细胞生物学：Chapter 9. Endomembrane System or Intracellular compartment-2s

1、(4)(4)ER functionSynthesis and transport of proteins- Secretory proteins (antibody, hormones, extracellular matrix component (Glycoprotein（糖蛋白）糖蛋白）)- integral membrane proteins such as translocon- Water-soluble proteins for residence within the endomembrane system Function of RERPolyribosomes are lo

2、cated to RER ?Polyribosomes or proteins ?1971 Blobel et al (USA): signal sequence? Research of Research of signal sequencesignal sequenceMilstein等(1972)-cell-free system synthesis of IgG light chain: proteins synthesized more 20-aa than proteins released into the extracellular. G.Blobel等等- In vitro

3、protein translation with cell-free system: signal sequence is existing. The role of signal sequence ？ RER? Transport direction of proteins synthesized on RERs1960s，Redman and Sabatini, RER microsomes放射性放射性嘌呤霉素嘌呤霉素? whether newly synthesized proteins enter RER?进入ER腔内First, synthesize secretary protei

4、ns with polyribosomes, then remove ER with detergent, continue protein synthesis Finally, analyze protein products collected at different time pointResults: the products collected : early or late ?Secretary protein synthesispolyribosome Transport way of proteins synthesized by RER Cell-free system a

5、dding RER microsomes - synthesize secretory proteins，adding proteinase Cell-free system（ RER microsomes) - synthesize secretory proteins，adding both proteinase and detergentResults: translating proteins are degradated ?Hydrolysis experiment using proteinaseco-translation translocation (共翻译转运或协同共翻译转运

6、或协同翻译翻译): 边合成边运输边合成边运输 Signal peptide Signal peptide （信号肽信号肽）the amino acid sequence at the N-terminus of the newly synthesizing polypeptide chain 15-35aa residues, N-terminus containing one or several positively charged residues, followed by 6-12 hydrophobic residues，directing polypeptide chains to

7、 ER lumen. 疏水核心疏水核心HIV-1型病毒的糖蛋白型病毒的糖蛋白 The signal sequence has no specificity for the polypeptide chain guided by using protein localization technique the signal sequencesequence is cut after the polypeptide chain enters to ER lumen Several protein complexes binding to ER membrane (Walter, Blobel 等等

8、1981） SRP（signal recognition particle): recognising and binding to signal sequence, directing ER Signal to the receptor DP (docking protein,停泊蛋白停泊蛋白): the receptor located on ER membrane. translocon (易位子易位子) SRP: a cytosolic ribonucleoprotein (RNP) particle- six different polypeptides -a small RNA m

9、olecule (300 nt)three domains translational pause domain (P9/P14)signal-sequence-binding site (P54)(P54)SRP SRP receptor binding site (P68/P72).(P68/P72). rodlike structure信号肽识别结信号肽识别结合位点合位点(P54)翻译暂停翻译暂停结构域结构域受体结受体结合位点合位点DP: an integral membrane protein complex.heterodimer: hydrophilicsubunit with G

10、TP-binding domains, hydrophobic subunit (embedded in RER membrane).SRP receptorSRP-DP识别的识别的GDP-GTP交换循环交换循环 translocon (易位子易位子) Several copies of the Sec61 complex assemble to form a cylinder translocon channel (56 nm high and 8.5 nm in diameter) a central aqueous pore, roughly 2 nm in diameter, the

11、pore is a dynamic gated structure three types of Sec61 in mammals: , and Signal hypothesis（信号假说）信号假说）1975年，年，Blobel等等 Proteins translocated by ER start synthesizing in cytosolic free ribosomes, the signal peptide at the N-terminus determine their translocation target direction.When the ER signal pep

12、tide emerges from the ribosomes, SRP binds to the signal peptide, thereby inducing a pause in translation. SRP of SRPribosome complex binds to the SRP receptor (DP). This interaction brings the SRPribosome complex to a protein translocator. GTP hydrolysis，SRP is released and the protein translocator

13、 opens，the ribosome then binds to the translocator， the translocator then inserts the polypeptide chain into the membrane. protein synthesis resumes， the translocator transfers the polypeptide chain across the lipid bilayer, the polypeptide chain enters ER lumen，the signal sequence is then cleaved o

14、ff by a signal peptidase in the ER membrane. protein synthesis ends，the mature protein is released into the ER lumen. the protein translocator is closed， the ribosome leaves ER membrane. 2021-7-1017 Orientation of transmembrane proteins into RER membrane singlepass transmembrane proteins (单次跨膜蛋单次跨膜蛋

15、白白)doublepass transmembrane protein into ER membrane multipass transmembrane proteins 18 Integration of singlepass transmembrane proteinsFigure 12-46 Molecular Biology of the Cell ( Garland Science 2008)-start transfer signal (起始转移信号起始转移信号-signal peptide ) + stop transfer sequence (停止转移序列停止转移序列): N-

16、terminal cleavable signal sequence -internal signal sequence (内部信号序列内部信号序列): noncleavable signal sequencesN-terminal in ER lumenC-terminal in cytosol信号序列两端氨基酸信号序列两端氨基酸带正电荷的多少，正带正电荷的多少，正电荷多的一边在胞质，电荷多的一边在胞质，另一端在另一端在ER腔腔转运时，信号序列转运时，信号序列N端始终朝向端始终朝向ER外侧外侧Integration of doublepass or multipass transmembra

