Lecture9 - Lecture 9 4/27/10 Background Reading: MBOC 5th...

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Lecture 9 4/27/10 Background Reading: MBOC 5 th edition: Chapter 13: pages 749-766 Chapter 16: pages 965-991 MBOC 4 th edition: Chapter 13: pages 711-726 Chapter 16: pages 907-929 Outline: Mechanism of vesicle transport Monomeric GTPases Organelle biogenesis Cytoskeleton – Overview Intermediate filaments Microtubules Microtubule dynamics
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Mechanism of vesicle transport: Two main approaches to study: 1) Reconstitution studies: Used cell free systems, such as discussed last time, to deter- mine requirements for vesicular transport. Fractionated cytosol to find what components were required. Found required GTP, ATP and a number of specific proteins in the cytosolic fraction. Transport occurred only when ATP and cytosol were added. Can substitute cytosol from wide variety of sources. Thus, highly conserved process during evolution.
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2) Molecular Genetic approaches: Schekman was pioneer in these studies. Used yeast. Can easily grow, mutagenize and screen. ince secretory system so important, many mutants of it are lethal under ordinary conditions. Therefore used tempera- ture sensitive mutants. Cells grow normally at 25°C but lethal at 37°C. Using such mutants, Identified more than 25 genes. sing above biochemical and genetic approaches,identified many of the components involved in vesicular transport. Can divide into three separate processes: 1) Budding – vesicle formation 2) Docking – vesicle recognition 3) Fusion – fusion of vesicle with target compartment
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Lecture9 - Lecture 9 4/27/10 Background Reading: MBOC 5th...

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