By James E. Rothman (Eds.)
This quantity comprises sections on reconstitution in cell-free extracts, reconstitution utilizing semi-intact and perforated cells, and identity of shipping intermediates
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Additional info for Reconstitution of Intracellular Transport
32 R E C O N S T I T U T I O N IN C E L L - F R E E E X T R A C T S   R e c o n s t i t u t i o n o f E n d o s o m e F u s i o n : I d e n t i f i c a t i o n o f Factors Necessary for Fusion Competency By MARIA ISABEL COLOMBO, JAMES M. LENHARD, LuIs S. MAYORGA, and PHILIP D. STAHL Protein transport through the endocytic and exocytic pathways requires fusion of cartier vesicles with the appropriate target membrane? -3 The requirements for vesicle fusion have been partially delineated by the development of cell-free systems.
Keller and O. Touster, Z Biol. Chem. 250, 4765 (1975). ~5 F. L. Otsuka, M. J. Welch, K. D. McElvany, R. A. Nicolotti, and J. B. Fleischman, J. Nucl. Med. 25, 1343 (1984). 24 R E C O N S T I T U T I O N IN C E L L - F R E E E X T R A C T S  As shown in Fig. l, on internalization 125I-labeled anti-DNP IgG remains associated with the S. a u r e u s for approximately 5 - l0 min. Thereafter, the majority of 125I-labeled anti-DNP IgG quickly dissociates from the particle. The low concurrent rise in trichloroacetic acid (TCA)-precipitable radioactivity indicates that the majority of the 125I-labeled anti-DNP IgG that leaves the S.
2. Homogenize the cells by either the syringe method as previously described 7 or by use of a ball-bearing homogenizer. Phagosomes are more fragile than endosomes and more prone to breakage during homogenization. Thus, to prevent overhomogenization, stop homogenizing when 80% of the cells are broken (15- 30 passes through either device). 3. Remove nuclei by centrifuging homogenate at 200 g for 3 min at 4 °. 4. Postnuclear supernatants (200-#1 aliquots) may be frozen in liquid nitrogen for storage.
Reconstitution of Intracellular Transport by James E. Rothman (Eds.)