13-3. Synthesis-Secretory pathway: Sort lumenal proteins, Secrete proteins, Sort membrane proteins

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1 13-3. Synthesis-Secretory pathway: Sort lumenal proteins, Secrete proteins, Sort membrane proteins Molecular sorting: specific budding, vesicular transport, fusion 1. Why is this important? A. Form and maintain membrane identity: Formation of ER, Golgi, PM, Nuc envelope assembly b. Endocytic pathway: recycle membrane, transduce signals, nutrient uptake, defense against invading microbes. C. Exocytosis: synaptic transmission in neuron, polarized distribution of PM proteins Many cells show polarity. E.g. Intestinal epithelial cells Protein sorting in polarized epithelial cells. Cells can take up nutrients by endocytosis and sort the nutrients to the right place. E.g Receptor-mediated endocytosis. To bring cholesterol into cells. Yeast cell cycle Polarized secretion and cell surface growth 1

2 I. Vesicular Transport: 1. Budding: specific 2. Transport: targeted vesicle transport 3. Fusion: Recognition, binding and fusion II. Approaches/ Methods 1. Biochem: In vitro (reconstitution) assays to identify the players. by removing one at a time. (e.g. mutants, inhibitors, or physical removal), or adding one 2. Genetic: Yeast sec mutants deficient in secretion 3. Cell biol.- endocytosis of labeled ligand by living cell, and visualize internalized ligand by Immuno-Gold or flourescence. [To visualize, cell is fixed] 4. Transfection with GFP-tagged proteins! [living cell] Movie later Specificity? A. Markers on the cytosolic side of the membrane b. Sometimes it is not only 1 specific marker but a combinations of markers serve as the unique address. Budding with specificity Three types of coated vesicles 13-5 Subclasses within each type: each specialized for a different transport step Process: Assembly and disassembly of clathrin coat , Formation of clathrin-coated pit and vesicle. Gold-labeled anti-dynamin of nerve terminals treated with GTP-g-S 1. Adaptin binds coat 2. Adaptin binds to cargo receptor 3. Coat assembly introduce curvature to vesicle 4. Coat is nearly assembled 5. Vesicle pinches off with help of dynamin 6. After budding, coat disassembles Dynamin Mutant flies are paralyzed 2

3 ARF or Sar proteins : Coat recruitment GTPases. Uncoating: also depends on GTPase ARF-GTP --> ARF-GDP Coat dissociates and are released Budding: How does it start? steps for all types of coats: 1. Initiated by recruitment of GTP-binding protein ARF to membrane (due to Receptor or lipid). 2. GTP-bound protein recruits coat protein and adapter to form complex 3. [Coat protein or assembly factors binds to membrane cargo, and membrane receptor proteins] 4. Coat is assembled 5. Vesicle buds, pinches off Clathrin coat Clathrin: heavy chain 180 kd; and light chain 40 kd Assembly particles (adapters): proteins that link clathrin to membrane proteins. E.g. AP1, AP2, AP3. They bind to specific integral proteins. Dynamin: GTP-binding protein of kd How does sorting occur during budding?? Tab Sorting signals and receptors How does a transport vesicle recognize the right target membrane?? -SNARE -Targeting GTPases = Rabs Snares: provide specificity and catalyze fusion Rabs: control specificity of docking and tethering V-SNARE pairs with its complementary t-snare The pair must be separated before they can function again. > 20 SNARES Rab GTPases and their effectors determine compartment specificity Additional layer of regulation to ensure that t-snare fuse membranes only at the right time and in the correct place. Effectors: Protein(s) that bind a Rab-GTPase directly and is required for downstream function determined by that Rab. [e.g. tethering, motor proteins] Rabs Organelle Rab Effectors Function Rab1 ER-G p115 tethering Rab3a synaptic ves rabphilin 3 potentiates fusion Rab4 early endosome Rabaptin/NEF sorting, recycling Rab 6 G ->ER, intra G Rabkinesin6 vesicle motility 3

