细胞生物学第五章 细胞内膜系统与囊泡转运

COPI
COPI有被囊泡的主要功能
Bidirectional transport between the ER and the Golgi
网格蛋白有被囊泡（CCV）
Initially a clathrin The cargo coated pit forms. receptors extend
产生于高尔基复合体、细胞膜的网格蛋白有被囊泡
特征：
直径在50～100nm之间，外被以由网格蛋白纤维构成的网架结构 在网格蛋白结构外框与囊膜之间填充、覆盖有大量的衔接蛋白
功能：
介导从高尔基复合体向溶酶体、胞内体或质膜外的物质转运 将外来物质转送到细胞质或溶酶体
网格蛋白
网格蛋白有被小泡形态结构
三、囊 泡 转 运 的 分 子机 制
As the process continues, the vesicles rounds up and pinches off.
网格蛋白有被囊泡（CCV）的分子基础 CCV有被囊泡
Clathrin网格蛋白
Clathrin网格蛋白
Clathrin “heavy chain”
“Light chain”
Addition of M6P to lysosomal enzymes in cis-Golgi M6P receptor in TGN directs transport of enzymes to lysosome via clathrin-coated vesicles
How do cells take up specific macromolecules? “Receptor-mediated endocytosis”
Summary of “receptor-mediated” endocytosis of LDL
Low density lipoprotein (LDL)
pH ~7-.7.2
A single receptor makes hundreds of trips (~10 min/cycle)
Adaptin complex binds endocytosis
LDL
signal in cytoplasmic domain of
receptor:
LDL-R
-NPXY- (Asn-Pro-Val-Tyr) in
Plasma membrane
LDL-R At least three different adaptin
George Palade’s Noble Prize Lecture 1974
Pulse-chase 分析发现了分泌蛋白的转运途径
放射自显影技术进一步确定转运定位
囊泡卸货需要细胞膜融合
细胞内运输模型
George Palade’s Noble Prize Lecture 1974
囊泡释放
A single coated pit has many different receptors and cargos
Low density lipoprotein (LDL)
LDL-R
1,000s of receptors of many types per coated pit…
Same coated pits used for pinocytosis!
Randy Shekman1975s年代开始对 囊 泡 转 运的机制感兴趣
选择酵母细胞作为模式生物
开展了被许多科学家认为是“愚蠢的实验”
HMSF 1
24 ºC
37 ºC for 1 hr
Secretion-defective mutant strain (sec 1-1)
发现23个与细胞分泌相关基因
Domains in LDL receptor
Based on MBoC (3) figure 13-53
NH2
LDL
N terminus of LDL receptor
LDL-R
binds apoprotein B in LDL
Plasma membrane
HOOC
Tyr Asn Val Pro
the morphology of a COPII vesicle budded from microsomes using purified coat proteins
COPII 有被小泡的组装激活
COPII 组装
COPII
COPII介导的内质网---高尔基复合体物质运输
COPI的分子基础
COPI= Coatomer Coatomer is a stable protein complex of seven subunits.
高尔基复合体的分选
Mannose-6-P targets proteins from Golgi to lysosome
Cis Golgi Network (CGN)
Addition of M6P
RER
Lysosomal hydrolase (precursor)
Trans Golgi Network (TGN)
Example: Low-density lipoprotein (LDL), structure in which cholesterol is transported through our bodies
Lipid micelle: ~800 phospholipids… ~500 molecules of cholesterol…
Hydrolytic enzymes cleave LDL, releasing cholesterol to cytoplasm for continued membrane biosynthesis in smooth ER
Receptor is recycled back to surface (cycles about every 10 min!)
Transport via clathrin-coated vesicles to… Lysosome
Clathrin coat
Uncoupling Mature (pH 5) hydrolase
M6P receptor
M6P receptor recycling back to Golgi
Removal of phosphate & proteolytic processing…
GDP
Explains why non-hydrolyzable GTP analogues block endocytosis
经由胞吞作用的网格蛋白有被小泡之形成过程
衣被组装及 货运选择
货运分子
有被小窝 的形成
有被小泡 的分离
有被小泡 的形成
脱衣被 释放小泡
网格蛋白有被小泡之来源与形成
网格蛋白
HOOC
Tyr Asn Val Pro
Endocytosis complexes; bind different signal endocytosis signals on receptors
Adaptin complex (four
polypeptides)
Adaptins recruit clathrin and initiate coated pit/vesicle formation
1. COPII有被囊泡 （coat protein complex II vesicle， COPII）
2. COPI 有被囊泡（ coat protein complex I vesicle，COPI ）
3. 网格蛋白有被囊泡（clathrin-coated vesicle， CCV）
1. COPII有被囊泡
~1500 molecules of cholesterol ester
1 copy of apoprotein B…
Total mass: ~ 3 x 106 Da
Overview of receptor-mediated uptake of LDL
ECB 15-32
Low pH of endosome (~6) causes LDL to dissociate from receptor LDL is transferred to lysosome (fusion of vesicles from TGN)
细胞内外物质交换和信号传递 细胞内物质定向运输
电镜观察发现囊泡系统
cv, condensing vacuoles gc, Golgi cisternae
gv, Golgi vesicles; te, transitional elements rer, rough endoplasmic reticulum
产生于内质网
特征：非网格蛋白有被囊泡 功能： 介导从内质网到高尔基复合体的物质转运
2. COPⅠ有被囊泡 特征：亦属于非网格蛋白有被囊泡类型 功能：负责内质网逃逸蛋白的捕捉、回收转运及高尔基复合
体膜内蛋白的逆向运输，行使从高尔基复合体到内质网的转移
产生于高尔基复合体 的COPⅠ有被囊泡
3. 网格蛋白有被囊泡 Clathrincoated vesicles (CCV)
囊泡转运
细胞的物流机制
一. George Emil Palade 1960s 发现了囊泡转运
囊泡转运（ vesicular transport）
囊泡（vesicle）：由细胞器膜外凸或质膜内凹芽生而成， 囊泡形成是一个主动的自我装配过程。
囊泡卸货: 囊泡移动，定向地与另一种细胞器膜或质膜
融合， 释放货物, 实现物质运输。
Electron micrographs of negatively stained Sec13/31 complex.
Schematic view of the 15 nm Sec23/24–Sar1 complex adhered to the surface of a 60 nm vesicle