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. 2002 Apr 1;157(1):45–62. doi: 10.1083/jcb.200112127

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Copyright © 2002, The Rockefeller University Press

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Figure 9. — Resolution of vesicle tethering and SNARE assembly during NSF-driven Golgi reassembly. (A) Morphology of NSF reaction products in the presence of various inhibitors. Note the presence of stacks of cisternae in the absence of inhibitor compared with dispersed tubules and vesicles in the MGFs (ice + fix) or in the presence of GM130 NT, and the highly clustered tubules and vesicles in the presence of His–GOS-28. Bar, 0.5 μm. (B) MGFs were incubated at 37°C for 1 h with NSF, SNAPs, and p115 (0.13 μM). Various inhibitors were added: GM130 NT (13 μM), Gtn1-448 (13 μM), GDI (14 μM), chrysin (120 μM), CC1 (13 μM), His–GOS-28 (6 μM), or His–syntaxin-5 (6 μM). Dilution (10×) was with reaction buffer containing NSF, SNAPs, and p115. Reactions were terminated by fixation, processed for EM, and the amount of relative cisternal regrowth was determined. 100% relative cisternal regrowth represents an increase from 25 to 75% of the total membrane present as cisternae. Values represent means ± SEM (n = 3). (C) Quantitation of density of tubules/vesicles per μm² for the reactions described in B. (D) Kinetic sensitivity of NSF reaction to various inhibitors. NSF reactions were performed as in B except that at the indicated times reactions were either stopped by fixation or treated as indicated and incubated for a total of 1 h at 37°C. Reactions were processed as in B. Values represent means ± SEM (n = 4). (E) A model depicting the proposed sequence of events during GOS-28–syntaxin-5 complex assembly in NSF-driven Golgi reassembly (see Discussion).