生物化学教学课件：Cytoplasm (细胞质)

1、5. Cytoplasm (细胞质)2Cytoplasm (细胞质)Cytoplasm: All of semitransparent(半透明的), gel-like(胶状的) and granular(颗粒状) substances except for nucleus (细胞核) or nuclear zone (核区) enclosed by plasma membrane3Components(成分) of cytoplasmCytosol (细胞质基质或胞质溶胶):dissolved nutrients (amino acids, fatty acids, saccharides)

2、and waste productsdissolved macromolecules, such as RNAs and proteins (enzymes-酶，cytoskeleton-细胞骨架)many salts (无机离子) and waterOrganelles (细胞器): Membrane-bounded organelles and Ribosomes （prokaryotes-原核生物）？Cytoplasmic inclusions(包涵物): granules with typical shapes and without metabolic activity.-glyco

3、gen (糖原)granules storing energy- fat drop storing fatty acids and sterols（固醇,甾酮）- secretory granules storing proenzyme（酶原）4胰腺外分泌腺细胞内大量胰酶原分泌颗粒 肝细胞 分泌类固醇激素的细胞内脂滴 Cytoplasmic inclusions5Metabolic pathways, biochemical reactions and Ubiquitin-proteasome system（泛素-蛋白酶体系统）Maintenance of cell shape and mov

4、ementstabilizing the intracellular environment Participating in cell signal transduction Functions of organelles Storage（贮存）2) Functions of cytoplasm6The secretory pathway: L. Caro and G. Palade (1969) pancreatic tissue（胰腺组织）: culturing in the culture medium adding amino acids(3H-Leu) by isotope（同位素

5、） labeling pulse-chase experiment and Autoradiography 胰腺外分泌细胞豚鼠胰腺组织块yeast mutants relative with endomembrane functionsMutationsEndomembrane System(内膜系统)9OUTLINEConcepts of Endomembrane System Component of Endomembrane System Endoplasmic Reticulum（ER） Golgi complex (GC) Lysosomes(溶酶体)Characteristics

6、of endomembrane systemBiological significance of endomembrane systems101.The concepts of Endomembrane System: all membrane-enclosed compartments in the cytosol, which are correlative in structure, function and biogenesis(生物发生) of membrane, including Endoplasmic Reticulum, Golgi complex, lysosomes, e

7、ndosomes (胞内体或内体) and secretory vesicles(分泌泡). 11Membrane-bounded organelles: all organelles with membrane structure in the cytosol.细胞膜、生物膜、细胞内膜系统121) Endoplasmic Reticulum（ER）ER discovery：Garnier(1897)-Optical Microscope: bar-like(条形) or silk-like structure in cytoplasm-K.R.Porter et al(1945)electr

8、on microscope:mouse fibroblast(成纤维细胞)cultured: endoplasmic region(内质区)-a net-like labyrinth (迷宫).13(1) Structure ER：a network of membrane-enclosed branching tubules and flattened sacs（潴泡）- extend from the nuclear membrane throughout the cytoplasm. ER is the largest organelle in most eukaryotic cells

9、.cytosolic space (胞质溶胶面)(潴泡面)14ER cisternal space often occupies about 10% of the total cell volume. 15ER membrane may account for about half of the total area of all cell membranes.16(2)Types of endoplasmic reticulum Rough endoplasmic reticulum (RER)-flattened sacs, membrane-bound ribosomes bound t

10、o its cytosolic surface.Smooth endoplasmic reticulum (SER) - branching tubules, no ribosomes.17Special types of ER sarcoplasmic reticulum (肌质网:心肌和骨骼肌) microsome (微粒体): ER fragments reseal to form small closed vesicles. Microsome(微粒体)：- protein synthesis and translocation, protein glycosylation, Ca2+

11、 uptake and release, and lipid synthesis. 差速离心19(3)Chemical component of ER ER: proteins (60%), Lipids (40%). Proteins: Glycoprotein (糖蛋白), enzymes, the component of electron transport chain(电子传递链)such as Cytochrome P450-glucose-6-phosphatasemarker enzyme-the distribution of ER proteins is heterogen

