基質(zhì)與內(nèi)膜

1、Chapter 6,Cytoplasmic matrix, Endomembrane system, Protein Sorting and membrane trafficking,Learning objective,1. Compartmentalization in Eukaryotic Cells; The structural and functional relationship among the ER, Gol

2、gi 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 synthesiz

3、ed;Types of vesicle transport and their functions.,1. The Compartmentalization in Eukaryotic Cells,Membranes divide the cytoplasm of eukaryotic cells into distinct compartments. Three categories in eukaryotic cells:

4、 (1) the endomembrane system: ER, Golgi complex, Lys., secretory vesicles. (2) the cytosol. (3) mitochondria, chloroplasts, peroxisomes, and the nucleus.,Membrane-bound structures (organelle

5、s) are found in all eukaryotic cells.,Cytoplasmic matrix and its functions,Cytoplasmic Matrix: The region of fluid content of the cytoplasm outside of the membranous organelles. Aqueous solution of large and small molecu

6、les 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.,C

7、ytoplasmic matrix (Cytosol) and Endomembrane System,Functions of cytoplasmic matrix:,The protein synthesis, degradation and modification.,,Cells carefully monitor the amount of misfolded proteins. An accumulation

8、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.,B. Endomembrane System,Endomembrane System

9、: The structural and functional relationship organelles including ER,Golgi complex, lysosome, endosomes, secretory vesicles.Membrane-bound structures (organelles) are found in all eukaryotic cells.,Relative volumes occu

10、pied by the major intracellular compartments in Liver Cell,,C. The Dynamic Nature of the Endomembrane System,Most organelles are part of a dynamic system in which vesicles move between compartments.Biosynthetic parthway

11、s 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

12、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;Insights gained from the study of geneti

13、c 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 Plrize,2. The structure and functions of

14、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 me

15、mbrane elements.,Microsome(100-200nm),rER of pancreatic cells,Microsomes are heterogeneous mixtures of similar-sized vesicles, formed from membranes of the ER and Golgi complex. Microsomes retain activity during purifica

16、tion, allowing studies of function and composition.,,A. Functions of the rER,Proteins synthesized on ribosomes of rER include: secretory proteins, integral membrane proteins, soluble proteins of organelles.,Modificati

17、on 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

18、 precursor of 14 residues is the same in plants, animals, and single-celled eukaryotes,then remove 3 glucoses and 1 mannose in the ER,Quality control of of newly synthesized proteins---The role of N-linked glycosylation

19、in ER protein folding,Quality control: ensuring that misfolded proteins do not leave ER,The lumen of rER contains:Bip and calnexin (chaperones) : that recognize and bind to unfolded or misfolded proteins and give them c

20、orrect 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 lipids,M

21、ost 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 (thems

22、elf).The membranes of different 0rganelles have markedly different lipids composition.Transport by budding：ER→GC、Ly、PMTransport by phospholipid exchange proteins(PEP)：ER→other organelles（including Mit and Chl）,The rol

23、e of phospholipid translocators in lipid bilayer synthesis,phospholipid translocators =Scramblase(ABC transporter Family),,B. Functions of the sER,Synthesis of steroids in endocrine cells.Detoxification of organic co

24、mpounds in liver cells. System of oxygenases---cytochrome p450 familyRelease of glucose 6-phosphate in liver cells.Sequestration of Ca2+. Ca2+-ATPase,,3. The structure and functions of Golgi complex,,A.The pola

25、rity of Golgi complex,a) 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

26、 trans cisternae.,,Regional differences in membrane composition across the Golgi stack,B. The Functions of Golgi complex,Glycosylation in the Golgi complex,Golgi complex plays a key role in the assembly of the carboh

27、ydrate 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-link

28、ed oligosaccharides takes place in Golgi complex.,Structure of typical O- and N-linked oligosaccharides,Core Region,,,After R. Kornfeld and S. Kornfeld, 1985, Annu. Rev. Biochem. 45:631,What is the purpose of glycosylati

29、on?,,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 o

30、f 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 carbohydr

31、ate-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 c

32、ell-surface receptor Notch, to allows these cells to respond selectively to activating stimuli.,,The Golgi networks are processing and sorting stations where proteins are modified, segregated and then shipped in differen

33、t directions.,Golgi complex and cell’s secretion,Continual,unregulated discharge of material from the cells,The discharge of products stored in cytoplasmic granules, in response to appropriate stimuli.,Vesivular transpor

34、t within the Golgi apparatus: Two views: cisternal maturation model and vesicular transport model,Two possible models explaining the organization of the Golgi complex and the transport from one cisterna to th

35、e next.,,十,十,十,C. Golgi Biogenesis,Stages 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 c

36、ategories according to the number and size of the Golgi: a, single Golgi; b, single, elongated Golgi; c, two Golgi; d, Golgi, often more vesiculated, ineach nascent daughter cell, delineated by the growing inner membran

37、e 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 expres

38、sion 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 transgeni

39、c 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 o

40、f NAGTI–YFP that accumulates in the parasitophorous vacuole. Scale bars, 5mm.,Immunoelectron microscopy of transgenic parasites. a–c, Cryosections of GRASP–YFP (a, c) or NAGTI–YFP (b) transgenic parasites, pretreated for

41、 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 t

42、o 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, Qu

43、antification of images in a and b. Results are presented as mean ± s.d. gold particles /um2.,Biogenesis of the Golgi apparatus in living parasites. a–h, Transgenic parasites stably expressing IMC1–CFP (blue) were tr

44、ansfected 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. Repres

45、entative 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 paras

46、ites 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 eventu

47、al 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

48、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

49、 reconstructed partially and contains a single Golgi structure.,4. The structure and functions of Lysosomes,A. Characteristics of Lysosomes,① Lysosome is a heterogenous organelle:,Primary lysosomesSecond lysosomes h

50、eterophagic autophagicResidual body,Primary Lys.,Second Lys,,,,Figure 6-19 Histochemical visualization of lysosomes.    Electron micro-graphs of two sections of a cell stained to reveal

51、 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 th

52、e 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 thoug

53、ht to be carrying acid hydrolases from the Golgi apparatus are indicated by red arrows in the top panel. (Courtesy of Daniel S. Friend.),② Lysosomes contain plenty acid hydrolases that can digest every kind of biological

54、 molecule. ---the principal sites of intracellular digestion.Marker enzyme: acid phosphatase,③Lysosome membrane: H+-pumps: internal proton concentration is kept high by H+-ATPase

55、Glycosylated proteins: may protect the lysosome from self-digestion. Transport proteins: transporting digested materials.,Figure 13-18 The low pH in lysosomes and endosomes.    Protein

56、s 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 encount

57、ers 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

58、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 th

59、e 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:① phagocytosis; ② autophagy; ③ endocytosis.

60、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 fr

61、om 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

62、 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-Phosphotran

63、sferase 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. 神經(jīng)鞘脂貯積病,D. Biogenesi

64、s 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

65、 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 modifi

66、ed 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

67、 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 targ

68、et 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

69、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

70、 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

71、 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 the

72、n 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 lysosom

73、al 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