基质与内膜

nullChapter 6Chapter 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 proteins 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.null1. The Compartmentalization in Eukaryotic CellsMembranes divide the cytoplasm of eukaryotic cells into distinct compartments. 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.nullCytoplasmic matrix and its functionsCytoplasmic 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 modification. It also performs most of the cell’s intermediary metabolism.Cytoplasmic matrix (Cytosol) and Endomembrane SystemnullFunctions of cytoplasmic matrix:The protein synthesis, degradation and modification.Cells carefully monitor the amount of misfolded proteins. An accumulation of misfolded proteins in the cytosol triggers a heat-shock response, which stimulates the transcription of genes encoding cytosolic chaperones that help to refold the proteins.nullB. Endomembrane SystemB. Endomembrane SystemEndomembrane System : The structural and functional relationship organelles including ER,Golgi complex, lysosome, endosomes, secretory vesicles. Membrane-bound structures (organelles) are found in all eukaryotic cells.nullRelative volumes occupied by the major intracellular compartments in Liver CellnullC. 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 from cells. Endocytic parthways move materials into cells. Sorting signals are recognized by receptors and target proteins to specific sites.nullD. A few approaches to the study of cytomembranes Insights gained from autoradiography; Insights gained from the biochemical analysis of subcellular fractions; Insights gained from the study of genetic mutants; The dynamic activities of endomembrane systems are highly conserved despite the structural diversity of different cell types.nullDe Duve, A.Claude and G.Palade,1974 Nobel Plrizenull2. The structure and functions of Endoplasmic Reticulum(ER)Rough endoplasmic reticulum 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.nullMicrosome(100-200nm)rER of pancreatic cellsMicrosomes are heterogeneous mixtures of similar-sized vesicles, formed from membranes of the ER and Golgi complex. Microsomes retain activity during purification, allowing studies of function and composition.nullA. Functions of the rERProteins synthesized on ribosomes of rER include: secretory proteins, integral membrane proteins, soluble proteins of organelles. nullModification 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 eukaryotesthen remove 3 glucoses and 1 mannose in the ERnull 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 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.. nullSynthesis of membrane lipidsMost membrane lipids are synthesized 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：ER→GC、Ly、PM Transport by phospholipid exchange proteins(PEP)：ER→other organelles（including Mit and Chl）nullThe role of phospholipid translocators in lipid bilayer synthesisphospholipid translocators = Scramblase (ABC transporter Family)nullB. Functions of the sERSynthesis of steroids in endocrine cells. Detoxification of organic compounds in liver cells. System of oxygenases---cytochrome p450 family Release of glucose 6-phosphate in liver cells. Sequestration of Ca2+. Ca2+-ATPasenull3. The structure and functions of Golgi complexA.The polarity of Golgi complexnulla) Cis cisternae of Golgi complex: reduced osmium tetroxide(OsO4); b) 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 stacknullB. The Functions of Golgi complexGlycosylation in the Golgi complexGolgi complex plays a key role in the assembly of the carbohydrate component of glycoproteins and glycolipids.null 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.nullStructure of typical O- and N-linked oligosaccharidesCore RegionAfter R. Kornfeld and S. Kornfeld, 1985, Annu. Rev. Biochem. 45:631nullWhat is the purpose of glycosylation?N-linked glycosylation is prevalent in all eucaryotes, but is absent from procaryotes. It don’t require a template. There is an important 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-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 stimuli.nullThe Golgi networks are processing and sorting stations where proteins are modified, segregated and then shipped in different directions.nullGolgi complex and cell’s secretionContinual,unregulated discharge of material from the cellsThe discharge of products stored in cytoplasmic granules, in response to appropriate stimuli.null 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.null十 十 十 C. Golgi BiogenesisStages of Golgi 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 more vesiculated, in each 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.nullStable expression of mammalian Golgi proteins. a, b, Overlaid immunofluorescence and phase images of GRASP–YFP (a) and NAGTI–YFP (b) in stable, transgenic cell lines of Toxoplasma gondii. c–h, Immunofluorescence images of a transgenic cell line expressing both GRASP–CFP (green) and NAGTI–YFP (red) before (c–e) or after (f–h) treatment with 5mg /ml BFA for 10 min at 37ºC. Merged images are shown on the right. Asterisks indicate a secreted form of NAGTI–YFP that accumulates in the parasitophorous vacuole. Scale bars, 5mm.nullImmunoelectron microscopy of transgenic parasites. a–c, Cryosections of GRASP–YFP (a, c) or NAGTI–YFP (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, GRASP–YFP 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.nullBiogenesis of the Golgi apparatus in living parasites. a–h, Transgenic parasites stably expressing IMC1–CFP (blue) were transfected with plasmid DNA encoding GRASP–YFP (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 °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 NAGTI–YFP (green) were imaged over time and sample images 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 other 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 reconstructed partially and contains a single Golgi structure.null4. The structure and functions of LysosomesA. Characteristics of Lysosomes① Lysosome is a heterogenous organelle: Primary lysosomes Second lysosomes heterophagic autophagic Residual bodyPrimary Lys.Second Lysnull Figure 6-19 Histochemical visualization of lysosomes.    