1、1 A common feature for lysosomal enzymes： -containing mannose-6-phosphate (M6P, 6-磷酸甘露糖) in its oligosaccharide chain，-The receptor of lysosomal enzymes specifically binding to M6P(5) Lysosome biogenesis2N-乙酰葡萄糖胺磷酸轉移酶N-乙酰葡萄糖苷酶N-乙酰葡萄糖胺催化磷酸轉移Phosphorylation of mannose:N- GlcNAc phosphotransferase (N-乙

2、酰葡萄糖胺磷酸轉移酶) 3 signal patch (信號斑) ：存在于完成折疊的蛋白質中，由幾個信號序列折疊在一起構成的三維結構。N-乙酰葡萄糖胺磷酸轉移酶識別信號斑4溶酶體酶合成及糖基化修飾寡糖鏈甘露糖殘基磷酸化溶酶體酶分選磷酸基團被去除-初級溶酶體部分M6P受體也會到達細胞質膜M6P受體蛋白在pH6.57的條件下與M6P結合，在pH=6條件下脫落Mannose-6-phosphate（M6P） pathway 溶酶體酶的識別和分選缺損途徑（default pathway）受體介導低pH時晚期內體晚期內體與溶酶體融合，將溶酶體酶送入溶酶體在-細胞患者的某些細胞如肝細胞中，盡管由于N-乙酰

3、葡萄糖胺磷酸轉移酶的突變，溶酶體酶不能形成M6P標志，但溶酶體酶仍有正常溶酶體酶的補給-不依賴于M6P的分選途徑。5 PLANT CELL VACUOLES62.Characteristics of endomembrane systemIndependent enclosed compartments specialized functionDynamic and InterdependentBiosynthetic pathway Secretory pathway Endocytosis pathway:7Vesicle transport-transport way of protei

4、ns and lipids*a dynamic, interconnected network：protein sortingProtein sorting inside cell83.Biological significance of endomembrane systemsforming some functionally specialized compartments -provide microenvironment within the cell for varieties of biochemical reactions.-raising reactive efficiency

5、 by improving important molecular concentration. improving its regulatory capacity by enlarging membrane area of metabolism, which provides attachment bracket for the enzyme 9Function as a intracellular membrane system -all the membrane is synthesized and assembled uniformly, thereby keeping intrace

6、llular membrane structure in agreement. -intercoordination in functiongroup-like management- membrane traffic (膜流) by the secretory and endocytic pathways keeps cells youthful-Membrane transformation.膜流（membrane flow or membrane traffic ）：細胞的各種膜結構相互聯系和轉移現象稱為膜流。內質網至高爾基體膜流，高爾基體至內質網的反向膜流不僅有物質運輸，還存在膜的轉換

7、。10思考題簡述細胞質的概念及組成成分區分生物膜、細胞膜和內膜系統的概念簡述內質網的類型簡述內質網和高爾基體的結構與功能舉例高爾基體的極性體現在哪些方面簡述溶酶體類型和功能簡述內膜系統的特征和生物學意義8. 簡述細胞分泌的概念和途徑,并分析胰酶原顆粒和溶酶體酶在胞質中如何合成并轉運至靶位點的Chapter 6Ribosome， synthesis and transport of proteins Ribosome: structure and function Transport of proteinsFate of proteinsOUTLINE1. Ribosome (核糖體)The r

8、ibosome is a small, campact ribonucleoprotein particle (核糖核蛋白顆粒) composed of RNAs and proteins，and involved in protein synthesis. Ribosome for shortProkaryotes1)Ribosome structure(1)Shape of ribosome -no membrane structure-two subunits(2)Chemical component of ribosomes Each subunit consists of rRNAs

9、 and proteins RNAs inside the subunit, proteins over the surface (The proteins of the small subunit is named as S1, S2, . and the proteins of the large subunit is named as L1, L2, .).(31)Main types of ribosomes3.9103-4.5103kDacomponentProkaryotes(70S)Eukaryotes(80S)component ratio two-thirds rRNA(2/

10、3)one-third protein (1/3)three-fifths rRNA(3/5)two-fifths r protein (2/5)rRNAs ? 16S rRNA：1457-1544 nt，the conserved sequence of ACCUCCUUA at the 3end is complementary with the purine-rich SD sequence. 18S rRNA: the homologous nucleotide sequence to 16S rRNA at the 3end.Sequence characteristics of r

11、RNAs 5S rRNA (細菌)：120 or 116 nt, two highly conserved sequences, one CGAAC sequence may recognise tRNA, the other may interact with GTCG sequence of TC loop. 5.8S rRNA：160nt，the CGAAC sequence similar to highly conserved one of 5S rRNA. 23S rRNA：2904 nt, the sequence complementary with tRNAfMet sequ

