8.2 a few approaches to study of endomembranes Autoradiography and* The use of GFP Palade& Jamieson: in the autoradiographic experiment of pancreatic tissue, using this approach, it was proved that er is the site of synthesis of secretory proteins and then pulse-chase experiment first defined the biosynthetic (secretory) pathway and tied a number of seemingly separate membranous compartments into an integrated functional unit "Biochemical analysis of subcellular fractions Vesicles are isolated from endomembranes by cell fractionation and then their composition and functional preperties can be determined by biochemical analysis and immuno-electromicroscopy. For example, enzymatic activities and the capacities of transporting newly synthesized proteins of the microsomes and the role of golgi complex in the stepwise assembly of complex carbohydrates *The use of genetic mutants in study of EMS a secretion study in yeast using temperature-sensitive mutants
8.2 a few approaches to study of endomembranes *Autoradiography and * The use of GFP Palade& Jamieson: in the autoradiographic experiment of pancreatic tissue, using this approach, it was proved that ER is the site of synthesis of secretory proteins and then “pulse-chase” experiment first defined the biosynthetic (secretory) pathway and tied a number of seemingly separate membranous compartments into an integrated functional unit. *Biochemical analysis of subcellular fractions Vesicles are isolated from endomembranes by cell fractionation and then their composition and functional preperties can be determined by biochemical analysis and immuno-electromicroscopy. For example, enzymatic activities and the capacities of transporting newly synthesized proteins of the microsomes and the role of Golgi complex in the stepwise assembly of complex carbohydrates. *The use of genetic mutants in study of EMS A secretion study in yeast using temperature-sensitive mutants
Rough endoplasmic reticulum Golgi vesicles Secretory Time of chase(min) o◎ Golgi complex Endoplasmic 3 20 min
容 015pm Figure 8 19 Regional differences in membrane compo- the medial cisternae. (c) The enzyme nucleoside diphos. sition across the Golgi stack. (a) Reduced osmium tetroxide phatase, which splits dinucleotides(e. g, UDP) after they preferentially impregnates the cis cisternae of the golgi ave donated their sugar, is preferentially localized in the complex. (b) The enzyme mannosidase Il, which is involved trans cisternae. (a, c: From Robert S. Decker, J Cell Biol. 61: 603 in trimming the mannose residues from the core oligosac- 1974; b: from Angel Velasco et al., J Cell Biol. 122: 41, 1993: all charide as described in the text, is preferentially localized in y copyright permission of thte Rockefeller LIniversity Press
司 The use of green fluorescent protein(GFP)reveals proteins within ali ing elf, t m p eite she ysw icmgrapab rc a the cultus 0 gene newly synthesized vsvG pcin fmm leaving the ER when kept at 40'C. The green hsed to GFP gene a live infected cell that was held at ane to allow the VSVG Protein to accumuaein the er and
8 Centrifug (b) Homogenate Whole cells, nuclei 动 Transfer postnuclear △ Figure 8.4 Isolation of a microsomal fraction by differ- somes)in the supernatant(step 2). The microsomes can be cal homogenization(step 1), the various membranous or ni. membranous vesicles Vesicles derived from different vesicle types in subsequent steps.(b) Electron micrograph is first subjected to low-speed centrifugation to pellet the vesicles lack ribosomes. (c) Electron micrograph of a rough microsomal fraction containing ribosome-studded larger, denser particles, leaving the smaller vesicles(micro- branes. (b, c: Courtesy of J. A. Higgins and R. I. Barnett)