17、ne protein into ER membrane Processing and Modification of newly synthesized proteins in ERN-linked glycosylation (Asn-X-Ser/Thr)The formation of disulfide bonds between the side chains of cysteine residues : protein disulfide isomerase (二硫键异构酶) Hydroxylation: Pro, Lys Addition of glycosylphosphatid

18、ylinositol (GPI,糖基磷脂酰肌醇)anchors Export of proteins from the ERAssembly of oligosaccharide chain：saccharide molecules（供体为核甘糖）-glycosyltransferase-dolichol phosphate (磷酸多萜醇) Glycosylation: oligosaccharyl -oligosaccharyl transferase NH2 group of Asn residue N-Linked glycosylation第一个糖第一个糖基：基：N-乙酰葡萄乙酰葡萄糖

19、胺糖胺Asn-X-Ser/Thr Folding and assembly of nascent polypeptides Bip (heavy-chain Binding protein): a Hsp70-like chaperone protein binds to the unfolded polypeptide chain as it crosses the ER membrane and mediates protein folding and the assembly of multisubunit proteins within the ER Functions of SERv

20、Glycogen metabolism v Lipid （phospholipid and cholesterol） synthesis and transportvDetoxification: oxygenases (cytochrome P450 family) vCa2+ storage Ca2+-ATPases in the sarcoplasmic reticulum membrane vSynthesis of steroid hormones(类固醇激素类固醇激素) vExport of Lipids from the SER Glycogen decomposition葡萄糖

21、磷酸变位酶糖原颗粒附着于SER的胞质面G-1-P不能通过扩散进入血液ER的标志酶是的标志酶是葡萄糖葡萄糖-6-磷酸酶磷酸酶 Synthesis and translocation of phospholipids -The enzymes for lipid synthesis lie in the SER membrane, their activity sites face to the cytosol；substrates from the cytosol，phospholipid translocators or flippases (转位酶) transfer the phospho

22、lipids into ER lumen.卵磷脂卵磷脂CoA转移酶转移酶3-磷酸甘油磷酸甘油乙酰转移酶乙酰转移酶2) 2) Golgi complex (Golgi complex (GC) or or Golgi apparatusDiscovered by Camillo Golgi (Italian scientist) in 1898(1) Structure Shape- 4-8 flattened membrane vesicles or disclike sacs (cisternea) surrounded by a number of different sizes of s

23、pherical membrane vesicles.ER budding into small vesicles -medial cisternae(中间中间潴泡潴泡)：flattened cisternae, bow-shaped-forming face or cis face(顺面顺面) )toward the nucleus-Cis-Golgi Network, CGN(高尔基高尔基内侧网络内侧网络): -mature face or trans face toward plasma membrane- Trans Golgi Network, TGN (高尔基外侧网络高尔基外侧网络

24、)convex sideconcave side Structure of GCFunction of CGN: GC entrance, its membrane similar to that of ER. acceptance, identification and classification of substances newly synthesized from the ER and dispatches them to GC medial cisternae.Function of medial cisternae：glycosyl modification and the fo

25、rmation of glycolipids and polysaccharidesFunction of TGN: GC exit -protein sorting, packaging and exportingN乙酰葡糖胺乙酰葡糖胺GC is a polar organelle (Polarity)： -GC structure (Cis region (close to RER); medial region and trans region)-biochemical properities -direction of substance transportation (2) The

26、number and chemical composition Number: more GCs in secretory cells. Chemical composition -proteins (60%) and lipids(40%) marker enzyme: glycosyltransferase（糖基转移酶糖基转移酶）(3) (3) Functions of GCGCProtein modification and processing:O- linked GlycosylationProtein and lipid transport Protein sorting Cell

27、 secretion Formation of plant cell wall Proteoglycan(蛋白聚糖蛋白聚糖) assemblyparticipating in lysosome formation protein processing O-linked glycosylation: The glycosyl is transferred to the OH group of ser (Thr or hyp羟赖氨酸羟赖氨酸) residue of polypeptide chainO-linked glycosylation. The first glycosyl is N- a

28、cetyl galactosamine(N-乙酰半乳糖), and then one after another monosacchavide is added. Modifications to N-linked oligosaccharides in Golgi成熟的成熟的糖蛋白糖蛋白N-乙酰葡萄糖胺乙酰葡萄糖胺半乳糖半乳糖岩藻糖岩藻糖 唾液酸唾液酸 The biological functions of glycosylation Protect proteins from enzyme digestion and increase protein stabilityendow the

29、proteins with signaling function in cell-cell adhesion ； help proteins to fold Properly(胰岛素胰岛素) Proprotein processing in Golgi：inactive proprotein（GC）removing the sequence at the N-terminus or two terminusactive polypeptide ( Processing of Proinsulin ) Protein transport through Golgi Protein Sorting and Protein Sorting and ExportExportER retention proteinslysosome proteins and membrane proteins Secretory proteins and extracellular matrix component-Sorting and Export : mainly depend on the interaction between signal sequence（or signal patch）of proteins sorted and . ER protein retention in ERE