Lect. 10 F10 - Vesicular Transport TODAY-VESICULAR...

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Vesicular Transport
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TODAY-VESICULAR TRANSPORT!! GATED TRANSPORT VESICULAR TRANSPORT TRANSMEMBRANE TRANSPORT
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Vesicular Transport Pathways protein transport between series of membrane bound organelles and the extracellular world 1) Biosynthetic/Secretory: ER Golgi Lysosome or ER Golgi Plasma membrane 2) Endocytosis: Plasma membrane Endosome Lysosome 3) Retrieval: Membrane/protein returns to compartment of origin, maintains unique identity
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George Palade: Combined pulse/chase autoradiography with electron microscopy to follow pathway as proteins secreted from cell. Pancreatic acinar cells-virtually all newly synthesized protein will be secreted. Shared the Nobel Prize in 1974. 1.Pulse-Incubate cells in radioactive amino acids for several minutes. Label incorporated into growing polypeptide chains, making new proteins radioactive. After short pulse labeled proteins will be at or near site of synthesis. 2.Chase-incubate cells in non-radioactive growth medium. Replace radioactive amino acids with nonradioactive amino acids. Only radiolabeled protein is that made during the pulse. 3.Follow protein movement through the cell. Radioactive proteins detected using autoradiography-particle emissions hitting photographic emulsion produce small black silver grains. Tissue preparation for EM overlaid with photographic emulsion-can now see both internal structure of cells and site of radiolabeled protein. Experimental demonstration of protein movement through the secretory pathway
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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 min 20 min 120 min Jamieson & Palade: Results of their classic 1967 pulse/chase experiments. J. Cell Biology 34: 577-596 3 min: After 3 min pulse, labeled protein (red) visualized in ER 20 min: After 3 min pulse and 17 min chase, label protein predominately in Golgi. 120 min: After 3 min pulse and 117 min chase, labeled protein accumulates in secretory vesicles underneath plasma membrane.
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Visualization of protein trafficking using GFP-tagged proteins. Vesicular stomatitis virus coat protein fused to CFP-normally goes to plasma membrane This movie shows mutant VSV coat protein that only leaves ER at low temperature. After temperature shift, CFP fusion first leaves ER to Golgi, then from Golgi to cell surface. Microtubule tracks required for both ER Golgi and Golgi PM transit. QuickTimeª and a Sorenson Video 3 decompressor are needed to see this picture.
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How do vesicles pinch off (bud)?    How are vesicles targeted to their destination?   How do they fuse with their target membrane? Transport vesicles ferry specific cargo to specific compartments
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Lect. 10 F10 - Vesicular Transport TODAY-VESICULAR...

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