Labrousse A M, Shurland D L, van der Bliek A M
Department of Biological Chemistry, University of California Los Angeles School of Medicine, Los Angeles, California 90095, USA.
Mol Biol Cell. 1998 Nov;9(11):3227-39. doi: 10.1091/mbc.9.11.3227.
Caenorhabditis elegans dynamin is expressed at high levels in neurons and at lower levels in other cell types, consistent with the important role that dynamin plays in the recycling of synaptic vesicles. Indirect immunofluorescence showed that dynamin is concentrated along the dorsal and ventral nerve cords and in the synapse-rich nerve ring. Green fluorescent protein (GFP) fused to the N terminus of dynamin is localized to synapse-rich regions. Furthermore, this chimera was detected along the apical membrane of intestinal cells, in spermathecae, and in coelomocytes. Dynamin localization was not affected by disrupting axonal transport of synaptic vesicles in the unc-104 (kinesin) mutant. To investigate the alternative mechanisms that dynamin might use for translocation to the synapse, we systematically tested the localization of different protein domains by fusion to GFP. Localization of each chimera was measured in one specific neuron, the ALM. The GTPase, a middle domain, and the putative coiled coil each contribute to synaptic localization. Surprisingly, the pleckstrin homology domain and the proline-rich domain, which are known to bind to coated-pit constituents, did not contribute to synaptic localization. The GFP-GTPase chimera was most strongly localized, although the GTPase domain has no known interactions with proteins other than with dynamin itself. Our results suggest that different dynamin domains contribute to axonal transport and the sequestration of a pool of dynamin molecules in synaptic cytosol.
秀丽隐杆线虫的发动蛋白在神经元中高表达，在其他细胞类型中低表达，这与发动蛋白在突触小泡循环中所起的重要作用相一致。间接免疫荧光显示，发动蛋白集中在背侧和腹侧神经索以及富含突触的神经环中。与发动蛋白N端融合的绿色荧光蛋白（GFP）定位于富含突触的区域。此外，在肠细胞的顶端膜、受精囊和体腔细胞中也检测到了这种嵌合体。在unc-104（驱动蛋白）突变体中，破坏突触小泡的轴突运输并不影响发动蛋白的定位。为了研究发动蛋白可能用于转运到突触的其他机制，我们通过与GFP融合，系统地测试了不同蛋白结构域的定位。在一个特定的神经元ALM中测量了每个嵌合体的定位。GTP酶、中间结构域和假定的卷曲螺旋结构域都对突触定位有贡献。令人惊讶的是，已知与包被小窝成分结合的普列克底物蛋白同源结构域和富含脯氨酸的结构域对突触定位没有贡献。GFP-GTP酶嵌合体的定位最强，尽管GTP酶结构域除了与发动蛋白本身外，与其他蛋白质没有已知的相互作用。我们的结果表明，不同的发动蛋白结构域有助于轴突运输以及发动蛋白分子在突触胞质溶胶中的隔离。
