复旦大学 2013现代生物学基础与前言i【课件全】9203361.ppt

How is membrane traffic studied? -Microscopy -Yeast genetics -Biochemistry How is membrane traffic studied? -Microscopy -Biochemistry -purification of neural synaptic vesicles -in vitro reconstitution -Yeast genetics Studying vesicular transport in test tubes(Biochemistry) Biochemistry: in vitro reconstitution Compartment A + Compartment B (donor) (acceptor) + “goodies” in the cytosol reconstitute transport (monitored by protein (e.g.viral protein) glycosylation) In vitro reconstitution of Golgi vesicle transport In vitro reconstitution of Golgi vesicle transport Lipid Mixing Assay Experimental procedures: (1) Purify v-/t-SNAREs (2) Mix SNAREs with lipid mixture: t:85%PC+15%PS, v:82%PC/15%PS/1.5%NBD-PE/1.5%Rh-PE (3) Dilute and remove detergent to form vesicles (4) Isolate proteoliposomes by Nycodenz gradient (5) Mix v-SNARE vesicle and t-SNARE vesicle and monitor NBD fluorescence (Ex:485nm/Em:530nm) under different conditions (6) Terminate reaction by adding detergent to reach maximum signals, for normalization How is membrane traffic studied? -Microscopy -Biochemistry -purification of neural synaptic vesicles -in vitro reconstitution -Yeast genetics Membrane Traffic in Eukaryotic Cells Nucleus Budding Yeast The budding yeast Saccharomyces cerevisiae as a model system to study membrane trafficking Easy to culture. Powerful genetics. Conserved membrane trafficking pathways. Yeast secretion Cell, S116:S13-S15. Electron microscopy of wild-type yeast cells sec mutants accumulate post-Golgi secretory vesicles Protein Transport in Living Cells Studying the secretory pathway Biochemistry (Rothman, Blobel, Scheller, Jahn…) Genetics (Schekman, Novick, Emr, Wickner….) Imaging (Palade, DeCamilli…..) Green Fluorescent Protein (GFP) Isolated from fluorescent jellyfish Used for live imaging of intracellular molecules for more than 15 years now X Fluorescent Protein (XFP) R