Chapter 6 Cytoplasmic matrix, Endomembrane system, Protein Sorting and membrane trafficking Cytoplasmic matrix and its functions Functions of cytoplasmic matrix: B. Endomembrane System Gated transport: Vesicular transport 作业膜泡表面标志与膜泡运输过氧化物酶体中蛋白质的来源及输入机制COPI-coated vasicles transporting Escaped ER resident Proteins Back to the ER. The assembly of a COPI-coat is mediated by ADP-ribosylation factor(ARF), GTP binding protein, which is required for vesicle transfer between cisternae. COPI coated vesicles may select specific cargo. ER is an open prison. Soluble ER protein bear Retrieving signal—KDEL(Lys-Asp-Glu-Leu)in mammal and HDEL in yeast, whereas ER membrane proteins bear the signal KKXX . The KDEL receptor present in vesicular tubular clusters and the Golgi apparatus.. (3) COPI-coated vesicle were first identified by treatment of GTP analogues --- COPI-coated vesicle accumulated within the cell and could be isolated by centrifugation. A model for the retrieval of ER resident proteins. The KDEL receptor captures the soluble ER resident proteins and carries them in COPI-coated transport vesicles back to the ER. Neutral pH: dissociate from the KDEL; low pH: binding the KDEL Clathrin-coated vesicle: Transporting Cargo from the TGN to endosomes, Lysosomes, and plant vacuoles and also move materials from the PM to cytoplasmic compartments along the endocytic pathway. The TGN of Golgi is the Sourse of Clathrin-coated vesicle. (2) Clathrin-coats contain: protein clathrin-----which forms a structural scaffold, adaptors---- multisubunit,which forms an inner shell. Biogenesis 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. 4. The structure and functions of Lysosomes A. Characteristics of Lysosomes ①Lysosome is a heterogenous organelle: Primary lysosomes Second lysosomes heterophagic autophagic Residual body Primary Lys. Second Lys  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.)  ②Lysosomes contain plenty acid hydrolases that can digest every kind of biological molecule. ---the principal sites of intracellular digestion. Marker enzyme: acid phosphatase ③Lysosome 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. Figure 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.) 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 called the tonoplast. (Courtesy of J. Burgess.) B. The Functions of Lysosomes Lysosomes are involved in three major cell functions: ①phagocytosis; ②autophagy; ③endocytosis. 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 Diseases Disorders 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). ②Lysosomal 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. 表1. 神经鞘脂贮积病疾病缺失酶类主要贮积底物后果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Farber’s 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Krabbe’s 病半乳糖脑苷酯酶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 Lysosomes Figure 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. 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 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 phosphorylated enzymes bind to M6P receptors, which direct the enzymes into vesicles coated with the clathrin. The clathrin lattice surrounding these vesicles is rapidly