4 Docking by a specific Rab and its effector Rab Effector- docking protein Intracellular location of Rab proteins. e.g. Rab1-ER P115- Golgi Zerial & McBride 2001 Nat Rev MCB Rab domains on endosomes Snares may mediate fusion similar to viral fusion proteins. Zerial & McBride 2001 Summary How is vesicular transport specificity determined? 1. Budding a. Recognition of membrane proteins exposed to cytosolic side by ARF or Sar proteins b. Type of coat protein recruited and assembled 2. Transport motor -cargo recognition by motor receptor on the membrane 3. Docking and fusion a. Rab GTPases (distinct) and their specific effectors b. SNARES pairs Arf-, Sar-, Rab-GTPases serve as membrane organizers Movies from J. Lippicott-Schwartz. ER to Golgi Presley et al 1997 Nature Kinetics of Golgi to PM Hirschberg et al 1998 JCB VSVG-viral glycoprotein synthesized at ER, traffic to G, to PM. 1. VSVG-GFP gene is transfected into cell. 2. Hold cell at 40 C, VSVG-GFP synthesized at ER (but cannot move at this temp) or at 15 C, VSVG-GFP stay in pre-golgi 3. Shift to 32 C, VSVG will move to G and to PM 4

5 Fig. 1. Kinetics of VSVG- GFP transport. (A) VSVG- GFP expressing cells were incubated for 20 h at 40 C, shifted to 32 C, and then imaged every 30 s for 3 h using a confocal microscope. Shown are images at 0, 40, and 180 min after temperature shift. See Quicktime movie sequence at b7labob.html. The waves of VSVG-GFP seen propagating out from the Golgi region 60 min after temperature shift in the movie represent plasma membrane ruffling, possibly in response to the arrival at the cell surface of VSVG-GFP. Hirschberg et al J Cell Biol. Fig. 2. Kinetic modeling of VSVG- GFP transport through the secretory pathway. (A) The three compartment model for trafficking of VSVG- GFP. (B) VSVG-GFP-expressing cells incubated for 20 h at 40 C were shifted to 32 C and imaged every 120 s for 10 h. The fluorescent intensities with time within the Golgi ROI (solid circles) and total cell ROI (open circles) are plotted. The smooth curves show fit by the model solution. Hirschberg et al J Cell Biol Fig. 3. Effect of pharmacologic reagents on VSVG- GFP trafficking kinetics. (A) VSVG-GFP-expressing cells were incubated for 20 h at 40 C and shifted to 32 C in the presence of cytochalasin-b (10 µm). (C) Cells expressing VSVG-GFP were shifted to 32 C. AlF (AlCl3, 60 µm and NaF, 20 mm) or nocodazole (33.3 µm) was added after 40 min at 32 C. The images were collected at this time (40 min) or 140 min later (180 min). (D) Fluorescent intensities associated with the Golgi region in the experiment described in C are plotted against time. Fig. 6. Translocation of post-golgi carriers A. velocity B. Route of a single PGC C. Nocodazole inhibits. I.e. Microtubule-dep. Hirschberg et al ER align with microtubules in frog fibroblast. Evidence that ER vesicles move along microtubules. ER stained with DiOC Different kinesins transport different cargo. Antitubulin Kinesinmotored transport 5

6 Nature 2001-News about Fusion needs more than SNARES. Peters et al Nature. A. V-ATPase and Vo b. Dimer of Vo may form during vacuole fusion. Model of vacuole fusion by Peters et al Nature. A. Two fusing membranes are pinned together. B. Ring opens as c subunits separate. The fusion pore forms. C. Fusion pore expands 3-5. Structure of Ras bound to GDP. Molecular basis for specific binding interactions lies in the uneven distribution of + (blue) and -- (red) charges over the surface of the protein Release of neurotransmitter and recycling of synaptic vesicle GDP Rab GTPases represent a large family of Ras-like enzymes 6