Figure 5.

The polybasic motif in the C2A domain of Syt IV functions in calcium-regulated secretion. (A) The indicated recombinant GST fusion proteins were immobilized on glutathione–agarose and incubated with 800 μg of rat brain synaptosomes in the presence (+) or absence (−) of 3 mM calcium. Protein complexes were isolated, fractionated by SDS-PAGE, and examined by Western analysis. Stx 1a binding was detected with HPC-1, followed by enhanced chemiluminescence. One hundred nanograms of soluble Stx 1a (Con) were used as a Western control. (B) NGF-differentiated PC12 cells were coinjected with Texas Red–conjugated dextran and the indicated soluble recombinant Syt IV or Nedd4 fragments. Control cells were injected with Texas Red–conjugated dextran only (Texas Red) or coinjected with a control GST extract (GST). One hour after microinjection, the cells were K⁺ depolarized in the presence of calcium, and DβH surface immunoreactivity was detected with a fluorescein-labeled secondary antibody. The numbers of individual fluorescent particles were counted, regardless of their size, and are presented as a percent of the total number of cells injected. Data are shown as the mean of two independent experiments ± SD with the total number of injected cells (n). Significant differences (p ≤ 0.01, χ² analysis) between experimental and control treatments are indicated with an asterisk.