12、ence. 28S rRNA：3890-4700nt，unknown function.TC臂負責和核糖體上的rRNA 識別結合Compositions of ribosome from different sources SourceRibosomeSubunits ribosomal RNAs Cytoplasm 80S 60S (大亞基) 28S(Eukaryote) 40S (小亞基) 18S, 5.8S, 5S Cytoplasm 70S 50S (大亞基) 23S(Prokaryote) 30S (小亞基) 16S, 5S Mitochondrion 55-60S 45S (大亞基

13、) 16S(Mamalian) 35S (小亞基) 12S Mitochondrion 75S 53S (大亞基) 21S(Yeast) 35S (小亞基) 14S Mitochondrion 78S 60S (大亞基) 26S(Plant) 45S (小亞基) 18S, 5SChloroplast 70S 50S (大亞基) 23S 30S (小亞基) 16S, 5S2) Biogenesis of RibosomeSynthesis of ribosomal proteins and rRNAs Assembly of subunits(1)Synthesis and processing

14、 of rRNAs Eukaryotic cells: rRNA genes of 18S, 5.8S and 28S rRNA, as a transcription unit, are tandemly(串聯地) repeated 1001,000 times on chromosomes. Transcription RNA polymerase I primary transcript:34S45S18S5.8S28S23 pre-rRNA in different species Species pre-rRNA Drosophila (果蠅)34S S. pombe (裂殖酵母)3

15、7S Tobacco 38S Frog 40S Chicken 45S Mouse 45S Human 45S24 Processing of 45S pre-rRNA Cleavage(剪切)25Modification of pre-rRNAsMethylation(甲基化)（核仁小RNA）假尿（嘧啶核）苷 Synthesis and Processing of 5S rRNAThe 5S rRNA genes are located outside the nucleolus(核仁) and have over 50000 copies. Transcription RNA polyme

16、rase III : binds to a promoter site (內部啟動子) located within the transcribed portion of the gene. Prokaryotic cells: all rRNA genes (E.coli) as a transcription unit of 16S-23S-5S，7 copies，several types of tRNA molecules lie in every transcription unit simultaneously. Transcription: RNA polymerase rRNA

17、s purified and ribosomal proteins isolated using ion exchange resin are mixed: -a self-assembly processRibosomal proteins combine with rRNAs in sequence (temporally and spatially) -primary binding protein-secondary binding protein.(2)Assembly of ribosomal subunits The relationship between rRNA and r

18、ibosomal proteins in small subunit of E.coli ribosome. The lines denote the interaction, which the thick lines represent stronger role, the thin ones represent weaker role（Alberts et al，1989）. Even if ribosomal proteins (E.coli) are mutated or deleted, there are no effect of “all” or “none” on prote

19、in synthesis. If rRNA genes are mutated, protein synthesis will be controlled.If rRNAs are removed, ribosome can not keep in normal appearance rRNAs were structurally central component and proteins play auxiliary (輔助)roles in keep rRNA conformation in ribosome. The effect of rRNAs and proteins on th

20、e role of the ribosomeL11-rRNA復合物的三維結構(引自Porse et.al.,1999)less than 1mM higher than 10mM70S ribosome3) Function of Ribosome33氨基酸活化,翻譯起始,延伸,終止(1)Relationship between its structure and Function There are a series of key active sites relative to protein synthesis. Four binding sites for RNA molecules:

21、 one is for the mRNA and three for tRNAs. The binding site for mRNA澳大利亞科學家約翰夏因與琳達爾加諾 Three binding sites for tRNAsaminoacyl-tRNA（acceptor site）peptidyl-tRNA(donor site )spent tRNA-binding site(exit site)APEA catalytic site for a peptidyl transferase(肽酰轉移酶)23S rRNA catalyzes the formation of peptide

22、bond. Harry Noller等(1992) 50S ribosomal proteins, treatment with protease K、SDS、phenol（苯酚）: no effect on protein synthesis 50S rRNA treatment with RNase (核酸酶): inhibiting protein synthesis 23S rRNA forms a highly structured pocket that, through a network of hydrogen bonds, precisely orients the two

23、reactants and thereby greatly accelerates their covalent joining. The main role of the ribosomal proteins seems to stabilize the RNA core. 23S rRNA owns enzyme activity-Ribozyme. Ribozymes: RNA molecules capable of catalyzing chemical reactions in the absence of proteins. Figure 671 Structure of the

24、 L15 protein in the large subunit of the bacterial ribosome. The globular domain of the protein lies on the surface of the ribosome and an extended regionpenetrates deeply into the RNA core of the ribosome. The L15 protein is shown in yellow and a portion of the ribosomalRNA core is shown in red. (F

25、rom D. Klein,P.B. Moore and T.A. Steitz, J. Mol. Biol.340:141147, 2004. other binding sites The binding sites for translation initiation factors (IF1, IF2, IF3) elongation factors (EF-Tu and EF-Gin bacteria, and EF1 and EF2 in eukaryotes), and release factors The binding sites for the antibiotics(潮霉

