Overview of the secretory pathway
Overview of the secretory pathway
Lodish et al, 2000
Deducing Protein Function A. Temporal and spatial correlations - provide first clues 1. RNA localization- Northern blot analysis, RT-PCR, in situ hybridization 2. Protein localization- Western blot analysis, immunofluorescence, epitope tagging, chimeric GFP B. Reconstitution in vitro - requires purified components C. Gene Manipulation 1. Overexpression- transfection, viral infection-- limitations 2. Misexpression- viral infection, oocyte injection 3. Gene disruption -Yeast -homologous recombination in mice- standard and conditional -RNA interference -dominant negative alleles 4. Mutational studies
Three major experimental systems contributed to our current understanding of the mechanism of membrane secretion. 1. Study of fusion of endosomal and Golgi membranes and Vesicles 1. Earliest reconstitution assay 2. Purification of first proteins
2. Study of synaptic vesicle release 1. Purification and Biochemical Analysis of Proteins 2. Mouse Genetics 3. Electrophysiology (measurement of fusion in real time)
3. Study of Yeast Vacuolar System 1. Genetic Screens and rapid Genetic Analysis 2. Biochemical Reconstitution Assays
Analysis of yeast mutants defined the major steps in the secretory pathway
Lodish et al, 2000
Regulated exocytosis underlies neural communication
Lodish et al, 2000
Overview of the secretory pathway
cisternal progression
Lodish et al, 2000
A cell-free assay for fusion between Golgi cisternae
Lodish et al, 2000
Early model for vesicle fusion
More current model for vesicle fusion
Lodish et al., 2000
SNARE complex formation can mix lipids
Protein free vesicles
Weber et al (1998). Cell 92: 759
Lodish et al, 2000
Deducing Protein Function A. Temporal and spatial correlations - provide first clues 1. RNA localization- Northern blot analysis, RT-PCR, in situ hybridization 2. Protein localization- Western blot analysis, immunofluorescence, epitope tagging, chimeric GFP B. Reconstitution in vitro - requires purified components C. Gene Manipulation 1. Overexpression- transfection, viral infection-- limitations 2. Misexpression- viral infection, oocyte injection 3. Gene disruption -Yeast -homologous recombination in mice- standard and conditional -RNA interference -dominant negative alleles 4. Mutational studies
Three major experimental systems contributed to our current understanding of the mechanism of membrane secretion. 1. Study of fusion of endosomal and Golgi membranes and Vesicles 1. Earliest reconstitution assay 2. Purification of first proteins
2. Study of synaptic vesicle release 1. Purification and Biochemical Analysis of Proteins 2. Mouse Genetics 3. Electrophysiology (measurement of fusion in real time)
3. Study of Yeast Vacuolar System 1. Genetic Screens and rapid Genetic Analysis 2. Biochemical Reconstitution Assays
Analysis of yeast mutants defined the major steps in the secretory pathway
Lodish et al, 2000
Regulated exocytosis underlies neural communication
Lodish et al, 2000
Overview of the secretory pathway
cisternal progression
Lodish et al, 2000
A cell-free assay for fusion between Golgi cisternae
Lodish et al, 2000
Early model for vesicle fusion
More current model for vesicle fusion
Lodish et al., 2000
SNARE complex formation can mix lipids
Protein free vesicles
Weber et al (1998). Cell 92: 759
