复旦大学细胞生物学课件07内膜系统、蛋白质分类和膜运输

1、Chapter 7 Endomembrane system, Protein Sorting and membrane trafficingLearning objective1. Compartmentalization in Eukaryotic Cells; The structural and functional relationship between the ER, Golgi complexes, lysosomes and plasma membranes of eukaryotic cells;The pathways of proteins targeting and s

2、orting, 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 compartments. Tree categorie

3、s 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.Cytosol: The region of fluid content of the cytopla

4、sm 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 cells intermediary metabolism.A.

5、 Cytosol and its functionsB. 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.Intracellular compartment % of tota

6、l cell volumeCytosol 54Mittchondria 22Rough ER cisternae 9Smooth ER cisternae plus Golgi cisternae 6Nucleus 6Peroxisome 1Lysosomes 1Endosomes 1Relative volumes occupied by the major intracellular compartments in Liver CellC. The Dynamic Nature of the Endomembrane SystemMost organelles are part of a

7、dynamic system in which vesicles move between compartments.Biosynthetic pathways move proteins, carbohydrates and lipids within the cell.Secretary pathways discharge proteins from cells.Endocytic pathways move materials into cells.Sorting signals are recognized by receptors and target proteins to sp

8、ecific 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 genetic mutants;The dynamic activities of endomembrane systems are highly conserved despite th

9、e structural diversity of different cell types.De Duve, A.Claude and G.Palade,1974 Nobel Plrize2. The structure and functions of Endoplasmic Reticulum(ER)A. Functions of the rERProteins synthesized on ribosomes of rER include: secretory proteins, integral membrane proteins, soluble proteins of organ

10、elles. Modification 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-

11、celled eukaryotesthen remove 3 glucoses and 1 mannose in the ER Quality control of newly synthesized proteins-The role of N-linked glycosylation in ER protein foldingSynthesis 0f membrane lipidsMost membrane lipids are synthesized enterely within the ER. There are two exceptions:(1) sphingomyelin an

12、d 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 markdly different lipids composition.Transport by budding：ERGC、Ly、PMTransport by phospholipid exchange proteins(PEP)：ERother organelles（

13、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 organic compounds in liver cells. System of oxygenases-cytochrome p45

14、0 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;b)Reaction for enzyme mannosidaseII , localized in the medial;c) Reaction

15、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 complex Golgi complex plays a key role in the assembly of the carbohydrate component of glycoproteins

16、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.Structure of typical O- and N-linked oligosaccharidesCore RegionWhat is the

17、purpose of glycosylation?N-linked glycosylation is prevalent in all eukaryotes, but is absent from prokaryotes.It doesnt 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 sus

18、pect 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

19、digestion by proteases. (3) Regulatory roles in signaling through the cell-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 different direction

20、s.中考题之一Golgi complex and cells secretionContinual,unregulated discharge of material from the cellsThe discharge of products stored in cytoplasmic granules, in response to appropriate stimuli. Vesivular transport within the Golgi apparatus: Two views: cisternal maturation model and vesicular transpor

21、t modelTwo possible models explaining the organization of the Golgi complex and the transport from one cisterna to the next.4. The structure and functions of LysosomesA. Characteristics of Lysosomes Lysosome is a heterogenous organelle: Primary lysosomesSecond lysosomes heterophagic autophagicResidu

22、al bodyPrimary Lys.Second Lys 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+-ATPase Glycosylated p

23、roteins: may protect the lysosome from self-digestion. Transport proteins: transporting digested materials.B. The Functions of LysosomesLysosomes are involved in three major cell functions: phagocytosis; autophagy; endocytosis.Primary lys fuse with either phagocytic or autophagic vesicles, forming r

24、esidual bodies that either undergo exocytosis or are retained in the cell as lipofuscin granules.C. Lysosomes 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 ti

25、ssues (Silicosis). Lysosomal storage diseases are due to the absence of one or more lysosomal enzymes, and resulting in accumulation of material in lysosomes as large inclusios. One severe type of the disease is I-cell disease (inclusion cell disease, GlcNAc-Phosphotransferase gene mutant). Tay-Sach

26、s 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

27、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Farbers 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Krabbes 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 LysosomesThe transport of newly synthesized lysosomal hydrolases to lyso

28、somes.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 patchGlcNAc phosphotransferasephosphodiester

29、aseThe mannose 6-phosphate pathway: M-6-P receptor binds its specific oligo. at pH6.5-6.7 in TGN and releases it at pH6.0 in late endosomes.pH6.0pH6.5-6.7pH5.05. Protein Sorting Overview of sorting of nuclear-encoded proteins in eukaryotic cellsProteins are imported into organelles by three mechanis

30、ms:Gated Transport: Transport through nuclear poresTransmembrane transport: ER, Mit, Chl, PerVesicular transport: ER-Golgi-PM-Lys, Endosome Road map of protein sorting Protein sorting: Protein molecules move from the cytosol to their target organelles or cell surface directed by the sorting signals

31、in the proteins.Signal peptides and Signal patchesGated transport: Through gated poresNuclear 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;Signal and Unfold f

32、orm; Helped by molecular chaperonsVesicular 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 change

33、d during transporting.B. Signal Hypothesis -G.Blobel & D.Sabatini,1971. A model for the Signal Mechanism of Cotranslational ImportMilstein 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

34、 this system leads to the production of an IgG light chain of the correct size.Signal-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 re

35、gion, 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 offSRP receptor (GTP binding protein)SRP have three main active sites:One that r

36、ecognizes 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)The Orientation of Nascent Polypeptide The Nascent polypeptide is oriented within translocon so that the positively charged flanking sequence

37、 faces the cytosolCorrect folding of newly made proteins is facilitated by several ER proteinsProteins without any signal sequence are cytosol residual proteins6. Types of Vesicle Transport and Their FunctionsA. The three different types of coated vesicles.Different coat proteins select different ca

38、rgo and shape the transport vesicles that mediate the various steps in the biosynthetic-secretory and endocytic pathways.COPII-coated vesicles move materials from the ER to the Golgi.The assembly of a COPII-coated vesicles. SarGTP binding protein: Sar-GTP binds to the ER; Sar-GDP dissociates from th

39、e ERAntibodies is able to block the budding of vesicle from ER but have no effect on vesicle transport from one Golgi compartment to another in mammalian cell.COPI-coated vasicles transporting Escaped ER resident Proteins Back to the ER.The assembly of a COPI-coat is mediated by ADP-ribosylation fac

40、tor(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 signalKDEL(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 treatmentof GTP analogues - COPI-coated vesicle accumulated within the cell and could be isolated by centrifugation.Clathrin