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Vesicles Transport Proteins from the Plasma Membrane and Trans-Golgi Network to Late Endosomes

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Vesicles Transport Proteins from the Plasma Membrane and Trans-Golgi Network to Late Endosomes Lodish • Berk • Kaiser • Krieger • Scott • Bretscher •Ploegh • Matsudaira MOLECULARMOLECULAR CELLCELL BIOLOGYBIOLOGY SIXTHSIXTH EDITIONEDITION CHAPTERCHAPTER 1414 VesicularVesicular Traffic,Traffic, Secretion,Secretion, andand EndocytosisEndocytosis ©Copyright 2008 W. 2008 H. © FreemanW. H. Freeman and and Company Company Outline: 1. Techniques for studying the secretory pathway 2. Molecular mechanisms of vesicular traffic 3. Vesicular trafficking in the early stages of the secretory pathways 4. Protein sorting and processing in late stages of the secretory pathways 5. Receptor mediated endocytosis SEM of the formation of 6. Directing membrane proteins and clathrin-coated vesicles on the cytosolic face of the plasma cytosolic materials to the lysosome membrane Secretory pathway: protein to various organelles by transport vesicles Anterograde: forward moving Retrograde: backward moving Trans position: farthest from the ER Cis position: nearest the ER Cisternal progression: cis-Golgi cisterna → cargo of protein → move form cis → medial → trans ; anterograde transport vesicle; normal TGN (trans Golgi network): proteins not transport to ER or Golgi, are destined for compartment to others (by different types of vesicles) 1. from trans → fuses membrane → trnasport → exocytosis 2. from trans → stored inside → formation of secretory vesicles; release by signal for exocytosis 3. from trans → late endosome → lysosome (intracellular degradation of organelle) the mechanism not well know endosome had endocytic pathway, from the plasma membrane bringing membrane proteins and their bound ligands into the cell Overview of major protein-sorting pathways in eukaryote (protein targeting) No signal peptide Overview of secretory & endocytic pathways: Transport vesicles transport vesicle cargo proteins same orientation anterograde transport vesicles retrograde transport vesicles cisternal progression trans-Golgi network (TGN) secretory vesicle (regulated..) constitutive secretion-exocytosis transport vesicle-late endosome endocytosis Techniques for studying the secretory pathway: Pulse-chase labeling & EM autoradiography Animal + radio AA → different time → kill → chemical fix → autoradiography Tissue sections of pancreas acinar cells -> a brief incubation (3 min) with H3-Leucine -> transfer to unlabeled medium & incubate for a period of time (0, 7, 37, 117 min) -> cover tissue sections with photographic emulsion - > EM Pulse-chase exp Low density lipoprotein receptor 脈搏 補捉 To investigate the fate of a specific newly synthesized protein Cell + isotope for 0.5h degrade ↓ wash <0.5h, protein convert to mature Different time point PTM ↓ Glyco.. Immunoprecipitation ↓ Specific protein ↓ SDS-PAGE ↓ Techniques for studying the secretory pathway: Use of temperature-sensitive mutant proteins (e.g. vesicular stomatitis virus 水疱口炎病毒 VSV G protein) At restrictive temp. of 40oC, newly made G protein is misfolded & retained within ER. At permissive temp. of 32oC, accumulated G protein is correctly folded & transported through secretory pathway. Different time course → change Temp → misfolded → stop transport Palade’s early exp had found that in mammalian, vesicle mediated transport of a protein molecule from ER to membrane about 30-60 min. Techniques for studying the secretory pathway: by living cells 1. Transport of a protein through the secretory pathway can be assayed in living cells: 1) Microscopy of GFP-labeled VSV G protein 2) Detection of compartment-specific oligosaccharide modifications 2. Yeast mutants define major stages and many components in vesicular transport 3. Cell-free transport assays allow dissection of individual steps in vesicular transport Microscopy of GFP-labeled VSV G protein Use temperature-sensitive mutant, Plasma membrane VSVG-GFP. 40oC the protein in ER 32oC move → Golgi → plasma membrane→ Form ER to Golgi about 60min Protein transport through the secretory pathway can be visualized by fluorescence microscopy of cells producing a GFP-tagged membrane protein: VSV G protein Techniques for studying the secretory pathway: 1. Transport of a protein through the secretory pathway can be assayed in living cells: 1) Microscopy of GFP-labeled VSV G protein 2) Detection of compartment-specific oligosaccharide modifications 2. Yeast mutants define major stages and many components in vesicular transport 3. Cell-free transport assays allow dissection of individual steps in vesicular transport Transport of a membrane glycoprotein from the ER to golgi can be assayed based on sensitivity to cleavage by endoglycosidaseD 電泳分離時 分子量大 分子量小 分子量大 分子量小 Cleavage by endoglycosidase D Cell expression VSV G protein → at Temp 40 → link radioactive aa and protein keep in ER → Tem 32 C → VSV From ER to golgi about 60 min G extracted → digested by endoglycosidase (about cis Golgi protein) → SDS electrophoresis Endoglycosidase can not cleavage ER’s ER to golgi protein. 