12、eous(异质性) ribophorin (核糖体结合蛋白): RER P450 enzymes（SER标志酶）: SER translocon（易位子）protein complex: RERLipids：Phospholipids (Principal lipid ingredient)20(4)ER functionSynthesis and transport of proteins- Secretory proteins (antibody, hormones, extracellular matrix component (Glycoprotein（糖蛋白）)- integral

13、membrane proteins such as translocon- Water-soluble proteins for residence within the endomembrane system Function of RERPolyribosomes are located to RER ?1971 Blobel et al (USA): signal sequence?21 Research of signal sequenceMilstein等(1972)-cell-free system synthesis of IgG light chain: proteins sy

14、nthesized more 20-aa than proteins released into the extracellular. G.Blobel等- In vitro protein translation with cell-free system: signal sequence is existing. The role of signal sequence ？ RER?22 Transport direction of proteins synthesized on RERs1960s，Redman and Sabatini, RER microsomes放射性嘌呤霉素? wh

15、ether newly synthesized proteins enter RER?进入ER腔内23（1）First, synthesize secretary proteins with polyribosomes （+ER）, （2）then remove ER with detergent, continue protein synthesis analyze protein products collected at different time pointResults: the products collected : early or late ?Secretary prote

16、in synthesispolyribosome24 Transport way of proteins synthesized by RER Cell-free system adding RER microsomes - synthesize secretory proteins，adding proteinase Cell-free system（ RER microsomes) - synthesize secretory proteins，adding both proteinase and detergentResults: proteins are degradated ?Hyd

17、rolysis experiment using proteinaseco-translation translocation (共翻译转运或协同翻译): 边合成边运输25 Signal peptide （信号肽）the amino acid sequence at the N-terminus of the newly synthesizing polypeptide chain 15-35 amino acid residues, N-terminus containing one or several positively charged residues, followed by 6-

18、12 hydrophobic residues，directing polypeptide chains to ER lumen（腔）. 疏水核心HIV-1型病毒的糖蛋白26 The signal sequence has no specificity for the polypeptide chain guided by using protein localization technique the signal sequence is cut after the polypeptide chain enters ER lumen27 Several protein complexes b

19、inding to ER membrane (Walter, Blobel 等1981） SRP（signal recognition particle信号识别颗粒): recognising and binding to signal sequence, directing ER Signal to the receptor DP (docking protein,停泊蛋白): the receptor located on ER membrane. translocon (易位子)28 SRP: a cytosolic ribonucleoprotein (RNP) particle- s

20、ix different polypeptides -a small RNA molecule (300 nt)three domains（功能域）signal-sequence-binding site (P54)translational pause domain (P9/P14)SRP receptor binding site (P68/P72). rodlike structure信号肽识别结合位点(P54)翻译暂停结构域受体结合位点29DP: an integral membrane protein complex.heterodimer: hydrophilic subunit

21、with GTP-binding domains, hydrophobic subunit (embedded in RER membrane).SRP receptorSRP-DP识别的GDP-GTP交换循环30 translocon (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 po

22、re is a dynamic gated structure three types of Sec61 in mammals: , and 31Signal hypothesis（信号假说）1975年，Blobel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 peptide eme

23、rges from the ribosomes, SRP binds to the signal peptide, thereby inducing a pause in translation. 32 SRPribosome complex binds to the SRP receptor (DP). This interaction brings the SRPribosome complex to a protein translocator. 33GTP hydrolysis，SRP is released and the protein translocator opens，the

24、 ribosome then binds to the translocator， the translocator then inserts the polypeptide chain into the membrane. 34protein synthesis resumes， the translocator transfers the polypeptide chain across the lipid bilayer, the polypeptide chain enters ER lumen，the signal sequence is then cleaved off by a

25、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. 35 Processing and Modification of newly synthesized proteins in ERN-linked glycosylation (Asn-X-Ser/Thr)The formation of

26、disulfide bonds between the side chains of cysteine residues : protein disulfide isomerase (PDI-二硫键异构酶) Hydroxylation(羟基化): Pro, Lys36Assembly of oligosaccharide chain：saccharide molecules（供体为核甘糖）-glycosyltransferase-dolichol phosphate (磷酸多萜醇) N-Linked glycosylation第一个糖基：N-乙酰葡萄糖胺Glycosylation: oligosaccharyl -oligosaccharyl transferase NH2 group of Asn residue第