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 amount 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 in the top panel. (Courtesy of Daniel S. Friend.) null② Lysosomes contain plenty acid hydrolases that can digest every kind of biological molecule. ---the principal sites of intracellular digestion. Marker enzyme: acid phosphatase③Lysosome membrane: H+-pumps: internal proton concentration is kept high by H+-ATPase Glycosylated proteins: may protect the lysosome from self-digestion. Transport proteins: transporting digested materials.nullFigure 13-18 The 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 the 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.) nullFigure 13-20 The 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.) nullB. The Functions of LysosomesLysosomes are involved in three major cell functions: ① phagocytosis; ② autophagy; ③ 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.nullC. Lysosomes and DiseasesDisorders resulting from defects in lysosomal function: ①Autolysis: A break or leak in the membrane of lys releases digestive enzymes into the cell which damages the surrounding tissues (Silicosis). ② 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 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 gangliosides, a major component of brain cell membranes.null 表 关于同志近三年现实表现材料材料类招标技术评分表图表与交易pdf视力表打印pdf用图表说话 pdf 1. 神经鞘脂贮积病nullD. Biogenesis of LysosomesFigure 6-23 The transport of newly synthesized lysosomal hydrolases to lysosomes. The precursors of lysosomal hydrolases are covalently modified by the addition of mannose 6-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 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 return to the Golgi apparatus with the receptor. nullMannose 6-phosphate residues target proteins to lysosomesTargeting of soluble lysosomal enzymes to endosomes and lysosomes by M-6-P tagnull Phosphorylation of mannose residues on lysosomal enzymes catalyzed by two enzymesRecognition site binds to Signal patchGlcNAc phosphotransferasephosphodiesterasenullFigure 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 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 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 transport 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. Griffiths et al., Cell 52:329; S. Kornfeld, Annu. Rev. Biochem. 61:307; and G. Griffiths and J. Gruenberg, Trends Cell Biol. 1:5]null5. Protein Sorting Overview of sorting of nuclear-encoded proteins in eukaryotic cellsProteins are imported into organelles by three mechanisms: Gated Transport: Transport through nuclear pores Transmembrane transport: ER, Mit, Chl, Per Vesicular transport: ER-Golgi-PM-Lys, Endosomenull Road map of protein sorting nullProtein sorting: Protein molecules move from the cytosol to their target organelles or cell surface directed by the sorting signals in the proteins.nullSignal peptides and Signal patchesFigure 6-8 Two ways that a sorting signal can be built into a protein. (A) The signal resides in a single discrete stretch 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 separated 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. nullGated transport: Through gated pores—Nuclear pores; Nuclear localization 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 chaperonsnullVesicular 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.nullB. Signal Hypothesis --G.Blobel & D.Sabatini,1971. A model for the Signal Mechanism of Cotranslational ImportEvidence That Protein Synthesized on Ribosomes Attached to ER Membranes Pass Directly into the ER Lumen (D.Sabatini) Milstein : IgGnullMilstein et al: Studying the synthesis of light chain of IgG (in cell-free systems, 20 Aa longer at N-terminal end than the authentic light chain )Adding ER membranes to this system leads to the production of an IgG light chain of the correct size.null A Schematic model for the synthesis of a secretory protein on a membrane-bound ribosome of the rough ERnullSignal-recognition particle, SRP: Six different polypeptides complexed with a 300-nucleotide (7S)molecule of RNA. ER signal sequence: Typically 15-30 amino acids: Consist of three domains: a positively charged N-terminal region, a central hydrophobic region, and a polar region adjoining the site where cleavage from the mature protein will take place. A signal sequence on nascent seretory proteins targets them to the ER and is then cleaved off SRP receptor (GTP binding protein)SRP have three main active sites: One that recognizes and binds to ER signal sequence; One that interacts with the ribosome to block further translation; One that binds to the ER membrane (docking protein))nullThe sorting signal of VSV glycoproteins : Asp-X-Gln或DXEFigure 6-43. The sorting of proteins destined for the apical and basolateral plasma membranes of epithelial cells. When cultured MDCK cells are infected simultaneously with VSV and influenza virus, the VSV glycoprotein is found only on the basolateral membrane, whereas the HA glycoprotein of the influenza virus is found only on the apical membrane. Like these viral proteins, some cellular proteins are sorted directly to the apical membrane and others to the basolateral membrane via specific transport vesicles that bud from the trans-Golgi network. In certain other polarized cells, some apical and basolateral proteins are transported together to the basolateral surface; the apical proteins then move selectively, by endocytosis and transcytosis, to