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1、Biogenesis of the Golgi 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 eve
2、ry 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 NAGTIYFP (green) were imaged ov
3、er 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
4、 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 daugh
5、ter was only reconstructed partially and contains a single Golgi structure.,4. The structure and functions of Lysosomes,A. Characteristics of Lysosomes, Lysosome is a heterogenous organelle:,Primary lysosomes Second lysosomes heterophagic autophagic Residual body,Primary Lys.,Second Lys,Figure 6-19
6、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 morpholog
7、y 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 appa
8、ratus are indicated by red arrows 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 phosphatase,Lysosome membrane: H+-pumps: internal prot
9、on concentration is kept high by H+-ATPase Glycosylated proteins: may protect the lysosome from self-digestion. Transport proteins: transporting digested materials.,Figure 13-18 The low pH in lysosomes and endosomes. Proteins labeled with a pH-sensitive fluorescent probe (fluorescein) and then endoc
10、ytosed 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
11、 of Fred Maxfield and Kenneth Dunn.),Figure 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 calle
12、d the tonoplast. (Courtesy of J. Burgess.),B. The Functions of Lysosomes,Lysosomes 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
13、in the cell as lipofuscin granules.,C. Lysosomes and Diseases,Disorders 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 th
14、e 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-hexo
15、saminidase A) whose function is to degrade gangliosides, a major component of brain cell membranes.,表1. 神经鞘脂贮积病,D. Biogenesis of Lysosomes,Figure 6-23 The transport of newly synthesized lysosomal hydrolases to lysosomes. The precursors of lysosomal hydrolases are covalently modified by the addition
16、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 subsequentl
17、y 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
18、do not return to the Golgi apparatus with the receptor.,Mannose 6-phosphate residues target proteins to lysosomes,Targeting of soluble lysosomal enzymes to endosomes and lysosomes by M-6-P tag,Phosphorylation of mannose residues on lysosomal enzymes catalyzed by two enzymes,Recognition site binds to
19、 Signal patch,GlcNAc phosphotransferase,phosphodiesterase,Figure 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
20、 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
21、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
22、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 B
23、iol. 1:5,5. Protein Sorting,Overview of sorting of nuclear-encoded proteins in eukaryotic cells,Proteins 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, Endosome,Road m
24、ap of protein sorting,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 patches,Figure 6-8 Two ways that a sorting signal can be built into a protein. (A) The signal resides
25、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 regi
26、ons 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.,Gated transport:,Through gated poresNuclear pores; Nuclear localization signal (NLS); Folded and assembly for
27、m to transport.,Transmembrane transport,ER signal sequence, Mit, Chl, Per: Leader sequence; Through translocon on the membrane; Single and Unfold form; Helped by molecular chaperons,Vesicular transport,Budding, 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.,