The function and expression-mechanism of growth-associated protein GAP-43 in neuron

GAP-43是一种神经特异性蛋白，主要定位于轴突生长锥和突触前终末膜，但其确切的功能是核，它通过两个位于3和4位的酰化的Cys附着在突触前膜上。各质粒转染COS-1或PC12细胞后，通过细胞分离和免疫荧光染色检测野生型和突变型（C3C4→A3A4和C3→A3） GAP-43的定位。突变体（C3C4→A3A4）主要在细胞质部分和细胞质部分恢复，并在神经突染色。相比之下，在这两种细胞中都检测到突变体（C3→A3）。因此，这些Cys的脂肪酰化对于将GAP-43靶向到生长锥或突触前膜是必不可少的。此外，为了鉴定与GAP-43相互作用的组分，我使用了酵母双杂交系统。然后我在这个屏幕上得到钙调蛋白和肌凝蛋白轻链，并通过再转化确认为阳性克隆。此外，体外实验表明，GAP-43与p115/TAP相互作用，参与细胞内囊泡胞吞。综上所述，这些结果表明，由于我们已经报道了GAP-43与SNARE复合物的相互作用，因此GAP-43参与突触囊泡胞吐和突触前膜活性区神经递质释放的调节。

GAP-43 is a nerve-specific protein that is a predominantly localized in the axonal growth cones and presynaptic terminal membrane, but its precise function is nuclear, GAP-43 is attached to the presynaptic membrane by two acylated Cys at positions 3 and 4. The localization of wild and mutated (C3C4→A3A4 and C3→A3) GAP-43 was examined by cell-fractionation and immunofluorescence staining after transfection with each plasmid in COS-1 or PC12 cells. The mutant (C3C4→A3A4) was mainly recovered in the cytoplasmic fraction and fraction and stained at the neurites. In contrast, the mutant (C3→A3) was detected in both of them. Therefore it was shown that the fatty acylation of these Cys is essential for targeting GAP-43 to the growth cones or presynaptic membrane. Moreover, to identify the components interacted with GAP-43 I have used the yeast two-hybrid system. Then I obtained calmodulin and myosin light-chain in this screen and confirmed as the positive clones by retransformation. In addition, the experiments in vitro revealed that GAP-43 interacted p115/TAP which is implicated in the intracellular vesicular transcytosis. Taken together, these results suggested that GAP-43 is participated in the regulation of synaptic vesicle exocytosis with neurotransmitter release at the active zone of presynaptic membrane, since we have reported the interaction of GAP-43 with SNARE complex.