26、素)（四環素）（壯觀霉素）（鏈霉素）（鏈陽性菌素）（氯霉素）（紅霉素）(嘌呤霉素)(苯丙氨酰基-tRNA)（苯丙氨酸）the existence forms of ribosomes in CytosolProkaryotesEukaryotes70S80Spolyribosome or polysomefree ribosome, polysome,membrane-bound polyribosome 4) Polyribosome (多聚核糖體)Polyribosome (or polysomes) : Many ribosomes connected by a single of mR

27、NA molecule-ribosomes spaced as close as 80 nucleotides apart（每隔80核苷酸有一個核糖體） The synthesis of most proteins: between 20 seconds and several minutes Biological significance for polyribosomesFor synthesis of a variety of intracellular polypeptides: the number of the polypeptide molecules synthesized p

28、er unit time are roughly equal, regardless of their molecular size or the length of the mRNA. These multiple initiations allow the cell to make more protein molecules in a given time. These multiple initiations improve the utilization efficiency for mRNAs.(一條mRNA可同時合成多條多肽鏈)452. Sorting and and targe

29、ting of proteins46two types of protein translocation ways(共翻譯轉運或協同翻譯)（翻譯后轉運）47-signal sequence of proteins -sorting receptor in target organelles(1）Targeting of proteins 48核定位信號（nuclear-localization signal, NLS）Gunter Blobel 1999年49Gated transport(門控運輸)Transmembrane transport (穿（跨）膜運輸：胞質中合成的肽鏈進入線粒體、

30、內質網時，肽鏈去折疊)Vesicle transport (膜泡運輸)(2）Transport pathways of proteins in cytoplasmic matrix 都需能量 Vesicle transport (膜泡運輸)Proteins located in different compartments were sorted？Transport routes for different vesicles ？5151Known Sorting Signals That Direct Proteins to Specific Transport VesiclesSignal

31、Sequence* Proteins with Signal Signal Receptor Vesicles Lys-Asp-Glu-Leu ER-resident luminal proteins KDEL receptor in COPI (KDEL) cis-Golgi membrane Lys-Lys-X-X ER-resident membrane COPI _and _subunits COPI (KKXX) proteins (cytosolic domain) Di-acidic Cargo membrane proteins in COPII Sec24 subunit C

32、OPII (e.g., Asp-X-Glu) ER (cytosolic domain) Mannose 6-phosphate Soluble lysosomal enzymes M6P receptor in trans-Clathrin/AP1 (M6P) after processing in cis-Golgi Golgi membrane Secreted lysosomal enzymes M6P receptor in plasma Clathrin/AP2 membrane Asn-Pro-X-Tyr LDL receptor in the plasma AP2 comple

33、x Clathrin/AP2 (NPXY) membrane (cytosolic domain) Tyr-X-X- _ Membrane proteins in trans-AP1 (_1 subunit) Clathrin/AP1 (YXX_) Golgi (cytosolic domain) Plasma membrane proteins AP2 (_2 subunit) Clathrin/AP2 (cytosolic domain) Leu-Leu Plasma membrane proteins AP2 complexes Clathrin/AP2 (LL) (cytosolic

34、domain) *X, any amino acid; _hydrophobic amino acid. Single-letter amino acid abbreviations are in parentheses.52521）Types of coated vesicles（衣被小泡）clathrin-coated vesicle (網格（成籠）蛋白有被小泡)COPII-coated vesicles (COPII有被小泡)COPI-coated vesicles (COPI有被小泡)53532)Transport routes of coated vesicles in cellsE

35、RGolgiLysosomePlasma membraneCOPIIClathrinClathrinplant vacuoleCOPIendosomeCGN TGNCOPIClathrinClathrinClathrin5454543）Formation of coated vesicles Clathrin(網格蛋白或籠形蛋白) coated vesicles -Clathrin: formation in TGN of Golgi complex and plasma membrane. clathrin Dimer (重鏈和輕鏈: 網格蛋白) clathrin triskelion （包

36、被的結構單位）Clathrin coated pits三個Clathrin二聚體三腳蛋白復合體Clathrin coated pits三個Clathrin triskelion(三腳蛋白復合體)Assembly pathway of clathrin coat subunits565656（發動蛋白縊斷蛋白）發動蛋白(dynamin) 是一種GTPase一個hsp70家族的伴侶蛋白作為一個脫被的ATPase參與了成籠蛋白的脫被過程ARF（assembly response factor-裝配反應因子：一種單體GTPase） is involved in formation of clathrin coat in Golgi complex, as coat-recruitment GTPase.衣被成分主要為網格蛋白和銜接蛋白銜接蛋白識別受體5757 COPI-coated vesicles: COPI-coated proteins: eight types of subunits. COPI: f