32 C: protein move from ER → Golgi (modification) → membrane In ER 40 C: in ER not move. Did not cleavage by endoglycosidase Protein folding ok → move → golgi → can cleavage Addition & processing of N-linked oligosaccharides in R-ER of vertebrate cells 酶的反應是有其專一性,其反應物必需是特定的,缺一不可 Remove 2 mannose Add Remove 3 mannose In cis, specific glycosidase Cleavage by endoglycosidase D. • glycosidases (cis-) • endoglycosidase D Processing of N-linked oligosaccharide chains on glycoproteins within cis-, medial-, and trans-Golgi cisternae in vertebrate cells Techniques for studying the secretory pathway: 1. Transport of a protein through the secretory pathway can be assayed in living cells: 1) Microscopy of GFP-labeled VSV G protein 2) Detection of compartment-specific oligosaccharide modifications 2. Yeast mutants define major stages and many components in vesicular transport 3. Cell-free transport assays allow dissection of individual steps in vesicular transport Yeast sec (secretion) mutants protein The temperature sensitive mutant → grouped into 5 classes Combination of different mutant → for research of protein transport pathway, ie BD → protein in ER not Golgi → so ER is before, and Golgi is after. 利用到達的時間去計算 These studies confirmed that: cytosol → RER → ER-to Golgi transport vesiceles → Golgi cisternce → secretory → exocytosed Phenotypes of yeast sec mutants identified stages in the secretory pathway Techniques for studying the secretory pathway: 1. Transport of a protein through the secretory pathway can be assayed in living cells: 1) Microscopy of GFP-labeled VSV G protein 2) Detection of compartment-specific oligosaccharide modifications 2. Yeast mutants define major stages and many components in vesicular transport 3. Cell-free transport assays allow dissection of individual steps in vesicular transport Cell-free transport assay To plasma membrane Can not add Protein transport from Golgi cisternae to another can be assayed in a cell-free system Protein need modification in Golgi Proof: golgi can retrograde vesicular transport for modification Normal expression it demonstrated protein transport from one golgi cisterna to another Tradional Model - Golgi is a static organelle. Secretory proteins move forward in small vesicles. Golgi resident proteins stay where they are. Two Models For Cis to Trans-Golgi Progression “Radical” Model - Golgi is a dynamic structure. It only exists as a steady-state representation of transport intermediates. Secreted molecules move ahead with a cisterna. Golgi resident proteins move backward to stay in the same relative position. 問題: 到底細胞內利用vesicle的方式的機轉是什麼? Molecular mechanisms of vesicular traffic Vesicle transport: from organelle (Donor) target organelle (a) Coated vesicle: From membrane interaction with integral (b) Uncoated vesicle: Target membrane vSNARE: Crucial to fusion of the vesicle with correct target membrane tSNARE: specific joining of vSNARE Overview of vesicle budding and fusion with target membrane Assembly of a protein coat drives vesicle formation & selection of cargo molecules. A conserved set of GTPase switch proteins controls assembly of different vesicle coats Three types of coated vesicles have been characterized. All need GTP binding antrograde retrograde To endosome GTPase superfamily ARF (ADP Ribosylation Factor) Different coated proteins Clathrin and adapter protein (AP): vesicles transport proteins from the plasma membrane and trans-Golgi network to late endosomes – With AP1: Golgi to endosome – With AP2: Endocytosis (PM to endosome) – With AP3: Golgi to lysosome and other vesicles COPI: Golgi to ER (retrograde transport) COPII: ER to Golgi (antrograde trnasport) AP: complex consists of four different subunits Vesicle buds can be visualized during in vitro budding reactions. Coated vesicles Artifical membranes and purified coat protein (COP II) → polymerization of coat protein onto the cytosolic face of the parent membrane A conserved set of GTPase switch proteins controls assembly of different vesicle coats. All three coated vesicles contain a small GTP-binding protein COP I and clathrin vesicle: ARF (ADP-ribosylation factors) COP II vesicle: Sar I protein ARF and Sar I protein can switch GTP (GDP-protein → GTP-protein active; GTPase) There two sets of small GTP-binding proteins for vesicle secretion. One is ARF and Sar I; another is Rab protein ARF (ADP Ribosylation Factor) protein exchanges bound GDP for GTP and then binds to its receptor on Golgi membrane A conserved set of GTPase switch proteins controls assembly of different vesicle coats. COPII coated formation GTP → Sar1 conformational change →Sar1-GTP binding to membrane → polymerization
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