of lysosomal enzymes from secretory protein

54、s 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. Griffiths et al., Cell 52:329; S. Kornfeld, Annu. Rev. Biochem. 61:307; and G. Griffiths and J. Gruenberg, Trends Cell Biol. 1:55. Protei

55、n Sorting Overview of sorting of nuclear-encoded proteins in eukaryotic cellsProteins are imported into organelles by three mechanisms:Gated Transport: Transport through nuclear poresTransmembrane transport: ER, Mit, Chl, PerVesicular transport: ER-Golgi-PM-Lys, Endosome Road map of protein sorting

56、Protein sorting: Protein molecules move from the cytosol to their target organelles or cell surface directed by the sorting signals in the proteins.Signal peptides and Signal patchesFigure6-8Two ways that a sorting signal can be built into a protein.(A) The signal resides in a single discrete stretc

57、h of amino acid sequence, called a signal peptide, that is exposed in the folded protein. Signal peptides often occur at the end of the polypeptide chain, but they can also be located elsewhere. (B) A signal patch can be formed by the juxtaposition of amino acids from regions that are physically sep

58、arated before the protein folds; alternatively, separate patches on the surface of the folded protein that are spaced a fixed distance apart could form the signal. Gated transport: Through gated poresNuclear pores;Nuclear localization signal (NLS);Folded and assembly form to transport.Transmembrane

59、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

60、COPI);Only Properly folded and assembled proteins;The orientation of transported proteins and lipids is not changed during transporting.B. Signal Hypothesis -G.Blobel & D.Sabatini,1971. A model for the Signal Mechanism of Cotranslational ImportEvidence That Protein Synthesized on Ribosomes Attached