FIGURE 4.

In vitro post-Golgi vesicle clustering depends on the presence of GTP-Sec4 on vesicles. A, HSP fraction obtained from a sec6-4 mutant strain was treated with GDP (0.5 mm), MgCl₂ (3.6 mm, and GDI (8 mm) or a mock buffer for 30 min on ice. The vesicles were then purified on parallel 20–40% sorbitol velocity gradients. Vesicle-containing fractions from each column were subject to a second centrifugation to generate two concentrated vesicle fractions. GDI-treated and mock-treated samples were then incubated with MgCl₂ (3 mm) and GTPγS (1 mm) on ice for 30 min and then with Sro7 (1 μm) for 60 min at 27 °C. Results of the clustering assay were analyzed by fluorescence microscopy (A), and quantitation of clustering is shown in B. Scale bar, 2 μm. Vesicle amount used was monitored by Western blot analysis (C) using polyclonal antibody to vesicle markers Sec4 and Snc1/2. D, HSP fraction obtained from a sec6-4 mutant strain was incubated with equal amount of monoclonal anti-Sec4 or control monoclonal anti-Myc IgG for 1 h on ice before treating with MgCl₂ (3 mm) and GTPγS (1 mm) for 30 additional min on ice. The vesicles were then incubated with Sro7 (1 μm) for 20 min at 27 °C. The reaction was analyzed by fluorescence microscopy and quantitated as described previously. Scale bar, 2 μm.